Insecticidal proteins and methods of use thereof

Abstract

This disclosure provides compositions and methods for controlling pests. The methods involve transforming organisms with nucleic acid sequences encoding insecticidal proteins. Specifically, the nucleic acid sequences can be used to prepare plants and microorganisms with insecticidal activity. Therefore, this disclosure provides transformed bacteria, plants, plant cells, plant tissues, and seeds. The compositions are insecticidal nucleic acids and proteins of bacterial strains. The sequences can be used to construct expression vectors for subsequent transformation into organisms of interest, including plants, and as probes for isolating other homologous (or partially homologous) genes. The insecticidal proteins can be used to control, kill, or inhibit the growth of the following pest populations: Lepidoptera, Coleopteran, Dipteran, fungi, Hemipteran, and nematodes, and can also be used to produce compositions with insecticidal activity.

Description

[0001] References to sequence lists submitted electronically

[0002] A sequence list named "5345PCT_sequence_listing.txt", created on August 28, 2014, and measuring 576 kilobytes in size, is submitted with this specification in a computer-readable form. This sequence list is part of this specification and is incorporated herein by reference in its entirety. Technical Field

[0003] This disclosure relates to the field of molecular biology. This disclosure provides novel genes encoding insecticidal proteins. These insecticidal proteins, along with the nucleic acid sequences encoding them, can be used to prepare insecticides and to produce transgenic insect-resistant plants. Background Technology

[0004] Biological control of agriculturally significant insect pests using microbial agents such as fungi, bacteria, or other insects offers an environmentally friendly and commercially attractive alternative to synthetic chemical pesticides. Generally, the use of biopesticides poses a lower risk of pollution and environmental harm, and they provide higher target specificity than traditional broad-spectrum chemical insecticides. Furthermore, biopesticides are often cheaper to produce, thus increasing the economic yield of many crops.

[0005] Certain species of Bacillus microorganisms are known to possess insecticidal activity against a range of insect pests, including Lepidoptera, Diptera, Coleoptera, Hemiptera, and others. Bacillus thuringiensis (Bt) and Bacillus popilliae are representative of the most successful biocontrol agents discovered to date. Insect pathogenicity has long been thought to be caused by strains of bacteria such as Bacillus larvae, Bacillus lentimorbus, Bacillus sphaericus, and Bacillus cereus. Microbial insecticides, particularly those derived from Bacillus strains, have played a significant role in agriculture as an alternative to chemical pest control.

[0006] In this field, crops have been genetically engineered to produce insecticidal proteins secreted by Bacillus subtilis, thereby developing crops with enhanced insect resistance. For example, corn and cotton plants have been genetically engineered to produce insecticidal proteins isolated from Bt strains. These genetically engineered crops are now widely used in agriculture, providing farmers with an environmentally friendly alternative to traditional insect control methods. While these genetically engineered insect-resistant crops have proven commercially successful, they are resistant only to a narrow range of economically important insect pests. In some cases, insects can develop resistance to different insecticidal compounds, necessitating the search for alternative biological control agents to manage pests.

[0007] Therefore, there is still a need for new insecticidal proteins with different ranges of insecticidal activity against insect pests, such as insecticidal proteins that are active against a variety of insects in the Lepidoptera and Coleoptera orders, including but not limited to insect pests that have developed resistance to existing insecticides. Summary of the Invention

[0008] This disclosure provides compositions and methods for conferring insecticidal activity to bacteria, plants, plant cells, tissues, and seeds. The compositions comprise nucleic acid molecules encoding sequences of insecticidal and insecticidal polypeptides, a vector containing those nucleic acid molecules, and a host cell containing said vector. The compositions also comprise insecticidal polypeptide sequences and antibodies against those polypeptides. The nucleic acid sequences can be used in DNA constructs or expression cassettes for transformation and expression in organisms, including microorganisms and plants. The nucleotide or amino acid sequences can be synthetic sequences designed for expression in organisms, including but not limited to microorganisms or plants. The compositions also comprise transformed bacteria, plants, plant cells, tissues, and seeds.

[0009] Specifically, this disclosure provides isolated or recombinant nucleic acid molecules encoding a Pseudomonas insecticidal protein-72 (PIP-72) polypeptide, the polypeptide comprising amino acid substitutions, amino acid deletions, amino acid insertions, amino acid fragments, and combinations thereof. Furthermore, this disclosure also covers the amino acid sequence corresponding to the PIP-72 polypeptide. This disclosure provides isolated or recombinant nucleic acid molecules capable of encoding the PIP-72 polypeptide shown in SEQ ID NO: 849, and amino acid substitutions, amino acid deletions, amino acid insertions, amino acid fragments, and combinations thereof. This disclosure also covers nucleic acid sequences complementary to the nucleic acid sequences in the embodiments herein, or nucleic acid sequences hybridized to the sequences in the embodiments herein. This disclosure also provides isolated or recombinant PIP-72 polypeptide shown in SEQ ID NO: 849, and amino acid substitutions, amino acid deletions, amino acid insertions, amino acid fragments, and combinations thereof.

[0010] This disclosure provides methods for producing the aforementioned polypeptides, and methods for using these polypeptides to control or kill lepidopteran, coleopteran, nematode, fungal, and / or dipteran pests. The transgenic plants of the embodiments express one or more of the insecticidal sequences disclosed herein. In various embodiments, the transgenic plants also contain one or more additional genes for insect resistance, for example, one or more additional genes for controlling coleopteran, lepidopteran, hemiptera, or nematode pests. Those skilled in the art will understand that the transgenic plants may contain any genes conferring agronomic traits of interest.

[0011] The disclosure also includes methods for detecting nucleic acids and peptides of the embodiments described above in a sample. A kit is also provided for detecting the presence of the PIP-72 peptide, or the presence of a nucleotide sequence encoding the PIP-72 peptide, in a sample. The kit is provided together with all reagents and control samples required to perform the method for detecting the expected factor, as well as instructions for use.

[0012] The compositions and methods described herein can be used to produce organisms with enhanced resistance or tolerance to pests. These organisms and compositions containing them are of great value to agricultural production. The compositions described herein can also be used to produce modified or improved proteins with insecticidal activity, or to detect the presence of PIP-72 peptides or nucleic acids in products or organisms. Attached Figure Description

[0013] Figure 1The amino acid sequence alignments of the following substances are shown: PIP-72Aa (SEQ ID NO: 2), PIP-72Ba (SEQ ID NO: 4), PIP-72Ca (SEQ ID NO: 6), PIP-72Cb (SEQ ID NO: 8), PIP-72Da (SEQ ID NO: 10), PIP-72Db (SEQ ID NO: 12), PIP-72Dc (SEQ ID NO: 14), PIP-72Ea (SEQ ID NO: 16), PIP-72Fa (SEQ ID NO: 18), GBP_A3175 (SEQ ID NO: 20), SRBS_294080 (SEQ ID NO: 22), JG43047 (SEQ ID NO: 24), Swirh_4910 (SEQ ID NO: 26), PIP-72Ff (SEQ ID NO: 28), PFL_6283 (SEQ ID NO: 28). The sequences identified are PIP-72Aa (SEQ ID NO: 30), PIP-72Gb (SEQ ID NO: 32), XBJ1_1078 (SEQ ID NO: 34), plu2373 (SEQ ID NO: 36), and PIP-72Ge (SEQ ID NO: 38). Sequence differences are highlighted in the figure. Amino acids 37 to 51 (motif 1) in PIP-72Aa (SEQ ID NO: 2) are underlined.

[0014] Figure 2 The amino acid sequence alignments of the following substances are shown: PIP-72Aa (SEQ ID NO: 2), PIP-72Ab (SEQ ID NO: 927), PIP-72Ba (SEQ ID NO: 4), PIP-72Bb (SEQ ID NO: 928), PIP-72Ca (SEQ ID NO: 6), PIP-72Cb (SEQ ID NO: 8), WP_030131237 (SEQ ID NO: 929), PIP-72Da (SEQ ID NO: 10), PIP-72Db (SEQ ID NO: 12), PIP-72Dc (SEQ ID NO: 14), PIP-72Fa (SEQ ID NO: 18), and GBP_A3175 (SEQ ID NO: 20). Amino acid differences between PIP-72Aa (SEQ ID NO: 2) and its homologs are highlighted with shading.

[0015] Figure 3The amino acid sequence alignments of the following substances are shown: PIP-72Aa (SEQ ID NO: 2), PIP-72Ba (SEQ ID NO: 4), PIP-72Ca (SEQ ID NO: 6), PIP-72Cb (SEQ ID NO: 8), PIP-72Da (SEQ ID NO: 10), PIP-72Db (SEQ ID NO: 12), and PIP-72Dc (SEQ ID NO: 14). Amino acid differences between PIP-72Aa (SEQ ID NO: 2) and its homologs are highlighted with shading.

[0016] Figure 4 The amino acid sequence alignments of the following substances are shown: WP_030131237 (SEQ ID NO: 929), PIP-72Ca (SEQ ID NO: 6), PIP-72Cb (SEQ ID NO: 8), PIP-72Da (SEQ ID NO: 10), PIP-72Db (SEQ ID NO: 12), and PIP-72Dc (SEQ ID NO: 14). The amino acid differences between PIP-72Da (SEQ ID NO: 10) and its homologs are highlighted with shading.

[0017] Figure 5 The amino acid sequence alignments of the following substances are shown: PIP-72Fh (SEQ ID NO: 932), PIP-72Gi (SEQ ID NO: 941), PIP-72Fi (SEQ ID NO: 933), PIP-72Gl (SEQ ID NO: 944), and PIP-72Fa (SEQ ID NO: 14). The amino acid differences between PIP-72Ca (SEQ ID NO: 2) and its homologs are highlighted with shading.

[0018] Figure 6The TOGH efficacy results of events generated by the constructs PHP61664, PHP61666, PHP61668, PHP64465, PHP64468, PHP64471, and PHP69828 are shown. The efficacy of events derived from these constructs compared to negative control events was observed based on measurements of root protection against western maize rootworm damage. Root protection was measured using the method developed by Oleson et al. 2005 [J. Econ Entomol. 98(1): 1-8], based on the number of damaged root nodes (CRWNIS = maize rootworm damage score). Root damage scores were measured from “0” to “3”, where “0” indicates no visible root damage, “1” indicates one damaged root node, “2” indicates two damaged root nodes, and “3” indicates three damaged root nodes (highest score). Each symbol (triangle, square, or circle) in the figure represents a single event. Detailed Implementation

[0019] It should be understood that this disclosure is not limited to the specific methods, protocols, cell lines, genera, and reagents described, as these can vary. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of this disclosure.

[0020] Unless the context clearly specifies otherwise, the singular forms “a,” “an,” and “the” used herein include multiple referents. Thus, for example, reference to “a cell” includes multiple such cells, and reference to “the protein” includes reference to one or more proteins, as well as their equivalents known to those skilled in the art, etc. Unless otherwise expressly stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains.

[0021] This disclosure relates to compositions and methods for controlling pests. These methods involve transforming organisms with a nucleic acid sequence encoding a PIP-72 polypeptide. Specifically, the nucleic acid sequence of the embodiments can be used to prepare plants and microorganisms with insecticidal activity. Therefore, this disclosure provides transformed bacteria, plants, plant cells, plant tissues, and seeds. The compositions are insecticidal nucleic acids and proteins of bacterial strains. The nucleic acid sequence can be used to construct expression vectors for subsequent transformation into organisms of interest, can be used as a probe for isolating other homologous (or partially homologous) genes, and can be used to produce altered PIP-72 polypeptides by methods known in the art (e.g., site-directed mutagenesis, domain exchange, or DNA shuffling). The PIP-72 polypeptide can be used to control or kill populations of lepidopteran, coleopteran, dipteran, fungal, hemiptera, and nematode pests, and can be used to produce compositions with insecticidal activity. Insect pests of interest include, but are not limited to, Lepidoptera species, including but not limited to: diamondback moths, such as the grain cutworm (Helicoverpa zea Boddie); soybean cutworms, such as the soybean looper (Pseudoplusia includens Walker); soybean hairy moths, such as the soybean cutworm (Anticarsia gemmatalis Hübner); and Coleoptera species, including but not limited to: western corn rootworm (Diabrotica virgifera) - WCRW, southern corn rootworm (Diabrotica undecimpunctata howardi) - SCRW, and northern corn rootworm (Diabrotica barberi) - NCRW.

[0022] The term "insecticide toxin" or "insecticide protein" as used herein refers to a toxin or protein homologous to one or more pests (including, but not limited to, members of the orders Lepidoptera, Diptera, Hemiptera, and Coleoptera or Nematoda). Insecticide proteins have been isolated from organisms including, for example, species of Bacillus, Pseudomonas, Photorhabdus, Xenorhabdus, Clostridium bifermentans, and Paenibacillus popilliae. Insecticidal proteins include, but are not limited to: insecticidal proteins from Pseudomonas species, such as PSEEN3174 (Monalysin; (2011) PLoS Pathogens 7: 1-13); insecticidal proteins from biocontrol Pseudomonas protegens strains CHA0 and Pf-5 (formerly known as fluorescent Pseudomonas) (Pechy-Tarr, (2008) Environmental Microbiology 10: 2368-2386; GenBank accession number EU400157); insecticidal proteins from Pseudomonas Taiwanensis (Liu et al., (2010) J. Agric. Food Chem., 58: 12343-12349); and insecticidal proteins from Pseudomonas pseudoalcligenes (Zhang et al., (2009) Annals of Microbiology 59:45-50; and Li et al., (2007) Plant Cell Tiss. Organ Cult. 89:159-168); Insecticidal proteins from species of the genera *Proteobacterium* and *Bacillus* (Hinchliffe et al., (2010) The Open Toxicology Journal, 3:101-118; and Morgan et al., (2001) Applied andEnvir.Micro.67:2062-2069); US Patent 6,048,838, US Patent 6,379,946; PIP-1 peptide in US Patent Serial No. 13 / 792861; AfIP-1A and / or AfIP-1B peptides in US Patent Serial No. 13 / 800233; PHI-4 peptide in US Patent Serial No. 13 / 839702; PIP-47 peptide in US Patent Serial No. 61 / 866747; insecticidal proteins in US Patent Serial Nos. 61 / 863761 and 61 / 863763;And delta-endotoxins, including but not limited to Cry1, Cry2, Cry3, Cry4, Cry5, Cry6, Cry7, Cry8, Cry9, Cry10, Cry11, Cry12, Cry13, Cry14, Cry15, Cry16, Cry17, Cry18, Cry19, Cry20, Cry21, Cry22, Cry23, Cry24, Cry25, Cry26, Cry27, Cry28, Cry29, Cry30, Cry31, Cry32, Cry33, Cry34, Cry35, Cry36, Cry37, Cry38, C The Cry39, Cry40, Cry41, Cry42, Cry43, Cry44, Cry45, Cry46, Cry47, Cry49, Cry51, Cry52, Cry53, Cry54, Cry55, Cry56, Cry57, Cry58, Cry59, Cry60, Cry61, Cry62, Cry63, Cry64, Cry65, Cry66, Cry67, Cry68, Cry69, Cry70 and Cry71 type δ-endotoxin genes, as well as the Bacillus thuringiensis cytolysis genes cyt1 and cyt2. Members of these categories of Bacillus thuringiensis insecticidal proteins include, but are not limited to, Cry1Aa1 (accession number AAA22353); Cry1Aa2 (accession number AAA22552); Cry1Aa3 (accession number BAA00257); Cry1Aa4 (accession number CAA31886); Cry1Aa5 (accession number BAA04468); Cry1Aa6 (accession number AAA86265); Cry1Aa7 (accession number AAD46139); Cry1Aa8 (accession number I26149); Cry1Aa9 (accession number BAA77213); Cry1Aa10 (accession number AAD55382); Cry1Aa11 (accession number CAA70856); Cry 1Aa12 (Login ID AAP80146); Cry1Aa13 (Login ID AAM44305); Cry1Aa14 (Login ID AAP40639); Cry1Aa15 (Login ID AAY66993); Cry1Aa16 (Login ID HQ439776); Cry1Aa17 (Login ID HQ439788); Cry1Aa18 (Login ID HQ439790); Cry1Aa19 (Login ID HQ685121); Cry1Aa20 (Login ID JF340156); Cry1Aa21 (Login ID JN651496); Cry1Aa22 (Login ID KC158223); Cry1Ab1 (Login ID AAA22330);Cry1Ab2 (Login ID AAA22613); Cry1Ab3 (Login ID AAA22561); Cry1Ab4 (Login ID BAA00071); Cry1Ab5 (Login ID CAA28405); Cry1Ab6 (Login ID AAA22420); Cry1Ab7 (Login ID CAA31620); Cry1Ab8 (Login ID AAA22551); Cry1Ab9 (Login ID CAA38701); Cry1Ab10 (Login ID A29125); Cry1Ab11 (Login ID I12419); Cry1Ab12 (Login ID AAC64003); Cry1Ab13 (Login ID AAN7) 6494); Cry1Ab14 (Login ID AAG16877); Cry1Ab15 (Login ID AAO13302); Cry1Ab16 (Login ID AAK55546); Cry1Ab17 (Login ID AAT46415); Cry1Ab18 (Login ID AAQ88259); Cry1Ab19 (Login ID AAW31761); Cry1Ab20 (Login ID ABB72460); Cry1Ab21 (Login ID ABS18384); Cry1Ab22 (Login ID ABW87320); Cry1Ab23 (Login ID HQ439777); Cry1Ab24 (Login ID HQ439778) Cry1Ab25 (Login ID HQ685122); Cry1Ab26 (Login ID HQ847729); Cry1Ab27 (Login ID JN135249); Cry1Ab28 (Login ID JN135250); Cry1Ab29 (Login ID JN135251); Cry1Ab30 (Login ID JN135252); Cry1Ab31 (Login ID JN135253); Cry1Ab32 (Login ID JN135254); Cry1Ab33 (Login ID AAS93798); Cry1Ab34 (Login ID KC156668); Cry1Ab (Login ID AAK14336); Cry1A Sample b (Grant No. AAK14337); Sample Cry1Ab (Grant No. AAK14338); Sample Cry1Ab (Grant No. ABG88858); Cry1Ac1 (Grant No. AAA22331); Cry1Ac2 (Grant No. AAA22338); Cry1Ac3 (Grant No. CAA38098); Cry1Ac4 (Grant No. AAA73077); Cry1Ac5 (Grant No. AAA22339); Cry1Ac6 (Grant No. AAA86266); Cry1Ac7 (Grant No. AAB46989); Cry1Ac8 (Grant No. AAC44841); Cry1Ac9 (Grant No. AAB49768);Cry1Ac10 (Accession number CAA05505); Cry1Ac11 (Accession number CAA10270); Cry1Ac12 (Accession number I12418); Cry1Ac13 (Accession number AAD38701); Cry1Ac14 (Accession number AAQ06607); Cry1Ac15 (Accession number AAN07788); Cry1Ac16 (Accession number AAU87037); Cry1Ac17 (Accession number AAX18704); Cry1Ac18 (Accession number AAY88347); Cry1Ac19 (Accession number ABD37053); Cry1Ac20 (Accession number ABB89046); Cry1Ac21 (Accession number AAY66992); Cry1Ac22 (Accession number ABZ01836); Cry1Ac23 (Accession number CAQ30431); Cry1Ac24 (Accession number ABL01535); Cry1Ac25 (Accession number FJ513324); Cry1Ac26 (Accession number FJ617446); Cry1Ac27 (Accession number FJ617447); Cry1Ac28 (Accession number ACM90319); Cry1Ac29 (Accession number DQ438941); Cry1Ac30 (Accession number GQ227507); Cry1Ac31 (Accession number GU446674); Cry1Ac32 (Accession number HM061081); Cry1Ac33 (Accession number GQ866913); Cry1Ac34 (Accession number HQ230364); Cry1Ac35 (Accession number JF340157); Cry1Ac36 (Accession number JN387137); Cry1Ac37 (Accession number JQ317685); Cry1Ad1 (Accession number AAA22340); Cry1Ad2 (Accession number CAA01880); Cry1Ae1 (Accession number AAA22410); Cry1Af1 (Accession number AAB82749); Cry1Ag1 (Accession number AAD46137); Cry1Ah1 (Accession number AAQ14326); Cry1Ah2 (Accession number ABB76664); Cry1Ah3 (Accession number HQ439779); Cry1Ai1 (Accession number AAO39719); Cry1Ai2 (Accession number HQ439780); Cry1A-like (Accession number AAK14339); Cry1Ba1 (Accession number CAA29898); Cry1Ba2 (Accession number CAA65003); Cry1Ba3 (Accession number AAK63251); Cry1Ba4 (Accession number AAK51084); Cry1Ba5 (Accession number ABO20894); Cry1Ba6 (Accession number ABL60921); Cry1Ba7 (Accession number HQ439781);Cry1Bb1 (Login ID AAA22344); Cry1Bb2 (Login ID HQ439782); Cry1Bc1 (Login ID CAA86568); Cry1Bd1 (Login ID AAD10292); Cry1Bd2 (Login ID AAM93496); Cry1Be1 (Login ID AAC32850); Cry1Be2 (Login ID AAQ52387); Cry1Be3 (Login ID ACV96720); Cry1Be4 (Login ID HM070026); Cry1Bf1 (Login ID CAC50778); Cry1Bf2 (Login ID AAQ52380); Cry1Bg1 (Login ID AAO39 720); Cry1Bh1 (login number HQ589331); Cry1Bi1 (login number KC156700); Cry1Ca1 (login number CAA30396); Cry1Ca2 (login number CAA31951); Cry1Ca3 (login number AAA22343); Cry1Ca4 (login number CAA01886); Cry1Ca5 (login number CAA65457); Cry1Ca6[1] (login number AAF37224); Cry1Ca7 (login number AAG50438); Cry1Ca8 (login number AAM00264); Cry1Ca9 (login number AAL79362); Cry1Ca10 (Login ID AAN16462); Cry1Ca11 (Login ID AAX53094); Cry1Ca12 (Login ID HM070027); Cry1Ca13 (Login ID HQ412621); Cry1Ca14 (Login ID JN651493); Cry1Cb1 (Login ID M97880); Cry1Cb2 (Login ID AAG35409); Cry1Cb3 (Login ID ACD50894); Cry1Cb sample (Login ID AAX63901); Cry1Da1 (Login ID CAA38099); Cry1Da2 (Login ID I76415); Cry1Da3 (Login ID HQ439784); Cr y1Db1 (Login ID CAA80234); Cry1Db2 (Login ID AAK48937); Cry1Dc1 (Login ID ABK35074); Cry1Ea1 (Login ID CAA37933); Cry1Ea2 (Login ID CAA39609); Cry1Ea3 (Login ID AAA22345); Cry1Ea4 (Login ID AAD04732); Cry1Ea5 (Login ID A15535); Cry1Ea6 (Login ID AAL50330); Cry1Ea7 (Login ID AAW72936); Cry1Ea8 (Login ID ABX11258); Cry1Ea9 (Login ID HQ439785);Cry1Ea10 (Login ID ADR00398); Cry1Ea11 (Login ID JQ652456); Cry1Eb1 (Login ID AAA22346); Cry1Fa1 (Login ID AAA22348); Cry1Fa2 (Login ID AAA22347); Cry1Fa3 (Login ID HM070028); Cry1Fa4 (Login ID HM439638); Cry1Fb1 (Login ID CAA80235); Cry1Fb2 (Login ID BAA25298); Cry1Fb3 (Login ID AAF21767); Cry1Fb4 (Login ID AAC10641); Cry1Fb5 (Login ID A AO13295); Cry1Fb6 (Grant No. ACD50892); Cry1Fb7 (Grant No. ACD50893); Cry1Ga1 (Grant No. CAA80233); Cry1Ga2 (Grant No. CAA70506); Cry1Gb1 (Grant No. AAD10291); Cry1Gb2 (Grant No. AAO13756); Cry1Gc1 (Grant No. AAQ52381); Cry1Ha1 (Grant No. CAA80236); Cry1Hb1 (Grant No. AAA79694); Cry1Hb2 (Grant No. HQ439786); Cry1H (Grant No. AAF01213); Cry1Ia Cry1Ia1 (Login ID CAA44633); Cry1Ia2 (Login ID AAA22354); Cry1Ia3 (Login ID AAC36999); Cry1Ia4 (Login ID AAB00958); Cry1Ia5 (Login ID CAA70124); Cry1Ia6 (Login ID AAC26910); Cry1Ia7 (Login ID AAM73516); Cry1Ia8 (Login ID AAK66742); Cry1Ia9 (Login ID AAQ08616); Cry1Ia10 (Login ID AAP86782); Cry1Ia11 (Login ID CAC85964); Cry1Ia12 (Login ID AAV533) 90); Cry1Ia13 (Login ID ABF83202); Cry1Ia14 (Login ID ACG63871); Cry1Ia15 (Login ID FJ617445); Cry1Ia16 (Login ID FJ617448); Cry1Ia17 (Login ID GU989199); Cry1Ia18 (Login ID ADK23801); Cry1Ia19 (Login ID HQ439787); Cry1Ia20 (Login ID JQ228426); Cry1Ia21 (Login ID JQ228424); Cry1Ia22 (Login ID JQ228427); Cry1Ia23 (Login ID JQ228428);Cry1Ia24 (Accession number JQ228429); Cry1Ia25 (Accession number JQ228430); Cry1Ia26 (Accession number JQ228431); Cry1Ia27 (Accession number JQ228432); Cry1Ia28 (Accession number JQ228433); Cry1Ia29 (Accession number JQ228434); Cry1Ia30 (Accession number JQ317686); Cry1Ia31 (Accession number JX944038); Cry1Ia32 (Accession number JX944039); Cry1Ia33 (Accession number JX944040); Cry1Ib1 (Accession number AAA82114); Cry1Ib2 (Accession number ABW88019); Cry1Ib3 (Accession number ACD75515); Cry1Ib4 (Accession number HM051227); Cry1Ib5 (Accession number HM070028); Cry1Ib6 (Accession number ADK38579); Cry1Ib7 (Accession number JN571740); Cry1Ib8 (Accession number JN675714); Cry1Ib9 (Accession number JN675715); Cry1Ib10 (Accession number JN675716); Cry1Ib11 (Accession number JQ228423); Cry1Ic1 (Accession number AAC62933); Cry1Ic2 (Accession number AAE71691); Cry1Id1 (Accession number AAD44366); Cry1Id2 (Accession number JQ228422); Cry1Ie1 (Accession number AAG43526); Cry1Ie2 (Accession number HM439636); Cry1Ie3 (Accession number KC156647); Cry1Ie4 (Accession number KC156681); Cry1If1 (Accession number AAQ52382); Cry1Ig1 (Accession number KC156701); Cry1I-like (Accession number AAC31094); Cry1I-like (Accession number ABG88859); Cry1Ja1 (Accession number AAA22341); Cry1Ja2 (Accession number HM070030); Cry1Ja3 (Accession number JQ228425); Cry1Jb1 (Accession number AAA98959); Cry1Jc1 (Accession number AAC31092); Cry1Jc2 (Accession number AAQ52372); Cry1Jd1 (Accession number CAC50779); Cry1Ka1 (Accession number AAB00376); Cry1Ka2 (Accession number HQ439783); Cry1La1 (Accession number AAS60191); Cry1La2 (Accession number HM070031); Cry1Ma1 (Accession number FJ884067); Cry1Ma2 (Accession number KC156659); Cry1Na1 (Accession number KC156648);Cry1Nb1 (Accession number KC156678); Cry1-like (Accession number AAC31091); Cry2Aa1 (Accession number AAA22335); Cry2Aa2 (Accession number AAA83516); Cry2Aa3 (Accession number D86064); Cry2Aa4 (Accession number AAC04867); Cry2Aa5 (Accession number CAA10671); Cry2Aa6 (Accession number CAA10672); Cry2Aa7 (Accession number CAA10670); Cry2Aa8 (Accession number AAO13734); Cry2Aa9 (Accession number AAO13750); Cry2Aa10 (Accession number AAQ04263); Cry2Aa11 (Accession number AAQ52384); Cry2Aa12 (Accession number ABI83671); Cry2Aa13 (Accession number ABL01536); Cry2Aa14 (Accession number ACF04939); Cry2Aa15 (Accession number JN426947); Cry2Ab1 (Accession number AAA22342); Cry2Ab2 (Accession number CAA39075); Cry2Ab3 (Accession number AAG36762); Cry2Ab4 (Accession number AAO13296); Cry2Ab5 (Accession number AAQ04609); Cry2Ab6 (Accession number AAP59457); Cry2Ab7 (Accession number AAZ66347); Cry2Ab8 (Accession number ABC95996); Cry2Ab9 (Accession number ABC74968); Cry2Ab10 (Accession number EF157306); Cry2Ab11 (Accession number CAM84575); Cry2Ab12 (Accession number ABM21764); Cry2Ab13 (Accession number ACG76120); Cry2Ab14 (Accession number ACG76121); Cry2Ab15 (Accession number HM037126); Cry2Ab16 (Accession number GQ866914); Cry2Ab17 (Accession number HQ439789); Cry2Ab18 (Accession number JN135255); Cry2Ab19 (Accession number JN135256); Cry2Ab20 (Accession number JN135257); Cry2Ab21 (Accession number JN135258); Cry2Ab22 (Accession number JN135259); Cry2Ab23 (Accession number JN135260); Cry2Ab24 (Accession number JN135261); Cry2Ab25 (Accession number JN415485); Cry2Ab26 (Accession number JN426946); Cry2Ab27 (Accession number JN415764); Cry2Ab28 (Accession number JN651494); Cry2Ac1 (Accession number CAA40536);Cry2Ac2 (Login ID AAG35410); Cry2Ac3 (Login ID AAQ52385); Cry2Ac4 (Login ID ABC95997); Cry2Ac5 (Login ID ABC74969); Cry2Ac6 (Login ID ABC74793); Cry2Ac7 (Login ID CAL18690); Cry2Ac8 (Login ID CAM09325); Cry2Ac9 (Login ID CAM09326); Cry2Ac10 (Login ID ABN15104); Cry2Ac11 (Login ID CAM83895); Cry2Ac12 (Login ID CAM83896); Cry2Ad1 (Login ID AAG35410); Cry2Ac3 (Login ID CAM83896); Cry2Ac1 (Login ID AAG35410); Cry2Ac3 (Login ID AAG35410); Cry2Ac3 (Login ID AAG35410); Cry2Ac4 ... AAF09583); Cry2Ad2 (login number ABC86927); Cry2Ad3 (login number CAK29504); Cry2Ad4 (login number CAM32331); Cry2Ad5 (login number CAO78739); Cry2Ae1 (login number AAQ52362); Cry2Af1 (login number ABO30519); Cry2Af2 (login number GQ866915); Cry2Ag1 (login number ACH91610); Cry2Ah1 (login number EU939453); Cry2Ah2 (login number ACL80665); Cry2Ah3 (login number GU073380); Cry2A h4 (Login ID KC156702); Cry2Ai1 (Login ID FJ788388); Cry2Aj (Login ID); Cry2Ak1 (Login ID KC156660); Cry2Ba1 (Login ID KC156658); Cry3Aa1 (Login ID AAA22336); Cry3Aa2 (Login ID AAA22541); Cry3Aa3 (Login ID CAA68482); Cry3Aa4 (Login ID AAA22542); Cry3Aa5 (Login ID AAA50255); Cry3Aa6 (Login ID AAC43266); Cry3Aa7 (Login ID CAB41411); Cry3Aa8 (Login ID AAS79487); Cry3Aa9 (Login ID AAW05659); Cry3Aa10 (Login ID AAU29411); Cry3Aa11 (Login ID AAW82872); Cry3Aa12 (Login ID ABY49136); Cry3Ba1 (Login ID CAA34983); Cry3Ba2 (Login ID CAA00645); Cry3Ba3 (Login ID JQ397327); Cry3Bb1 (Login ID AAA22334); Cry3Bb2 (Login ID AAA74198); Cry3Bb3 (Login ID I15475); Cry3Ca1 (Login ID CAA42469);Cry4Aa1 (Registration No. CAA68485); Cry4Aa2 (Registration No. BAA00179); Cry4Aa3 (Registration No. CAD30148); Cry4Aa4 (Registration No. AFB18317); Cry4A sample (Registration No. AAY96321); Cry4Ba1 (Registration No. CAA30312); Cry4Ba2 (Registration No. CAA30114); Cry4Ba3 (Registration No. AAA22337); Cry4Ba4 (Registration No. BAA00178); Cry4Ba5 (Registration No. CAD30095); Cry4Ba sample (Registration No. ABC47686); Cry4Ca1 (Registration No. EU64620) 2); Cry4Cb1 (Login ID FJ403208); Cry4Cb2 (Login ID FJ597622); Cry4Cc1 (Login ID FJ403207); Cry5Aa1 (Login ID AAA67694); Cry5Ab1 (Login ID AAA67693); Cry5Ac1 (Login ID I34543); Cry5Ad1 (Login ID ABQ82087); Cry5Ba1 (Login ID AAA68598); Cry5Ba2 (Login ID ABW88931); Cry5Ba3 (Login ID AFJ04417); Cry5Ca1 (Login ID HM461869); Cry5Ca2 (Login ID ZP_04) 123426); Cry5Da1 (Login ID HM461870); Cry5Da2 (Login ID ZP_04123980); Cry5Ea1 (Login ID HM485580); Cry5Ea2 (Login ID ZP_04124038); Cry6Aa1 (Login ID AAA22357); Cry6Aa2 (Login ID AAM46849); Cry6Aa3 (Login ID ABH03377); Cry6Ba1 (Login ID AAA22358); Cry7Aa1 (Login ID AAA22351); Cry7Ab1 (Login ID AAA21120); Cry7Ab2 (Login ID AAA21121); Cry 7Ab3 (Login ID ABX24522); Cry7Ab4 (Login ID EU380678); Cry7Ab5 (Login ID ABX79555); Cry7Ab6 (Login ID ACI44005); Cry7Ab7 (Login ID ADB89216); Cry7Ab8 (Login ID GU145299); Cry7Ab9 (Login ID ADD92572); Cry7Ba1 (Login ID ABB70817); Cry7Bb1 (Login ID KC156653); Cry7Ca1 (Login ID ABR67863); Cry7Cb1 (Login ID KC156698); Cry7Da1 (Login ID ACQ99547);Cry7Da2 (Login ID HM572236); Cry7Da3 (Login ID KC156679); Cry7Ea1 (Login ID HM035086); Cry7Ea2 (Login ID HM132124); Cry7Ea3 (Login ID EEM19403); Cry7Fa1 (Login ID HM035088); Cry7Fa2 (Login ID EEM19090); Cry7Fb1 (Login ID HM572235); Cry7Fb2 (Login ID KC156682); Cry7Ga1 (Login ID HM572237); Cry7Ga2 (Login ID KC156669); Cry7Gb1 (Login ID KC15 6650); Cry7Gc1 (KC156654); Cry7Gd1 (KC156697); Cry7Ha1 (KC156651); Cry7Ia1 (KC156665); Cry7Ja1 (KC156671); Cry7Ka1 (KC156680); Cry7Kb1 (BAM99306); Cry7La1 (BAM99307); Cry8Aa1 (AAA21117); Cry8Ab1 (EU044830); Cry8Ac1 (KC156662); Cry8Ad1 (KC156662); Cry8 (KC156684); Cry8Ba1 (AAA21118); Cry8Bb1 (CAD57542); Cry8Bc1 (CAD57543); Cry8Ca1 (AAA21119); Cry8Ca2 (AAR98783); Cry8Ca3 (EU625349); Cry8Ca4 (ADB54826); Cry8Da1 (BAC07226); Cry8Da2 (BD133574); Cry8Da3 (BD133575); Cry8Db1 (BAF93483); Cry8E a1 (Login ID AAQ73470); Cry8Ea2 (Login ID EU047597); Cry8Ea3 (Login ID KC855216); Cry8Fa1 (Login ID AAT48690); Cry8Fa2 (Login ID HQ174208); Cry8Fa3 (Login ID AFH78109); Cry8Ga1 (Login ID AAT46073); Cry8Ga2 (Login ID ABC42043); Cry8Ga3 (Login ID FJ198072); Cry8Ha1 (Login ID AAW81032); Cry8Ia1 (Login ID EU381044); Cry8Ia2 (Login ID GU073381);Cry8Ia3 (login ID HM044664); Cry8Ia4 (login ID KC156674); Cry8Ib1 (login ID GU325772); Cry8Ib2 (login ID KC156677); Cry8Ja1 (login ID EU625348); Cry8Ka1 (login ID FJ422558); Cry8Ka2 (login ID ACN87262); Cry8Kb1 (login ID HM123758); Cry8Kb2 (login ID KC156675); Cry8La1 (login ID GU325771); Cry8Ma1 (login ID HM044665); Cry8Ma2 (login ID EEM) 86551); Cry8Ma3 (Grant No. HM210574); Cry8Na1 (Grant No. HM640939); Cry8Pa1 (Grant No. HQ388415); Cry8Qa1 (Grant No. HQ441166); Cry8Qa2 (Grant No. KC152468); Cry8Ra1 (Grant No. AFP87548); Cry8Sa1 (Grant No. JQ740599); Cry8Ta1 (Grant No. KC156673); Cry8sample (Grant No. FJ770571); Cry8sample (Grant No. ABS53003); Cry9Aa1 (Grant No. CAA41122); Cry9Aa2 (Grant No. C AA41425); Cry9Aa3 (GQ249293); Cry9Aa4 (GQ249294); Cry9Aa5 (JX174110); Cry9Aa (AAQ52376); Cry9Ba1 (CAA52927); Cry9Ba2 (GU299522); Cry9Bb1 (AAV28716); Cry9Ca1 (CAA85764); Cry9Ca2 (AAQ52375); Cry9Da1 (BAA19948); Cry9Da2 (AAB97923); Cry9Da 3 (Login ID GQ249293); Cry9Da4 (Login ID GQ249297); Cry9Db1 (Login ID AAX78439); Cry9Dc1 (Login ID KC156683); Cry9Ea1 (Login ID BAA34908); Cry9Ea2 (Login ID AAO12908); Cry9Ea3 (Login ID ABM21765); Cry9Ea4 (Login ID ACE88267); Cry9Ea5 (Login ID ACF04743); Cry9Ea6 (Login ID ACG63872); Cry9Ea7 (Login ID FJ380927); Cry9Ea8 (Login ID GQ249292);Cry9Ea9 (Accession number: JN651495); Cry9Eb1 (Accession number: CAC50780); Cry9Eb2 (Accession number: GQ249298); Cry9Eb3 (Accession number: KC156646); Cry9Ec1 (Accession number: AAC63366); Cry9Ed1 (Accession number: AAX78440); Cry9Ee1 (Accession number: GQ249296); Cry9Ee2 (Accession number: KC156664); Cry9Fa1 (Accession number: KC156692); Cry9Ga1 (Accession number: KC156699); Cry9-like (Accession number: AAC63366); Cry10Aa1 (Accession number: AAA22614); Cry10Aa2 (Accession number: E00614); Cry10Aa3 (Accession number: CAD30098); Cry10Aa4 (Accession number: AFB18318); Cry10A-like (Accession number: DQ167578); Cry11Aa1 (Accession number: AAA22352); Cry11Aa2 (Accession number: AAA22611); Cry11Aa3 (Accession number: CAD30081); Cry11Aa4 (Accession number: AFB18319); Cry11Aa-like (Accession number: DQ166531); Cry11Ba1 (Accession number: CAA60504); Cry11Bb1 (Accession number: AAC97162); Cry11Bb2 (Accession number: HM068615); Cry12Aa1 (Accession number: AAA22355); Cry13Aa1 (Accession number: AAA22356); Cry14Aa1 (Accession number: AAA21516); Cry14Ab1 (Accession number: KC156652); Cry15Aa1 (Accession number: AAA22333); Cry16Aa1 (Accession number: CAA63860); Cry17Aa1 (Accession number: CAA67841); Cry18Aa1 (Accession number: CAA67506); Cry18Ba1 (Accession number: AAF89667); Cry18Ca1 (Accession number: AAF89668); Cry19Aa1 (Accession number: CAA68875); Cry19Ba1 (Accession number: BAA32397); Cry19Ca1 (Accession number: AFM37572); Cry20Aa1 (Accession number: AAB93476); Cry20Ba1 (Accession number: ACS93601); Cry20Ba2 (Accession number: KC156694); Cry20-like (Accession number: GQ144333); Cry21Aa1 (Accession number: I32932); Cry21Aa2 (Accession number: I66477); Cry21Ba1 (Accession number: BAC06484); Cry21Ca1 (Accession number: JF521577); Cry21Ca2 (Accession number: KC156687);Cry21Da1 (logo JF521578); Cry22Aa1 (logo I34547); Cry22Aa2 (logo CAD43579); Cry22Aa3 (logo ACD93211); Cry22Ab1 (logo AAK50456); Cry22Ab2 (logo CAD43577); Cry22Ba1 (logo CAD43578); Cry22Bb1 (logo KC156672); Cry23Aa1 (logo AAF76375); Cry24Aa1 (logo AAC61891); Cry24Ba1 (logo BAD32657); Cry24Ca 1 (Login ID CAJ43600); Cry25Aa1 (Login ID AAC61892); Cry26Aa1 (Login ID AAD25075); Cry27Aa1 (Login ID BAA82796); Cry28Aa1 (Login ID AAD24189); Cry28Aa2 (Login ID AAG00235); Cry29Aa1 (Login ID CAC80985); Cry30Aa1 (Login ID CAC80986); Cry30Ba1 (Login ID BAD00052); Cry30Ca1 (Login ID BAD67157); Cry30Ca2 (Login ID ACU24781); Cry30Da1 (Login ID... EF095955); Cry30Db1 (login number BAE80088); Cry30Ea1 (login number ACC95445); Cry30Ea2 (login number FJ499389); Cry30Fa1 (login number ACI22625); Cry30Ga1 (login number ACG60020); Cry30Ga2 (login number HQ638217); Cry31Aa1 (login number BAB11757); Cry31Aa2 (login number AAL87458); Cry31Aa3 (login number BAE79808); Cry31Aa4 (login number BAF32571); Cry31Aa5 (login number BAF32) 572); Cry31Aa6 (login number BAI44026); Cry31Ab1 (login number BAE79809); Cry31Ab2 (login number BAF32570); Cry31Ac1 (login number BAF34368); Cry31Ac2 (login number AB731600); Cry31Ad1 (login number BAI44022); Cry32Aa1 (login number AAG36711); Cry32Aa2 (login number GU063849); Cry32Ab1 (login number GU063850); Cry32Ba1 (login number BAB78601); Cry32Ca1 (login number BAB78602);Cry32Cb1 (login ID KC156708); Cry32Da1 (login ID BAB78603); Cry32Ea1 (login ID GU324274); Cry32Ea2 (login ID KC156686); Cry32Eb1 (login ID KC156663); Cry32Fa1 (login ID KC156656); Cry32Ga1 (login ID KC156657); Cry32Ha1 (login ID KC156661); Cry32Hb1 (login ID KC156666); Cry32Ia1 (login ID KC156667); Cry32Ja1 (login ID KC156685); Cry32 Ka1 (KC156688); Cry32La1 (KC156689); Cry32Ma1 (KC156690); Cry32Mb1 (KC156704); Cry32Na1 (KC156691); Cry32Oa1 (KC156703); Cry32Pa1 (KC156705); Cry32Qa1 (KC156706); Cry32Ra1 (KC156707); Cry32Sa1 (KC156709); Cry32Ta1 (KC156710); Cry32Ua ...Ra1 (KC156707); Cry32Sa1 (KC156709); Cry32Ta1 (KC156710); Cry32Ua1 (KC156688); Cry32Ra1 (KC156689); Cry32Ra1 (KC156709); Cry32Ta1 (KC156710); Cry32Ua1 (KC156688); Cry32Ra1 (KC Cry33Aa1 (logo AAL26871); Cry34Aa1 (logo AAG50341); Cry34Aa2 (logo AAK64560); Cry34Aa3 (logo AAT29032); Cry34Aa4 (logo AAT29030); Cry34Ab1 (logo AAG41671); Cry34Ac1 (logo AAG50118); Cry34Ac2 (logo AAK64562); Cry34Ac3 (logo AAT29029); Cry34Ba1 (logo AAK64565); Cry34Ba2 (logo AAT2) 9033); Cry34Ba3 (logo AAT29031); Cry35Aa1 (logo AAG50342); Cry35Aa2 (logo AAK64561); Cry35Aa3 (logo AAT29028); Cry35Aa4 (logo AAT29025); Cry35Ab1 (logo AAG41672); Cry35Ab2 (logo AAK64563); Cry35Ab3 (logo AY536891); Cry35Ac1 (logo AAG50117); Cry35Ba1 (logo AAK64566); Cry35Ba2 (logo AAT29027);Cry35Ba3 (Accession number AAT29026); Cry36Aa1 (Accession number AAK64558); Cry37Aa1 (Accession number AAF76376); Cry38Aa1 (Accession number AAK64559); Cry39Aa1 (Accession number BAB72016); Cry40Aa1 (Accession number BAB72018); Cry40Ba1 (Accession number BAC77648); Cry40Ca1 (Accession number EU381045); Cry40Da1 (Accession number ACF15199); Cry41Aa1 (Accession number BAD35157); Cry41Ab1 (Accession number BAD35163); Cry41Ba1 (Accession number HM461871); Cry41Ba2 (Accession number ZP_04099652); Cry42Aa1 (Accession number BAD35166); Cry43Aa1 (Accession number BAD15301); Cry43Aa2 (Accession number BAD95474); Cry43Ba1 (Accession number BAD15303); Cry43Ca1 (Accession number KC156676); Cry43Cb1 (Accession number KC156695); Cry43Cc1 (Accession number KC156696); Cry43-like (Accession number BAD15305); Cry44Aa (Accession number BAD08532); Cry45Aa (Accession number BAD22577); Cry46Aa (Accession number BAC79010); Cry46Aa2 (Accession number BAG68906); Cry46Ab (Accession number BAD35170); Cry47Aa (Accession number AAY24695); Cry48Aa (Accession number CAJ18351); Cry48Aa2 (Accession number CAJ86545); Cry48Aa3 (Accession number CAJ86546); Cry48Ab (Accession number CAJ86548); Cry48Ab2 (Accession number CAJ86549); Cry49Aa (Accession number CAH56541); Cry49Aa2 (Accession number CAJ86541); Cry49Aa3 (Accession number CAJ86543); Cry49Aa4 (Accession number CAJ86544); Cry49Ab1 (Accession number CAJ86542); Cry50Aa1 (Accession number BAE86999); Cry50Ba1 (Accession number GU446675); Cry50Ba2 (Accession number GU446676); Cry51Aa1 (Accession number ABI14444); Cry51Aa2 (Accession number GU570697); Cry52Aa1 (Accession number EF613489); Cry52Ba1 (Accession number FJ361760); Cry53Aa1 (Accession number EF633476);Cry53Ab1 (Login ID FJ361759); Cry54Aa1 (Login ID ACA52194); Cry54Aa2 (Login ID GQ140349); Cry54Ba1 (Login ID GU446677); Cry55Aa1 (Login ID ABW88932); Cry54Ab1 (Login ID JQ916908); Cry55Aa2 (Login ID AAE33526); Cry56Aa1 (Login ID ACU57499); Cry56Aa2 (Login ID GQ483512); Cry56Aa3 (Login ID JX025567); Cry57Aa1 (Login ID ANC87261); Cry5 8Aa1 (Grant No. ANC87260); Cry59Ba1 (Grant No. JN790647); Cry59Aa1 (Grant No. ACR43758); Cry60Aa1 (Grant No. ACU24782); Cry60Aa2 (Grant No. EAO57254); Cry60Aa3 (Grant No. EEM99278); Cry60Ba1 (Grant No. GU810818); Cry60Ba2 (Grant No. EAO57253); Cry60Ba3 (Grant No. EEM99279); Cry61Aa1 (Grant No. HM035087); Cry61Aa2 (Grant No. HM132125); Cry61Aa3 (Login ID EEM19308); Cry62Aa1 (Login ID HM054509); Cry63Aa1 (Login ID BAI44028); Cry64Aa1 (Login ID BAJ05397); Cry65Aa1 (Login ID HM461868); Cry65Aa2 (Login ID ZP_04123838); Cry66Aa1 (Login ID HM485581); Cry66Aa2 (Login ID ZP_04099945); Cry67Aa1 (Login ID HM485582); Cry67Aa2 (Login ID ZP_04148882); Cry68Aa1 (Login ID HQ113114); Cry 69Aa1 (Login ID HQ401006); Cry69Aa2 (Login ID JQ821388); Cry69Ab1 (Login ID JN209957); Cry70Aa1 (Login ID JN646781); Cry70Ba1 (Login ID ADO51070); Cry70Bb1 (Login ID EEL67276); Cry71Aa1 (Login ID JX025568); Cry72Aa1 (Login ID JX025569); Cyt1Aa (GenBank Login ID X03182); Cyt1Ab (GenBank Login ID X98793); Cyt1B (GenBank Login ID U37196);Cyt2A (GenBank accession number Z14147); and Cyt2B (GenBank accession number U52043).

[0023] Examples of delta-endotoxins include, but are not limited to: the Cry1A protein in U.S. Patents 5,880,275, 7,858,849, 8,530,411, 8,575,433, and 8,686,233; and DIG-3 or DIG-11 toxins (α-helix 1 and / or α-helix 2 of cry proteins such as Cry1A, Cry3A) in U.S. Patents 8,304,604, 8,304,605, and 8,476,226. (N-terminal deletion of variants); Cry1B in U.S. Patent Application Serial No. 10 / 525,318; Cry1C in U.S. Patent 6,033,874; Cry1F in U.S. Patents 5,188,960 and 6,218,188; Cry1A / F chimeras in U.S. Patents 7,070,982, 6,962,705 and 6,713,063; Cry2 proteins, such as Cry in U.S. Patent 7,064,249. 2Ab protein; Cry3A protein, including but not limited to engineered hybrid insecticidal protein (eHIP) formed by a unique combination of variable and conserved blocks of at least two different Cry proteins (US Patent Application Publication 2010 / 0017914); Cry4 protein; Cry5 protein; Cry6 protein; Cry8 protein as described in US Patents 7,329,736, 7,449,552, 7,803,943, 7,476,781, 7,105,332, 7,378,499, and 7,462,760; Cry9 protein, such as members of the Cry9A, Cry9B, Cry9C, Cry9D, Cry9E, and Cry9F families, including but not limited to the Cry9D protein as described in US Patent 8,802,933 and the Cry9B protein as described in US Patent 8,802,934; Naimov et al., (2008) Applied The Cry15 protein in *and Environmental Microbiology*, 74:7145-7151; the Cry22 and Cry34Ab1 proteins in US patents 6,127,180, 6,624,145, and 6,340,593; the CryET33 and CryET34 proteins in US patents 6,248,535, 6,326,351, 6,399,330, 6,949,626, 7,385,107, and 7,504,229; and US patent publications 2006 / 0191034, 2012 / 0278954 and PCT publication WO. Homologs of CryET33 and CryET34 in 2012 / 139004; Cry35Ab1 protein in U.S. Patents 6,083,499, 6,548,291 and 6,340,593; Cry46 protein, Cry51 protein, Cry binary toxin; TIC901 or related toxins;TIC807 in US Patent Application Publication 2008 / 0295207; ET29, ET37, TIC809, TIC810, TIC812, TIC127, and TIC128 in PCT US2006 / 033867; TIC853 toxin in US Patent 8,513,494; AXMI-027, AXMI-036, and AXMI-038 in US Patent 8,236,757; AXMI-031, AXMI-039, AXMI-040, and AXMI-049 in US Patent 7,923,602; AXMI-018, AXMI-020, and AXMI-021 in WO 2006 / 083891; WO AXMI-010 in WO 2005 / 038032; AXMI-003 in WO 2005 / 021585; AXMI-008 in U.S. Patent Application Publication 2004 / 0250311; AXMI-006 in U.S. Patent Application Publication 2004 / 0216186; AXMI-007 in U.S. Patent Application Publication 2004 / 0210965; AXMI-009 in U.S. Patent Application Publication 2004 / 0210964; AXMI-014 in U.S. Patent Application Publication 2004 / 0197917; AXMI-004 in U.S. Patent Application Publication 2004 / 0197916; WO AXMI-028 and AXMI-029 in 2006 / 119457; AXMI-007, AXMI-008, AXMI-0080rf2, AXMI-009, AXMI-014, and AXMI-004 in WO2004 / 074462; AXMI-150 in U.S. Patent 8,084,416; AXMI-205 in U.S. Patent Application Publication 2011 / 0023184; U.S. Patent Application Publication 2011 / 0263 AXMI-011, AXMI-012, AXMI-013, AXMI-015, AXMI-019, AXMI-044, AXMI-037, AXMI-043, AXMI-033, AXMI-034, AXMI-022, AXMI-023, AXMI-041, AXMI-063, and AXMI-064 in 488; AXMI-R1 and related proteins in U.S. Patent Application Publication 2010 / 0197592; AXMI221Z, AXMI222z, AXMI223z, AXMI224z, and AXMI225z in WO 2011 / 103248;AXMI218, AXMI219, AXMI220, AXMI226, AXMI227, AXMI228, AXMI229, AXMI230, and AXMI231 in WO2011 / 103247 and U.S. Patent 8,759,619; AXMI-115, AXMI-113, AXMI-005, AXMI-163, and AXMI-184 in U.S. Patent 8,334,431; AXMI-001, AXMI-002, AXMI-030, AXMI-035, and AXMI-045 in U.S. Patent Application Publication 2010 / 0298211; AXMI-066 and AXMI-076 in U.S. Patent Application Publication 2009 / 0144852; and AXMI128, AXMI130, AXMI131, and AXMI133 in U.S. Patent 8,318,900. AXMI140, AXMI141, AXMI142, AXMI143, AXMI144, AXMI146, AXMI148, AXMI149, AXMI152, AXMI15 3. AXMI154, AXMI155, AXMI156, AXMI157, AXMI158, AXMI162, AXMI165, AXMI166, AXMI167, AXMI 168, AXMI169, AXMI170, AXMI171, AXMI172, AXMI173, AXMI174, AXMI175, AXMI176, AXMI177, AX MI178, AXMI179, AXMI180, AXMI181, AXMI182, AXMI185, AXMI186, AXMI187, AXMI188, AXMI189;AXMI079, AXMI080, AXMI081, AXMI082, AXMI091, AXMI092, AXMI096, AXMI097, AXMI098, AXMI099, AXMI100, AXMI101, AXMI102, AXMI103, AXMI104, AXMI107, AXMI108, AXMI109, AXMI110, AXMI111, AXMI112, AXMI114, AXMI116, AXMI117, AXMI118, AXMI119, AXMI1 20, AXMI121, AXMI122, AXMI123, AXMI124, AXMI1257, AXMI1268, AXMI127, AXMI129, AXMI164, AXMI151, AXMI161, AXMI183, AXMI132, AXMI138, AXMI137, AXMI270 in US Patent Application Publication US20140223598, AXMI279 in US Patent Application Publication US20140223599, cry proteins, such as Cry1A and Cry3A with modified proteolytic sites in US Patent 8,319,019;Cry1Ac, Cry2Aa, and Cry1Ca toxin proteins from Bacillus thuringiensis strain VBTS 2528, as published in U.S. Patent Application Publication 2011 / 0064710. Other Cry proteins are also well known to those skilled in the art (see Crickmore et al., “Bacillus thuringiensis toxin nomenclature” (2011) at lifesci.sussex.ac.uk / home / Neil_Crickmore / Bt / (accessible via the “www” prefix on the World Wide Web). The insecticidal activity of Cry proteins is well known to those skilled in the art (for a review, see van Frannkenhuyzen, (2009) J. Invert. Path. 101: 1-16). The use of Cry proteins as a trait in transgenic plants is well known to those skilled in the art, and Cry transgenic plants (including, but not limited to, plants expressing Cry1Ac, Cry1Ac+Cry2Ab, Cry1Ab, Cry1A.105, Cry1F, Cry1Fa2, Cry1F+Cry1Ac, Cry2Ab, Cry3A, mCry3A, Cry3Bb1, Cry34Ab1, Cry35Ab1, Vip3A, mCry3A, Cry9c, and CBI-Bt) have been subject to regulatory approval (see Sanahuja, (2011) Plant Biotech Journal 9: 283-300; and cera-gmc.org / index.php?action=gm_crop_database (accessible on the World Wide Web with the prefix "www") at CERA (2010) GM Crop Database Center for Environmental Risk Assessment (CERA), ILSI Research Foundation, Washington DC). More than one insecticidal protein well known to those skilled in the art can also be expressed in plants, such as: Vip3Ab and Cry1Fa (US2012 / 0317682); Cry1BE and Cry1F (US2012 / 0311746); Cry1CA and Cry1AB (US2012 / 0311745); Cry1F and CryCa (US2012 / 0317681); Cry1DA and Cry1BE (US2012 / 0331590); Cry1DA and Cry1Fa (US2012 / 0331589); Cry1AB and Cry1BE (US2012 / 0324606);Cry1Fa and Cry2Aa, Cry1I and Cry1E (US2012 / 0324605); Cry34Ab / 35Ab and Cry6Aa (US20130167269); Cry34Ab / VCry35Ab and Cry3Aa (US20130167268); Cry1Ab and Cry1F (US20140182018); Cry3A and Cry1Ab or Vip3Aa (US20130116170). Insecticidal proteins also include insecticidal lipases (including acyl hydrolases in US Patent 7,491,869) and cholesterol oxidases, such as cholesterol oxidases from Streptomyces (Purcell et al. (1993) Biochem Biophys Res Commun 15:1406-1413). Insecticidal proteins also include VIP (plant-derived insecticidal protein) toxins, as described in U.S. Patents 5,877,012, 6,107,279, 6,137,033, 7,244,820, 7,615,686, and 8,237,020. Other VIP proteins are well known to those skilled in the art (see lifesci.sussex.ac.uk / home / Neil_Crickmore / Bt / vip.html (accessible via the World Wide Web with the prefix "www")). Insecticidal proteins also include toxin complex (TC) proteins, which are obtained from organisms such as pathogenic bacteria, luminescent bacteria, and Paenibacillus (see U.S. Patents 7,491,698 and 8,084,418). Some TC proteins have "independent" insecticidal activity, while others enhance the activity of independent toxins produced by the same given organism. The toxicity of “independent” TC proteins (from, for example, *Proteobacterium*, *Pathobacterium*, or *Bacillus* species) can be enhanced by one or more TC protein “synergists” from organisms of different genera. There are three main types of TC proteins. As mentioned herein, type A proteins (“protein A”) are independent toxins. Type B proteins (“protein B”) and type C proteins (“protein C”) enhance the toxicity of type A proteins. Examples of type A proteins are TcbA, TcdA, XptA1, and XptA2. Examples of type B proteins are TcaC, TcdB, XptB1Xb, and XptC1Wi. Examples of type C proteins are TccC, XptC1Xb, and XptB1Wi. Insecticidal proteins also include spider, snake, and scorpion venom proteins. Examples of spider venom peptides include, but are not limited to, lycotoxin-1 peptide and its mutants (US Patent 8,334,366).

[0024] In some embodiments, the PIP-72 polypeptide comprises an amino acid sequence deduced from the full-length nucleic acid sequence disclosed herein, as well as an amino acid sequence shorter than the full-length sequence due to the use of an alternative downstream starting site or due to processing to produce a shorter protein with insecticidal activity. Processing can occur in the organism expressing the protein or in the pest after protein ingestion.

[0025] Therefore, this paper provides novel isolated or recombinant nucleic acid sequences conferring insecticidal activity. The amino acid sequence of the PIP-72 polypeptide is also provided. Cells containing proteins translated from these PIP-72 polypeptide genes can control or kill pests that feed on these cells.

[0026] bacterial strains

[0027] This disclosure relates, in one aspect, to bacterial strains expressing the PIP-72 polypeptide. In some embodiments, the bacterial strains are species of the genera *Halomonas*, *Luminobacterium*, *Pathobacterium*, *Burkholderia*, *Paludibacterium*, or *Pseudomonas*. In some embodiments, the bacterial strains are *Halomonas anticariensis*, *Photorhabdus luminescens*, *Xenorhabdus bovienii*, *Burkholderia pseudomallei*, *Burkholderia multivorans*, *Burkholderia thailandensis*, *Paludibacterium yongneupense*, *Pseudomonas rhodesiae*, *Pseudomonas entomophila*, *Pseudomonas chlororaphis*, *Pseudomonas mandelii*, and *Pseudomonas glabripennis*. *Pseudomonas congelans*; *Pseudomonas meningitidis*; *Pseudomonas plecoglossicida*, *Pseudomonas biocontrolicarum*, *Pseudomonas ficuserectae*; *Pseudomonas mosselii* or *Pseudomonas brassicacearum* strains. In some embodiments, the bacterial strain is a pure biological culture of *Pseudomonas aeruginosa* strain SS143D5, which was deposited on February 7, 2013, at the Agricultural Research Culture Collection (NRRL) (1815 North University Street, Peoria, Illinois 616040) (nrrl.ncaur.usda.gov, accessible via the World Wide Web using the "www" prefix). This collection will be preserved in accordance with the terms of the Budapest Treaty on the Preservation of Microorganisms, which is internationally recognized for use in patent proceedings. These deposits are kept solely for the convenience of those skilled in the art and do not constitute an admission that the deposits are required under 35 U.S.SC § 112. During the pending period of this application, the Director of the United States Patent and Trademark Office and authorized persons may access the deposits upon his request.Upon grant of any claim of this application, the applicant will, in accordance with 37C.FR §1.808, make samples of the deposits held at the Agricultural Research Culture Collection (NRRL), 1815 North University Street, Peoria, Illinois 61604, publicly available. These deposits will be held at the NRRL, a public depository, for 30 years, or 5 years after the most recent request, or until the expiry of this patent, whichever is longer, and will be replaced if they become inactive during this period. Upon patent grant, the deposits will be irrevocably and unrestrictedly available to the public. Furthermore, the applicant has satisfied all the requirements of 37C.FR §§1,801-1,809, including providing indicators of the viability of the samples at the time of deposit. The applicant has no right to exempt itself from any legal restrictions on the transfer or commercial transport of biological material. The applicant does not exempt itself from any infringement of the rights granted to them under this patent. However, it should be understood that the availability of the deposits should not be regarded as a license to practice the invention in violation of the patent rights granted by government decree.

[0028] Nucleic acid molecules and their variants and fragments

[0029] This disclosure relates, in one aspect, to isolated or recombinant nucleic acid molecules comprising a nucleic acid sequence encoding a PIP-72 polypeptide or its biologically active portion; and to nucleic acid molecules sufficient to be used as hybridization probes for identifying nucleic acid molecules encoding proteins having regions of sequence homology. As used herein, the term “nucleic acid molecule” refers to DNA molecules (e.g., recombinant DNA, cDNA, genomic DNA, plastid DNA, mitochondrial DNA) and RNA molecules (e.g., mRNA), as well as analogs of DNA or RNA produced using nucleotide analogs. Nucleic acid molecules can be single-stranded or double-stranded, but double-stranded DNA is preferred.

[0030] "Isolated" nucleic acid molecule (or DNA) is used herein to refer to a nucleic acid sequence (or DNA) that is no longer in its natural environment, such as in vitro. "Recombinant" nucleic acid molecule (or DNA) is used herein to refer to a nucleic acid sequence (or DNA) in a recombinant bacterial or plant host cell. In some embodiments, the "isolated" or "recombinant" nucleic acid does not contain a sequence naturally located flanking the nucleic acid in the genomic DNA of the source organism (i.e., the sequence located at the 5′ and 3′ ends of the nucleic acid) (preferably a protein-coding sequence). For the purposes of this disclosure, "isolated" or "recombinant" excludes isolated chromosomes when used to refer to nucleic acid molecules. For example, in various embodiments, a recombinant nucleic acid molecule encoding the PIP-72 polypeptide may contain a nucleic acid sequence of less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb, or 0.1 kb, which is naturally located flanking the nucleic acid molecule in the genomic DNA of the source cell of the nucleic acid.

[0031] In some embodiments, the isolated nucleic acid molecule encoding the PIP-72 polypeptide has one or more alterations in its nucleic acid sequence compared to the natural or genomic nucleic acid sequence. In some embodiments, the alterations in the natural or genomic nucleic acid sequence include, but are not limited to: alterations in the nucleic acid sequence due to genetic code degeneracy; alterations in the nucleic acid sequence due to amino acid substitutions, insertions, deletions, and / or additions compared to the natural or genomic sequence; removal of one or more introns; deletion of one or more upstream or downstream regulatory regions; and deletion of 5′ and / or 3′ untranslated regions associated with the genomic nucleic acid sequence. In some embodiments, the nucleic acid molecule encoding the PIP-72 polypeptide is a non-genomic sequence.

[0032] This disclosure envisions various polynucleotides encoding the PIP-72 polypeptide or related proteins. These polynucleotides, when operatively linked to suitable promoters, transcription termination sequences, and / or polyadenylation sequences, can be used to generate the PIP-72 polypeptide in host cells. These polynucleotides can also be used as probes to isolate homologous or substantially homologous polynucleotides encoding the PIP-72 polypeptide or related proteins.

[0033] The sources of polynucleotides encoding the PIP-72 polypeptide or related proteins include, but are not limited to, *Haloxylon anticlocatea*, *Luminobacterium luminiferum*, *Burkholderia burgdorferi*, *Burkholderia melioides*, *Burkholderia polyphaga*, *Burkholderia thamnip*, *Paludibacterium yongneupense*, *Pseudomonas rozebladder*, *Pseudomonas tinctoria*, *Pseudomonas monnieri*, *Pseudomonas icarius*, *Pseudomonas monnieri*, *Pseudomonas proteus*, *Pseudomonas biocontrolensis*, *Pseudomonas sphaeroides*, *Pseudomonas moss*, or *Pseudomonas brassicae* strains. The sources of polynucleotides encoding the PIP-72 polypeptide or related proteins include, but are not limited to: *Pseudomonas aeruginosa* strains containing the PIP-72Aa polypeptide encoded by SEQ ID NO: 2, and the PIP-72Aa polynucleotide shown in SEQ ID NO: 1; *Pseudomonas repens* strains containing the PIP-72Ba polypeptide encoded by SEQ ID NO: 4, and the PIP-72Ba polynucleotide shown in SEQ ID NO: 3; *Pseudomonas aeruginosa* strains containing the PIP-72Ca polypeptide encoded by SEQ ID NO: 6, and the PIP-72Ca polynucleotide shown in SEQ ID NO: 5; *Pseudomonas meningitidis* strains containing the PIP-72Cb polypeptide encoded by SEQ ID NO: 8, and the PIP-72Cb polynucleotide shown in SEQ ID NO: 7; and *Pseudomonas icariina* strains containing the PIP-72Da polypeptide encoded by SEQ ID NO: 10, and the PIP-72Cb polynucleotide shown in SEQ ID NO: 10. PIP-72Da polynucleotide shown in SEQ ID NO: 9; *Pseudomonas meningitidis* strain containing the PIP-72Db polypeptide shown in SEQ ID NO: 12, and the PIP-72Db polynucleotide shown in SEQ ID NO: 11; *Pseudomonas serrata* strain containing the PIP-72Dc polypeptide shown in SEQ ID NO: 14, and the PIP-72Dc polynucleotide shown in SEQ ID NO: 13; *Pseudomonas moschata* strain containing the PIP-72Fa polypeptide shown in SEQ ID NO: 18, and the PIP-72Fa polynucleotide shown in SEQ ID NO: 17; *Pseudomonas aeruginosa* strain containing the PIP-72Ff polypeptide shown in SEQ ID NO: 28, and the PIP-72Ff polynucleotide shown in SEQ ID NO: 27; *Pseudomonas aeruginosa* strain containing the PIP-72Gb polypeptide shown in SEQ ID NO: 32, and the PIP-72Db polynucleotide shown in SEQ ID NO: 9; PIP-72Gb polynucleotide shown in NO: 31; Pseudomonas aeruginosa strain containing the PIP-72Ab polypeptide shown in SEQ ID NO: 927, and the PIP-72Ab polynucleotide shown in SEQ ID NO: 949.A *Pseudomonas rapeus* strain containing the polypeptide encoding PIP-72Ab (SEQ ID NO: 928) and the polynucleotide encoding PIP-72Bb (SEQ ID NO: 950); a *Pseudomonas entomopathogenicus* strain containing the polypeptide encoding PIP-72AFh (SEQ ID NO: 932) and the polynucleotide encoding PIP-72Fh (SEQ ID NO: 954); a *Pseudomonas entomopathogenicus* strain containing the polypeptide encoding PIP-72AFh (SEQ ID NO: 933) and the polynucleotide encoding PIP-72Fh (SEQ ID NO: 955); a *Pseudomonas aeruginosa* strain containing the polypeptide encoding PIP-72Fj (SEQ ID NO: 934) and the polynucleotide encoding PIP-72Fj (SEQ ID NO: 956); and a *Pseudomonas aeruginosa* strain containing the polypeptide encoding PIP-72Fk (SEQ ID NO: 935). PIP-72Fk polynucleotide shown in SEQ ID NO: 957; Burkholderia polyphaga strain containing PIP-72Fl polypeptide shown in SEQ ID NO: 936, PIP-72Fl polynucleotide shown in SEQ ID NO: 958; Pseudomonas aeruginosa strain containing PIP-72Gg polypeptide shown in SEQ ID NO: 939, PIP-72Gg polynucleotide shown in SEQ ID NO: 961; Pseudomonas aeruginosa strain containing PIP-72Gh polypeptide shown in SEQ ID NO: 940, PIP-72Gh polynucleotide shown in SEQ ID NO: 962; Pseudomonas moschata strain containing PIP-72Gi polypeptide shown in SEQ ID NO: 941, PIP-72Gi polynucleotide shown in SEQ ID NO: 963; Pseudomonas biocontrol strain containing PIP-72Gk polypeptide shown in SEQ ID NO: 943, PIP-72Gi polynucleotide shown in SEQ ID NO: 963. PIP-72Gk polynucleotide shown in ID NO: 965; Pseudomonas proteus strain containing the PIP-72Gl polypeptide shown in SEQ ID NO: 944, PIP-72Gl polynucleotide shown in SEQ ID NO: 966;A strain of *Pseudomonas aeruginosa* containing the PIP-72Gn polypeptide shown in SEQ ID NQ: 946 and the PIP-72Gn polynucleotide shown in SEQ ID NO: 968. These polynucleotide sequences have been isolated from hosts such as *Haloxymonas*, *Luminobacterium*, *Pathobacterium*, *Burkholderia*, *Paludibacterium*, or *Pseudomonas*, and are therefore suitable for expression of the encoded PIP-72 polypeptide in other bacterial hosts. For example, SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 17, SEQ ID NO: 27 and SEQ ID NO: 31, SEQ ID NO: 949, SEQ ID NO: 950, SEQ ID NO: 955, SEQ ID NO: 956, SEQ ID NO: 957, SEQ ID NO: 958, SEQ ID NO: 961, SEQ ID NO: 962, SEQ ID NO: 963, SEQ ID NO: 965, SEQ ID NO: 966, SEQ ID NO: 967, SEQ ID NO: 968 can be used to express the PIP-72 polypeptide in bacterial host cells including but not limited to the following: Agrobacterium, Bacillus, Escherichia, Salmonella, Pseudomonas, and Rhizobium. The polynucleotides can also be used as probes to isolate homologous or substantially homologous polynucleotides encoding the PIP-72 polypeptide or related proteins. Such probes can be used to identify homologous or substantially homologous polynucleotides derived from the following bacteria or other related bacteria: *Haliotis*, *Luminobacterium*, *Pathogenic Bacillus*, *Burkholderia*, *Paludibacterium*, or *Pseudomonas*.

[0034] The polynucleotide encoding the PIP-72 polypeptide can also be synthesized de novo from the PIP-72 polypeptide sequence. The sequence of the polynucleotide gene can be deduced from the PIP-72 polypeptide sequence using the genetic code. Computer programs such as "BackTranslate" (GCG) can be used. TMThe software package (Acclerys, Inc., San Diego, Calif.) converts peptide sequences into corresponding nucleotide sequences encoding such peptides. Examples of PIP-72 polypeptide sequences that can be used to obtain the corresponding nucleotide-encoding sequences include, but are not limited to, PIP-72 polypeptides having the following sequences: SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 18, SEQ ID NO: 28 and SEQ ID NO: 32, SEQ ID NO: 927, SEQ ID NO: 928, SEQ ID NO: 932, SEQ ID NO: 933, SEQ ID NO: 934, SEQ ID NO: 935, SEQ ID NO: 936, SEQ ID NO: 939, SEQ ID NO: 940, SEQ ID NO: 941, SEQ ID NO: 943, SEQ ID NO: 944, SEQ ID NO: 945 or SEQ ID NO: 946. Furthermore, the synthetic PIP-72 polynucleotide sequence disclosed herein can be engineered for expression in plants. U.S. Patent 5,500,365 describes a method for synthesizing a plant gene to improve the expression level of a protein encoded by that synthetic gene. This method involves modifying the structural gene sequence of a foreign transgene to make it more efficiently transcribed, processed, translated, and expressed by the plant. A gene adequately expressed in a plant is characterized by the elimination of sequences that could cause undesirable intron splicing or polyadenylation in the coding region of the gene transcript, while substantially preserving the amino acid sequence of the toxic portion of the insecticidal protein. A similar method for obtaining enhanced expression of transgenes in monocotyledonous plants is disclosed in U.S. Patent 5,689,052.

[0035] In some embodiments, the nucleic acid molecule encoding the PIP-72 polypeptide is a polynucleotide having the sequences shown in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 17, SEQ ID NO: 27, SEQ ID NO: 31, SEQ ID NO: 949, SEQ ID NO: 950, SEQ ID NO: 955, SEQ ID NO: 956, SEQ ID NO: 957, SEQ ID NO: 958, SEQ ID NO: 961, SEQ ID NO: 962, SEQ ID NO: 963, SEQ ID NO: 965, SEQ ID NO: 966, SEQ ID NO: 967, and SEQ ID NO: 968, as well as variants, fragments, and complementary sequences thereof. "Complementary sequence" is used herein to refer to a nucleic acid sequence that is sufficiently complementary to a given nucleic acid sequence such that it can hybridize to that given nucleic acid sequence to form a stable duplex. In this article, "polynucleotide sequence variant" refers to a nucleic acid sequence that encodes the same polypeptide except for genetic code degeneracy.

[0036] In some embodiments, the nucleic acid molecule encoding the PIP-72 polypeptide is a non-genomic nucleic acid sequence. As used herein, a “non-genomic nucleic acid sequence” or “non-genomic nucleic acid molecule” refers to a nucleic acid molecule that has one or more alterations in its nucleic acid sequence compared to a native or genomic nucleic acid sequence. In some embodiments, alterations in a native or genomic nucleic acid molecule include, but are not limited to: alterations in the nucleic acid sequence due to genetic code degeneracy; codon optimization of the nucleic acid sequence for expression in plants; alterations in the nucleic acid sequence compared to a native or genomic sequence to introduce at least one amino acid substitution, insertion, deletion, and / or addition; removal of one or more introns associated with a genomic nucleic acid sequence; insertion of one or more heterologous introns; deletion of one or more upstream or downstream regulatory regions associated with a genomic nucleic acid sequence; insertion of one or more heterologous upstream or downstream regulatory regions; deletion of 5′ and / or 3′ untranslated regions associated with a genomic nucleic acid sequence; insertion of heterologous 5′ and / or 3′ untranslated regions; and modification of polyadenylation sites. In some embodiments, the non-genomic nucleic acid molecule is cDNA. In some embodiments, the non-genomic nucleic acid molecule is a synthetic nucleic acid sequence. In some embodiments, the non-genomic nucleic acid molecule is not one of the following nucleic acid sequences: SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 17, SEQ ID NO: 27, SEQ ID NO: 31, SEQ ID NO: 949, SEQ ID NO: 950, SEQ ID NO: 955, SEQ ID NO: 956, SEQ ID NO: 957, SEQ ID NO: 958, SEQ ID NO: 961, SEQ ID NO: 962, SEQ ID NO: 963, SEQ ID NO: 965, SEQ ID NO: 966, SEQ ID NO: 967, SEQ ID NO: 968.

[0037] In some embodiments, the non-genomic nucleic acid molecule encodes a PIP-72 polypeptide comprising an amino acid sequence having at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 78%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the following amino acid sequences: SEQ ID NO: 2, SEQ ID SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 18, SEQ ID NO: 28, SEQ ID NO: 32, SEQ ID NO: 927, SEQ ID NO: 928, SEQ ID NO: 932, SEQ ID NO: 933, SEQ ID NO: 934, SEQ ID NO: 935, SEQ ID NO: 936, SEQ ID NO: 939, SEQ ID NO: 940, SEQ ID NO: 941, SEQ ID NO: 943, SEQ ID NO: 944, SEQ ID NO: 945 or SEQ ID NO: 946, wherein the polypeptide has insecticidal activity.

[0038] In some embodiments, the non-genomic nucleic acid molecule encodes a PIP-72 polypeptide, the PIP-72 polypeptide comprising an amino acid sequence identical to SEQ ID NO. The amino acid sequence shown in NO:2 has at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 78%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, wherein the polypeptide has insecticidal activity.

[0039] In some embodiments, the amino acid sequence of the non-genomic nucleic acid molecule has at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 78%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 4, wherein the polypeptide has insecticidal activity.

[0040] In some embodiments, the non-genomic nucleic acid molecule encodes a PIP-72 polypeptide, the PIP-72 polypeptide comprising an amino acid sequence identical to SEQ ID NO. The amino acid sequence shown in NO:6 has at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 78%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, wherein the polypeptide has insecticidal activity.

[0041] In some embodiments, the non-genomic nucleic acid molecule encodes a PIP-72 polypeptide, the PIP-72 polypeptide comprising an amino acid sequence identical to SEQ ID NO. The amino acid sequence shown in NO:8 has at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 78%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, wherein the polypeptide has insecticidal activity.

[0042] In some embodiments, the non-genomic nucleic acid molecule encodes the PIP-72 polypeptide, the PIP-72 polypeptide comprising an amino acid sequence identical to SEQ ID NO. The amino acid sequence shown in NO: 10 has at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 78%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, wherein the polypeptide has insecticidal activity.

[0043] In some embodiments, the non-genomic nucleic acid molecule encodes the PIP-72 polypeptide, the PIP-72 polypeptide comprising an amino acid sequence identical to SEQ ID NO. The amino acid sequence shown in NO:12 has at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 78%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, wherein the polypeptide has insecticidal activity.

[0044] In some embodiments, the non-genomic nucleic acid molecule encodes the PIP-72 polypeptide, the PIP-72 polypeptide comprising an amino acid sequence identical to SEQ ID NO. The amino acid sequence shown in NO:14 has at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 78%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, wherein the polypeptide has insecticidal activity.

[0045] In some embodiments, the non-genomic nucleic acid molecule encodes a PIP-72 polypeptide, the PIP-72 polypeptide comprising an amino acid sequence identical to SEQ ID NO. The amino acid sequence shown in NO:18 has at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 78%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, wherein the polypeptide has insecticidal activity.

[0046] In some embodiments, the non-genomic nucleic acid molecule encodes a PIP-72 polypeptide, the PIP-72 polypeptide comprising an amino acid sequence identical to SEQ ID NO. The amino acid sequence shown in NO:28 has at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 78%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, wherein the polypeptide has insecticidal activity.

[0047] In some embodiments, the non-genomic nucleic acid molecule encodes a PIP-72 polypeptide, the PIP-72 polypeptide comprising an amino acid sequence identical to SEQ ID NO. The amino acid sequence shown in NO:32 has at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 78%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, wherein the polypeptide has insecticidal activity.

[0048] In some embodiments, the non-genomic nucleic acid molecule encodes a PIP-72 polypeptide, the PIP-72 polypeptide comprising an amino acid sequence identical to SEQ ID NO. The amino acid sequence shown in NO: 927 has at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 78%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, wherein the polypeptide has insecticidal activity.

[0049] In some embodiments, the non-genomic nucleic acid molecule encodes a PIP-72 polypeptide, the PIP-72 polypeptide comprising an amino acid sequence identical to SEQ ID NO. The amino acid sequence shown in NO: 928 has at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 78%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, wherein the polypeptide has insecticidal activity.

[0050] In some embodiments, the non-genomic nucleic acid molecule encodes a PIP-72 polypeptide, the PIP-72 polypeptide comprising an amino acid sequence identical to SEQ ID NO. The amino acid sequence shown in NO: 932 has at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 78%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, wherein the polypeptide has insecticidal activity.

[0051] In some embodiments, the non-genomic nucleic acid molecule encodes a PIP-72 polypeptide, the PIP-72 polypeptide comprising an amino acid sequence identical to SEQ ID NO. The amino acid sequence shown in NO: 933 has at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 78%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, wherein the polypeptide has insecticidal activity.

[0052] In some embodiments, the non-genomic nucleic acid molecule encodes a PIP-72 polypeptide, the PIP-72 polypeptide comprising an amino acid sequence identical to SEQ ID NO. The amino acid sequence shown in NO: 934 has at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 78%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, wherein the polypeptide has insecticidal activity.

[0053] In some embodiments, the non-genomic nucleic acid molecule encodes a PIP-72 polypeptide, the PIP-72 polypeptide comprising an amino acid sequence identical to SEQ ID NO. The amino acid sequence shown in NO: 935 has at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 78%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, wherein the polypeptide has insecticidal activity.

[0054] In some embodiments, the non-genomic nucleic acid molecule encodes a PIP-72 polypeptide, the PIP-72 polypeptide comprising an amino acid sequence identical to SEQ ID NO. The amino acid sequence shown in NO: 936 has at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 78%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, wherein the polypeptide has insecticidal activity.

[0055] In some embodiments, the non-genomic nucleic acid molecule encodes a PIP-72 polypeptide, the PIP-72 polypeptide comprising an amino acid sequence identical to SEQ ID NO. The amino acid sequence shown in NO: 939 has at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 78%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, wherein the polypeptide has insecticidal activity.

[0056] In some embodiments, the non-genomic nucleic acid molecule encodes a PIP-72 polypeptide, the PIP-72 polypeptide comprising an amino acid sequence identical to SEQ ID NO. The amino acid sequence shown in NO: 940 has at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 78%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, wherein the polypeptide has insecticidal activity.

[0057] In some embodiments, the non-genomic nucleic acid molecule encodes a PIP-72 polypeptide, the PIP-72 polypeptide comprising an amino acid sequence identical to SEQ ID NO. The amino acid sequence shown in NO: 941 has at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 78%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, wherein the polypeptide has insecticidal activity.

[0058] In some embodiments, the non-genomic nucleic acid molecule encodes a PIP-72 polypeptide, the PIP-72 polypeptide comprising an amino acid sequence identical to SEQ ID NO. The amino acid sequence shown in NO: 943 has at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 78%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, wherein the polypeptide has insecticidal activity.

[0059] In some embodiments, the non-genomic nucleic acid molecule encodes a PIP-72 polypeptide, the PIP-72 polypeptide comprising an amino acid sequence identical to SEQ ID NO. The amino acid sequence shown in NO: 944 has at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 78%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, wherein the polypeptide has insecticidal activity.

[0060] In some embodiments, the non-genomic nucleic acid molecule encodes a PIP-72 polypeptide, the PIP-72 polypeptide comprising an amino acid sequence identical to SEQ ID NO. The amino acid sequence shown in NO: 945 has at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 78%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, wherein the polypeptide has insecticidal activity.

[0061] In some embodiments, the non-genomic nucleic acid molecule encodes a PIP-72 polypeptide, the PIP-72 polypeptide comprising an amino acid sequence identical to SEQ ID NO. The amino acid sequence shown in NO: 946 has at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 78%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, wherein the polypeptide has insecticidal activity.

[0062] In some embodiments, the non-genomic nucleic acid molecule encodes a PIP-72 polypeptide containing an amino acid sequence that shares at least 50% identity with the following amino acid sequences: SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 18, SEQ ID NO: 28, SEQ ID NO: 32, SEQ ID NO: 927, SEQ ID NO: 928, SEQ ID NO: 932, SEQ ID NO: 933, SEQ ID NO: 934, SEQ ID NO: 935, SEQ ID NO: 936, SEQ ID NO: 939, SEQ ID NO: 940, SEQ ID NO: 941, SEQ ID NO: 943, SEQ ID NO: 944, SEQ ID NO: 945, or SEQ ID NO: 946, wherein the PIP-72 polypeptide has at least one amino acid change compared to the following sequences: SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 18, SEQ ID NO: 28, SEQ ID NO: 32, SEQ ID NO: 927, SEQ ID NO: 928, SEQ ID NO: 932, SEQ ID NO: 933, SEQ ID NO: 934, SEQ ID NO: 935, SEQ ID NO: 936, SEQ ID NO: 939, SEQ ID NO: 940, SEQ ID NO: 941, SEQ ID NO: 943, SEQ ID NO: 944, SEQ ID NO: 945, or SEQ SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 18, SEQ ID NO: 28, SEQ ID NO: 32, SEQ ID NO: 927, SEQ ID NO: 928, SEQ ID NO: 932, SEQ ID NO: 933, SEQ ID NO: 934, SEQ ID NO: 935, SEQ ID NO: 936, SEQ ID NO: 939, SEQ ID NO: 940, SEQ ID NO: 941, SEQ ID NO: 943, SEQ ID NO: 944, SEQ ID NO: 945 or SEQ ID NO: 946, wherein the PIP-72 polypeptide has insecticidal activity.

[0063] In some embodiments, the non-genomic nucleic acid molecule encodes a PIP-72 polypeptide containing an amino acid sequence that has at least 50% identity with the amino acid sequence shown in SEQ ID NO: 2, wherein the PIP-72 polypeptide has at least one amino acid change compared to SEQ ID NO: 2, and the PIP-72 polypeptide has insecticidal activity.

[0064] In some embodiments, the non-genomic nucleic acid molecule encodes a PIP-72 polypeptide containing an amino acid sequence that has at least 50% identity with the amino acid sequence shown in SEQ ID NO: 4, wherein the PIP-72 polypeptide has at least one amino acid change compared to SEQ ID NO: 4, and the PIP-72 polypeptide has insecticidal activity.

[0065] In some embodiments, the non-genomic nucleic acid molecule encodes a PIP-72 polypeptide containing an amino acid sequence that has at least 50% identity with the amino acid sequence shown in SEQ ID NO: 6, wherein the PIP-72 polypeptide has at least one amino acid change compared to SEQ ID NO: 6, and the PIP-72 polypeptide has insecticidal activity.

[0066] In some embodiments, the non-genomic nucleic acid molecule encodes a PIP-72 polypeptide containing an amino acid sequence that has at least 50% identity with the amino acid sequence shown in SEQ ID NO: 8, wherein the PIP-72 polypeptide has at least one amino acid change compared to SEQ ID NO: 8, and the PIP-72 polypeptide has insecticidal activity.

[0067] In some embodiments, the non-genomic nucleic acid molecule encodes a PIP-72 polypeptide containing an amino acid sequence that has at least 50% identity with the amino acid sequence shown in SEQ ID NO: 10, wherein the PIP-72 polypeptide has at least one amino acid change compared to SEQ ID NO: 10, and the PIP-72 polypeptide has insecticidal activity.

[0068] In some embodiments, the non-genomic nucleic acid molecule encodes a PIP-72 polypeptide containing an amino acid sequence that has at least 50% identity with the amino acid sequence shown in SEQ ID NO: 12, wherein the PIP-72 polypeptide has at least one amino acid change compared to SEQ ID NO: 12, and the PIP-72 polypeptide has insecticidal activity.

[0069] In some embodiments, the non-genomic nucleic acid molecule encodes a PIP-72 polypeptide containing an amino acid sequence that has at least 50% identity with the amino acid sequence shown in SEQ ID NO: 14, wherein the PIP-72 polypeptide has at least one amino acid change compared to SEQ ID NO: 14, and the PIP-72 polypeptide has insecticidal activity.

[0070] In some embodiments, the non-genomic nucleic acid molecule encodes a PIP-72 polypeptide containing an amino acid sequence that has at least 60% identity with the amino acid sequence shown in SEQ ID NO: 18, wherein the PIP-72 polypeptide has at least one amino acid change compared to SEQ ID NO: 18, and the PIP-72 polypeptide has insecticidal activity.

[0071] In some embodiments, the non-genomic nucleic acid molecule encodes a PIP-72 polypeptide containing an amino acid sequence that has at least 85% identity with the amino acid sequence shown in SEQ ID NO: 28, wherein the PIP-72 polypeptide has at least one amino acid change compared to SEQ ID NO: 28, and the PIP-72 polypeptide has insecticidal activity.

[0072] In some embodiments, the non-genomic nucleic acid molecule encodes a PIP-72 polypeptide containing an amino acid sequence that has at least 95% identity with the amino acid sequence shown in SEQ ID NO: 32, wherein the PIP-72 polypeptide has at least one amino acid change compared to SEQ ID NO: 32, and the PIP-72 polypeptide has insecticidal activity.

[0073] In some embodiments, the non-genomic nucleic acid molecule encodes a PIP-72 polypeptide containing an amino acid sequence that has at least 95% identity with the amino acid sequence shown in SEQ ID NO: 927, wherein the PIP-72 polypeptide has at least one amino acid change compared to SEQ ID NO: 927, and the PIP-72 polypeptide has insecticidal activity.

[0074] In some embodiments, the non-genomic nucleic acid molecule encodes a PIP-72 polypeptide containing an amino acid sequence that has at least 95% identity with the amino acid sequence shown in SEQ ID NO: 928, wherein the PIP-72 polypeptide has at least one amino acid change compared to SEQ ID NO: 928, and the PIP-72 polypeptide has insecticidal activity.

[0075] In some embodiments, the non-genomic nucleic acid molecule encodes a PIP-72 polypeptide containing an amino acid sequence that has at least 95% identity with the amino acid sequence shown in SEQ ID NO: 932, wherein the PIP-72 polypeptide has at least one amino acid change compared to SEQ ID NO: 932, and the PIP-72 polypeptide has insecticidal activity.

[0076] In some embodiments, the non-genomic nucleic acid molecule encodes a PIP-72 polypeptide containing an amino acid sequence that has at least 95% identity with the amino acid sequence shown in SEQ ID NO: 933, wherein the PIP-72 polypeptide has at least one amino acid change compared to SEQ ID NO: 933, and the PIP-72 polypeptide has insecticidal activity.

[0077] In some embodiments, the non-genomic nucleic acid molecule encodes a PIP-72 polypeptide containing an amino acid sequence that has at least 95% identity with the amino acid sequence shown in SEQ ID NO: 934, wherein the PIP-72 polypeptide has at least one amino acid change compared to SEQ ID NO: 934, and the PIP-72 polypeptide has insecticidal activity.

[0078] In some embodiments, the non-genomic nucleic acid molecule encodes a PIP-72 polypeptide containing an amino acid sequence that has at least 95% identity with the amino acid sequence shown in SEQ ID NO: 935, wherein the PIP-72 polypeptide has at least one amino acid change compared to SEQ ID NO: 935, and the PIP-72 polypeptide has insecticidal activity.

[0079] In some embodiments, the non-genomic nucleic acid molecule encodes a PIP-72 polypeptide containing an amino acid sequence that has at least 95% identity with the amino acid sequence shown in SEQ ID NO: 936, wherein the PIP-72 polypeptide has at least one amino acid change compared to SEQ ID NO: 936, and the PIP-72 polypeptide has insecticidal activity.

[0080] In some embodiments, the non-genomic nucleic acid molecule encodes a PIP-72 polypeptide containing an amino acid sequence that has at least 95% identity with the amino acid sequence shown in SEQ ID NO: 939, wherein the PIP-72 polypeptide has at least one amino acid change compared to SEQ ID NO: 939, and the PIP-72 polypeptide has insecticidal activity.

[0081] In some embodiments, the non-genomic nucleic acid molecule encodes a PIP-72 polypeptide containing an amino acid sequence that has at least 95% identity with the amino acid sequence shown in SEQ ID NO: 940, wherein the PIP-72 polypeptide has at least one amino acid change compared to SEQ ID NO: 940, and the PIP-72 polypeptide has insecticidal activity.

[0082] In some embodiments, the non-genomic nucleic acid molecule encodes a PIP-72 polypeptide containing an amino acid sequence that has at least 95% identity with the amino acid sequence shown in SEQ ID NO: 941, wherein the PIP-72 polypeptide has at least one amino acid change compared to SEQ ID NO: 941, and the PIP-72 polypeptide has insecticidal activity.

[0083] In some embodiments, the non-genomic nucleic acid molecule encodes a PIP-72 polypeptide containing an amino acid sequence that has at least 95% identity with the amino acid sequence shown in SEQ ID NO: 943, wherein the PIP-72 polypeptide has at least one amino acid change compared to SEQ ID NO: 943, and the PIP-72 polypeptide has insecticidal activity.

[0084] In some embodiments, the non-genomic nucleic acid molecule encodes a PIP-72 polypeptide containing an amino acid sequence that has at least 95% identity with the amino acid sequence shown in SEQ ID NO: 944, wherein the PIP-72 polypeptide has at least one amino acid change compared to SEQ ID NO: 944, and the PIP-72 polypeptide has insecticidal activity.

[0085] In some embodiments, the non-genomic nucleic acid molecule encodes a PIP-72 polypeptide containing an amino acid sequence that is at least 95% identical to the amino acid sequence shown in SEQ ID NO: 945, wherein the PIP-72 polypeptide has at least one amino acid change compared to SEQ ID NO: 945, and the PIP-72 polypeptide has insecticidal activity.

[0086] In some embodiments, the non-genomic nucleic acid molecule encodes a PIP-72 polypeptide containing an amino acid sequence that has at least 95% identity with the amino acid sequence shown in SEQ ID NO: 946, wherein the PIP-72 polypeptide has at least one amino acid change compared to SEQ ID NO: 946, and the PIP-72 polypeptide has insecticidal activity.

[0087] In some embodiments, the non-genomic nucleic acid molecule encodes the PIP-72 polypeptide, which comprises the amino acid sequences SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 18, SEQ ID NO: 28, SEQ ID NO: 32, SEQ ID NO: 927, SEQ ID NO: 928, SEQ ID NO: 932, SEQ ID NO: 933, SEQ ID NO: 934, SEQ ID NO: 935, SEQ ID NO: 936, SEQ ID NO: 939, SEQ ID NO: 940, SEQ ID NO: 941, SEQ ID NO: 943, SEQ ID NO: 944, SEQ ID NO: 945, or SEQ ID NO: 946, but not SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 74, SEQ ID NO: 8, SEQ ID NO: 945, SEQ ID NO: 946, but not SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 74, SEQ ID NO: 946, SEQ ID NO: 947, SEQ ID NO: 948, SEQ ID NO: 949, SEQ ID NO: 940, SEQ ID NO: 941, SEQ ID NO: 943, SEQ ID NO: 944, SEQ ID NO: 945, or SEQ ID NO: 946. NO: 12, SEQ ID NO: 14, SEQ ID NO: 18, SEQ ID NO: 28, SEQ ID NO: 32, SEQ ID NO: 927, SEQ ID NO: 928, SEQ ID NO: 932, SEQ ID NO: 933, SEQ ID NO: 934, SEQ ID NO: 935, SEQ ID NO: 936, SEQ ID NO: 939, SEQ ID NO:940, SEQ ID NO:941, SEQ ID NO:943, SEQ ID NO:944, SEQ ID NO:945 or SEQ ID Compared to the natural amino acids at the corresponding positions in NO: 946, it has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, or 45 amino acid substitutions.

[0088] In some embodiments, the non-genomic nucleic acid molecule encodes a PIP-72 polypeptide containing the amino acid sequence SEQ ID NO: 2, but having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, or 45 amino acid substitutions compared to the native amino acids at the corresponding positions in SEQ ID NO: 2.

[0089] In some embodiments, the non-genomic nucleic acid molecule encodes a PIP-72 polypeptide containing the amino acid sequence SEQ ID NO: 4, but having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, or 45 amino acid substitutions compared to the native amino acids at the corresponding positions in SEQ ID NO: 4.

[0090] In some embodiments, the non-genomic nucleic acid molecule encodes a PIP-72 polypeptide containing the amino acid sequence SEQ ID NO: 6, but having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, or 45 amino acid substitutions compared to the native amino acids at the corresponding positions in SEQ ID NO: 6.

[0091] In some embodiments, the non-genomic nucleic acid molecule encodes a PIP-72 polypeptide containing the amino acid sequence SEQ ID NO: 8, but having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, or 45 amino acid substitutions compared to the native amino acids at the corresponding positions in SEQ ID NO: 8.

[0092] In some embodiments, the non-genomic nucleic acid molecule encodes a PIP-72 polypeptide containing the amino acid sequence SEQ ID NO: 10, but having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, or 45 amino acid substitutions compared to the native amino acids at the corresponding positions in SEQ ID NO: 10.

[0093] In some embodiments, the non-genomic nucleic acid molecule encodes a PIP-72 polypeptide containing the amino acid sequence SEQ ID NO: 12, but having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, or 45 amino acid substitutions compared to the native amino acids at the corresponding positions in SEQ ID NO: 12.

[0094] In some embodiments, the non-genomic nucleic acid molecule encodes a PIP-72 polypeptide containing the amino acid sequence SEQ ID NO: 14, but having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, or 45 amino acid substitutions compared to the native amino acids at the corresponding positions in SEQ ID NO: 14.

[0095] In some embodiments, the non-genomic nucleic acid molecule encodes a PIP-72 polypeptide containing the amino acid sequence SEQ ID NO: 18, but with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 amino acid substitutions compared to the native amino acids at the corresponding positions in SEQ ID NO: 18.

[0096] In some embodiments, the non-genomic nucleic acid molecule encodes a PIP-72 polypeptide containing the amino acid sequence SEQ ID NO: 28, but with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 amino acid substitutions compared to the native amino acid at the corresponding position in SEQ ID NO: 28.

[0097] In some embodiments, the non-genomic nucleic acid molecule encodes a PIP-72 polypeptide containing the amino acid sequence SEQ ID NO: 32, but having 1, 2, 3, 4 or 5 amino acid substitutions compared to the native amino acid at the corresponding position in SEQ ID NO: 32.

[0098] In some embodiments, the non-genomic nucleic acid molecule encodes a PIP-72 polypeptide containing the amino acid sequence SEQ ID NO: 927, but with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, or 45 amino acid substitutions compared to the native amino acids at the corresponding positions in SEQ ID NO: 927.

[0099] In some embodiments, the non-genomic nucleic acid molecule encodes a PIP-72 polypeptide containing the amino acid sequence SEQ ID NO: 928, but with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, or 45 amino acid substitutions compared to the native amino acids at the corresponding positions in SEQ ID NO: 928.

[0100] In some embodiments, the non-genomic nucleic acid molecule encodes a PIP-72 polypeptide containing the amino acid sequence SEQ ID NO: 932, but having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, or 45 amino acid substitutions compared to the native amino acids at the corresponding positions in SEQ ID NO: 932.

[0101] In some embodiments, the non-genomic nucleic acid molecule encodes a PIP-72 polypeptide containing the amino acid sequence SEQ ID NO: 933, but having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, or 45 amino acid substitutions compared to the native amino acids at the corresponding positions in SEQ ID NO: 933.

[0102] In some embodiments, the non-genomic nucleic acid molecule encodes a PIP-72 polypeptide containing the amino acid sequence SEQ ID NO: 934, but with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, or 45 amino acid substitutions compared to the native amino acids at the corresponding positions in SEQ ID NO: 934.

[0103] In some embodiments, the non-genomic nucleic acid molecule encodes a PIP-72 polypeptide containing the amino acid sequence SEQ ID NO: 935, but having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, or 45 amino acid substitutions compared to the native amino acids at the corresponding positions in SEQ ID NO: 935.

[0104] In some embodiments, the non-genomic nucleic acid molecule encodes a PIP-72 polypeptide containing the amino acid sequence SEQ ID NO: 936, but with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, or 45 amino acid substitutions compared to the native amino acids at the corresponding positions in SEQ ID NO: 936.

[0105] In some embodiments, the non-genomic nucleic acid molecule encodes a PIP-72 polypeptide containing the amino acid sequence SEQ ID NO: 939, but with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, or 45 amino acid substitutions compared to the native amino acids at the corresponding positions in SEQ ID NO: 939.

[0106] In some embodiments, the non-genomic nucleic acid molecule encodes a PIP-72 polypeptide containing the amino acid sequence SEQ ID NO: 940, but having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, or 45 amino acid substitutions compared to the native amino acids at the corresponding positions in SEQ ID NO: 940.

[0107] In some embodiments, the non-genomic nucleic acid molecule encodes a PIP-72 polypeptide containing the amino acid sequence SEQ ID NO: 941, but with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, or 45 amino acid substitutions compared to the native amino acids at the corresponding positions in SEQ ID NO: 941.

[0108] In some embodiments, the non-genomic nucleic acid molecule encodes a PIP-72 polypeptide containing the amino acid sequence SEQ ID NO: 943, but having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, or 45 amino acid substitutions compared to the native amino acids at the corresponding positions in SEQ ID NO: 943.

[0109] In some embodiments, the non-genomic nucleic acid molecule encodes a PIP-72 polypeptide containing the amino acid sequence SEQ ID NO: 944, but with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, or 45 amino acid substitutions compared to the native amino acids at the corresponding positions in SEQ ID NO: 944.

[0110] In some embodiments, the non-genomic nucleic acid molecule encodes a PIP-72 polypeptide containing the amino acid sequence SEQ ID NO: 945, but having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, or 45 amino acid substitutions compared to the native amino acids at the corresponding positions in SEQ ID NO: 945.

[0111] In some embodiments, the non-genomic nucleic acid molecule encodes a PIP-72 polypeptide containing the amino acid sequence SEQ ID NO: 946, but with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, or 45 amino acid substitutions compared to the native amino acids at the corresponding positions in SEQ ID NO: 946.

[0112] In some embodiments, the non-genomic nucleic acid molecule encodes a PIP-72 polypeptide comprising the amino acid sequence SEQ ID NO: 846, which, compared to the native amino acid at the corresponding position in SEQ ID NO: 2, has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, or 45 amino acid substitutions at the positions indicated by Xaa.

[0113] In some embodiments, the non-genomic nucleic acid molecule encodes a PIP-72 polypeptide comprising the amino acid sequence SEQ ID NO: 847, which, compared to the native amino acid at the corresponding position in SEQ ID NO: 2, has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, or 45 amino acid substitutions at the positions indicated by Xaa.

[0114] In some embodiments, the non-genomic nucleic acid molecule encodes a PIP-72 polypeptide comprising the amino acid sequence SEQ ID NO: 848, which, compared to the native amino acid at the corresponding position in SEQ ID NO: 2, has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, or 45 amino acid substitutions at the positions indicated by Xaa.

[0115] In some embodiments, the non-genomic nucleic acid molecule encodes a PIP-72 polypeptide comprising the amino acid sequence SEQ ID NO: 849, which, compared to the native amino acid at the corresponding position in SEQ ID NO: 2, has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, or 45 amino acid substitutions at the positions indicated by Xaa.

[0116] In some embodiments, the nucleic acid molecule encodes a PIP-72 polypeptide, the PIP-72 polypeptide comprising the amino acid sequence SEQ ID. NO: 846, where the second Xaa is Gly, Ala, Cys, Asp, Glu, Ile, Lys, Leu, Asn, Arg, Ser, Thr, Val, Trp, or Tyr; the third Xaa is Ile or Trp; the fourth Xaa is Thr, Ala, Asp, Glu, His, Ile, Lys, Leu, Arg, Ser, Val, Trp, or Tyr; the fifth Xaa is Val, Ala, Cys, Gly, His, Ile, or Tyr; the sixth Xaa is Thr, Ala, Cys, Phe, Gly, His, Ile, Lys, Met, Pro, Gln, Arg, Ser, Trp, or Tyr; the seventh Xaa... The first Xaa is Asn, Ala, or Val; the 8th Xaa is Asn, Ala, Cys, Asp, Glu, Gly, His, Ile, Lys, Leu, Met, Gln, Arg, Ser, Thr, or Val; the 9th Xaa is Ser, Ala, Cys, Gly, or Thr; the 10th Xaa is Ser, Ala, Glu, Phe, Gly, His, Ile, Lys, Leu, Asn, Pro, Gln, Arg, Thr, or Trp; the 11th Xaa is Asn, Ala, Cys, Asp, Glu, Gly, His, Ile, Lys, Leu, Met, Gln, Ser, Thr, Val, or Tyr; the 12th Xaa is Pr. oThe Xaa at position 13 is Ile, Asn, Gln, Leu, Asn, Gln, Arg, Ser, Thr, Val, Trp, or Tyr; the Xaa at position 14 is Glu, Ala, Cys, Phe, His, Lys, or Gln; the Xaa at position 15 is Val, Ala, Cys, Ile, Met, or Arg; the Xaa at position 17 is Ile, Glu, or Val; the Xaa at position 18 is Asn or Ser; the Xaa at position 19 is His, Ala, Glu, Lys, Leu, or Pro. The Xaa at position 20 is Trp, Ala, or Thr; the Xaa at position 22 is Ser, Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Thr, Val, or Tyr; the Xaa at position 23 is Asp, Ala, Gly, His, Lys, Met, Asn, Gln, Ser, Thr, or Val; the Xaa at position 24 is Gly, Asp, or Phe; the Xaa at position 25 is Asp, Ala, Glu, Phe, Asn, or Gln; the Xaa at position 26 is Thr. Glu or Pro; Xaa at position 27 is Ser, Ala, Cys, Asp, Glu, Phe, Gly, His, Asn, Gln, Arg, or Thr; Xaa at position 28 is Phe, Pro, Trp, or Tyr; Xaa at position 29 is Phe, Ala, Cys, Ile, Leu, Gln, Arg, Trp, or Tyr; Xaa at position 30 is Ser, Ala, Cys, Asp, Glu, Phe, Gly, His, Lys, Leu, Met, Asn, Pro, Gln, Arg, Thr, Val, Trp, or Tyr; Xaa at position 31 is V al, Ile, or Leu; Xaa at position 32 is Gly, Ala, Asp, Glu, Phe, His, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, or Tyr; Xaa at position 33 is Asn, Ala, Cys, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Pro, Gln, Arg, Ser, Thr, Val, or Tyr; Xaa at position 34 is Gly, Glu, Phe, His, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, or Tyr;The Xaa at position 35 is Lys, Ala, Cys, Asp, Gly, His, Ile, Leu, Met, Asn, Gln, Arg, Ser, Thr, or Val; the Xaa at position 36 is Gln, Ala, Cys, Glu, Gly, His, Ile, Lys, Leu, Asn, Pro, Arg, Ser, Thr, or Val; the Xaa at position 37 is Glu, Ala, Cys, Asp, Phe, Gly, Ile, Lys, Leu, Met, Asn, Ser, Thr, or yal; the Xaa at position 38 is Thr, Ala, Cys, Asp, Glu, Phe, Gly, His. Ile, Leu, Met, Asn, Gln, Arg, Ser, Val, Trp, or Tyr; Xaa at position 39 is Trp or Phe; Xaa at position 40 is Asp, Ala, Cys, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp, or Tyr; Xaa at position 42 is Ser, Ala, Cys, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Thr, Val, Trp, or Tyr; Xaa at position 44 is Ser, A The Xaa at position 45 is Arg, Lys, or Ser; the Xaa at position 46 is Gly, Ala, or Gln; the Xaa at position 47 is Phe, Cys, Val, or Tyr; the Xaa at position 48 is Val, Ile, or Leu; the Xaa at position 49 is Leu, Cys, Phe, Met, Arg, or Tyr; the Xaa at position 50 is Ser, Ala, Cys, Asp, Ile, Met, Pro, Gln, Thr, or Val; the Xaa at position 51 is Leu, Ala, Cys, Met. Or Val; Xaa at position 52 is Lys, Cys, Phe, His, Ile, Leu, Met, Asn, Arg, Ser, Thr, Trp, or Tyr; Xaa at position 53 is Lys, Ala, Cys, Asp, Glu, Phe, His, Ile, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, or Tyr; Xaa at position 54 is Asn, Cys, Asp, Glu, Phe, Gly, Lys, Met, Gln, Arg, Ser, or Trp; Xaa at position 56 is Ala, Gly, Leu, Asn, Pro, Gln, Arg, Ser, or Thr;Xaa at position 57 is Gln, Glu, Leu, Met, Ser, or Thr; Xaa at position 58 is His, Ala, Asp, Phe, Leu, Met, Asn, Arg, Trp, or Tyr; Xaa at position 60 is Tyr, Glu, or Phe; Xaa at position 63 is Gln, Cys, Gly, Ile, Leu, Met, Asn, Thr, Val, or Tyr; Xaa at position 64 is Ala, Phe, Gly, His, Arg, Ser, or Tyr; Xaa at position 65 is Ser, Ala, Cys, Asp, Glu, Phe, Gly, His, Ile, Leu, Asn, Trp, or Tyr. r or Val; Xaa at position 66 is Ser, Ala, or Gly; Xaa at position 67 is Lys, Ala, Cys, Asp, Phe, His, Ile, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp, or Tyr; Xaa at position 68 is Ile, Asp, Leu, or Val; Xaa at position 69 is Glu, Ala, Cys, Asp, Phe, His, Ile, Leu, Met, Gln, Arg, Ser, Thr, Val, or Tyr; Xaa at position 70 is Val, Cys, or Ile; Xaa at position 71 is Asp, Ala, Cys, or Gly. His, Ile, Leu, Met, Asn, Ser, Thr, Val, or Tyr; Xaa at position 72 is Asn, Ala, Cys, Asp, Glu, Gly, Lys, Met, Pro, Gln, Arg, Ser, Thr, Val, or Trp; Xaa at position 73 is Asn, Ala, Cys, Asp, Phe, Gly, His, Ile, Leu, Ser, Thr, Val, or Tyr; Xaa at position 74 is Ala, Cys, Asp, Phe, Gly, His, Ile, Leu, Asn, Gln, Arg, Ser, Thr, Val, or Tyr; Xaa at position 75 is Va. l, Cys, Ile, or Leu; Xaa at position 76 is Lys, Ala, Cys, Phe, His, Ile, Leu, Gln, Arg, Ser, Thr, Val, Trp, or Tyr; Xaa at position 77 is Asp or Tyr; Xaa at position 78 is Gln, Ala, Cys, Asp, Phe, Gly, His, Ile, Leu, Met, Asn, Arg, Ser, Thr, Val, or Tyr; Xaa at position 79 is Gly, Arg, Ala, Cys, Asp, Glu, Phe, His, Lys, Leu, Asn, Gln, Arg, Ser, Thr, Trp, or Tyr;The Xaa at position 80 is Arg, Ala, Cys, Asp, Phe, Gly, His, Ile, Leu, Asn, Ser, Thr, Val, or Tyr; the Xaa at position 81 is Leu, Ala, Cys, Asp, Phe, Gly, His, Ile, Asn, Pro, Arg, Ser, Thr, or Val; the Xaa at position 82 is Ile, Ala, Leu, Met, Arg, or Val; the Xaa at position 83 is Glu, Ala, Cys, Asp, P he, Gly, His, Ile, Lys, Leu, Asn, Pro, Arg, Ser, Thr, Val, or Tyr; Xaa at position 84 is Pro, Ala, Cys, Glu, Ile, Ser, Val, Trp, or Tyr; Xaa at position 85 is Leu, Cys, Gly, or Val; and Xaa at position 86 is Ser, Ala, Ile, Thr, or Val, wherein optionally 1 to 14 amino acids are deleted from the N-terminus and / or C-terminus of the PIP-72 polypeptide.

[0117] In some embodiments, the nucleic acid molecule encodes the PIP-72 polypeptide, which contains the amino acid sequence SEQ ID NQ: 847, wherein Xaa at position 2 is Gly, Lys, or Ala; Xaa at position 3 is Ile or Leu; Xaa at position 4 is Thr or Ser; Xaa at position 5 is Val or Ile; Xaa at position 6 is Thr or Lys; Xaa at position 8 is Asn, Lys, Gly, or Ser; Xaa at position 9 is Ser or Ala; Xaa at position 11 is Asn, Lys, His, or Thr; Xaa at position 12 is Pro, Thr, Lys, or Ser; Xaa at position 13 is Ile or Val; Xaa at position 14 is Glu or Asp; Xaa at position 15 is... The Xaa at position 1 is Val, Ala, or Ile; the Xaa at position 16 is Ala or Ser; the Xaa at position 17 is Ile or Val; the Xaa at position 18 is Asn or Ser; the Xaa at position 19 is His, Lys, Arg, Gln, or Ala; the Xaa at position 21 is Gly or Arg; the Xaa at position 22 is Ser, Lys, Asn, Asp, or Thr; the Xaa at position 25 is Asp or Asn; the Xaa at position 26 is Thr or Asp; the Xaa at position 27 is Ser, Thr, Asn, or Lys; the Xaa at position 28 is Phe, Tyr, or P ro; Xaa at position 29 is Phe or Tyr; Xaa at position 30 is Ser, Gly, or Lys; Xaa at position 31 is Val, Ile, or Met; Xaa at position 32 is Gly, Ala, or Asp; Xaa at position 33 is Asn, Ser, Gln, or Pro; Xaa at position 35 is Lys, Glu, or Ser; Xaa at position 36 is Gln, Asn, or Ser; Xaa at position 37 is Glu or Asp; Xaa at position 38 is Thr or Ser; Xaa at position 42 is Ser or Asn; Xaa at position 44 is Ser, Asp, or Al Xaa at position 47 is Phe or Tyr; Xaa at position 48 is Leu or Met; Xaa at position 49 is Leu or Met; Xaa at position 50 is Ser, Ala, or Tyr; Xaa at position 51 is Leu or Val; Xaa at position 52 is Lys or Gln; Xaa at position 53 is Lys, Arg, Met, or Leu; Xaa at position 54 is Asn, Lys, or Gly; Xaa at position 55 is Gly or Ser; Xaa at position 56 is Ala, Thr, Gln, or Ser; Xaa at position 57 is Gln, Val, or Ala.Xaa at position 58 is His, Ala, Lys, Tyr, or Thr; Xaa at position 59 is Pro or Thr; Xaa at position 62 is Val or Ile; Xaa at position 63 is Gln, Ser, or Leu; Xaa at position 64 is Ala, Gln, or Ser; Xaa at position 65 is Ser or Thr; Xaa at position 67 is Lys, Gln, Arg, or Asn; Xaa at position 69 is Glu, Lys, or Val; Xaa at position 70 is Val or Ile; Xaa at position 71 is Asp, Glu, or Tyr; Xaa at position 72 is Asn, His, Ser, or Asp; Xaa at position 73 is Asn, Ser, or Asp. Xaa at position 74 is Ala, Thr, Met, Ile, or Lys; Xaa at position 76 is Lys or Thr; Xaa at position 78 is Gln, His, or Ser; Xaa at position 80 is Arg, Glu, or Gln; Xaa at position 81 is Leu, Pro, Ala, or Thr; Xaa at position 82 is Ile or Leu; Xaa at position 83 is Glu, His, Asn, Gln, or Leu; Xaa at position 85 is Leu, Val, or Ala; and Xaa at position 86 is Ser, Ala, Tyr, or Asn, wherein optionally 1 to 14 amino acids are deleted from the N-terminus and / or C-terminus of the PIP-72 polypeptide, and / or an amino acid is inserted between residues at positions 24 and 25, compared to SEQ ID NO: 847.

[0118] In some embodiments, the nucleic acid molecule encodes the PIP-72 polypeptide, which contains the amino acid sequence SEQ ID NO: 848, wherein Xaa at position 2 is Gly, Lys, Ala, or Arg; Xaa at position 3 is Ile, Leu, or Val; Xaa at position 4 is Thr or Ser; Xaa at position 5 is Val, Ile, or Leu; Xaa at position 6 is Thr, Lys, Ser, or Arg; Xaa at position 8 is Asn, Lys, Gly, Ser, Gln, Arg, Thr, or Ala; Xaa at position 9 is Ser, Ala, or Thr; Xaa at position 11 is Asn, Lys, Thr, Gln, Arg, His, or Ser; and Xaa at position 12 is Pro, T hr, Lys, Ser, or Arg; Xaa at position 13 is Ile, Val, or Leu; Xaa at position 14 is Glu or Asp; Xaa at position 15 is Val, Ala, Ile, or Leu; Xaa at position 16 is Ala or Ser; Xaa at position 17 is Ile, Val, or Leu; Xaa at position 18 is Asn, Ser, Gln, or Thr; Xaa at position 19 is His, Lys, Ala, Gln, Asn, or Arg; Xaa at position 21 is Gly, Arg, or Lys; Xaa at position 22 is Ser, Lys, Asn, Thr, or Arg. Asp, Glu, or Gln; Xaa at position 25 is Asp, Asn, Glu, or Gln; Xaa at position 26 is Thr, Asp, Ser, or Glu; Xaa at position 27 is Ser, Thr, Lys, Asn, Gln, or Arg; Xaa at position 28 is Phe, Tyr, Pro, or Trp; Xaa at position 29 is Phe, Tyr, or Trp; Xaa at position 30 is Ser, Gly, Lys, Thr, or Arg; Xaa at position 31 is Val, Ile, Met, or Leu; Xaa at position 32 is Gly, Ala, Asp, or Glu; Xaa at position 33... The Xaa at position 35 is Asn, Ser, Gln, Pro, or Thr; the Xaa at position 36 is Gln, Ser, Asn, or Thr; the Xaa at position 37 is Glu or Asp; the Xaa at position 38 is Thr or Ser; the Xaa at position 42 is Ser, Asn, Thr, or Gln; the Xaa at position 44 is Ser, Asp, Ala, Leu, Thr, Glu, Ile, or Val; the Xaa at position 47 is Phe, Tyr, or Trp; the Xaa at position 48 is Leu, Met, Ile, or Val.Xaa at position 49 is Leu, Met, Ile, or Val; Xaa at position 50 is Ser, Ala, Tyr, or Thr; Xaa at position 51 is Leu, Val, or Ile; Xaa at position 52 is Lys, Gln, Arg, or Asn; Xaa at position 53 is Lys, Arg, Met, Leu, Ile, or Val; Xaa at position 54 is Asn, Lys, Gly, Gln, or Arg; Xaa at position 55 is Gly, Ser, or Thr; Xaa at position 56 is Ala, Thr, Gln, Ser, or Asn; Xaa at position 57 is Gln, Va The Xaa at position 58 is His, Ala, Asn, Leu, or Ile; the Xaa at position 59 is Pro, Thr, or Ser; the Xaa at position 62 is Val, Ile, or Leu; the Xaa at position 63 is Gln, Ser, Leu, Asn, Thr, Ile, or Val; the Xaa at position 64 is Ala, Gln, Ser, Asn, or Thr; the Xaa at position 65 is Ser or Thr; the Xaa at position 67 is Lys, Gln, Asn, or Arg; the Xaa at position 69 is Glu, Val, Asp, Lys, or Arg. The Xaa at position 70 is Val, Ile, or Leu; the Xaa at position 71 is Asp, Glu, Tyr, or Trp; the Xaa at position 72 is Asn, His, Ser, Asp, Gln, Thr, or Glu; the Xaa at position 73 is Asn, Ser, Asp, Gln, Thr, or Glu; the Xaa at position 74 is Ala, Thr, Met, Ile, Lys, Ser, Leu, Val, or Arg; the Xaa at position 76 is Lys, Thr, Arg, or Ser; the Xaa at position 78 is Gln, His, Ser, Asn, or Thr; the Xaa at position 79 is... Xaa at position 80 is Arg, Glu, Gln, Lys, Asp, or Asn; Xaa at position 81 is Leu, Pro, Thr, Ile, Val, Ala, or Ser; Xaa at position 82 is Ile, Leu, or Val; Xaa at position 83 is Glu, His, Asn, Leu, Gln, Ile, or Val; Xaa at position 85 is Leu, Val, or Ala; and Xaa at position 86 is Ser, Ala, Tyr, Asn, or Thr, wherein optionally 1 to 14 amino acids are deleted from the N-terminus and / or C-terminus of the PIP-72 polypeptide, and / or an amino acid is inserted between residues at positions 24 and 25, compared to SEQ ID NO: 848.

[0119] In some embodiments, the nucleic acid molecule encodes the PIP-72 polypeptide, which contains the amino acid sequence SEQ ID NO: 849, wherein Xaa at position 2 is Gly, Ala, Cys, Asp, Glu, Ile, Lys, Leu, Asn, Arg, Ser, Thr, Val, Trp, or Tyr; Xaa at position 3 is Ile, Leu, Val, or Trp; Xaa at position 4 is Thr, Ala, Asp, Glu, His, Ile, Lys, Leu, Arg, Ser, Val, Trp, or Tyr; Xaa at position 5 is Val, Ala, Cys, Gly, His, Ile, Leu, or Tyr; and Xaa at position 6 is Thr, Ala, Cys, Gly, As, Ile, Leu, or Tyr. Phe, Gly, His, Ile, Lys, Met, Pro, Gln, Arg, Ser, Trp, or Tyr; Xaa at position 7 is Asn, Ala, or Val; Xaa at position 8 is Asn, Lys, Gly, Ser, Gln, Arg, Thr, Ala, Cys, Asp, Glu, His, Ile, Leu, Met, or Val; Xaa at position 9 is Ser, Ala, Cys, Gly, or Thr; Xaa at position 11 is Asn, Lys, Thr, Gln, Arg, Ser, Ala, Cys, Asp, Glu, Gly, H is, Ile, Leu, Met, Val, or Tyr; Xaa at position 12 is Pro, Thr, Lys, Ser, Arg, Ala, Cys, Asp, Glu, Gly, His, Leu, Asn, Gln, Arg, Val, Trp, or Tyr; Xaa at position 13 is Ile, Asn, Gln, Leu, or Val; Xaa at position 14 is Glu, Ala, Cys, Phe, His, Lys, Asp, or Gln; Xaa at position 15 is Val, Ala, Ile, Leu, Cys, Met, or Arg; Xaa at position 16 is A The Xaa at position 17 is Ile, Glu, Leu, or Val; the Xaa at position 18 is Asn, Gln, Thr, or Ser; the Xaa at position 19 is His, Lys, Ala, Arg, Glu, Leu, Pro, Ser, or Tyr; the Xaa at position 20 is Trp, Ala, or Thr; the Xaa at position 21 is Gly, Arg, or Lys; the Xaa at position 22 is Ser, Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Thr, Val, or Tyr.The Xaa at position 23 is Asp, Ala, Gly, His, Lys, Met, Asn, Gln, Ser, Thr, or Val; the Xaa at position 24 is Gly, Asp, or Phe; the Xaa at position 25 is Asp, Ala, Glu, Phe, Asn, or Gln; the Xaa at position 26 is Thr, Glu, Asp, Ser, or Pro; the Xaa at position 27 is Ser, Thr, Lys, Arg, Ala, Cys, Asp, Glu, Phe, Gly, His, Asn, or Gln; the Xaa at position 28 is Phe, Tyr, Pro, or Trp; the 2 The Xaa at position 9 is Phe, Ala, Cys, Ile, Leu, Gln, Arg, Trp, or Tyr; the Xaa at position 30 is Ser, Gly, Lys, Thr, Arg, Ala, Cys, Asp, Glu, Phe, His, Leu, Met, Asn, Pro, Gln, Val, Trp, or Tyr; the Xaa at position 31 is Val, Ile, Met, or Leu; the Xaa at position 32 is Gly, Ala, Asp, Glu, Phe, His, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, or T rp or Tyr; Xaa at position 33 is Asn, Ser, Gln, Pro, Thr, Ala, Cys, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Arg, Val, or Tyr; Xaa at position 34 is Gly, Glu, Phe, His, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, or Tyr; Xaa at position 35 is Lys, Glu, Ala, Cys, Asp, Gly, His, Ile, Leu, Met, Asn, Gln, Arg, Ser, Thr, or Val; at position 36... Xaa is Gln, Ala, Cys, Glu, Gly, His, Ile, Lys, Leu, Asn, Pro, Arg, Ser, Thr, or Val; Xaa at position 37 is Glu, Asp, Ala, Cys, Phe, Gly, Ile, Lys, Leu, Met, Asn, Ser, Thr, or Val; Xaa at position 38 is Thr, Ser, Ala, Cys, Asp, Glu, Phe, Gly, His, Ile, Leu, Met, Asn, Gln, Arg, Val, Trp, or Tyr; Xaa at position 39 is Trp or Phe.The Xaa at position 40 is Asp, Ala, Cys, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp, or Tyr; the Xaa at position 42 is Ser, Asn, Thr, Ala, Cys, Asp, Glu, Phe, Gly, Ile, Lys, Leu, Met, Arg, Val, Trp, Tyr, or Gln; the Xaa at position 44 is Ser, Asp, Ala, Leu, Thr, Glu, Ile, Ala, Gly, Leu, Met, Asn, Pro, Gln, Val, Tyr, or Va. The Xaa at position 45 is Arg, Lys, or Ser; the Xaa at position 46 is Gly, Ala, or Gln; the Xaa at position 47 is Phe, Tyr, Cys, Val, or Trp; the Xaa at position 48 is Leu, Met, Ile, Cys, Phe, Met, Arg, Tyr, or Val; the Xaa at position 49 is Leu, Met, Ile, or Val; the Xaa at position 50 is Ser, Ala, Tyr, Cys, Asp, Ile, Met, Pro, Gln, Val, or Thr; the Xaa at position 51 is Leu, Val, Ala, Cys, Met, or Ile; the Xaa at position 52... The first Xaa is Lys, Cys, Phe, His, Ile, Leu, Met, Asn, Arg, Ser, Thr, Gln, Trp, or Tyr; the second Xaa at position 53 is Lys, Arg, Met, Leu, Ile, Ala, Cys, Asp, Glu, Phe, His, Asn, Gln, Ser, Thr, Tyr, or Val; the third Xaa at position 54 is Asn, Cys, Asp, Glu, Phe, Gly, Lys, Met, Gln, Arg, Ser, or Trp; the fourth Xaa at position 55 is Gly, Ser, or Thr; the fifth Xaa at position 56 is Ala, Thr, Gln, Ser, or Gly. The Xaa at position 57 is Gln, Glu, Leu, Met, Ser, Val, Ala, Asn, Ile, or Thr; the Xaa at position 58 is His, Ala, Lys, Asp, Phe, Leu, Met, Asn, Arg, Trp, Tyr, or Thr; the Xaa at position 59 is Pro, Thr, or Ser; the Xaa at position 60 is Tyr, Glu, or Phe; the Xaa at position 62 is Val, Ile, or Leu; the Xaa at position 63 is Gln, Ser, Cys, Gly, Ile, Leu, Met, Asn, Thr, Val, or Tyr.Xaa at position 64 is Ala, Gln, Asn, Phe, Gly, His, Arg, Ser, or Tyr; Xaa at position 65 is Ser, Ala, Cys, Asp, Glu, Phe, Gly, His, Ile, Leu, Asn, Val, or Thr; Xaa at position 66 is Ser, Ala, or Gly; Xaa at position 67 is Lys, Gln, Asn, or Arg; Xaa at position 68 is Lys, Ala, Cys, Asp, Phe, His, Ile, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp, or Tyr; Xaa at position 68 is Ile... Asp, Leu, or Val; Xaa at position 69 is Glu, Ala, Cys, Asp, Phe, His, Ile, Leu, Met, Gln, Arg, Ser, Thr, Val, or Tyr; Xaa at position 70 is Val, Ile, Cys, or Leu; Xaa at position 71 is Asp, Glu, Tyr, Ala, Cys, Gly, His, Ile, Leu, Met, Asn, Ser, Thr, Val, or Trp; Xaa at position 72 is Asn, Ala, Cys, Asp, Glu, Gly, Lys, Met, Pro, Gln, Arg, Ser, Thr, Val, His, or Trp; Xaa at position 73 is Asn, Ser, Asp, Gln, Thr, Ala, Cys, Phe, Gly, His, Ile, Leu, Val, Tyr, or Glu; Xaa at position 74 is Ala, Thr, Met, Ile, Lys, Ser, Leu, Val, Cys, Asp, Phe, Gly, His, Asn, Gln, Tyr, or Arg; Xaa at position 75 is Val, Cys, Ile, or Leu; Xaa at position 76 is Lys, Ala, Cys, Phe, His, Ile, Leu, Gln, Arg, Ser, Thr, Val, Trp, or Tyr; 77th position The Xaa at position 70 is Asp or Tyr; the Xaa at position 78 is Gln, His, Ser, Asn, Ala, Cys, Asp, Phe, Gly, Ile, Leu, Met, Asn, Arg, Val, Tyr, or Thr; the Xaa at position 79 is Gly, Arg, Ala, Cys, Asp, Glu, Phe, His, Lys, Leu, Asn, Gln, Arg, Ser, Thr, Trp, or Tyr; the Xaa at position 80 is Arg, Glu, Gln, Lys, Asp, Ala, Cys, Phe, Gly, His, Ile, Leu, Ser, Thr, Val, Tyr, or Asn.The Xaa at position 81 is Leu, Pro, Thr, Ile, Val, Ala, Cys, Asp, Phe, Gly, His, or Ser; the Xaa at position 82 is Ile, Ala, Leu, Met, Arg, and Val; the Xaa at position 83 is Glu, His, Asn, Leu, Gln, Ile, Ala, Cys, Asp, Phe, Gly, Lys, Pro, Arg, Ser, Thr, or Tyr. Or Val; Xaa at position 84 is Pro, Ala, Cys, Glu, Ile, Ser, Val, Trp, or Tyr; Xaa at position 85 is Leu, Val, Cys, Gly, or Ala; and Xaa at position 86 is Ser, Ala, Tyr, Asn, Ile, Val, or Thr, wherein optionally 1 to 14 amino acids are deleted from the N-terminus and / or C-terminus of the PIP-72 polypeptide, and / or an amino acid is inserted between residues at positions 24 and 25 compared to SEQ ID NO: 849.

[0120] In some embodiments, the nucleic acid molecule encodes a PIP-72 polypeptide, the amino acid motif of which is represented by positions 37 to 51 of the following sequences: SEQ ID NO: 846, SEQ ID NO: 847, SEQ ID NO: 848, or SEQ ID NO: 849.

[0121] In some embodiments, the nucleic acid molecule encodes a PIP-72 polypeptide containing an amino acid sequence that has at least 50% identity with the amino acid sequence shown in SEQ ID NO: 2.

[0122] In some embodiments, exemplary nucleic acid molecules encode the PIP-72 polypeptide shown in the following sequences: SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 18, SEQ ID NO: 28, SEQ ID NO: 32, any one of SEQ ID NO: 528 to SEQ ID NO: 768, any one of SEQ ID NO: 825 to SEQ ID NO: 844, SEQ ID NO: 771, SEQ ID NO: 772, SEQ ID NO: 852, any one of SEQ ID NO: 903 to SEQ ID NO: 914, SEQ ID NO: 927, SEQ ID NO: 928, SEQ ID NO: 932, SEQ ID NO: 933, SEQ ID NO: 934, SEQ ID NO: 935, SEQ ID NO: 936, SEQ ID NO: 939, SEQ ID NO: 940, SEQ ID NO: 941 ... SEQ ID NO: 943, SEQ ID NO: 944, SEQ ID NO: 945 or SEQ ID NO: 946, and amino acid substitutions, amino acid deletions, amino acid insertions, amino acid fragments and combinations thereof in these polypeptides.

[0123] In some embodiments, the nucleic acid molecule encodes the PIP-72 polypeptide shown in Tables 14, 17, 20, 23, 24, 26, 28 and / or 29, and combinations thereof with amino acid substitutions, amino acid deletions and / or amino acid insertions.

[0124] This disclosure also provides nucleic acid molecules encoding transcription and / or translation products, which are subsequently spliced ​​to ultimately produce a functional PIP-72 polypeptide. Splicing can be performed in vitro or in vivo and can involve cis or trans splicing. The substrate for splicing can be a polynucleotide (e.g., RNA transcript) or a polypeptide. An example of polynucleotide cis splicing is the removal of introns from the inserted coding sequence, followed by splicing two flanking exon regions to obtain the PIP-72 polypeptide coding sequence. An example of trans splicing would be the encryption of polynucleotides by splitting the coding sequence into two or more fragments, which can be transcribed individually and then spliced ​​to form a full-length insecticidal coding sequence. The use of splicing enhancer sequences that can be introduced into the construct can facilitate cis or trans splicing of the polypeptide (US Patents 6,365,377 and 6,531,316). Therefore, in some embodiments, the polynucleotide does not directly encode the full-length PIP-72 polypeptide, but rather encodes one or more fragments of the PIP-72 polypeptide. Functional PIP-72 peptides can be expressed with these polynucleotides via mechanisms involving splicing, where splicing can occur at the polynucleotide (e.g., intron / exon) and / or peptide (e.g., intron / exon) levels. This can be used, for example, to control the expression of insecticidal activity, since the functional insecticidal peptide will only be expressed if all the necessary fragments are expressed in an environment that allows for splicing, thus producing a functional product. Furthermore, the introduction of one or more insert sequences into the polynucleotide can facilitate recombination with low-homology polynucleotides; the use of introns or introns in the insert sequences facilitates the removal of the intercalated sequences, thereby restoring the function of the encoded variant.

[0125] Embodiments of this disclosure also cover nucleic acid molecules that are fragments of nucleic acid sequences encoding these PIP-72 polypeptides. As used herein, a “fragment” refers to a portion of a nucleic acid sequence encoding a PIP-72 polypeptide. Fragments of nucleic acid sequences may encode the biologically active portion of the PIP-72 polypeptide or may be used as hybridization probes or PCR primers when using the methods disclosed below. Nucleic acid molecules that are fragments of nucleic acid sequences encoding PIP-72 polypeptides contain at least about 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, or 260 consecutive nucleotides, the number of which is at most the total number of nucleotides present in the full-length nucleic acid sequence encoding the PIP-72 polypeptide disclosed herein, depending on the intended use. “Consecutive nucleotides” herein refers to nucleotide residues that are immediately adjacent to each other. Protein fragments encoded by fragments of nucleic acid sequences of embodiments of this disclosure will retain the biological activity of the PIP-72 polypeptide, and thus retain insecticidal activity. As used herein, "retained PIP-72 activity" refers to an insecticidal activity of the peptide of at least about 10%, at least about 30%, at least about 50%, at least about 70%, 80%, 90%, 95%, or higher of the full-length PIP-72Aa peptide shown in SEQ ID NO: 2. In one embodiment, the insecticidal activity is lepidopteran activity. In one embodiment, the insecticidal activity is activity against Coleoptera species. In one embodiment, the insecticidal activity is activity against species of the genus *Diabrotica*. In one embodiment, the insecticidal activity is activity against one or more of the following insect pests in the maize rootworm species assemblage: western maize rootworm (*Diabrotica virgifera virgifera*); northern maize rootworm (*D. barberi*); southern maize rootworm or the root-eating subspecies of the cucumber leaf beetle (*Diabrotica undecimpunctata howardi*); and Mexican maize rootworm (*D. virgifera zeae*). In one implementation, the insecticidal activity is the activity against the western maize root borer (Diabrotica virgifera virgifera).

[0126] In some embodiments, the fragment of the nucleic acid sequence encoding the PIP-72 polypeptide (the biologically active portion of the protein encoded by the PIP-72 polypeptide) encodes at least about 15, 20, 30, 40, 50, 60, 70, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84 or 85 consecutive amino acids, the number of consecutive nucleotides being at most the total number of amino acids present in the full-length PIP-72 polypeptide as shown in the embodiments of this disclosure. In some embodiments, the fragment is, for example, obtained by proteolytic digestion, insertion of a start codon, deletion of the codon encoding the missing amino acid accompanied by insertion of a stop codon, or insertion of a stop codon into the coding sequence, and is derived from any of the following sequences: SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 18, SEQ ID NO: 28, SEQ ID NO: 32, SEQ ID NO: 528 to SEQ ID NO: 768, any of the following sequences: SEQ ID NO: 825 to SEQ ID NO: 844, SEQ ID NO: 771, SEQ ID NO: 772, SEQ ID NO: 852, SEQ ID NO: 903 to SEQ ID NO: 914, SEQ ID NO: 927, SEQ ID NO: 928, SEQ ID NO: 932, SEQ ID NO: 933, SEQ ID NO: 934, SEQ ID NO: 935, SEQ ID NO: 936. The N-terminus and / or C-terminus of SEQ ID NO: 936, SEQ ID NO: 939, SEQ ID NO: 940, SEQ ID NO: 941, SEQ ID NO: 943, SEQ ID NO: 944, SEQ ID NO: 945 or SEQ ID NO: 946 or variants thereof are obtained by truncating at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or more amino acids.In some implementations, the fragments covered herein are, for example, obtained by proteolytic digestion or by inserting a start codon into the coding sequence, from any of the following sequences relative to SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 18, SEQ ID NO: 28, SEQ ID NO: 32, SEQ ID NO: 528 to SEQ ID NO: 768, any of the following sequences relative to SEQ ID NO: 825 to SEQ ID NO: 844, SEQ ID NO: 771, SEQ ID NO: 772, SEQ ID NO: 852, SEQ ID NO: 903 to SEQ ID NO: 914, SEQ ID NO: 927, SEQ ID NO: 928, SEQ ID NO: 932, SEQ ID NO: 933, SEQ ID NO: 934, SEQ ID NO: 935, SEQ ID NO: 936, SEQ ID NO: 939, SEQ ID NO: 939. The sequences obtained by removing 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or more amino acids from the N-terminus of SEQ ID NO: 940, SEQ ID NO: 941, SEQ ID NO: 943, SEQ ID NO: 944, SEQ ID NO: 945 or SEQ ID NO: 946 or variants thereof.In some implementations, the fragments covered herein are, for example, obtained by proteolytic digestion or by inserting a start codon into the coding sequence, from any of the following sequences relative to SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 18, SEQ ID NO: 28 or SEQ ID NO: 32, SEQ ID NO: 528 to SEQ ID NO: 768, any of the following sequences: SEQ ID NO: 825 to SEQ ID NO: 844, SEQ ID NO: 771, SEQ ID NO: 772, SEQ ID NO: 852, SEQ ID NO: 903 to SEQ ID NO: 914, SEQ ID NO: 927, SEQ ID NO: 928, SEQ ID NO: 932, SEQ ID NO: 933, SEQ ID NO: 934, SEQ ID NO: 935, SEQ ID NO: 936, SEQ ID NO: 939 ... The sequences NO: 940, SEQ ID NO: 941, SEQ ID NO: 943, SEQ ID NO: 944, SEQ ID NO: 945 or SEQ ID NO: 946 or variants thereof are obtained by removing 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 amino acids from the N-terminus.

[0127] In some embodiments, the PIP-72 polypeptide is encoded by a nucleic acid sequence that is sufficiently homologous to the following nucleic acid sequences: SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 17, SEQ ID NO: 27, SEQ ID NO: 31, SEQ ID NO: 949, SEQ ID NO: 950, SEQ ID NO: 954, SEQ ID NO: 955, SEQ ID NO: 956, SEQ ID NO: 957, SEQ ID NO: 958, SEQ ID NO: 961, SEQ ID NO: 962, SEQ ID NO: 963, SEQ ID NO: 965, SEQ ID NO: 966, SEQ ID NO: 967, or SEQ ID NO: 968. "Sufficient homology" herein refers to a sequence homology of at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher when compared with a reference sequence using one of the alignment procedures described herein and with standard parameters. Those skilled in the art will recognize that these values ​​can be appropriately adjusted to determine the corresponding homology of the proteins encoded by the two nucleic acid sequences by taking into account codon degeneracy, amino acid similarity, reading frame positioning, etc. In some embodiments, sequence homology is defined with respect to the full-length sequence of the polynucleotide encoding the PIP-72 polypeptide or to the full-length sequence of the PIP-72 polypeptide itself.In some embodiments, the PIP-72 peptide is associated with SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 18, SEQ ID NO: 28 or SEQ ID NO: 32, SEQ ID NO: 927, SEQ ID NO: 928, SEQ ID NO: 932, SEQ ID NO: 933, SEQ ID NO: 934, SEQ ID NO: 935, SEQ ID NO: 936, SEQ ID NO: 939, SEQ ID NO: 940, SEQ ID NO: 941, SEQ ID NO: 943, SEQ ID NO: 944, SEQ ID NO: 945 or SEQ ID NO: 946. NO: 946 has at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity. In some embodiments, sequence identity is defined with respect to the full-length sequence of the polynucleotide encoding the PIP-72 polypeptide or with respect to the full-length sequence of the PIP-72 polypeptide. In some embodiments, sequence identity is defined using a vector with all default parameters. Package (Invitrogen Corporation, Carlsbad, Calif.) The ClustalW algorithm in the module is used for calculation. In some implementations, sequence identity is calculated across the entire length of the peptide using the ClustalW algorithm in the ALIGNX module of the Vector NTI package (Invitrogen Corporation, Carlsbad, Calif.) with all default parameters.

[0128] To determine the percentage of identity between two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes. The percentage of identity between the two sequences is a function of the number of common positions shared by the sequences (i.e., percentage of identity = number of common positions / total number of positions (e.g., overlapping positions) × 100). In one embodiment, the two sequences are of the same length. In another embodiment, the comparison spans the entire reference sequence (e.g., spans the entirety of one of SEQ ID NO: 1 and SEQ ID NO: 2). The percentage of identity between the two sequences can be determined using techniques similar to those described below, with or without gaps. In calculating the percentage of identity, exact matches are typically counted.

[0129] Mathematical algorithms can be used to determine the percentage of identity between two sequences. A non-limiting example of a mathematical algorithm for comparing two sequences is the one proposed by Karlin and Altschul, (1990) Proc. Natl. Acad. Sci. USA 87:2264, which was improved by Karlin and Altschul, (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877. This algorithm was incorporated into the BLASTN and BLASTX programs proposed by Altschul et al., (1990) J. Mol. Biol. 215:403. BLAST nucleotide searches can be performed using the BLASTN program with a score of 100 and a word length of 12 to obtain nucleic acid sequences homologous to the insecticidal nucleic acid molecule of the embodiment described. BLAST protein searches can be performed using the BLASTX program with a score of 50 and a word length of 3 to obtain amino acid sequences homologous to the insecticidal protein molecule of the embodiment described. To obtain vacancy alignment results for comparison, vacancy BLAST (BLAST 2.0) can be used as described by Altschul et al., (1997) Nucleic Acids Res. 25: 3389. Alternatively, PSI-Blast can be used for iterative searching, which can detect distant relationships between molecules. See Altschul et al. (1997) (ibid.). When using BLAST, Gapped BLAST, and PSI-Blast programs, the default parameters of each program (e.g., BLASTX and BLASTN) can be used. Sequence alignment can also be performed manually.

[0130] Another non-limiting example of a mathematical algorithm for sequence comparison is the ClustalW algorithm (Higgins et al., (1994) Nucleic Acids Res. 22: 4673-4680). ClustalW compares sequences and aligns amino acid or DNA sequences as a whole, thus providing data on the sequence conservation of entire amino acid sequences. The ClustalW algorithm is used in several commercial DNA / amino acid analysis software packages, such as Vector. The package (Invitrogen Corporation, Carlsbad, Calif.) The module allows for the assessment of amino acid identity percentages after alignment using ClustalW. A non-limiting example of a software program that can be used to analyze ClustalW alignments is GENEDOC. TM GENEDOC TM (Karl Nicholas) can assess amino acid (or DNA) similarity and identity among a variety of proteins. Another non-limiting example of a mathematical algorithm for sequence comparison is the one proposed by Myers and Miller, (1988) CABIOS 4: 11-17. This type of algorithm is incorporated into the ALIGN program (version 2.0), which is part of the GCG WisconsinGenetics software package version 10 (Accelrys, Inc., 9685 Scranton Rd., San Diego, Calif., USA). When comparing amino acid sequences using the ALIGN program, a PAM120 weighted residue table, a vacancy length penalty of 12, and a vacancy penalty of 4 can be used.

[0131] Another non-limiting example of a mathematical algorithm for sequence comparison is the algorithm proposed by Needleman and Wunsch, (1970) J. Mol. Biol. 48(3): 443-453, which uses GAP version 10 software to determine sequence identity or similarity using the following default parameters: identity % and similarity % for nucleic acid sequences using GAP weight 50 and length weight 3 and the nwsgapdna.cmpii scoring matrix; identity % or similarity % for amino acid sequences using GAP weight 8 and length weight 2 and the BLOSUM62 scoring procedure. An equivalence procedure may also be used. “Equivalence procedure” is used herein to refer to any such sequence comparison procedure that, for any two considered sequences, produces alignments with the same nucleotide residue matches and the same percentage of sequence identity compared to the corresponding alignments produced by GAP version 10.

[0132] This disclosure also covers nucleic acid molecules encoding variants of the PIP-72 polypeptide. "Variants" of nucleic acid sequences encoding the PIP-72 polypeptide include those sequences encoding the PIP-72 polypeptide disclosed herein but exhibiting conserved differences due to genetic code degeneracy, as well as those sequences having sufficiently high identity with the sequences described above. Naturally occurring allelic variants can be identified using well-known molecular biology techniques, such as polymerase chain reaction (PCR) and hybridization techniques outlined below. Nucleic acid sequence variants also include synthetically obtained nucleic acid sequences, for example, generated by site-directed mutagenesis, but still encoding the disclosed PIP-72 polypeptide, as described below.

[0133] This disclosure provides isolated or recombinant polynucleotides encoding any of the PIP-72 polypeptides disclosed herein. Those skilled in the art will readily understand that, due to the degeneracy of the genetic code, there are numerous nucleotide sequences encoding the PIP-72 polypeptides of this disclosure. Table 1 is a codon table providing synonymous codons for each amino acid. For example, codons AGA, AGG, CGA, CGC, CGG, and CGU all encode the amino acid arginine. Therefore, at each position in the nucleic acid of this disclosure designated as arginine by a certain codon, that codon can be changed to any of the corresponding codons mentioned above without altering the encoded polypeptide. It should be understood that U in the RNA sequence corresponds to T in the DNA sequence.

[0134] Table 1

[0135]

[0136]

[0137] Those skilled in the art will also understand that alterations can be introduced by mutating nucleic acid sequences, thereby causing changes in the amino acid sequence of the encoded PIP-72 polypeptide without altering the protein's biological activity. Therefore, variant nucleic acid molecules can be formed by introducing one or more nucleotide substitutions, additions, and / or deletions into the corresponding nucleic acid sequences disclosed herein, such that one or more amino acid substitutions, additions, or deletions are introduced into the encoded protein. Mutations can be introduced using standard techniques such as site-directed mutagenesis and PCR-mediated mutagenesis. Such variant nucleic acid sequences are also covered by this disclosure.

[0138] Alternatively, mutant nucleic acid sequences can be obtained by randomly introducing mutations along all or part of the coding sequence, such as through saturation mutagenesis, and the resulting mutants can be screened for their insecticidal activity to identify those that retain the activity. After mutagenesis, the encoded protein can be expressed in a recombinant manner, and the activity of the protein can be determined using standard assay techniques.

[0139] In addition to the standard cloning methods described by Ausubel, Berger, and Sambrook, the polynucleotides and fragments thereof disclosed herein are optionally used as substrates for a variety of recombination and recurrent recombination reactions, i.e., to generate additional insecticidal polypeptide homologs and fragments thereof with desired properties. A variety of such reactions are known, including those developed by the inventors and colleagues. Methods for generating variants of any of the nucleic acids listed herein include recurrent recombination of such polynucleotides with a second (or more) polynucleotide to form a library of variant polynucleotides, as are embodiments of this disclosure, the resulting library, the cell containing the library, and any recombinant polynucleotides generated by such methods. Additionally, such methods optionally include selecting variant polynucleotides from such libraries based on insecticidal activity when such recurrent recombination is performed in vitro or in vivo.

[0140] A variety of diversity generation schemes (including nucleic acid recursive recombination schemes) are available and have been well described in the art. These procedures can be used alone and / or in combination to generate one or more variants of nucleic acids or nucleic acid sets, as well as variants of the proteins they encode. These procedures, individually and collectively, provide robust and widely applicable methods for generating diverse nucleic acids and nucleic acid sets (including, for example, nucleic acid libraries), which can be used for, for example, the engineering or rapid evolution of nucleic acids, proteins, pathways, cells, and / or organisms to acquire new and / or improved properties.

[0141] Although distinctions and classifications have been made in the subsequent discussion for clarity, it should be understood that these techniques are generally not mutually exclusive. In fact, the various methods described can be used alone or in combination, in parallel or sequentially, to obtain a wide variety of sequence variants.

[0142] The result of any diversity generation procedure described herein may be the production of one or more nucleic acids, which may be selected or screened to obtain nucleic acids having or conferring desired properties or nucleic acids encoding proteins having or conferring desired properties. Diversification can be performed using one or more methods described herein, or other methods available to those skilled in the art, and then any of the produced nucleic acids can be selected for desired activity or property (e.g., insecticidal activity or such insecticidal activity at a desired pH, etc.). This may include identifying any activity that can be detected by any assay in the art in, for example, an automated or automatable mode, see, for example, the discussion below on screening for insecticidal activity. Multiple related (or even unrelated) properties may be evaluated, either continuously or in parallel, at the discretion of the operator.

[0143] Descriptions of the diverse generation procedures for producing modified nucleic acid sequences (e.g., those sequences encoding polypeptides or fragments thereof with insecticidal activity) can be found in the following publications and the references cited therein: Soong et al., (2000) Nat Genet 25(4): 436-439; Stemmer et al., (1999) Tumor Targeting 4: 1-4; Ness et al., (1999) Nat Biotechnol 17: 893-896; Chang et al., (1999) Nat Biotechnol 17: 793-797; Minshull and Stemmer, (1999) Curr Opin Chem Biol 3: 284-290; Christians et al., (1999) Nat Biotechnol 17:259-264; Crameri et al., (1998) Nature 391:288-291; Crameri et al., (1997) Nat Biotechnol 15:436-438; Zhang et al., (1997) PNAS USA 94:4504-4509; Patten et al., (1997) Curr Opin Biotechnol 8:724-733; Crameri et al., (1996) Nat Med 2:100-103; Crameri et al., (1996) Nat Biotechnol 14:315-319; Gates et al., (1996) JMolBiol 255:373-386; Stemmer, (1996) "Sexual PCR and Assembly PCR", The Encyclopedia of Molecular Biology. VCH Publishers, New York. pp. 447-457; Crameri and Stemmer, (1995) BioTechniques 18: 194-195; Stemmer et al., (1995) Gene, 164: 49-53; Stemmer, (1995) Science 270: 1510; Stemmer, (1995) Bio / Technology 13: 549-553; Stemmer, (1994) Nature 370: 389-391; Stemmer, (1994) PNAS USA 91: 10747-10751.

[0144] Mutational methods that generate diversity include (for example): site-directed mutagenesis (Ling et al., (1997) Anal Biochem 254(2): 157-178; Dale et al., (1996) Methods Mol Biol 57: 369-374; Smith, (1985) Ann RevGenet 19: 423-462; Botstein and Shortle, (1985) Science 229: 1193-1201; Carter, (1986) Biochem J 237: 1-7; and Kunkel, (1987) “The efficiency of oligonucleotide directed mutagenesis”, Nucleic Acids & Molecular Biology (edited by Eckstein and Lilley, Springer Verlag, Berlin)); mutagenesis using uracil-containing templates (Kunkel, (1985) PNAS USA). 82:488-492; Kunkel et al., (1987) Methods Enzymol 154:367-382; and Bass et al., (1988) Science 242:240-245); Oligonucleotide site-directed mutagenesis (Zoller and Smith, (1983) Methods Enzymol 100:468-500; Zoller and Smith, (1987) Methods Enzymol 154:329-350 (1987); Zoller and Smith, (1982) Nucleic Acids Res 10:6487-6500); Phosphothioester modified DNA mutagenesis (Taylor et al., (1985) Nucleic Acids Res 13:8749-8764; Taylor et al., (1985) Nucleic Acids Res 13:8749-8764; Taylor et al., (1985) Nucleic Acids Res 13:8749-8764; Nucl Acids Res 13: 8765-8787 (1985); Nakamaye and Eckstein, (1986) Nucl Acids Res 14: 9679-9698; Sayers et al., (1988) Nucl Acids Res 16: 791-802; and Sayers et al., (1988) Nucl Acids Res 16: 803-814); Mutagenesis using double-stranded DNA with vacancy (Kramer et al., (1984) Nucl Acids Res 12: 9441-9456; Kramer and Fritz, (1987) Methods Enzymol 154: 350-367;Kramer et al., (1988) Nucl Acids Res 16: 7207; and Fritz et al., (1988) Nucl Acids Res 16: 6987-6999.

[0145] Other suitable methods include point mismatch repair (Kramer et al., (1984) Cell 38: 879-887), mutagenesis using repair-deficient host strains (Carter et al., (1985) Nucl Acids Res 13: 4431-4443; and Carter, (1987) Methods in Enzymol 154: 382-403), deletion mutagenesis (Eghtedarzadeh and Henikoff, (1986) Nucl Acids Res 14: 5115), restriction selection and restriction purification (Wells et al., (1986) Phil Trans RSoc Lond A 317: 415-423), and mutagenesis via whole-genome synthesis (Nambiar et al., (1984) Science 223: 1299-1301; Sakamar and Khorana, (1988) Nucl Acids Res 154: 382-403), 14:6361-6372; Wells et al., (1985) Gene 34:315-323; and (Mandek et al., (1985) Nucl Acids Res 13:3305-3316), double-strand break repair (Mandecki, (1986) PNAS USA, 83:7177-7181; and Arnold, (1993) Curr Opin Biotech 4:450-455). For more details on many of the methods mentioned above, see Methods Enzymol 154, which also describes controls for various mutagenesis methods that effectively eliminate uncertainties.

[0146] Further details regarding various methods of generating diversity can be found in the following U.S. patents, PCT publications and applications, and EPO publications: U.S. Patent 5,723,323, U.S. Patent 5,763,192, U.S. Patent 5,814,476, U.S. Patent 5,817,483, U.S. Patent 5,824,514, U.S. Patent 5,976,862, U.S. Patent 5,605,793, U.S. Patent 5,811,238, U.S. Patent 5,830,721, U.S. Patent 5,834,252, U.S. Patent 5,837,458, WO 1995 / 22625, WO 1996 / 33207, WO 1997 / 20078, WO 1997 / 35966, WO 1999 / 41402, WO 1999 / 41383, WO 1999 / 41369, WO 1999 / 41368、EP 752008、EP 0932670、WO 1999 / 23107、WO 1999 / 21979、WO 1998 / 31837、WO 1998 / 27230、WO 1998 / 27230、WO 2000 / 00632、WO 2000 / 09679、WO 1998 / 42832、WO1999 / 29902、WO 1998 / 41653、WO 1998 / 41622、WO 1998 / 42727、WO 2000 / 18906、WO 2000 / 04190、WO 2000 / 42561、WO 2000 / 42559、WO 2000 / 42560, WO 2001 / 23401 and PCT / US01 / 06775.

[0147] The nucleotide sequences of the embodiments disclosed herein can also be used to isolate corresponding sequences from other organisms, particularly other bacteria, especially species of the genus *Pseudomonas*, and more particularly strains of *Pseudomonas putida*, *Pseudomonas fulva*, or *Pseudomonas aeruginosa*. In this manner, such sequences can be identified based on their sequence identity with the sequences shown herein using methods such as PCR, hybridization, etc. The embodiments of this disclosure cover sequences selected based on their sequence identity with the complete sequences or fragments thereof shown herein. Such sequences include sequences that are orthologs of the disclosed sequences. The term "ortholog" refers to a gene derived from a common ancestral gene and present in different species due to speciation. Genes present in different species are considered orthologs when their nucleotide sequences and / or the protein sequences they encode have substantial identity as defined elsewhere herein. The function of orthologs is often highly conserved across species.

[0148] In PCR methods, oligonucleotide primers can be designed for PCR reactions to amplify corresponding DNA sequences from cDNA or genomic DNA extracted from any organism of interest. The methods for designing PCR primers and PCR cloning are well-known in the art and are disclosed in the following literature: Sambrook et al., (1989) Molecular Cloning: A Laboratory Manual (2nd edition, Cold Spring Harbor Laboratory Press, Plainview, New York), hereinafter referred to as "Sambrook". See also Innis et al., ed., (1990) PCR Protocols: A Guide to Methods and Applications (Academic Press, New York); Innis and Gelfand, ed., (1995) PCR Strategies (Academic Press, New York); and Innis and Gelfand, ed., (1999) PCR Methods Manual (Academic Press, New York). Known PCR methods include, but are not limited to, methods using paired primers, nested primers, single-specific primers, degenerate primers, gene-specific primers, vector-specific primers, and partially mismatched primers.

[0149] To identify potential PIP-72 peptides from bacterial deposits, bacterial cell lysates can be screened using Western blotting and / or ELISA methods with antibodies generated from PIP-72 peptides as antigens. This type of assay can be performed in high-throughput mode. Various techniques, such as antibody-based protein purification and identification techniques, can be used to further analyze positive samples. Methods for generating antibodies are well known in the art and are discussed below.

[0150] Alternatively, mass spectrometry-based protein identification methods can be used to identify homologs of the PIP-72 peptide using the protocol described in the literature (Scott Patterson, (1998), 10.22, 1-24, Current Protocol in Molecular Biology, John Wiley & Son Inc). Specifically, using an LC-MS / MS-based protein identification method, MS data of a given cell lysate or a sample rich in the desired molecular weight (excised from a PIP-72-related molecular weight band in an SDS-PAGE gel) are correlated with the sequence information of PIP-72 (e.g., SEQ ID NO: 2) and its homologs. Any match in the peptide sequence indicates the possible presence of a homolog in the sample. Further techniques (protein purification and molecular biology) can be used to separate the protein and identify the sequences of homologs.

[0151] When using hybridization methods, a portion or the entire insecticidal nucleic acid sequence can be used to screen cDNA or genomic libraries. Methods for constructing such cDNA and genomic libraries are well-known in the art and disclosed in Sambrook and Russell's 2001 publication (ibid.). Hybridization probes can be genomic DNA fragments, cDNA fragments, RNA fragments, or other oligonucleotides, and can be labeled with detectable groups such as 32P or any other detectable markers, such as other radioisotopes, fluorescent compounds, enzymes, or enzyme cofactors. Hybridization probes can be prepared by labeling synthetic oligonucleotides based on the known PIP-72 polypeptide-encoded nucleic acid sequence disclosed herein. Alternatively, degenerate primers designed based on conserved nucleotides or amino acid residues in the nucleic acid sequence or the encoded amino acid sequence can be used. The probe typically contains a nucleic acid sequence region that, under stringent conditions, hybridizes to at least about 12, at least about 25, at least about 50, 75, 100, 125, 150, 175, or 200 consecutive nucleotides of a nucleic acid sequence or fragment or variant thereof encoding the disclosed PIP-72 polypeptide. Methods for preparing the hybridization probe are well known in the art and are disclosed in Sambrook and Russell's 2001 publication (ibid.), which is incorporated herein by reference.

[0152] For example, the entire nucleic acid sequence encoding the PIP-72 polypeptide disclosed herein, or one or more portions thereof, can be used as a probe capable of specifically hybridizing to a corresponding nucleic acid sequence and messenger RNA encoding a PIP-72 polypeptide-like sequence. To achieve specific hybridization under various conditions, such probes comprise unique sequences and are preferably at least about 10 nucleotides or at least about 20 nucleotides in length. Such probes can be used to amplify the corresponding insecticidal sequence from selected organisms via PCR. This technique can be used to isolate additional coding sequences from desired organisms or as a diagnostic assay to determine the presence of coding sequences in organisms. Hybridization techniques include hybridization screening of DNA libraries inoculated on plates (plaques or colonies; see, for example, Sambrook et al., (1989) Molecular Cloning: A Laboratory Manual (2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY).

[0153] Hybridization of these sequences can be performed under stringent conditions. "Stringent conditions" or "stringent hybridization conditions" are used herein to refer to conditions where the probe hybridizes to a detectably higher degree with its target sequence than it hybridizes to other sequences (e.g., at least 2-fold higher than background). Stringent conditions are sequence-dependent and will vary in different situations. By controlling the stringency of hybridization and / or washing conditions, target sequences that are 100% complementary to the probe can be identified (homologous detection). Alternatively, stringent conditions can be adjusted to allow for some mismatches in the sequences, thereby detecting a lower degree of similarity (heterologous detection). Probe length is typically less than about 1000 nucleotides, preferably less than 500 nucleotides.

[0154] Typically, stringent conditions are as follows: a salt concentration below about 1.5 M sodium ions (typically about 0.01 to 1.0 M sodium ion concentration (or other salts)), a pH of 7.0 to 8.3, a usage temperature of at least about 30 °C for short probes (e.g., 10 to 50 nucleotides), and a usage temperature of at least about 60 °C for long probes (e.g., more than 50 nucleotides). Stringent conditions can also be achieved by adding a destabilizing agent such as formamide. Exemplary low-stringent conditions involve hybridization at 37 °C with a buffer of 30% to 35% formamide, 1 M NaCl, and 1% SDS (sodium dodecyl sulfate), followed by washing with 1 to 2 times the amount of SSC (20 times SSC = 3.0 M NaCl / 0.3 M trisodium citrate) at 50 to 55 °C. Exemplary moderate-stringent conditions involve hybridization at 37 °C with 40% to 45% formamide, 1.0 M NaCl, and 1% SDS, followed by washing with 0.5 to 1 times the amount of SSC at 55 to 60 °C. Exemplary high-toughness conditions include hybridization at 37°C in 50% formamide, 1M NaCl, and 1% SDS, followed by washing with 0.1-fold SSC at 60°C to 65°C. The wash buffer may optionally contain about 0.1% to about 1% SDS. The hybridization duration is typically less than about 24 hours, generally about 4 to about 12 hours.

[0155] Specificity typically varies with washing conditions after hybridization, with the key factors being the ionic strength and temperature of the final washing solution. For DNA-DNA hybrids, Tm can be calculated using the equation Tm = 81.5 °C + 16.6 (log M) + 0.41 (%GC) - 0.61 (%form) - 500 / L proposed by Meinkoth and Wahl, (1984) Anal. Biochem. 138: 267-284; where M is the molar concentration of monovalent cations, %GC is the percentage of guanine and cytosine nucleotides in the DNA, %form is the percentage of formamide in the hybridization solution, and L is the length of the hybrid (in base pairs). Tm is the temperature at which 50% of the complementary target sequence hybridizes with a perfectly matched probe (at a defined ionic strength and pH). For every 1% increase in mismatch rate, Tm decreases by approximately 1 °C; therefore, hybridization with sequences possessing the desired identity can be achieved by adjusting Tm, hybridization conditions, and / or washing conditions. For example, if a sequence with ≥90% identity is sought, Tm can be reduced by 10°C. Typically, stringent conditions are chosen to be approximately 5°C lower than the thermal desorption temperature (Tm) of the specific sequence and its complementary sequence at a given ionic strength and pH. However, extremely stringent conditions can be achieved using hybridization and / or washing at 1, 2, 3, or 4°C lower than the thermal desorption temperature (Tm); moderately stringent conditions can be achieved using hybridization and / or washing at 6, 7, 8, 9, or 10°C lower than the thermal desorption temperature (Tm); and low stringent conditions can be achieved using hybridization and / or washing at 11, 12, 13, 14, 15, or 20°C lower than the thermal desorption temperature (Tm). Using the aforementioned formulas, hybridization and washing compositions, and the desired Tm, those skilled in the art will recognize that variations in the stringency of the hybridization and / or washing solutions are inherently described. If the desired degree of mismatch results in a Tm below 45°C (aqueous solution) or 32°C (formamide solution), it is preferable to increase the SSC concentration to allow for the use of higher temperatures. Detailed instructions on nucleic acid hybridization can be found in Tijssen, (1993) Laboratory Techniques in Biochemistry and Molecular Biology - Hybridization with Nucleic Acid Probes, Part I, Chapter 2 (Elsevier, NY); and Ausubel et al., eds., Current Protocols in Molecular Biology, Chapter 2 (Greene Publishing and Wiley-Interscience, New York).See Sambrook et al., (1989) Molecular Cloning: A Laboratory Manual (2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY).

[0156] In some embodiments, a nucleic acid molecule encoding a polypeptide is provided, wherein the amino acid sequence comprising the polypeptide has at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or higher sequence identity with the following amino acid sequences: SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 30, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 929, SEQ ID NO: 930, SEQ ID NO: 931, SEQ ID NO: 937, SEQ ID NO: 938, SEQ ID NO: 942, SEQ ID NO: 947 or SEQ ID NO: 948, wherein the polypeptide has insecticidal activity.

[0157] Proteins and their variants and fragments

[0158] This disclosure also covers the PIP-72 polypeptide. The terms “Pseudomonas insecticidal protein-72,” “PIP-72 polypeptide,” or “PIP-72 protein” are used interchangeably herein and refer to a polypeptide with insecticidal activity that is sufficiently homologous to the protein shown in SEQ ID NO: 2, wherein the insecticidal activity includes, but is not limited to, insecticidal activity against one or more insect pests in the order Coleoptera. This disclosure envisions various PIP-72 polypeptides. The sources of polynucleotides encoding the PIP-72 polypeptide or related protein include, but are not limited to: *Pseudomonas aeruginosa* strains containing the PIP-72Aa polypeptide encoded by SEQ ID NO: 2, and the PIP-72Aa polynucleotide shown in SEQ ID NO: 1; *Pseudomonas repens* strains containing the PIP-72Ba polypeptide encoded by SEQ ID NO: 4, and the PIP-72Ba polynucleotide shown in SEQ ID NO: 3; *Pseudomonas aeruginosa* strains containing the PIP-72Ca polypeptide encoded by SEQ ID NO: 6, and the PIP-72Ca polynucleotide shown in SEQ ID NO: 5; *Pseudomonas meningitidis* strains containing the PIP-72Cb polypeptide encoded by SEQ ID NO: 8, and the PIP-72Cb polynucleotide shown in SEQ ID NO: 7; and *Pseudomonas icariina* strains containing the PIP-72Da polypeptide encoded by SEQ ID NO: 10, and the PIP-72Cb polynucleotide shown in SEQ ID NO: 10. PIP-72Da polynucleotide shown in SEQ ID NO: 9; *Pseudomonas meningitidis* strain containing the PIP-72Db polypeptide shown in SEQ ID NO: 12, and the PIP-72Db polynucleotide shown in SEQ ID NO: 11; *Pseudomonas serrata* strain containing the PIP-72Dc polypeptide shown in SEQ ID NO: 14, and the PIP-72Dc polynucleotide shown in SEQ ID NO: 13; *Pseudomonas moschata* strain containing the PIP-72Fa polypeptide shown in SEQ ID NO: 18, and the PIP-72Fa polynucleotide shown in SEQ ID NO: 17; *Pseudomonas aeruginosa* strain containing the PIP-72Ff polypeptide shown in SEQ ID NO: 28, and the PIP-72Ff polynucleotide shown in SEQ ID NO: 27; *Pseudomonas aeruginosa* strain containing the PIP-72Gb polypeptide shown in SEQ ID NO: 32, and the PIP-72Db polynucleotide shown in SEQ ID NO: 9; PIP-72Gb polynucleotide shown in NO: 31; Pseudomonas aeruginosa strain containing the PIP-72Ab polypeptide shown in SEQ ID NO: 927, and the PIP-72Ab polynucleotide shown in SEQ ID NO: 949.A *Pseudomonas rapeus* strain containing the polypeptide encoding PIP-72Ab (SEQ ID NO: 928) and the polynucleotide encoding PIP-72Bb (SEQ ID NO: 950); a *Pseudomonas entomopathogenicus* strain containing the polypeptide encoding PIP-72AFh (SEQ ID NO: 932) and the polynucleotide encoding PIP-72Fh (SEQ ID NO: 954); a *Pseudomonas entomopathogenicus* strain containing the polypeptide encoding PIP-72AFh (SEQ ID NO: 933) and the polynucleotide encoding PIP-72Fh (SEQ ID NO: 955); a *Pseudomonas aeruginosa* strain containing the polypeptide encoding PIP-72Fj (SEQ ID NO: 934) and the polynucleotide encoding PIP-72Fj (SEQ ID NO: 956); and a *Pseudomonas aeruginosa* strain containing the polypeptide encoding PIP-72Fk (SEQ ID NO: 935). PIP-72Fk polynucleotide shown in SEQ ID NO: 957; Burkholderia polyphaga strain containing PIP-72Fl polypeptide shown in SEQ ID NO: 936, PIP-72Fl polynucleotide shown in SEQ ID NO: 958; Pseudomonas aeruginosa strain containing PIP-72Gg polypeptide shown in SEQ ID NO: 939, PIP-72Gg polynucleotide shown in SEQ ID NO: 961; Pseudomonas aeruginosa strain containing PIP-72Gh polypeptide shown in SEQ ID NO: 940, PIP-72Gh polynucleotide shown in SEQ ID NO: 962; Pseudomonas moschata strain containing PIP-72Gi polypeptide shown in SEQ ID NO: 941, PIP-72Gi polynucleotide shown in SEQ ID NO: 963; Pseudomonas biocontrol strain containing PIP-72Gk polypeptide shown in SEQ ID NO: 943, PIP-72Gi polynucleotide shown in SEQ ID NO: 963. PIP-72Gk polynucleotide as shown in SEQ ID NO: 965; *Pseudomonas proteus* strain containing the PIP-72Gl polypeptide as shown in SEQ ID NO: 944, and the PIP-72Gl polynucleotide as shown in SEQ ID NO: 966; *Pseudomonas aeruginosa* strain containing the PIP-72Gn polypeptide as shown in SEQ ID NO: 946, and the PIP-72Gn polynucleotide as shown in SEQ ID NO: 968. In some embodiments, the insecticidal activity is activity against the western maize rootworm (*Diabrotica virgifera virgifera*).

[0159] In some embodiments, the PIP-72 peptide is sufficiently homologous to the following amino acid sequences: SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 18, SEQ ID NO: 28, SEQ ID NO: 32, SEQ ID NO: 927, SEQ ID NO: 928, SEQ ID NO: 932, SEQ ID NO: 933, SEQ ID NO: 934, SEQ ID NO: 935, SEQ ID NO: 936, SEQ ID NO: 939, SEQ ID NO: 940, SEQ ID NO: 941, SEQ ID NO: 943, SEQ ID NO: 944, SEQ ID NO: 945, or SEQ ID NO: 946. As used in this article, “sufficient homology” means that when an amino acid sequence is compared with a reference sequence using one of the alignment procedures described herein with standard parameters, the amino acid sequence has at least approximately 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 78%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence homology with the reference sequence. Those skilled in the art will recognize that these values ​​can be appropriately adjusted to determine the corresponding homology of a protein by taking into account amino acid similarity, etc. In some embodiments, sequence homology is homology relative to the full-length sequence of the PIP-72 polypeptide.In some embodiments, the PIP-72 peptide is compared with SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 18, SEQ ID NO: 28, SEQ ID NO: 32, SEQ ID NO: 927, SEQ ID NO: 928, SEQ ID NO: 932, SEQ ID NO: 933, SEQ ID NO: 934, SEQ ID NO: 935, SEQ ID NO: 936, SEQ ID NO: 939, SEQ ID NO: 940, SEQ ID NO: 941, SEQ ID NO: 943, SEQ ID NO: 944, SEQ ID NO: 945 or SEQ ID NO: 946. NO: 946, having at least about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 78%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity. In some embodiments, sequence identity is identity relative to the full-length sequence of the PIP-72 polypeptide. In some implementations, sequence identity is achieved using a Vector with all default parameters. Package (Invitrogen Corporation, Carlsbad, Calif.) The ClustalW algorithm in the module is used for computation. In some implementations, sequence identity is calculated using a Vector with all default parameters. Package (Invitrogen Corporation, Carlsbad, Calif.) The ClustalW algorithm in the module performs calculations across the entire length of the polypeptide.

[0160] As used herein, the terms “protein,” “peptide molecule,” or “polypeptide” include any molecule comprising five or more amino acids. It is well known in the art that protein, peptide, or polypeptide molecules can be modified, including post-translational modifications, such as, but not limited to, the formation of disulfide bonds, glycosylation, phosphorylation, or oligomerization. Therefore, as used herein, the terms “protein,” “peptide molecule,” or “polypeptide” include any protein modified by any biological or non-biological process. The term “amino acid” refers to all naturally occurring L-amino acids.

[0161] “Recombinant protein” is used herein to refer to a protein that is no longer in its natural environment, such as in vitro or in the cells of a recombinant bacterial or plant host. PIP-72 polypeptides that are substantially free of cellular material include protein preparations containing less than about 30%, 20%, 10%, or 5% (on dry weight) of non-insecticidal protein (also referred to herein as “polluting protein”).

[0162] The “fragment” or “bioactive portion” includes polypeptide fragments that contain an amino acid sequence with sufficient identity to the PIP-72 polypeptide and exhibit insecticidal activity. The “fragments” or “bioactive portions” of the PIP-72 polypeptide include the following fragments: the amino acid sequences of these fragments are associated with any one of the sequences in SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 18, SEQ ID NO: 28, SEQ ID NO: 32, SEQ ID NO: 528 to SEQ ID NO: 768, any one of the sequences in SEQ ID NO: 825 to SEQ ID NO: 844, SEQ ID NO: 771, SEQ ID NO: 772, SEQ ID NO: 852, SEQ ID NO: 903 to SEQ ID NO: 914, SEQ ID NO: 927, SEQ ID NO: 928, SEQ ID NO: 932, SEQ ID NO: 933, SEQ ID NO: 934, SEQ ID NO: 935, SEQ ID NO: 936, SEQ ID NO: 939, SEQ ID NO: 940, SEQ ID NO: 941, SEQ ID NO: 939, SEQ ID NO: 940, SEQ ID NO: 941, SEQ ID NO: 932, SEQ ID NO: 933, SEQ ID NO: 934, SEQ ID NO: 935, SEQ ID NO: 936 ... The amino acid sequences shown in SEQ ID NO: 943, SEQ ID NO: 944, SEQ ID NO: 945, or SEQ ID NO: 946 have sufficiently high identity. The bioactive portion of the PIP-72 peptide can be a peptide of length (e.g.) 10, 25, 50, 55, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, or 85 amino acids. Such bioactive portions can be prepared using recombinant techniques and their insecticidal activity can be evaluated. As used herein, the fragment contains at least eight consecutive amino acids of the PIP-72 peptide.In some embodiments, the PIP-72 polypeptide fragment comprises at least eight consecutive amino acids from the following sequences: any one of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 18, SEQ ID NO: 28, SEQ ID NO: 32, any one of SEQ ID NO: 528 to SEQ ID NO: 768, any one of SEQ ID NO: 825 to SEQ ID NO: 844, any one of SEQ ID NO: 771, SEQ ID NO: 772, SEQ ID NO: 852, any one of SEQ ID NO: 903 to SEQ ID NO: 914, SEQ ID NO: 927, SEQ ID NO: 928, SEQ ID NO: 932, SEQ ID NO: 933, SEQ ID NO: 934, SEQ ID NO: 935, SEQ ID NO: 936, SEQ ID NO: 939, SEQ ID NO: 940, SEQ ID NO: 939 ...39, SEQ ID NO: 932, SEQ ID NO: 933, SEQ ID NO: 934, SEQ ID NO: 935, SEQ ID NO: NO:941, SEQ ID NO:943, SEQ ID NO:944, SEQ ID NO:945 or SEQ ID NO:946.In some embodiments, the PIP-72 polypeptide fragment is, for example, obtained by proteolytic digestion, insertion of a start codon, deletion of the codon encoding the missing amino acid accompanied by the insertion of a start codon, and / or insertion of a stop codon, from any sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 18, SEQ ID NO: 28, SEQ ID NO: 32, SEQ ID NO: 528 to SEQ ID NO: 768, any sequence of SEQ ID NO: 825 to SEQ ID NO: 844, any sequence of SEQ ID NO: 771, SEQ ID NO: 772, SEQ ID NO: 852, SEQ ID NO: 903 to SEQ ID NO: 914, SEQ ID NO: 927, SEQ ID NO: 928, SEQ ID NO: 932, SEQ ID NO: 933, SEQ ID NO: 934, SEQ ID NO: 935, SEQ ID NO: 936, ...937, SEQ ID NO: 938, SEQ ID NO: 932, SEQ ID NO: The N-terminus and / or C-terminus of SEQ ID NO: 936, SEQ ID NO: 939, SEQ ID NO: 940, SEQ ID NO: 941, SEQ ID NO: 943, SEQ ID NO: 944, SEQ ID NO: 945 or SEQ ID NO: 946 are obtained by truncating at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or more amino acids.

[0163] In some embodiments, the PIP-72 polypeptide fragments covered herein are, for example, obtained by proteolytic digestion, insertion of a start codon, deletion of the codon encoding the missing amino acid, and the insertion of a start codon, and are derived from any of the following sequences: SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 18, SEQ ID NO: 28, SEQ ID NO: 32, SEQ ID NO: 528 to SEQ ID NO: 768, any of the following sequences: SEQ ID NO: 825 to SEQ ID NO: 844, SEQ ID NO: 771, SEQ ID NO: 772, SEQ ID NO: 852, SEQ ID NO: 903 to SEQ ID NO: 914, SEQ ID NO: 927, SEQ ID NO: 928, SEQ ID NO: 932, SEQ ID NO: 933, SEQ ID NO: 934, SEQ ID NO: 935, SEQ ID NO: 936, SEQ ID NO: 768, SEQ ID NO: 825 to SEQ ID NO: 844, SEQ ID NO: 771, SEQ ID NO: 772, SEQ ID NO: 852, SEQ ID NO: 903 to SEQ ID NO: 914, SEQ ID NO: 927, SEQ ID NO: 928, SEQ ID NO: 932, SEQ ID NO: 933, SEQ ID NO: 934, SEQ ID NO: 935, SEQ ID NO: 936. The N-terminus of SEQ ID NO: 936, SEQ ID NO: 939, SEQ ID NO: 940, SEQ ID NO: 941, SEQ ID NO: 943, SEQ ID NO: 944, SEQ ID NO: 945 or SEQ ID NO: 946 is obtained by removing 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids.

[0164] In some embodiments, the PIP-72 polypeptide fragments covered herein are obtained by removing 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids from the N-terminus of the following fragments: SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, or variants of these sequences, including but not limited to any one of SEQ ID NO: 528 to SEQ ID NO: 768, any one of SEQ ID NO: 825 to SEQ ID NO: 844, SEQ ID NO: 771, SEQ ID NO: 772, SEQ ID NO: 852, any one of SEQ ID NO: 903 to SEQ ID NO: 914, SEQ ID NO: 927, SEQ ID NO: 928, SEQ ID NO: 932, SEQ ID NO: 933, SEQ ID NO: 934, SEQ ID NO: 935, SEQ ID NO: 936, SEQ ID NO: 939, SEQ ID NO: 940, SEQ ID NO: 941, SEQ ID NO: 942, SEQ ID NO: 933, SEQ ID NO: 934, SEQ ID NO: 935, SEQ ID NO: 936, SEQ ID NO: 939, SEQ ID NO: 940, SEQ ID NO: 941, SEQ ID NO: 93 ... NO:943, SEQ ID NO:944, SEQ ID NO:945 or SEQ ID NO:946. In some embodiments, the first four amino acids of the following sequences are truncated: SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 18, SEQ ID NO: 28, SEQ ID NO: 32, or variants of these sequences, including but not limited to any one of SEQ ID NO: 528 to SEQ ID NO: 768, any one of SEQ ID NO: 825 to SEQ ID NO: 844, SEQ ID NO: 771, SEQ ID NO: 772, SEQ ID NO: 852, any one of SEQ ID NO: 903 to SEQ ID NO: 914, SEQ ID NO: 927, SEQ ID NO: 928, SEQ ID NO: 932, SEQ ID NO: 933, SEQ ID NO: 934, SEQ ID NO: 935, SEQ ID NO: 936, SEQ ID NO: 939, SEQ ID NO: 940, SEQ ID NO: 939, SEQ ID NO: 936, SEQ ID NO: 939, SEQ ID NO: 939, SEQ ID NO: 930 ...2, SEQ ID NO: 933, SEQ ID NO: 934, SEQ ID NO: 935, SEQ ID NO: 936, SEQ ID NO: 939, SEQ ID NO: 930, SEQ ID NO: NO:941, SEQ ID NO:943, SEQ ID NO:944, SEQ ID NO:945 or SEQ ID NO:946.

[0165] As used in this article, “variants” refers to proteins or polypeptides that have an amino acid sequence that is at least about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 78%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the parent amino acid sequence. The term “approximately” used in this article for the percentage of sequence identity (%) means a deviation limit of ±0.5%, in increments of 0.1%. For example, “approximately 90%” sequence identity includes sequence identity of 89.5%, 89.6%, 89.7%, 89.8%, 89.9%, 90%, 90.1%, 90.2%, 90.3%, 90.4%, and 90.5%.

[0166] In some embodiments, the PIP-72 polypeptide has at least about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 78%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the full length of the following amino acid sequences: SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 79%. NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 18, SEQ ID NO: 28, SEQ ID NO: 32, SEQ ID NO: 927, SEQ ID NO: 928, SEQ ID NO: 932, SEQ ID NO: 933, SEQ ID NO: 934, SEQ ID NO: 935, SEQ ID NO: 936, SEQ ID NO:939, SEQ ID NO:940, SEQ ID NO:941, SEQ ID NO:943, SEQ ID NO:944, SEQ ID NO:945 or SEQ ID NO:946.

[0167] In some embodiments, the PIP-72 polypeptide has at least about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 78%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity with the full length of the amino acid sequence shown in SEQ ID NO: 2.

[0168] In some embodiments, the PIP-72 polypeptide has at least about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 78%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity with the full length of the amino acid sequence shown in SEQ ID NO: 4.

[0169] In some embodiments, the PIP-72 polypeptide has at least about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 78%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity with the full length of the amino acid sequence shown in SEQ ID NO: 6.

[0170] In some embodiments, the PIP-72 polypeptide has at least about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 78%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity with the full length of the amino acid sequence shown in SEQ ID NO: 8.

[0171] In some embodiments, the PIP-72 polypeptide has at least about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 78%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity with the full length of the amino acid sequence shown in SEQ ID NO: 10.

[0172] In some embodiments, the PIP-72 polypeptide has at least about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 78%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity with the full length of the amino acid sequence shown in SEQ ID NO: 12.

[0173] In some embodiments, the PIP-72 polypeptide has at least about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 78%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity with the full length of the amino acid sequence shown in SEQ ID NO: 14.

[0174] In some embodiments, the PIP-72 polypeptide has at least about 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 78%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity with the full length of the amino acid sequence shown in SEQ ID NO: 18.

[0175] In some embodiments, the PIP-72 polypeptide has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity with the full length of the amino acid sequence shown in SEQ ID NO: 28.

[0176] In some embodiments, the PIP-72 polypeptide has at least about 95%, 96%, 97%, 98%, 99% or higher identity with the full length of the amino acid sequence shown in SEQ ID NO: 32.

[0177] In some embodiments, the PIP-72 polypeptide has at least about 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 78%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity with the full length of the amino acid sequence shown in SEQ ID NO: 927.

[0178] In some embodiments, the PIP-72 polypeptide has at least about 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 78%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity with the full length of the amino acid sequence shown in SEQ ID NO: 928.

[0179] In some embodiments, the PIP-72 polypeptide has at least about 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 78%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity with the full length of the amino acid sequence shown in SEQ ID NO: 932.

[0180] In some embodiments, the PIP-72 polypeptide has at least about 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 78%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity with the full length of the amino acid sequence shown in SEQ ID NO: 933.

[0181] In some embodiments, the PIP-72 polypeptide has at least about 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 78%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity with the full length of the amino acid sequence shown in SEQ ID NO: 934.

[0182] In some embodiments, the PIP-72 polypeptide has at least about 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 78%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity with the full length of the amino acid sequence shown in SEQ ID NO: 935.

[0183] In some embodiments, the PIP-72 polypeptide has at least about 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 78%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity with the full length of the amino acid sequence shown in SEQ ID NO: 936.

[0184] In some embodiments, the PIP-72 polypeptide has at least about 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 78%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity with the full length of the amino acid sequence shown in SEQ ID NO: 939.

[0185] In some embodiments, the PIP-72 polypeptide has at least about 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 78%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity with the full length of the amino acid sequence shown in SEQ ID NO: 940.

[0186] In some embodiments, the PIP-72 polypeptide has at least about 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 78%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity with the full length of the amino acid sequence shown in SEQ ID NO: 941.

[0187] In some embodiments, the PIP-72 polypeptide has at least about 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 78%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity with the full length of the amino acid sequence shown in SEQ ID NO: 943.

[0188] In some embodiments, the PIP-72 polypeptide has at least about 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 78%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity with the full length of the amino acid sequence shown in SEQ ID NO: 944.

[0189] In some embodiments, the PIP-72 polypeptide has at least about 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 78%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity with the full length of the amino acid sequence shown in SEQ ID NO: 945.

[0190] In some embodiments, the PIP-72 polypeptide has at least about 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 78%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity with the full length of the amino acid sequence shown in SEQ ID NO: 946.

[0191] In some embodiments, the PIP-72 polypeptide contains an amino acid sequence that is at least 50% identical to the following amino acid sequences: SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12 or SEQ ID NO: 14, wherein the polypeptide has insecticidal activity.

[0192] In some embodiments, the PIP-72 polypeptide contains an amino acid sequence that is at least 70% identical to the following amino acid sequences: SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12 or SEQ ID NO: 14, wherein the polypeptide has insecticidal activity.

[0193] In some embodiments, the PIP-72 peptide contains an amino acid sequence that is at least 50% identical to the amino acid sequence shown in SEQ ID NO: 2, wherein the peptide has insecticidal activity.

[0194] In some embodiments, the PIP-72 polypeptide contains an amino acid sequence that is at least 50% identical to the amino acid sequence shown in SEQ ID NO: 4, wherein the polypeptide has insecticidal activity.

[0195] In some embodiments, the PIP-72 polypeptide contains an amino acid sequence that is at least 50% identical to the amino acid sequence shown in SEQ ID NO: 6, wherein the polypeptide has insecticidal activity.

[0196] In some embodiments, the PIP-72 polypeptide contains an amino acid sequence that is at least 50% identical to the amino acid sequence shown in SEQ ID NO: 8, wherein the polypeptide has insecticidal activity.

[0197] In some embodiments, the PIP-72 polypeptide contains an amino acid sequence that is at least 50% identical to the amino acid sequence shown in SEQ ID NO: 10, wherein the polypeptide has insecticidal activity.

[0198] In some embodiments, the PIP-72 peptide contains an amino acid sequence that is at least 50% identical to the amino acid sequence shown in SEQ ID NO: 12, wherein the peptide has insecticidal activity.

[0199] In some embodiments, the PIP-72 polypeptide contains an amino acid sequence that is at least 50% identical to the amino acid sequence shown in SEQ ID NO: 14, wherein the polypeptide has insecticidal activity.

[0200] In some implementations, sequence identity is achieved using a Vector with all default parameters. Package (Invitrogen Corporation, Carlsbad, Calif.) The ClustalW algorithm in the module performs calculations across the entire length of the polypeptide.

[0201] In some embodiments, the amino acid motif of the PIP-72 polypeptide is represented by amino acid residues from position 37 to position 51 of the following sequences: SEQ ID NO: 846, SEQ ID NO: 847, SEQ ID NO: 848 or SEQ ID NO: 849.

[0202] In some embodiments, the PIP-72 polypeptide comprises the amino acid sequence SEQ ID NO: 2, but selected from the 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, 11th, 12th, 13th, 14th, 15th, 16th, 17th, 18th, 19th, 20th, 21st, 22nd, 23rd, 24th, 25th, 26th, 27th, 28th, 29th, 30th, 31st, 32nd, 33rd, 34th, 35th, 36th, 37th, 38th, 39th, 40th, 42nd, 44th, 45th, 46th, 47th, 48th, 49th, 50th, and 51st amino acids in SEQ ID NO: 2. The PIP-72 polypeptide has an amino acid substitution at one or more residues at positions 52, 53, 54, 56, 57, 58, 59, 60, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, or 86, and optionally, compared to SEQ ID NO: 2, a deletion of 1 to 5 amino acids, an insertion of 1 to 5 amino acids, an addition of one or more amino acids at the N-terminus, and / or an addition of one or more amino acids at the C-terminus (these changes can be combined in any way).

[0203] In some embodiments, the PIP-72 polypeptide comprises the amino acid sequence SEQ ID NO: 2, but selected from the 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, 11th, 12th, 13th, 14th, 15th, 17th, 18th, 19th, 20th, 22nd, 23rd, 24th, 25th, 26th, 27th, 28th, 29th, 30th, 31st, 32nd, 33rd, 34th, 35th, 36th, 37th, 38th, 39th, 40th, 42nd, 44th, 45th, 46th, 47th, 48th, 49th, 50 ... The PIP-72 polypeptide has an amino acid substitution at one or more residues at positions 1, 52, 53, 54, 56, 58, 60, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, or 86, and optionally, compared to SEQ ID NO: 2, a deletion of 1 to 5 amino acids, an insertion of 1 to 5 amino acids, an addition of one or more amino acids at the N-terminus, or an addition of one or more amino acids at the C-terminus (these changes can be combined in any way).

[0204] In some embodiments, the PIP-72 polypeptide comprises the amino acid sequence SEQ ID NO: 2, but selected from the 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, 11th, 12th, 13th, 14th, 15th, 16th, 17th, 18th, 19th, 20th, 21st, 22nd, 23rd, 24th, 25th, 26th, 27th, 28th, 29th, 30th, 31st, 32nd, 33rd, 34th, 35th, 36th, 37th, 38th, 39th, 40th, 42nd, 44th, 45th, 46th, 47th, 48th, 49th, 50th, and 51st amino acids in SEQ ID NO: 2. The PIP-72 polypeptide has amino acid substitutions at residues 52, 53, 54, 56, 57, 58, 59, 60, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, and 86, in any combination of 1 to 45 residues, and optionally, compared to SEQ ID NO: 2, the PIP-72 polypeptide also has 1 to 5 amino acids missing, 1 to 5 amino acids inserted, one or more amino acids added at the N-terminus, and / or one or more amino acids added at the C-terminus (these changes are in any combination).

[0205] In some embodiments, the PIP-72 polypeptide comprises the amino acid sequence SEQ ID NO: 2, which, compared to the natural amino acid shown in SEQ ID NO: 2, is selected from the 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, 11th, 12th, 13th, 14th, 15th, 17th, 18th, 19th, 20th, 22nd, 23rd, 24th, 25th, 26th, 27th, 28th, 29th, 30th, 31st, 32nd, 33rd, 34th, 35th, 36th, 37th, 38th, 39th, 40th, 42nd, 44th, 45th, 46th, 47th, 48th, 49th, 50 ... The PIP-72 polypeptide has amino acid substitutions at residues 1 to 45, in any combination of residues 1, 52, 53, 54, 56, 58, 60, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, and 86, and optionally, compared to SEQ ID NO: 2, it also has 1 to 5 amino acid deletions, 1 to 5 amino acid insertions, one or more amino acid additions at the N-terminus, and / or one or more amino acid additions at the C-terminus (these changes are in any combination).

[0206] In specific implementations, the substitution is made by replacing the natural amino acid with alanine at the listed positions. Also covered are nucleic acid sequences encoding variant proteins or polypeptides.

[0207] In some embodiments, the PIP-72 polypeptide comprises the amino acid sequence SEQ ID NO: 846, wherein Xaa at position 2 is Gly, Ala, Cys, Asp, Glu, Ile, Lys, Leu, Asn, Arg, Ser, Thr, Val, Trp, or Tyr; Xaa at position 3 is Ile or Trp; Xaa at position 4 is Thr, Ala, Asp, Glu, His, Ile, Lys, Leu, Arg, Ser, Val, Trp, or Tyr; Xaa at position 5 is Val, Ala, Cys, Gly, His, Ile, or Tyr; and Xaa at position 6 is Thr, Ala, Cys, Phe, Gly, His, Ile, Lys, Me t, Pro, Gln, Arg, Ser, Trp, or Tyr; Xaa at position 7 is Asn, Ala, or Val; Xaa at position 8 is Asn, Ala, Cys, Asp, Glu, Gly, His, Ile, Lys, Leu, Met, Gln, Arg, Ser, Thr, or Val; Xaa at position 9 is Ser, Ala, Cys, Gly, or Thr; Xaa at position 10 is Ser, Ala, Glu, Phe, Gly, His, Ile, Lys, Leu, Asn, Pro, Gln, Arg, Thr, or Trp; Xaa at position 11 is Asn, Ala, Pro, Gln, Arg, Thr, or Trp. Cys, Asp, Glu, Gly, His, Ile, Lys, Leu, Met, Gln, Ser, Thr, Val, or Tyr; Xaa at position 12 is Pro, Ala, Cys, Asp, Glu, Gly, His, Lys, Leu, Asn, Gln, Arg, Ser, Thr, Val, Trp, or Tyr; Xaa at position 13 is Ile, Asn, Gln, or Val; Xaa at position 14 is Glu, Ala, Cys, Phe, His, Lys, or Gln; Xaa at position 15 is Val, Ala, Cys, Ile, Met, or Arg; Xaa at position 17 is... The Xaa at position 1 is Ile, Glu, or Val; the Xaa at position 18 is Asn or Ser; the Xaa at position 19 is His, Ala, Glu, Lys, Leu, Pro, Arg, Ser, or Tyr; the Xaa at position 20 is Trp, Ala, or Thr; the Xaa at position 22 is Ser, Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Thr, Val, or Tyr; the Xaa at position 23 is Asp, Ala, Gly, His, Lys, Met, Asn, Gln, Ser, Thr, or Val.Xaa at position 24 is Gly, Asp, or Phe; Xaa at position 25 is Asp, Ala, Glu, Phe, Asn, or Gln; Xaa at position 26 is Thr, Glu, or Pro; Xaa at position 27 is Ser, Ala, Cys, Asp, Glu, Phe, Gly, His, Asn, Gln, Arg, or Thr; Xaa at position 28 is Phe, Pro, Trp, or Tyr; Xaa at position 29 is Phe, Ala, Cys, Ile, Leu, Gln, Arg, Trp, or Tyr; Xaa at position 30 is Ser, Ala, Cys, or Asp. The following are the possible values ​​for Xaa: Glu, Phe, Gly, His, Lys, Leu, Met, Asn, Pro, Gln, Arg, Thr, Val, Trp, or Tyr; Xaa at position 31 is Val, Ile, or Leu; Xaa at position 32 is Gly, Ala, Asp, Glu, Phe, His, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, or Tyr; Xaa at position 33 is Asn, Ala, Cys, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Pro, Gln, Arg, Ser The Xaa at position 34 is Gly, Glu, Phe, His, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, or Tyr; the Xaa at position 35 is Lys, Ala, Cys, Asp, Gly, His, Ile, Leu, Met, Asn, Gln, Arg, Ser, Thr, or Val; the Xaa at position 36 is Gln, Ala, Cys, Glu, Gly, His, Ile, Lys, Leu, Asn, Pro, Arg, Ser, Thr, or Val; the Xaa at position 37 is Glu, Ala, Cys, Asp, Phe, Gly, Ile, Lys, Leu, Met, Asn, Ser, Thr, or Val; Xaa at position 38 is Thr, Ala, Cys, Asp, Glu, Phe, Gly, His, Ile, Leu, Met, Asn, Gln, Arg, Ser, Val, Trp, or Tyr; Xaa at position 39 is Trp or Phe; Xaa at position 40 is Asp, Ala, Cys, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp, or Tyr;The Xaa at position 42 is Ser, Ala, Cys, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Thr, Val, Trp, or Tyr; the Xaa at position 44 is Ser, Ala, Asp, Glu, Gly, Leu, Met, Asn, Pro, Gln, Thr, Val, or Tyr; the Xaa at position 45 is Arg, Lys, or Ser; the Xaa at position 46 is Gly, Ala, or Gln; the Xaa at position 47 is Phe, Cys, Val, or Tyr; the Xaa at position 48 is Val, Ile, or Leu; the Xaa at position 49 is... Xaa at position 9 is Leu, Cys, Phe, Met, Arg, or Tyr; Xaa at position 50 is Ser, Ala, Cys, Asp, Ile, Met, Pro, Gln, Thr, or Val; Xaa at position 51 is Leu, Ala, Cys, Met, or Val; Xaa at position 52 is Lys, Cys, Phe, His, Ile, Leu, Met, Asn, Arg, Ser, Thr, Trp, or Tyr; Xaa at position 53 is Lys, Ala, Cys, Asp, Glu, Phe, His, Ile, Leu, Met, Asn, Gln, Arg, Ser, Thr, or Val. The Xaa at position 54 is either l or Tyr; the Xaa at position 54 is Asn, Cys, Asp, Glu, Phe, Gly, Lys, Met, Gln, Arg, Ser, or Trp; the Xaa at position 56 is Ala, Gly, Leu, Asn, Pro, Gln, Arg, Ser, or Thr; the Xaa at position 57 is Gln, Glu, Leu, Met, Ser, or Thr; the Xaa at position 58 is His, Ala, Asp, Phe, Leu, Met, Asn, Arg, Trp, or Tyr; the Xaa at position 60 is Tyr, Glu, or Phe; the Xaa at position 63 is Gln, Cys, Gly, Ile, Leu. Met, Asn, Thr, Val, or Tyr; Xaa at position 64 is Ala, Phe, Gly, His, Arg, Ser, or Tyr; Xaa at position 65 is Ser, Ala, Cys, Asp, Glu, Phe, Gly, His, Ile, Leu, Asn, Thr, or Val; Xaa at position 66 is Ser, Ala, or Gly; Xaa at position 67 is Lys, Ala, Cys, Asp, Phe, His, Ile, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp, or Tyr; Xaa at position 68 is Ile, Asp, Leu, or Val.Xaa at position 69 is Glu, Ala, Cys, Asp, Phe, His, Ile, Leu, Met, Gln, Arg, Ser, Thr, Val, or Tyr; Xaa at position 70 is Val, Cys, or Ile; Xaa at position 71 is Asp, Ala, Cys, Gly, His, Ile, Leu, Met, Asn, Ser, Thr, Val, or Tyr; Xaa at position 72 is Asn, Ala, Cys, Asp, Glu, Gly, Lys, Met, Pro, Gln, Arg, Ser, Thr, Val, or Trp; Xaa at position 73... The first Xaa is Asn, Ala, Cys, Asp, Phe, Gly, His, Ile, Leu, Ser, Thr, Val, or Tyr; the second Xaa at position 74 is Ala, Cys, Asp, Phe, Gly, His, Ile, Leu, Asn, Gln, Arg, Ser, Thr, Val, or Tyr; the third Xaa at position 75 is Val, Cys, Ile, or Leu; the fourth Xaa at position 76 is Lys, Ala, Cys, Phe, His, Ile, Leu, Gln, Arg, Ser, Thr, Val, Trp, or Tyr; the fifth Xaa at position 77 is Asp or Tyr; the sixth Xaa at position 78 is Asn, Ala, Cys, Phe, His, Ile, Leu, Gln, Arg, Ser, Thr, Val, Trp, or Tyr. Xaa at position 78 is Gln, Ala, Cys, Asp, Phe, Gly, His, Ile, Leu, Met, Asn, Arg, Ser, Thr, Val, or Tyr; Xaa at position 79 is Gly, Arg, Ala, Cys, Asp, Glu, Phe, His, Lys, Leu, Asn, Gln, Arg, Ser, Thr, Trp, or Tyr; Xaa at position 80 is Arg, Ala, Cys, Asp, Phe, Gly, His, Ile, Leu, Asn, Ser, Thr, Val, or Tyr; Xaa at position 81 is Leu, Al a, Cys, Asp, Phe, Gly, His, Ile, Asn, Pro, Arg, Ser, Thr, or Val; Xaa at position 82 is Ile, Ala, Leu, Met, Arg, or Val; Xaa at position 83 is Glu, Ala, Cys, Asp, Phe, Gly, His, Ile, Lys, Leu, Asn, Pro, Arg, Ser, Thr, Val, or Tyr; Xaa at position 84 is Pro, Ala, Cys, Glu, Ile, Ser, Val, Trp, or Tyr; Xaa at position 85 is Leu, Cys, Gly, or Val.Furthermore, Xaa at position 86 is Ser, Ala, Ile, Thr, or Val, and optionally, 1 to 14 amino acids are deleted from the N-terminus and / or C-terminus of the PIP-72 polypeptide.

[0208] In some embodiments, the PIP-72 polypeptide comprises the amino acid sequence SEQ ID NO: 846, which, compared to the native amino acid at the corresponding position in SEQ ID NO: 2, has any combination of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, or 45 amino acid substitutions at the residues marked by Xaa in SEQ ID NO: 846.

[0209] In some embodiments, the PIP-72 polypeptide comprises the amino acid sequence SEQ ID NO: 846, which, compared with the native amino acid at the corresponding position in SEQ ID NO: 2, has any combination of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29 amino acid substitutions at the residues marked by Xaa in SEQ ID NO: 846.

[0210] In some embodiments, the PIP-72 peptide comprises the amino acid sequence SEQ ID NO: 847, wherein Xaa at position 2 is Gly, Lys, or Ala; Xaa at position 3 is Ile or Leu; Xaa at position 4 is Thr or Ser; Xaa at position 5 is Val or Ile; Xaa at position 6 is Thr or Lys; Xaa at position 8 is Asn, Lys, Gly, or Ser; Xaa at position 9 is Ser or Ala; Xaa at position 11 is Asn, Lys, His, or Thr; Xaa at position 12 is Pro, Thr, Lys, or Ser; Xaa at position 13 is Ile or Val; Xaa at position 14 is Glu or Asp; and Xaa at position 15 is V al, Ala, or Ile; Xaa at position 16 is Ala or Ser; Xaa at position 17 is Ile or Val; Xaa at position 18 is Asn or Ser; Xaa at position 19 is His, Lys, Arg, Gln, or Ala; Xaa at position 21 is Gly or Arg; Xaa at position 22 is Ser, Lys, Asn, Asp, or Thr; Xaa at position 25 is Asp or Asn; Xaa at position 26 is Thr or Asp; Xaa at position 27 is Ser, Thr, Asn, or Lys; Xaa at position 28 is Phe, Tyr, or Pro; Xaa at position 29 is Ph e or Tyr; Xaa at position 30 is Ser, Gly, or Lys; Xaa at position 31 is Val, Ile, or Met; Xaa at position 32 is Gly, Ala, or Asp; Xaa at position 33 is Asn, Ser, Gln, or Pro; Xaa at position 35 is Lys, Glu, or Ser; Xaa at position 36 is Gln, Asn, or Ser; Xaa at position 37 is Glu or Asp; Xaa at position 38 is Thr or Ser; Xaa at position 42 is Ser or Asn; Xaa at position 44 is Ser, Asp, Ala, or Leu; Xaa at position 47 is Phe or Tyr. The Xaa at position 48 is Leu or Met; the Xaa at position 49 is Leu or Met; the Xaa at position 50 is Ser, Ala, or Tyr; the Xaa at position 51 is Leu or Val; the Xaa at position 52 is Lys or Gln; the Xaa at position 53 is Lys, Arg, Met, or Leu; the Xaa at position 54 is Asn, Lys, or Gly; the Xaa at position 55 is Gly or Ser; the Xaa at position 56 is Ala, Thr, Gln, or Ser; the Xaa at position 57 is Gln, Val, or Ala; the Xaa at position 58 is His, Ala, Lys, Tyr, or Thr.Xaa at position 59 is Pro or Thr; Xaa at position 62 is Val or Ile; Xaa at position 63 is Gln, Ser, or Leu; Xaa at position 64 is Ala, Gln, or Ser; Xaa at position 65 is Ser or Thr; Xaa at position 67 is Lys, Gln, Arg, or Asn; Xaa at position 69 is Glu, Lys, or Val; Xaa at position 70 is Val or Ile; Xaa at position 71 is Asp, Glu, or Tyr; Xaa at position 72 is Asn, His, Ser, or Asp; Xaa at position 73 is Asn, Ser, or Asp; Xaa at position 74 is Ala, T The Xaa at position 76 is Lys or Thr; the Xaa at position 78 is Gln, His, or Ser; the Xaa at position 80 is Arg, Glu, or Gln; the Xaa at position 81 is Leu, Pro, Ala, or Thr; the Xaa at position 82 is Ile or Leu; the Xaa at position 83 is Glu, His, Asn, Gln, or Leu; the Xaa at position 85 is Leu, Val, or Ala; and the Xaa at position 86 is Ser, Ala, Tyr, or Asn, wherein optionally 1 to 14 amino acids are deleted from the N-terminus and / or C-terminus of the PIP-72 polypeptide, and / or an amino acid is inserted between residues at positions 24 and 25, compared to SEQ ID NO: 847.

[0211] In some embodiments, the PIP-72 polypeptide comprises the amino acid sequence SEQ ID NO: 847, which, compared to the native amino acid at the corresponding position in SEQ ID NO: 2, has any combination of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, or 45 amino acid substitutions at the residues marked by Xaa in SEQ ID NO: 847.

[0212] In some embodiments, the PIP-72 polypeptide comprises the amino acid sequence SEQ ID NO: 847, which, compared to the native amino acid at the corresponding position in SEQ ID NO: 2, has any combination of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29 amino acid substitutions at the residues marked by Xaa in SEQ ID NO: 847.

[0213] In some embodiments, the PIP-72 peptide comprises the amino acid sequence SEQ ID NO: 848, wherein Xaa at position 2 is Gly, Lys, Ala, or Arg; Xaa at position 3 is Ile, Leu, or Val; Xaa at position 4 is Thr or Ser; Xaa at position 5 is Val, Ile, or Leu; Xaa at position 6 is Thr, Lys, Ser, or Arg; Xaa at position 8 is Asn, Lys, Gly, Ser, Gln, Arg, Thr, or Ala; Xaa at position 9 is Ser, Ala, or Thr; Xaa at position 11 is Asn, Lys, Thr, Gln, Arg, His, or Ser; and Xaa at position 12 is Pro, Thr, or Lys. The Xaa at position 13 is Ile, Val, or Leu; the Xaa at position 14 is Glu or Asp; the Xaa at position 15 is Val, Ala, Ile, or Leu; the Xaa at position 16 is Ala or Ser; the Xaa at position 17 is Ile, Val, or Leu; the Xaa at position 18 is Asn, Ser, Gln, or Thr; the Xaa at position 19 is His, Lys, Ala, Gln, Asn, or Arg; the Xaa at position 21 is Gly, Arg, or Lys; the Xaa at position 22 is Ser, Lys, Asn, Thr, Arg, Asp, Glu, or Gln; the Xaa at position 3 is Ser, Lys, Asn, Thr, Arg, Asp, Glu, or Gln; the Xaa at position 4 is Ser, Ala, Ile, or Leu; the Xaa at position 5 is Val, Ala, Ile, or Leu; the Xaa at position 6 is Ala or Ser; the Xaa at position 7 is Ile, Val, or Leu; the Xaa at position 8 is Asn, Ser, Gln, or Thr; the Xaa at position 9 is Asn, Ser, Gln, or Thr; the Xaa at position 10 is Asn, Ser, Gln, or Thr; the Xaa at position 11 is Asn, Lys, Ala, Gln, Asp, or Gln; the Xaa at position 12 is Ser, Lys, Asn, Thr, Arg, Asp, Glu, or Gln; the Xaa at position 13 is Ile, Val, or Leu; the Xaa at position 14 is Glu or Asp; the Xaa at position 15 is Val, Ala, Ile, or Leu; the Xaa at position 16 is Ala or Ser; the Xaa at position Xaa at position 25 is Asp, Asn, Glu, or Gln; Xaa at position 26 is Thr, Asp, Ser, or Glu; Xaa at position 27 is Ser, Thr, Lys, Asn, Gln, or Arg; Xaa at position 28 is Phe, Tyr, Pro, or Trp; Xaa at position 29 is Phe, Tyr, or Trp; Xaa at position 30 is Ser, Gly, Lys, Thr, or Arg; Xaa at position 31 is Val, Ile, Met, or Leu; Xaa at position 32 is Gly, Ala, Asp, or Glu; Xaa at position 33 is Asn, Ser, Gln, or Pr o or Thr; Xaa at position 35 is Lys, Glu, Ser, Arg, or Thr; Xaa at position 36 is Gln, Ser, Asn, or Thr; Xaa at position 37 is Glu or Asp; Xaa at position 38 is Thr or Ser; Xaa at position 42 is Ser, Asn, Thr, or Gln; Xaa at position 44 is Ser, Asp, Ala, Leu, Thr, Glu, Ile, or Val; Xaa at position 47 is Phe, Tyr, or Trp; Xaa at position 48 is Leu, Met, Ile, or Val; Xaa at position 49 is Leu, Met, Ile, or Val.Xaa at position 50 is Ser, Ala, Tyr, or Thr; Xaa at position 51 is Leu, Val, or Ile; Xaa at position 52 is Lys, Gln, Arg, or Asn; Xaa at position 53 is Lys, Arg, Met, Leu, Ile, or Val; Xaa at position 54 is Asn, Lys, Gly, Gln, or Arg; Xaa at position 55 is Gly, Ser, or Thr; Xaa at position 56 is Ala, Thr, Gln, Ser, or Asn; Xaa at position 57 is Gln, Val, Ala, Asn, Leu, or Ile; Xaa at position 58 is His, Ala, Lys, Tyr, or Thr; Xaa at position 59 is Pr; o The Xaa at position 62 is Val, Ile, or Leu; the Xaa at position 63 is Gln, Ser, Leu, Asn, Thr, Ile, or Val; the Xaa at position 64 is Ala, Gln, Ser, Asn, or Thr; the Xaa at position 65 is Ser or Thr; the Xaa at position 67 is Lys, Gln, Asn, or Arg; the Xaa at position 69 is Glu, Va The Xaa at position 70 is Val, Ile, or Leu; the Xaa at position 71 is Asp, Glu, Tyr, or Trp; the Xaa at position 72 is Asn, His, Ser, Asp, Gln, Thr, or Glu; the Xaa at position 73 is Asn, Ser, Asp, Gln, Thr, or Glu; the Xaa at position 74 is Ala, Thr The Xaa at position 76 is Lys, Thr, Arg, or Ser; the Xaa at position 78 is Gln, His, Ser, Asn, or Thr; the Xaa at position 80 is Arg, Glu, Gln, Lys, Asp, or Asn; the Xaa at position 81 is Leu, Pro, Thr, Ile, Val, Ala, or Ser; the Xaa at position 82 is Met, Ile, Lys, Ser, Leu, Val, Ala, or Ser; the Xaa at position 83 is Met, Ile, Lys, Ser, Leu, Val, or Arg; the Xaa at position 84 is Met, Ile, Lys, Ser, Leu, Val, or Arg; the Xaa at position 85 is Met, Ile, Lys, Ser, Leu, Val, Val, or Arg; the Xaa at position 86 is Lys, Thr, Arg, Ser, Ser, Ser, Leu, Val, or Arg; the Xaa at position 87 is Met, Ile, Lys, Ser, Leu, Val, Val, or Arg; the Xaa at position 88 is Met, Ile, Lys, Ser, Leu, Val, Val, or Arg; the Xaa at position 89 is Met, Ile, Lys, Ser, Leu, Val, Val, Val, or Arg; the Xaa at position 80 is Met, Ile, Lys, Asn, Asn, Ser, Leu, Val ... Xaa at position 2 is Ile, Leu, or Val; Xaa at position 83 is Glu, His, Asn, Leu, Gln, Ile, or Val; Xaa at position 85 is Leu, Val, or Ala; and Xaa at position 86 is Ser, Ala, Tyr, Asn, or Thr, wherein optionally 1 to 14 amino acids are deleted from the N-terminus and / or C-terminus of the PIP-72 polypeptide, and / or an amino acid is inserted between residues at positions 24 and 25 compared to SEQ ID NO: 848.

[0214] In some embodiments, the PIP-72 polypeptide comprises the amino acid sequence SEQ ID NO: 848, which, compared to the native amino acid at the corresponding position in SEQ ID NO: 2, has any combination of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, or 45 amino acid substitutions at the residues marked by Xaa in SEQ ID NO: 848.

[0215] In some embodiments, the PIP-72 polypeptide comprises the amino acid sequence SEQ ID NO: 848, which, compared to the native amino acid at the corresponding position in SEQ ID NO: 2, has any combination of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29 amino acid substitutions at the residues marked by Xaa in SEQ ID NO: 848.

[0216] In some embodiments, the PIP-72 polypeptide comprises the amino acid sequence SEQ ID NO: 849, wherein Xaa at position 2 is Gly, Ala, Cys, Asp, Glu, Ile, Lys, Leu, Asn, Arg, Ser, Thr, Val, Trp, or Tyr; Xaa at position 3 is Ile, Leu, Val, or Trp; Xaa at position 4 is Thr, Ala, Asp, Glu, His, Ile, Lys, Leu, Arg, Ser, Val, Trp, or Tyr; Xaa at position 5 is Val, Ala, Cys, Gly, His, Ile, Leu, or Tyr; and Xaa at position 6 is Thr, Ala, Cys, Phe, Gly, His, I le, Lys, Met, Pro, Gln, Arg, Ser, Trp, or Tyr; the 7th position Xaa is Asn, Ala, or Val; the 8th position Xaa is Asn, Lys, Gly, Ser, Gln, Arg, Thr, Ala, Cys, Asp, Glu, His, Ile, Leu, Met, or Val; the 9th position Xaa is Ser, Ala, Cys, Gly, or Thr; the 11th position Xaa is Asn, Lys, Thr, Gln, Arg, Ser, Ala, Cys, Asp, Glu, Gly, His, Ile, Leu, Met, Val, or Tyr; the 12th position... The Xaa at position 1 is Pro, Thr, Lys, Ser, Arg, Ala, Cys, Asp, Glu, Gly, His, Leu, Asn, Gln, Arg, Val, Trp, or Tyr; the Xaa at position 13 is Ile, Asn, Gln, Leu, or Val; the Xaa at position 14 is Glu, Ala, Cys, Phe, His, Lys, Asp, or Gln; the Xaa at position 15 is Val, Ala, Ile, Leu, Cys, Met, or Arg; the Xaa at position 16 is Ala or Ser; the Xaa at position 17 is Ile, Glu, Leu, or Val; the Xaa at position 18... 'a' is Asn, Gln, Thr, or Ser; Xaa at position 19 is His, Lys, Ala, Arg, Glu, Leu, Pro, Ser, or Tyr; Xaa at position 20 is Trp, Ala, or Thr; Xaa at position 21 is Gly, Arg, or Lys; Xaa at position 22 is Ser, Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Thr, Val, or Tyr; Xaa at position 23 is Asp, Ala, Gly, His, Lys, Met, Asn, Gln, Ser, Thr, or Val.Xaa at position 24 is Gly, Asp, or Phe; Xaa at position 25 is Asp, Ala, Glu, Phe, Asn, or Gln; Xaa at position 26 is Thr, Glu, Asp, Ser, or Pro; Xaa at position 27 is Ser, Thr, Lys, Arg, Ala, Cys, Asp, Glu, Phe, Gly, His, Asn, or Gln; Xaa at position 28 is Phe, Tyr, Pro, or Trp; Xaa at position 29 is Phe, Ala, Cys, Ile, Leu, Gln, Arg, Trp, or Tyr; Xaa at position 30 is Ser, Gly, ... Lys, Thr, Arg, Ala, Cys, Asp, Glu, Phe, His, Leu, Met, Asn, Pro, Gln, Val, Trp, or Tyr; Xaa at position 31 is Val, Ile, Met, or Leu; Xaa at position 32 is Gly, Ala, Asp, Glu, Phe, His, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, or Tyr; Xaa at position 33 is Asn, Ser, Gln, Pro, Thr, Ala, Cys, Asp, Glu, Phe, Gly, His, Ile, L The Xaa at position 34 is Gly, Glu, Phe, His, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, or Tyr; the Xaa at position 35 is Lys, Glu, Ala, Cys, Asp, Gly, His, Ile, Leu, Met, Asn, Gln, Arg, Ser, Thr, or Val; the Xaa at position 36 is Gln, Ala, Cys, Glu, Gly, His, Ile, Lys, Leu, Asn, Pro, Arg, Ser, Thr, or Val; the Xaa at position 37 is Glu. The Xaa at position 38 is Thr, Ser, Ala, Cys, Phe, Gly, Ile, Lys, Leu, Met, Asn, Ser, Thr, or Val; the Xaa at position 39 is Trp or Phe; the Xaa at position 40 is Asp, Ala, Cys, Glu, Phe, Gly, His, Ile, Leu, Met, Asn, Gln, Arg, Val, Trp, or Tyr.The Xaa at position 42 is Ser, Asn, Thr, Ala, Cys, Asp, Glu, Phe, Gly, Ile, Lys, Leu, Met, Arg, Val, Trp, Tyr, or Gln; the Xaa at position 44 is Ser, Asp, Ala, Leu, Thr, Glu, Ile, Ala, Gly, Leu, Met, Asn, Pro, Gln, Val, Tyr, or Val; the Xaa at position 45 is Arg, Lys, or Ser; the Xaa at position 46 is Gly, Ala, or Gln; the Xaa at position 47 is Phe, Tyr, Cys, Val, or Trp; the Xaa at position 48 is Leu, Me The Xaa at position 49 is Leu, Met, Ile, Phe, Met, Arg, Tyr, or Val; the Xaa at position 50 is Ser, Ala, Tyr, Cys, Asp, Ile, Met, Pro, Gln, Val, or Thr; the Xaa at position 51 is Leu, Val, Ala, Cys, Met, or Ile; the Xaa at position 52 is Lys, Cys, Phe, His, Ile, Leu, Met, Asn, Arg, Ser, Thr, Gln, Trp, or Tyr; the Xaa at position 53 is Lys, Arg, Met, Leu, Ile, Ala, Cys, As p, Glu, Phe, His, Asn, Gln, Ser, Thr, Tyr, or Val; Xaa at position 54 is Asn, Cys, Asp, Glu, Phe, Gly, Lys, Met, Gln, Arg, Ser, or Trp; Xaa at position 55 is Gly, Ser, or Thr; Xaa at position 56 is Ala, Thr, Gln, Ser, Gly, Leu, Pro, Arg, or Asn; Xaa at position 57 is Gln, Glu, Leu, Met, Ser, Val, Ala, Asn, Ile, or Thr; Xaa at position 58 is His, Ala, Lys, Asp, Phe, Leu, Me t, Asn, Arg, Trp, Tyr, or Thr; Xaa at position 59 is Pro, Thr, or Ser; Xaa at position 60 is Tyr, Glu, or Phe; Xaa at position 62 is Val, Ile, or Leu; Xaa at position 63 is Gln, Ser, Cys, Gly, Ile, Leu, Met, Asn, Thr, Val, or Tyr; Xaa at position 64 is Ala, Gln, Asn, Phe, Gly, His, Arg, Ser, or Tyr; Xaa at position 65 is Ser, Ala, Cys, Asp, Glu, Phe, Gly, His, Ile, Leu, Asn, Val, or Thr;The Xaa at position 66 is Ser, Ala, or Gly; the Xaa at position 67 is Lys, Gln, Asn, or Arg; the Xaa at position 67 is Lys, Ala, Cys, Asp, Phe, His, Ile, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp, or Tyr; the Xaa at position 68 is Ile, Asp, Leu, or Val; the Xaa at position 69 is Glu, Ala, Cys, Asp, Phe, His, Ile, Leu, Met, Gln, Arg, Ser, Thr, Val, or Tyr; the Xaa at position 70 is Val, Ile, Cys, or Leu; Xaa at position 71 is Asp, Glu, Tyr, Ala, Cys, Gly, His, Ile, Leu, Met, Asn, Ser, Thr, Val, or Trp; Xaa at position 72 is Asn, Ala, Cys, Asp, Glu, Gly, Lys, Met, Pro, Gln, Arg, Ser, Thr, Val, His, or Trp; Xaa at position 73 is Asn, Ser, Asp, Gln, Thr, Ala, Cys, Phe, Gly, His, Ile, Leu, Val, Tyr, or Glu; Xaa at position 74 is Ala, Thr, Met, Ile, Lys, Ser, Leu, Val, Cys, Asp, Phe, Gly, His, Asn, Gln, Tyr, or Arg; Xaa at position 75 is Val, Cys, Ile, or Leu; Xaa at position 76 is Lys, Ala, Cys, Phe, His, Ile, Leu, Gln, Arg, Ser, Thr, Val, Trp, or Tyr; Xaa at position 77 is Asp or Tyr; Xaa at position 78 is Gln, His, Ser, Asn, Ala, Cys, Asp, Phe, Gly, Ile, Leu, Met, Asn, Arg, Val, Tyr, or Thr; Xaa at position 79... The Xaa at position 1 is Gly, Arg, Ala, Cys, Asp, Glu, Phe, His, Lys, Leu, Asn, Gln, Arg, Ser, Thr, Trp, or Tyr; the Xaa at position 80 is Arg, Glu, Gln, Lys, Asp, Ala, Cys, Phe, Gly, His, Ile, Leu, Ser, Thr, Val, Tyr, or Asn; the Xaa at position 81 is Leu, Pro, Thr, Ile, Val, Ala, Cys, Asp, Phe, Gly, His, or Ser; the Xaa at position 82 is Ile, Ala, Leu, Met, Arg, or Val.Xaa at position 83 is Glu, His, Asn, Leu, Gln, Ile, Ala, Cys, Asp, Phe, Gly, Lys, Pro, Arg, Ser, Thr, Tyr, or Val; Xaa at position 84 is Pro, Ala, Cys, Glu, Ile, Ser, Val, Trp, or Tyr; Xaa at position 85 is Leu, Val, Cys, Gly, or Ala; and Xaa at position 86 is Ser, Ala, Tyr, Asn, Ile, Val, or Thr, wherein, compared to SEQ ID NO: 849, 1 to 14 amino acids are optionally deleted from the N-terminus and / or C-terminus of the PIP-72 polypeptide, and / or an amino acid is inserted between residues 24 and 25.

[0217] In some embodiments, the PIP-72 polypeptide comprises the amino acid sequence SEQ ID NO: 849, which, compared to the native amino acid at the corresponding position in SEQ ID NO: 2, has any combination of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, or 45 amino acid substitutions at the residues marked by Xaa in SEQ ID NO: 849.

[0218] In some embodiments, the PIP-72 polypeptide comprises the amino acid sequence SEQ ID NO: 849, which, compared to the native amino acid at the corresponding position in SEQ ID NO: 2, has any combination of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29 amino acid substitutions at the residues marked by Xaa in SEQ ID NO: 849.

[0219] In some embodiments, the exemplary PIP-72 peptide is composed of any one of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 17, SEQ ID NO: 27, SEQ ID NO: 31, SEQ ID NO: 287 to SEQ ID NO: 527, any one of SEQ ID NO: 796 to SEQ ID NO: 815, SEQ ID NO: 769, SEQ ID NO: 770, SEQ ID NO: 850, SEQ ID NO: 852, SEQ ID NO: 853 to SEQ ID NO: 864, any one of SEQ ID NO: 915 to SEQ ID NO: 926, SEQ ID NO: 949, SEQ ID NO: 950, SEQ ID NO: 954, SEQ ID NO: 955, SEQ ID NO: 956, SEQ ID NO: 957, SEQ ID NO: 958, SEQ ID NO: 959 ... The polynucleotide sequences shown in SEQ ID NO: 961, SEQ ID NO: 962, SEQ ID NO: 963, SEQ ID NO: 965, SEQ ID NO: 966, SEQ ID NO: 967 or SEQ ID NO: 968 encode the polynucleotide sequences.

[0220] In some embodiments, the PIP-72 polypeptide is encoded by the polynucleotides shown in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 17, SEQ ID NO: 27, SEQ ID NO: 31, SEQ ID NO: 769, SEQ ID NO: 770, SEQ ID NO: 850, SEQ ID NO: 852, SEQ ID NO: 949, SEQ ID NO: 950, SEQ ID NO: 954, SEQ ID NO: 955, SEQ ID NO: 956, SEQ ID NO: 957, SEQ ID NO: 958, SEQ ID NO: 961, SEQ ID NO: 962, SEQ ID NO: 963, SEQ ID NO: 965, SEQ ID NO: 966, SEQ ID NO: 967, or SEQ ID NO: 968.

[0221] In some embodiments, exemplary PIP-72 polypeptides are set forth in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 18, SEQ ID NO: 28, SEQ ID NO: 32, SEQ ID NO: 528, SEQ ID NO: 529, SEQ ID NO: 530, SEQ ID NO: 531, SEQ ID NO: 532, SEQ ID NO: 533, SEQ ID NO: 534, SEQ ID NO: 535, SEQ ID NO: 536, SEQ ID NO: 537, SEQ ID NO: 538, SEQ ID NO: 539, SEQ ID NO: 540, SEQ ID NO: 541, SEQ ID NO: 542, SEQ ID NO: 543, SEQ ID NO: 544, SEQ ID NO: 545, SEQ ID NO: 546, SEQ ID NO: 547, SEQ ID NO: 548, SEQ ID NO: 549, SEQ ID NO: 550, SEQ ID NO: 551, SEQ ID NO: 552, SEQ ID NO: 553, SEQ ID NO: 554, SEQ ID NO: 555, SEQ ID NO: 556, SEQ ID NO: 557, SEQ ID NO: 558, SEQ ID NO: 559, SEQ ID NO: 560, SEQ ID NO: 561, SEQ ID NO: 562, SEQ ID NO: 563, SEQ ID NO: 564, SEQ ID NO: 565, SEQ ID NO: 566, SEQ ID NO: 567, SEQ ID NO: 568, SEQ ID NO: 569, SEQ ID NO: 570, SEQ ID NO: 571, SEQ ID NO: 572, SEQ ID NO: 573, SEQ ID NO: 574, SEQ ID NO: 575, SEQ ID NO: 576, SEQ ID NO: 577, SEQ ID NO: 578, SEQ ID NO: 579, SEQ ID NO: 580, SEQ ID NO: 581, SEQ ID NO: 582, SEQ ID NO: 583, SEQ ID NO: 584, SEQ ID NO: 585, SEQ ID NO: 586, SEQ ID NO: 587, SEQ ID NO: 588, SEQID NO:589、SEQ ID NO:590、SEQ ID NO:591、SEQ ID NO:592、SEQ ID NO:593、SEQ ID NO:594、SEQ ID NO:595、SEQ ID NO:596、SEQ ID NO:597、SEQID NO:598、SEQ ID NO:599、SEQ ID NO:600、SEQ ID NO:601、SEQ ID NO:602、SEQ ID NO:603、SEQ ID NO:604、SEQ ID NO:605、SEQ ID NO:606、SEQ ID NO:607、SEQ ID NO:608、SEQID NO:609、SEQ ID NO:610、SEQ ID NO:611、SEQ ID NO:612、SEQ ID NO:613、SEQ ID NO:614、SEQ ID NO:615、SEQ ID NO:616、SEQ ID NO:617、SEQ ID NO:618、SEQ ID NO:619、SEQID NO:620、SEQ ID NO:621、SEQ ID NO:622、SEQ ID NO:623、SEQ ID NO:624、SEQ ID NO:625、SEQ ID NO:626、SEQ ID NO:627、SEQ ID NO:628、SEQ ID NO:629、SEQ ID NO:630、SEQID NO:631、SEQ ID NO:632、SEQ ID NO:633、SEQ ID NO:634、SEQ ID NO:635、SEQ ID NO:636、SEQ ID NO:637、SEQ ID NO:638、SEQ ID NO:639、SEQ ID NO:640、SEQ ID NO:641、SEQID NO:642、SEQ ID NO:643、SEQ ID NO:644、SEQ ID NO:645、SEQ ID NO:646、SEQ ID NO:647、SEQ ID NO:648、SEQ ID NO:649、SEQ ID NO:650、SEQ ID NO:651、SEQ ID NO:652、SEQID NO:653、SEQ ID NO:654、SEQ ID NO:655、SEQ ID NO:656、SEQ ID NO:657、SEQ ID NO:658、SEQ ID NO:659、SEQ IDNO:660、SEQ ID NO:661、SEQ ID NO:662、SEQ ID NO:663、SEQID NO:664、SEQ ID NO:665、SEQ ID NO:666、SEQ ID NO:667、SEQ ID NO:668、SEQ ID NO:669、SEQ ID NO:670、SEQ ID NO:671、SEQ ID NO:672、SEQ ID NO:673、SEQ ID NO:674、SEQID NO:675、SEQ ID NO:676、SEQ ID NO:677、SEQ ID NO:678、SEQ ID NO:679、SEQ ID NO:680、SEQ ID NO:681、SEQ ID NO:682、SEQ ID NO:683、SEQ ID NO:684、SEQ ID NO:685、SEQID NO:686、SEQ ID NO:687、SEQ ID NO:688、SEQ ID NO:689、SEQ ID NO:690、SEQ ID NO:691、SEQ ID NO:692、SEQ ID NO:693、SEQ ID NO:694、SEQ ID NO:695、SEQ ID NO:696、SEQID NO:697、SEQ ID NO:698、SEQ ID NO:699、SEQ ID NO:700、SEQ ID NO:701、SEQ ID NO:702、SEQ ID NO:703、SEQ ID NO:704、SEQ ID NO:705、SEQ ID NO:706、SEQ ID NO:707、SEQID NO:708、SEQ ID NO:709、SEQ ID NO:710、SEQ ID NO:711、SEQ ID NO:712、SEQ ID NO:713、SEQ ID NO:714、SEQ ID NO:715、SEQ ID NO:716、SEQ ID NO:717、SEQ ID NO:718、SEQID NO:719、SEQ ID NO:720、SEQ ID NO:721、SEQ ID NO:722、SEQ ID NO:723、SEQ ID NO:724、SEQ ID NO:725、SEQ ID NO:726、SEQ ID NO:727、SEQ ID NO:728、SEQ ID NO:729、SEQID NO:730、SEQ ID NO:731、SEQ IDNO:732、SEQ ID NO:733、SEQ ID NO:734、SEQ ID NO:735、SEQ ID NO:736、SEQ ID NO:737、SEQ ID NO:738、SEQ ID NO:739、SEQ ID NO:740、SEQID NO:741、SEQ ID NO:742、SEQ ID NO:743、SEQ ID NO:744、SEQ ID NO:745、SEQ ID NO:746、SEQ ID NO:747、SEQ ID NO:748、SEQ ID NO:749、SEQ ID NO:750、SEQ ID NO:751、SEQID NO:752、SEQ ID NO:753、SEQ ID NO:754、SEQ ID NO:755、SEQ ID NO:756、SEQ ID NO:757、SEQ ID NO:758、SEQ ID NO:759、SEQ ID NO:760、SEQ ID NO:761、SEQ ID NO:762、SEQID NO:763、SEQ ID NO:764、SEQ ID NO:765、SEQ ID NO:766、SEQ ID NO:767、SEQ ID NO:768、SEQ ID NO:771、SEQ ID NO:772、SEQ ID NO:825、SEQ ID NO:826、SEQ ID NO:827、SEQID NO:828、SEQ ID NO:829、SEQ ID NO:830、SEQ ID NO:831、SEQ ID NO:832、SEQ ID NO:833、SEQ ID NO:834、SEQ ID NO:835、SEQ ID NO:836、SEQ ID NO:837、SEQ ID NO:838、SEQID NO:839、SEQ ID NO:840、SEQ ID NO:841、SEQ ID NO:842、SEQ ID NO:843、SEQ ID NO:844、SEQ ID NO:852、SEQ ID NO:853、SEQ ID NO:854、SEQ ID NO:855、SEQ ID NO:856、SEQID NO:857、SEQ ID NO:858、SEQ ID NO:859、SEQ ID NO:860、SEQ ID NO:861、SEQ ID NO:862、SEQ ID NO:863、SEQ ID NO:864、SEQID NO: 903, SEQ ID NO: 904, SEQ ID NO: 905, SEQ ID NO: 906, SEQ ID NO: 907, SEQ ID NO: 908, SEQ ID NO: 909, SEQ ID NO: 910, SEQ ID NO: 911, SEQ ID NO: 912, SEQ ID NO: 913, SEQ ID NO: 914, SEQ ID NO: 927, SEQ ID NO:928, SEQ ID NO:932, SEQ ID NO:933, SEQ ID NO:934, SEQ ID NO:935, SEQ ID NO:936, SEQ ID NO:939, SEQ ID NO:940, SEQ ID NO:941, SEQ ID NO:943, SEQ ID NO:944, SEQ ID NO:945 and SEQ ID NO:946 are shown.

[0222] In some embodiments, the PIP-72 polypeptide comprises the amino acid sequence shown in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 18, SEQ ID NO: 28, SEQ ID NO: 32, SEQ ID NO: 927, SEQ ID NO: 928, SEQ ID NO: 932, SEQ ID NO: 933, SEQ ID NO: 934, SEQ ID NO: 935, SEQ ID NO: 936, SEQ ID NO: 939, SEQ ID NO: 940, SEQ ID NO: 941, SEQ ID NO: 943, SEQ ID NO: 944, SEQ ID NO: 945, or SEQ ID NO: 946.

[0223] In some implementations, the exemplary PIP-72 polypeptide is any combination of the polypeptides shown in Tables 14, 17, 20, 23, 24, 26, 28 and / or 29, as well as their amino acid substitutions, deletions and / or insertions and fragments.

[0224] In some embodiments, the calculated molecular weight of the PIP-72 peptide is between about 6 kDa and about 13 kDa, between about 7 kDa and about 12 kDa, between about 8 kDa and about 11 kDa, between about 9 kDa and about 10 kDa, about 8.75 kDa, about 9 kDa, about 9.25 kDa, about 9.5 kDa, about 9.75 kDa, about 10 kDa, about 10.25 kDa, and about 10.5 kDa.

[0225] As used herein, the term "about" is used when the molecular weight of the PIP-72 peptide is an average ± 0.25 kDaltons. In some embodiments, the PIP-72 peptide has modified physical properties. As used herein, the term "physical property" refers to any parameter suitable for describing the physicochemical characteristics of a protein. As used herein, "physical property of interest" and "property of interest" are used interchangeably and refer to the physical properties of the protein being studied and / or modified. Examples of physical properties include, but are not limited to, net surface charge and charge distribution on the protein surface, net hydrophobicity and distribution of hydrophobic residues on the protein surface, surface charge density, surface hydrophobicity density, total number of ionizable groups on the surface, surface tension, protein size and its distribution in solution, melting temperature, heat capacity, and second virial coefficient. Examples of physical properties also include, but are not limited to, solubility, foldability, stability, and digestibility. In some embodiments, the PIP-72 peptide enhances the digestibility of the hydrolyzed protein fragments in the insect gut. Models of digestion by simulating gastric juice are known to those skilled in the art (Fuchs, RL and JD. Astwood. Food Technology 50: 83-88, 1996; Astwood, JD et al., Nature Biotechnology 14: 1269-1273, 1996; Fu TJ et al., J. Agric Food Chem. 50: 7154-7160, 2002).

[0226] In some embodiments, the variants include polypeptides whose amino acid sequences differ due to mutagenesis. The variant proteins covered by this disclosure possess biological activity, meaning they retain the desired biological activity (i.e., insecticidal activity) of the natural protein. In one embodiment, the variant will have at least about 10%, at least about 30%, at least about 50%, at least about 70%, at least about 80% or more of the insecticidal activity of the natural protein. In some embodiments, the variant may have improved activity compared to the natural protein.

[0227] Bacterial genes often have multiple methionine start codons near the start of the open reading frame. Typically, translation initiation at one or more of these start codons leads to the production of a functional protein. These start codons may include the ATG codon. However, bacteria such as Bacillus species also recognize the GTG codon as a start codon, and proteins translated at the GTG codon contain a methionine as their first amino acid. In rare cases, translation in bacterial systems may begin at the TTG codon, although in this case TTG encodes methionine. Furthermore, it is generally not possible to a priori determine which of these codons are naturally used in bacteria. Therefore, it should be understood that using one of the alternative methionine codons can also lead to the production of insecticidal proteins. These insecticidal proteins are covered in this disclosure and can be used in the methods of this disclosure. It should be understood that when expressed in plants, it is necessary to change the alternative start codon to ATG for correct translation.

[0228] In another aspect, the PIP-72 peptide can be expressed as a precursor protein with an intercalation sequence that catalyzes a multi-step post-translational splicing process. Splicing involves the excision of the intercalation sequence from the peptide, accompanied by the joining of flanking sequences, resulting in a new peptide (Chong et al., (1996) J. Biol. Chem., 271: 22159-22168). This intercalation sequence, or splicing element, is called an intipeptide, which catalyzes its own excision at the N-terminal and C-terminal splice junctions via three coordinated reactions: acyl rearrangement of the N-terminal cysteine ​​or serine; transesterification between the two ends to form a branched ester or thioester intermediate; and peptide bond cleavage accompanied by cyclization of the intipeptide's C-terminal asparagine, releasing the intipeptide (Evans et al., (2000) J. Biol. Chem., 275: 9091-9094). The elucidation of this protein splicing mechanism has led to a variety of integrin-based applications (Comb et al., US Patent 5,496,714; Comb et al., US Patent 5,834,247; Camarero and Muir, (1999) J. Amer. Chem. Soc. 121: 5597-5598; Chong et al., (1997) Gene 192: 271-281; ​​Chong et al., (1998) Nucleic Acids Res. 26: 5109-5115; Chong et al., (1998) J. Biol. Chem. 273: 10567-10577; Cotton et al., (1999) J. Am. Chem. Soc. 121: 1100-1101; Evans et al., (1999) J. Biol. Chem. 274: 18359-18363; Evans et al., (1999) J. Biol. Chem. 274: 3923-3926; Evans et al., (1998) Protein Sci. 7: 2256-2264; Evans et al., (2000) J. Biol. Chem. 275: 9091-9094; Iwai and Pluckthun, (1999) FEBS Lett. 459: 166-172; Mathys et al., (1999) Gene 231: 1-13; Mills et al., (1998) Proc. Natl. Acad. Sci. USA 95: 3543-3548; Muir et al., (1998) Proc. Natl. Acad. Sci. USA 95: 6705-6710; Otomo et al., (1999) Biochemistry 38: 16040-16044; Otomo et al., (1999) J. Biolmol.NMR14: 105-114; Scott et al., (1999) Proc. Natl. Acad. Sci. USA 96: 13638-13643; Severinov and Muir, (1998) J. Biol. Chem. 273: 16205-16209; Shingledecker et al., (1998) Gene 207: 187-195; Southworth et al., (1998) EMBO J. 17: 918-926; Southworth et al., (1999) Biotechniques 27: 110-120; Wood et al., (1999) Nat. Biotechnol. 17: 889-892; Wu et al., (1998a) Proc. Natl. Acad. Sci. USA 95: 9226-9231; Wu et al., (1998b) Biochim Biophys Acta 1387: 422-432; Xu et al., (1999) Proc. Natl. Acad. Sci. USA 96: 388-393; Yamazaki et al., (1998) J. Am. Chem. Soc., 120: 5591-5592. For information on the application of integrins in plant transgenics, see Yang et al., (Transgene Res 15: 583-593 (2006)) and Evans et al., (Annu. Rev. Plant Biol. 56: 375-392 (2005)).

[0229] In another aspect, the PIP-72 polypeptide can be encoded by two separate genes, in which the inteins of the precursor protein (called the fragmented inteins) originate from these two genes, and the two parts of the precursor are linked together by peptide bonds. This peptide bond formation is achieved through intein-mediated trans-splicing. For this purpose, first and second expression cassettes containing these two separate genes also encode inteins capable of mediating protein trans-splicing. Through trans-splicing, the protein and polypeptide encoded by the first and second fragments are linked together by peptide bonds. The trans-splicing inteins can be selected from the nucleolar and organelle genomes of various organisms, including eukaryotes, archaea, and bacteria. Usable inteins are listed at neb.com / neb / inteins.html (accessible via the World Wide Web using the "www" prefix). The nucleotide sequence encoding the inteins can be split into 5' and 3' portions encoding the 5' and 3' portions of the inteins, respectively. Sequence portions not required for intein splicing (e.g., homing endonuclease domains) can be deleted. The inteptide coding sequence is broken to allow for trans-splicing of the 5′ and 3′ portions. To select suitable breakpoints in the inteptide coding sequence, considerations can be followed as published by Southworth et al., (1998) EMBO J.17:918-926. The construction of the first and second expression cassettes is as follows: the 5′ inteptide coding sequence is ligated to the 3′ end of a first fragment encoding the N-terminal portion of the PIP-72 polypeptide, and the 3′ inteptide coding sequence is ligated to the 5′ end of a second fragment encoding the C-terminal portion of the PIP-72 polypeptide.

[0230] In general, any fragmented inteptide (including any naturally occurring or artificially fragmented fragmented inteptides) can be used to design trans-splicing chaperones. Several naturally occurring fragmented inteptides are known, such as the fragmented inteptide of the DnaE gene of Synechocystis sp. PCC6803 (see Wu et al., (1998) ProcNatl Acad Sci USA. 95(16): 9226-31 and Evans et al., (2000) J Biol Chem. 275(13): 9091-4), and the fragmented inteptide of the DnaE gene from Nostoc punctiforme (see Iwai et al., (2006) FEBS Lett. 580(7): 1853-8). New fragmented inteins have been artificially fragmented in the laboratory to form new fragmented inteins, such as: artificially fragmented Ssp DnaB inteins (see, Wu et al., (1998) Biochim Biophys Acta. 1387: 422-32), fragmented Sce VMA inteins (see, Brenzel et al., (2006) Biochemistry. 45(6): 1571-8), and artificially fragmented fungal microinteins (see, Elleuche et al., (2007) Biochem Biophys Res Commun. 355(3): 830-4). Inteins databases containing known inteins are also available (see, for example, the online database: bioinformatics.weizmann.ac.il / ~pietro / inteins / Inteinstable.html, accessible via the World Wide Web using the "www" prefix).

[0231] Naturally occurring non-fragmented inteins may possess endonuclease or other enzymatic activities, which can typically be removed when designing artificially fragmented inteins. Such miniature or minimized fragmented inteins are well known in the art and are typically less than 200 amino acid residues in length (see, Wu et al., (1998) Biochim Biophys Acta. 1387: 422-32). Suitable fragmented inteins may have other peptide elements added to their structure to achieve purification, provided that such elements do not inhibit the splicing of the fragmented inteins or are added in a manner that allows them to be removed before splicing. Protein splicing using proteins containing the following domains has been reported: bacterial intipeptide-like (BIL) domains (see, Amitai et al., (2003) Mol Microbiol. 47: 61-73), hedgehog protein (Hog) self-processing domains (the latter combined with intipeptides are called the Hog / intipeptide superfamily or HINT family (see, Dassa et al., (2004) J Biol Chem. 279: 32001-7), and domains such as these can also be used to prepare artificially cleaved intipeptides. Specifically, non-splicing members of such families can be modified using molecular biological methods to introduce or restore splicing activity in such related substances. Recent studies have shown that splicing can be observed when N-terminal cleaved intipeptide components are reacted with C-terminal cleaved intipeptide components that are not naturally occurring as their "partners". For example, splicing was observed when using a partner with as little as 30% to 50% homology to a "natural" splicing partner. Splicing has been demonstrated (see Dassa et al., (2007) Biochemistry. 46(1): 322-30). Other mixtures of such dissimilar fragmented integrin chaperones have been shown to be unreactive (see Brenzel et al., (2006) Biochemistry. 45(6): 1571-8). However, it is within the capabilities of those skilled in the art to determine whether a particular pair of peptides can link together to provide a functional integrin using conventional methods without the need for inventive techniques.

[0232] In another respect, the PIP-72 peptide is a variant with a cyclic arrangement. In some embodiments, the PIP-72 peptide is a variant of the cyclic arrangement of the following peptides: SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 18, SEQ ID NO: 28, SEQ ID NO: 32, any one of SEQ ID NO: 528 to SEQ ID NO: 768, any one of SEQ ID NO: 825 to SEQ ID NO: 844, SEQ ID NO: 771, SEQ ID NO: 772, SEQ ID NO: 852, any one of SEQ ID NO: 903 to SEQ ID NO: 914, SEQ ID NO: 927, SEQ ID NO: 928, SEQ ID NO: 932, SEQ ID NO: 933, SEQ ID NO: 934, SEQ ID NO: 935, SEQ ID NO: 936, SEQ ID NO: 939, SEQ ID NO: 940, SEQ ID NO: 935, SEQ ID NO: 936, SEQ ID NO: 939, SEQ ID NO: 940, SEQ ID NO: 936, SEQ ID NO: 939, SEQ ID NO: 936, SEQ ID NO: 937, SEQ ID NO: 938 ... NO:941, SEQ ID NO:943, SEQ ID NO:944, SEQ ID NO:945 or SEQ ID NO:946.

[0233] The development of recombinant DNA methods has enabled the study of the effects of sequence transposition on protein folding, structure, and function. Methods for forming new sequences are similar to those used for the linear reconstruction of naturally occurring protein pairs with their amino acid sequences (Cunningham et al., (1979) Proc. Natl. Acad. Sci. USA 76: 3218-3222; Teather and Erfle, (1990) J. Bacteriol. 172: 3837-3841; Schimming et al., (1992) Eur. J. Biochem. 204: 13-19; Yamiuchi and Minamikawa, (1991) FEBS Lett. 260: 127-130; MacGregor et al., (1996) FEBS Lett. 378: 263-266). This type of rearrangement was first applied in vitro to proteins as described by Goldenbenberg and Creighton (J. Mol. Biol. 165: 407-413, 1983). In variants forming a circular arrangement, a new N-terminus is selected at an internal site (breakpoint) of the initial sequence. From this breakpoint, the new sequence has the same amino acid sequence as the initial sequence until it reaches an amino acid at or near the initial C-terminus. At this point, the new sequence is directly or via another part of the sequence (linker) attached to the amino acid at or near the initial N-terminus, and the new sequence continues to follow the same sequence as the initial sequence until it reaches a point at or near the N-terminal amino acid at the breakpoint of the initial sequence, where this residue forms the new C-terminus of the chain. The amino acid sequence length of the linker can be selected empirically, under the guidance of structural information, or by using a combination of both methods. When no structural information is available, a small series of linkers can be prepared for testing using a design where the length varies across 0 to 1. The range of the designed sequence was selected to conform to surface exposure (hydrophilicity, Hopp and Woods, (1983) Mol. Immunol. 20: 483-489; Kyte and Doolittle, (1982) J. Mol. Biol. 157: 105-132; solvent-exposed surface region, Lee and Richards, (1971) J. Mol. Biol. 55: 379-400) and the ability to take the desired conformation without disturbing the configuration of the insecticidal peptide (conformal flexibility; Karplus and Schulz, (1985) Naturwissenschaften 72: 212-213). An average translation of 2.0 to [missing information] per residue was assumed. This means that the length to be tested will be between 0 and 30 residues, with 0 to 15 residues being the preferred range. An example of such an empirical series would be the use of cassette sequences such as Gly-Gly-Gly-Ser repeated n times (where n is 1, 2, 3, or 4) to construct linkers. Those skilled in the art will recognize that many such sequences exist that vary in length or composition and can be used as linkers, and the primary consideration is that they cannot be too long or too short (see Sandhu, (1992) Critical Rev. Biotech. 12: 437-462); if they are too long, entropy effects may compromise the stability of the three-dimensional fold and may also make the folding kinetically impractical, and if they are too short, they may compromise the stability of the molecule due to torsion or spatial strain. Those skilled in protein structural information analysis will recognize that the distance between chain ends (defined as the distance between c-α carbons) can be used to limit the length of the sequence to be used, or at least to limit the number of possibilities that must be tested in the empirical selection of linkers. They will also recognize that sometimes the position of the polypeptide chain terminus is not clearly defined in the structural model derived from X-ray diffraction or nuclear magnetic resonance spectroscopy data, and when this is the case, it must be taken into account in order to correctly estimate the required linker length. From those residues with clearly defined positions, two residues adjacent to the chain terminus in the sequence are selected, and the approximate length of the linker between them is calculated using the distance between their c-α carbons. Then, using the calculated length as a guide, a range of residue numbers (using 2 to 1 per residue) are selected. The linkers are calculated. These linkers can consist of an initial sequence, which can be shortened or lengthened as needed, and when lengthened, additional residues can be selected as flexible and hydrophilic as described above; or optionally, a series of linkers can be used instead of the initial sequence, an example being the Gly-Gly-Gly-Ser box method described above; or optionally, a combination of the initial sequence and a new sequence with an appropriate total length can be used. The sequence of an insecticidal polypeptide capable of folding into a biologically active state can be prepared by appropriately selecting start (amino terminus) and end (carboxyl terminus) positions from within the initial polypeptide chain while using the linker sequences described above. The amino terminus and carboxyl terminus are selected from a common segment of the sequence (called the breakpoint region) using the guidelines below. Thus, by selecting the amino terminus and carboxyl terminus from the same breakpoint region, a novel amino acid sequence is generated. In many cases, the selection of the new terminus will result in the initial position of the carboxyl terminus immediately preceding the initial position of the amino terminus. However, those skilled in the art will recognize that selecting the terminus at any position within this region can be effective, and these will effectively cause the deletion or addition of the amino or carboxyl portion of the new sequence. A core principle of molecular biology is that the primary amino acid sequence of a protein determines its folding into the three-dimensional structure required to express its biological function. Methods for obtaining and interpreting three-dimensional structural information using X-ray diffraction of a single protein crystal or nuclear magnetic resonance spectroscopy of a protein solution are known to those skilled in the art. Examples of structural information associated with breakpoint identification include the location and type of protein secondary structures (α-helices and 3–10 helices, parallel and antiparallel β-sheets, chain inversions and turns, and loops; Kabsch and Sander, (1983) Biopolymers 22: 2577–2637); the solvent exposure of amino acid residues, the degree and type of interaction between residues (Chothia, (1984) Ann. Rev. Biochem. 53: 537–572); and the static and dynamic conformational distribution along the polypeptide chain (Alber and Mathews, (1987) Methods). Enzymol. 154: 511-533). In some cases, additional information about residue solvent exposure is known; one example is the need for post-translational linking sites of carbohydrates on the protein surface. When experimental structural information is unavailable or impossible to obtain, various methods can be used to analyze the primary amino acid sequence to predict protein tertiary and secondary structures, solvent accessibility, and the presence of turns and loops. When direct structural methods are not feasible, biochemical methods can sometimes be applied empirically to determine surface exposure. For example, the identification of chain break sites after restriction protease digestion can be used to infer surface exposure (Gentile and Salvatore, (1993) Eur. J. Biochem. 218: 603-621).Therefore, experimentally derived structural information or predictive methods (e.g., Srinivisan and Rose, (1995) Proteins: Struct., Funct. & Genetics 22: 81-99) are used to examine parental amino acid sequences to classify regions based on their indispensability for maintaining secondary and tertiary structures. Sequences appearing in regions known to involve periodic secondary structures (α-helices and 3-10 helices, parallel and antiparallel β-sheets) are regions to be avoided. Similarly, regions observed or predicted to have amino acid sequences with low solvent exposure are more likely to be the so-called hydrophobic core of the protein and should also be avoided when selecting amino and carboxyl terms. In contrast, regions known or predicted to be in surface turns or loops, especially those known not to be required for biological activity, are preferred sites for polypeptide chain terminal localization. Continuous segments of amino acid sequences selected based on the above criteria are called breakpoint regions. Polynucleotides encoding a circularly arranged PIP-72 polypeptide having a novel N-terminus / C-terminus containing a linker region separating the initial C-terminus and N-terminus can be prepared substantially according to the methods described in the following literature: Mullins et al., (1994) J. Am. Chem. Soc. 116: 5529-5533. The DNA sequence encoding the primary amino acid sequence of the protein is rearranged using multiple steps of polymerase chain reaction (PCR) amplification. Polynucleotides encoding a circularly arranged PIP-72 polypeptide having a novel N-terminus / C-terminus containing a linker region separating the initial C-terminus and N-terminus can be prepared based on the tandem repeat method described in the following literature: Horlick et al., (1992) Protein Eng. 5: 427-431. Polymerase chain reaction (PCR) amplification of the novel N-terminal / C-terminal gene is performed using template DNA with tandem repeats.

[0234] In another aspect, a fusion protein is provided, the fusion protein comprising within its amino acid sequence the amino acid sequence constituting the PIP-72 polypeptide, the PIP-72 polypeptide including, but not limited to, any one of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 18, SEQ ID NO: 28, SEQ ID NO: 32, any one of SEQ ID NO: 528 to SEQ ID NO: 768, any one of SEQ ID NO: 825 to SEQ ID NO: 844, any one of SEQ ID NO: 771, SEQ ID NO: 772, SEQ ID NO: 846, SEQ ID NO: 847, SEQ ID NO: 848, SEQ ID NO: 849, SEQ ID NO: 852, any one of SEQ ID NO: 903 to SEQ ID NO: 914, any one of SEQ ID NO: 927 to SEQ ID NO: 948, and their active fragments.

[0235] Methods for designing and constructing fusion proteins (and the polynucleotides encoding them) are known to those skilled in the art. The polynucleotide encoding the PIP-72 polypeptide can be fused to a signal sequence that guides the PIP-72 polypeptide to a specific compartment of a prokaryotic or eukaryotic cell and / or guides the secretion of the PIP-72 polypeptide of the present disclosure from a prokaryotic or eukaryotic cell. For example, in *E. coli*, we may wish to express the guide protein in the periplasmic space. Examples of signal sequences or proteins (or fragments thereof) to which the PIP-72 polypeptide can be fused to guide polypeptide expression in the bacterial periplasmic space include, but are not limited to, the pelB signal sequence, the maltose-binding protein (MBP) signal sequence, MBP, the ompA signal sequence, the signal sequence of the periplasmic *E. coli* thermostable enterotoxin B subunit, and the signal sequence of alkaline phosphatase. Several vectors for constructing fusion proteins that localize the guide protein are commercially available, for example, from New England Biotech. The pMAL series vectors (particularly the pMAL-p series) are available from (240 County Road, Ipswich, MA 01938-2723). In a specific embodiment, the PIP-72 peptide can be fused to the pelB pectic acid lyase signal sequence to increase the efficiency of expression and purification of such peptides in Gram-negative bacteria (see U.S. Patents 5,576,195 and 5,846,818). Plant plasmid transport peptide / peptide fusions are well known in the art (see U.S. Patent 7,193,133). Apoplast transport peptides, such as rice or barley α-amylase secretion signals, are also well known in the art. The plasmid transport peptide is typically fused at its N-terminus to the peptide to be targeted (e.g., a fusion chaperone). In one embodiment, the fusion protein consists essentially of the plasmid transport peptide and the PIP-72 peptide to be targeted. In another embodiment, the fusion protein comprises the plasmid transport peptide and the peptide to be targeted. In such embodiments, the plasmid transport peptide is preferably located at the N-terminus of the fusion protein. However, additional amino acid residues may be at the N-terminus of the plasmid transport peptide, provided that the fusion protein at least partially targets the plasmid. In a particular embodiment, the plasmid transport peptide is located at the half, one-third, or one-quarter of the N-terminus of the fusion protein. Most or all of the plasmid transport peptides typically cleave from the fusion protein after insertion into the plasmid. The cleavage site may vary slightly between plant species and at different plant developmental stages due to specific intercellular conditions or the specific combination of transport peptides / fusion partners used. In one embodiment, the plasmid transport peptide cleavage is homologous, such that the cleavage site is identical across the fusion protein group. In another embodiment, the plasmid transport peptide cleavage is non-homologous, such that the cleavage site differs by 1 to 10 amino acids across the fusion protein group. The plasmid transport peptide can be recombinantly fused to a second protein by one of several methods. For example, a restriction endonuclease recognition site may be introduced into the nucleotide sequence of the transport peptide at a position corresponding to its C-terminus, and this site or compatibility site may be designed at the N-terminus of the nucleotide sequence of the protein to be targeted. These sites must be carefully designed to ensure that the coding sequences of the transport peptide and the second protein remain "within the box," thus allowing for the synthesis of the desired fusion protein. In some cases, when introducing new restriction sites, it may be preferable to remove the starter methionine codon of the second protein. The introduction of restriction endonuclease recognition sites on both parent molecules and their subsequent ligation via recombinant DNA technology can result in the addition of one or more extra amino acids between the transport peptide and the second protein. This typically does not affect targeting activity, provided that the transport peptide cleavage site remains accessible and the function of the second protein is not affected by the addition of these extra amino acids at its N-terminus.Alternatively, those skilled in the art can use gene synthesis (Stemmer et al., (1995) Gene 164: 49-53) or similar methods to form a precise cleavage site between the transport peptide and the second protein (with or without its initiator methionine). Furthermore, the transport peptide fusion may intentionally contain an amino acid downstream of the cleavage site. The amino acid at the N-terminus of the mature protein can affect the transport peptide's ability to target the protein to the plasmid and / or the efficiency of cleavage after protein input. This can depend on the protein to be targeted. See, for example, Comai et al., (1988) J. Biol. Chem. 263(29): 15104-9).

[0236] In some embodiments, a fusion protein is provided comprising a PIP-72 polypeptide and an insecticidal polypeptide linked together by an amino acid linker.

[0237] In some implementations, a fusion protein represented by a formula selected from the following is provided:

[0238] R 1 -LR 2 R 2 -LR 1 R 1 -R 2 Or R 2 -R 1

[0239] Where R 1It is a PIP-72 polypeptide or any one of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 18, SEQ ID NO: 28, SEQ ID NO: 32, SEQ ID NO: 528 to SEQ ID NO: 768, any one of SEQ ID NO: 825 to SEQ ID NO: 844, SEQ ID NO: 771, SEQ ID NO: 772, SEQ ID NO: 852, SEQ ID NO: 903 to SEQ ID NO: 914, SEQ ID NO: 927, SEQ ID NO: 928, SEQ ID NO: 932, SEQ ID NO: 933, SEQ ID NO: 934, SEQ ID NO: 935, SEQ ID NO: 936, SEQ ID NO: 939, SEQ ID NO: 940, SEQ ID NO: 941, SEQ ID NO: 943, SEQ ID NO: 944, SEQ ID NO: 943, SEQ ID NO: 944, SEQ ID NO: 945, SEQ ID NO: 936, SEQ ID NO: 939, SEQ ID NO: 940, SEQ ID NO: 941 ... The polypeptide represented by NO: 944, SEQ ID NO: 945 or SEQ ID NO: 946, R 2 It is an insecticidal polypeptide. R 1 The polypeptide is fused directly or via a linker (L) segment to R 2 Polypeptide. The term "directly" defines the fusion of polypeptides without peptide linkers. Therefore, "L" stands for R. 1 and R 2 Both are chemical bonds or polypeptide segments fused to them within the frame, most commonly, L is R. 1 and R 2 A linear peptide bound to it via an amide bond, the amide bond connecting R 1 The carboxyl terminus of L is attached to the amino terminus of L, and the carboxyl terminus of L is attached to R. 2 The amino terminus. The term "intra-frame fusion" refers to R... 1 With R 2 There is no translation termination or interruption between the reading frames. The linker group (L) is typically a polypeptide with a length between 1 and 500 amino acids. The linker connecting the two molecules is preferably designed to (1) allow the two molecules to fold and function independently of each other, (2) not tend to form ordered secondary structures that could interfere with the functional domains of the two proteins, (3) have very few hydrophobic or charged properties that could interact with the functional protein domains, and (4) provide R 1 With R 2 The steric separation makes R 1 and R2 It can simultaneously interact with its corresponding receptor on a single cell. Typically, the surface amino acids in the flexible protein region include Gly, Asn, and Ser. In fact, any arrangement of amino acid sequences containing Gly, Asn, and Ser is expected to meet the above criteria for the linker sequence. Other neutral amino acids such as Thr and Ala can also be used in the linker sequence. Additional amino acids can also be included in the linker, as unique restriction sites can be added to the linker sequence to facilitate the construction of fusion bodies.

[0240] In some implementations, the connector comprises a sequence selected from the following: (Gly3Ser) n (Gly4Ser) n (Gly5Ser) n (GlynSer) n Or (AlaGlySer) n Where n is an integer. An example of a highly flexible linker is the GlySer-rich spacer region present within the pIII protein of filamentous bacteriophages (e.g., phage M13 or fd) (Schaller et al., 1975). This region provides a long, flexible spacer between the two domains of the pIII surface protein. Linkers containing endopeptidase recognition sequences are also included. Such cleavage sites can be valuable for separating the individual components of a fusion to determine whether they are properly folded and active in vitro. Examples of various endopeptidases include, but are not limited to, plasminogen activator, enterokinase, kallikrein, urokinase, tissue plasminogen activator, clostridial protease, rennet, collagenase, viper venom protease, proline lyase, V8 protease, thrombin, and factor Xa. In some embodiments, the linker comprises the amino acid EEKKN (SEQ ID NO: 488) from a multigene expression vector (MGEV) cleaved by a vacuolar protease as disclosed in U.S. Patent Application Publication US2007 / 0277263. In other embodiments, peptide linker segments from the hinge regions of heavy chain immunoglobulins IgG, IgA, IgM, IgD, or IgE provide the angular relationship between the linked peptides. Hinge regions where cysteine ​​residues are replaced with serine residues are particularly useful. The linkers of this disclosure comprise sequences derived from the hinge regions of mouse IgG γ2b with cysteine ​​residues replaced by serine residues. Fusion proteins are not limited by the morphology, size, or number of linker sequences employed, and the only requirement for the linkers is that they do not adversely interfere with the folding and function of the individual molecules of the fusion protein.

[0241] In another aspect, a chimeric PIP-72 polypeptide is provided, which is generated by linking two or more parts of a PIP-72 gene that initially encodes a single PIP-72 protein to form a chimeric gene. Translation of the chimeric gene yields a single chimeric PIP-72 polypeptide having a region, motif, or domain derived from each initial polypeptide. In some embodiments, the chimeric protein comprises any combination of PIP-72Aa (SEQ ID NO: 2), PIP-72Ba (SEQ ID NO: 4), PIP-72Ca (SEQ ID NO: 6), PIP-72Cb (SEQ ID NO: 8), PIP-72Da (SEQ ID NO: 10), PIP-72Db (SEQ ID NO: 12), PIP-72Dc (SEQ ID NO: 14), PIP-72Fa (SEQ ID NO: 18), PIP-72Ff (SEQ ID NO: 28), and PIP-72Gb (SEQ ID NO: 32), PIP-72Ab (SEQ ID NO: 927), PIP-72Bb (SEQ ID NO: 928), PIP-72Fh (SEQ ID NO: 932), PIP-72Fi (SEQ ID NO: 933), PIP-72Fj (SEQ ID NO: 934), and PIP-72Fk (SEQ ID NO: 934). Parts, motifs, or domains of PIP-72Fl (SEQ ID NO: 936), PIP-72Gg (SEQ ID NO: 939), PIP-72Gh (SEQ ID NO: 940), PIP-72Gi (SEQ ID NO: 941), PIP-72Gk (SEQ ID NO: 943), PIP-72Gl (SEQ ID NO: 944), PIP-72Gm (SEQ ID NO: 945), and PIP-72Gn (SEQ ID NO: 946).

[0242] It has been recognized that DNA sequences can be altered through various methods, and that these alterations can result in proteins encoded by that DNA sequence having amino acid sequences different from those of wild-type (or natural) insecticidal proteins. In some embodiments, the PIP-72 peptide can be modified in various ways, including amino acid substitution, deletion, truncation, and insertion of one or more amino acids, compared to any one of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 18, SEQ ID NO: 28, SEQ ID NO: 32, any one of SEQ ID NO: 528 to SEQ ID NO: 768, any one of SEQ ID NO: 825 to SEQ ID NO: 844, SEQ ID NO: 771, SEQ ID NO: 772, SEQ ID NO: 852, SEQ ID NO: 846, SEQ ID NO: 847, SEQ ID NO: 848, SEQ ID NO: 849, any one of SEQ ID NO: 903 to SEQ ID NO: 914, SEQ ID NO: 927, SEQ ID NO: 928, SEQ ID NO: 932, SEQ ID NO: 933, SEQ ID NO: 949 ... SEQ ID NO: 934, SEQ ID NO: 935, SEQ ID NO: 936, SEQ ID NO: 939, SEQ ID NO: 940, SEQ ID NO: 941, SEQ ID NO: 943, SEQ ID NO: 944, SEQ ID NO: 945 or SEQ ID NO: 946, the modified polypeptide includes up to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45 or more amino acid substitutions, deletions and / or insertions or combinations thereof.

[0243] Methods of such manipulation are well known in the art. For example, amino acid sequence variants of the PIP-72 peptide can be prepared by mutation in DNA. This can also be accomplished through one of several forms of mutagenesis and / or in directed evolution. In some respects, the changes encoded in the amino acid sequence do not substantially affect the function of the protein. Such a variant will possess the desired insecticidal activity. However, it should be understood that the ability to confer insecticidal activity upon the PIP-72 peptide can be enhanced by using such techniques on the compositions disclosed herein.

[0244] For example, conserved amino acid substitutions can be made at one or more predicted non-essential amino acid residues. “Non-essential” amino acid residues are residues whose biological activity can be altered by the wild-type sequence of the PIP-72 polypeptide without changing its biological activity. “Conserved amino acid substitution” is an amino acid substitution in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids having the following side chains or residues: basic side chains (e.g., lysine, arginine, histidine); acidic side chains (e.g., aspartic acid, glutamic acid); polar, negatively charged residues and their amides (e.g., aspartic acid, asparagine, glutamic acid, glutamine); uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine); and less aliphatic, nonpolar, or weakly polar residues (e.g., alanine, serine, threonine). Acids, proline, glycine); nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan); larger aliphatic, nonpolar residues (e.g., methionine, leucine, isoleucine, valine, cysteine); β-branched side chains (e.g., threonine, valine, isoleucine); aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine); larger aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan).

[0245] Amino acid substitutions can be made in non-conserved regions that retain function. Generally, such substitutions are not made on conserved amino acid residues or amino acid residues located within conserved motifs, where such residues are essential for protein activity. Examples of conserved residues that may be essential for protein activity include, for example, residues identical in all proteins contained in a sequence alignment of similar or related toxins with embodiments of this disclosure (e.g., identical residues in a homolog alignment). Examples of conserved residues that allow for conserved amino acid substitutions and still retain activity include, for example, residues with only conserved substitutions in all proteins contained in a sequence alignment of similar or related toxins with embodiments of this disclosure (e.g., only conserved substitutions in a homolog alignment). However, those skilled in the art will understand that functional variants may have fewer conserved or non-conserved changes in conserved residues. Guidance on appropriate amino acid substitutions that do not affect the biological activity of the protein of interest can be found in the model described in the following literature: Dayhoff et al., (1978) Atlas of Protein Sequence and Structure (Natl. Biomed. Res. Found., Washington, DC), which is incorporated herein by reference.

[0246] When making such changes, the hydrophilicity index of amino acids can be considered. The importance of the hydrophilic amino acid index in conferring biological functions of interactions to proteins is well understood in the art (Kyte and Doolittle, (1982) J Mol Biol. 157(1): 105-32). It is generally accepted that the relative hydrophilicity of amino acids contributes to the secondary structure of the resulting protein, which in turn defines the interactions between the protein and other molecules such as enzymes, substrates, receptors, DNA, antibodies, antigens, etc.

[0247] It is known in the art that certain amino acids can be substituted with other amino acids having similar hydrophilicity indices or scores, and still produce proteins with similar biological activities, i.e., proteins that are still biologically equivalent. Hydrophilicity indices (Kyte and Doolittle, ibid.) have been assigned to each amino acid based on its hydrophobic and charged properties. The hydrophilicity indices of each amino acid are as follows: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine / cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamic acid (-3.5); glutamine (-3.5); aspartic acid (-3.5); asparagine (-3.5); lysine (-3.9) and arginine (-4.5). When making such changes, substitutions of amino acids with a hydrophilicity index within +2 are preferred, substitutions of amino acids with a hydrophilicity index within +1 are particularly preferred, and substitutions of amino acids with a hydrophilicity index within +0.5 are even more particularly preferred.

[0248] It should also be understood in the art that similar amino acid substitutions can be effectively made based on hydrophilicity. U.S. Patent 4,554,101 states that the maximum local average hydrophilicity of a protein (determined by the hydrophilicity of its adjacent amino acids) is associated with the protein's biological properties.

[0249] As detailed in U.S. Patent 4,554,101, the following hydrophilicity values ​​have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartic acid (+3.0, +0.1); glutamic acid (+3.0, +0.1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5, +0.1); alanine (-0.5); histidine (-0.5); cysteine ​​(-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4).

[0250] Alternatively, the protein sequence at the amino-terminus or carboxyl-terminus of many proteins can be altered without substantially affecting their activity. This can include insertions, deletions, or alterations introduced by modern molecular methods such as PCR, including PCR amplification that alters or lengthens the protein-coding sequence by including the amino acid-coding sequence in the oligonucleotides used in the PCR amplification. Alternatively, the added protein sequence can include all protein-coding sequences, such as those commonly used in the art to generate protein fusions. Such fusion proteins are generally used to (1) increase the expression of the protein of interest, (2) introduce binding domains, enzyme activities, or epitopes to facilitate protein purification, protein detection, or other experimental uses known in the art, and (3) target the secretion or translation of the protein to subcellular organelles, such as the pericyte space of Gram-negative bacteria, mitochondria or chloroplasts of plants, or the endoplasmic reticulum of eukaryotic cells, which typically leads to protein glycosylation.

[0251] The variant nucleotide and amino acid sequences disclosed herein also encompass sequences derived from procedures such as mutagenesis and recombination, such as DNA shuffling. When employing such procedures, one or more distinct PIP-72 polypeptide coding regions can be used to form novel PIP-72 polypeptides with desired properties. In this manner, libraries of recombinant polynucleotides are generated from a set of related polynucleotide sequences containing sequence regions with substantial sequence identity and capable of homologous recombination in vitro or in vivo. For example, using this method, sequence motifs encoding domains of interest can be shuffled between insecticidal genes and other known insecticidal genes to obtain novel genes encoding proteins with improved properties of interest, such as enhanced insecticidal activity. Such DNA shuffling strategies are known in the art. See, for example, Stemmer (1994) Proc. Natl. Acad. Sci. USA 91: 10747-10751; Stemmer, (1994) Nature 370: 389-391; Crameri et al., (1997) Nature Biotech. 15: 436-438; Moore et al., (1997) J. Mol. Biol. 272: 336-347; Zhang et al., (1997) Proc. Natl. Acad. Sci. USA 94: 4504-4509; Crameri et al., (1998) Nature 391: 288-291; and U.S. Patents 5,605,793 and 5,837,458.

[0252] Domain exchange or shuffling is another mechanism for generating altered PIP-72 peptides. Domains can be exchanged between PIP-72 peptides, resulting in hybrid or chimeric toxins with improved insecticidal activity or target profiles. The methods used to generate recombinant proteins and test their insecticidal activity are well known in the art (see, for example, Naimov et al., (2001) Appl. Environ. Microbiol. 67: 5328-5330; de Maagd et al., (1996) Appl. Environ. Microbiol. 62: 1537-1543; Ge et al., (1991) J. Biol. Chem. 266: 17954-17958; Schnepperf et al., (1990) J. Biol. Chem. 265: 20923-20930; Rang et al., (1999) Appl. Environ. Microbiol. 65: 2918-2925).

[0253] Both DNA shuffling and site-directed mutagenesis are used to define polypeptide sequences with insecticidal activity. In Examples 8 and 9, DNA shuffling was used to generate an active variant library through diverse recombinations present in GBP_A3175 (SEQ ID NO: 20) and PIP-72Da (SEQ ID NO: 10). Those skilled in the art will be able to further define the motifs by comparison with other protein or functional assays. High-throughput screening can be used to test variations in these motifs to determine the role of specific residues. If several motifs are known, the requirements for functional proteins can be defined. Understanding the motifs allows those skilled in the art to design sequence variations that do not affect function.

[0254] Homologous comparison among PIP-72 homologs Figure 1 , Figure 2 , Figure 3 , Figure 4 and Figure 5 This allows for the identification of conserved residues among homologs of this family. Figure 1 In Examples 10 and 11, saturation mutagenesis was used to induce substitutions at selected amino acid positions, and these substitutions were tested. The activity of these mutants was tested, and a variety of active substitutions not present in homologs were identified, providing an understanding of functional limitations at these residues.

[0255] In some embodiments, a polypeptide comprising an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or higher sequence identity with the amino acid sequences shown below: SEQ ID NO: 20, SEQ ID NO: 24, SEQ ID NO: 30, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 929, SEQ ID NO: 930, SEQ ID NO: 931, SEQ ID NO: 937, SEQ ID NO: 938, SEQ ID NO: 942, SEQ ID NO: 947 or SEQ ID NO: 948, wherein the polypeptide has insecticidal activity.

[0256] Composition

[0257] It also covers compositions containing the PIP-72 peptide. In some embodiments, the composition contains the PIP-72 peptide. In some embodiments, the composition contains a PIP-72 fusion protein.

[0258] Antibody

[0259] This also covers antibodies against the PIP-72 polypeptide or its variants or fragments according to embodiments of this disclosure. The antibodies of this disclosure include polyclonal and monoclonal antibodies and fragments thereof that retain their ability to bind to the PIP-72 protein present in the insect gut. An antibody, monoclonal antibody, or fragment thereof is considered capable of binding a molecule if it can specifically react with a molecule to bind that molecule to the antibody, monoclonal antibody, or fragment thereof. The terms “antibody” (Ab) or “monoclonal antibody” (Mab) are intended to include the complete molecule capable of binding a hapten as well as fragments or binding regions or domains thereof (e.g., Fab and F(ab).sub.2 fragments). Such fragments are typically produced by proteolytic cleavage (e.g., papain or pepsin). Alternatively, hapten-binding fragments may be produced by applying recombinant DNA technology or by synthetic chemistry. Methods for preparing the antibodies of this disclosure are well known in the art. See, for example, Antibodies, A Laboratory Manual, Ed Harlow and David Lane (eds.), Cold Spring Harbor Laboratory, NY (1988), and the references cited therein. Standard references explaining the general principles of immunology include: Klein, J. Immunology: The Science of Cell-Noncell Discrimination, John Wiley & Sons, NY (1982); Dennett et al., Monoclonal Antibodies, Hybridoma: A New Dimension in Biological Analyses, Plenum Press, NY (1980); and Campbell, “Monoclonal Antibody Technology,” Laboratory Techniques in Biochemistry and Molecular Biology, Vol. 13, Burdon et al. (eds.), Elsevier, Amsterdam (1984). See also U.S. Patents 4,196,265, 4,609,893, 4,713,325, 4,714,681, 4,716,111, 4,716,117, and 4,720,459. PIP-72 peptides, peptide antibodies, or their antigen-binding moieties can be produced using a variety of techniques, including conventional monoclonal antibody methods, such as the standard somatic cell hybridization technique used by Kohler and Milstein (1975) Nature 256:495. Other techniques for producing monoclonal antibodies can also be employed, such as viral or oncogenic transformation of B lymphocytes.The animal system used for hybridoma preparation is the mouse system. Immunoassay protocols and techniques for isolating immune spleen cells for fusion are known in the art. Fusion partners (e.g., mouse myeloma cells) and fusion procedures are also known. The antibodies and monoclonal antibodies disclosed herein can be prepared by using the PIP-72 peptide as an antigen.

[0260] This disclosure also provides a kit for detecting the presence of a PIP-72 peptide or a nucleotide sequence encoding a PIP-72 peptide in a sample. In one embodiment, the kit provides antibody-based reagents for detecting the presence of a PIP-72 peptide in a tissue sample. In another embodiment, the kit provides labeled nucleic acid probes for detecting the presence of one or more polynucleotides encoding a PIP-72 peptide. The kit is provided together with appropriate reagents and controls for performing the detection method and instructions for use.

[0261] Receptor identification and isolation

[0262] This also covers receptors for the PIP-72 peptide or for its variants or fragments according to embodiments of this disclosure. Methods for identifying receptors are well known in the art (see, Hofmann et al., (1988) Eur. J. Biochem. 173: 85-91; Gill et al., (1995) J. Biol. Chem. 27277-27282), which can be used to identify and isolate receptors for the PIP-72 peptide using brush border vesicles from susceptible insects. In addition to the radiolabeling methods listed in the cited literature, the PIP-72 peptide can also be labeled with fluorescent dyes and other common tags such as streptavidin. Brush border vesicles (BBMV) of susceptible insects such as soybean looper and stink bug can be prepared according to the protocols listed in the references, isolated on an SDS-PAGE gel, and imprinted onto a suitable membrane. The labeled PIP-72 peptide can be incubated with the imprinted membrane of the BBMV, and the labeled PIP-72 peptide can be identified using a labeled reporter gene. The identity of protein bands interacting with the PIP-72 peptide can be detected by N-terminal amino acid gas chromatography or mass spectrometry-based protein identification methods (Patterson, (1998) 10.22, 1-24, Current Protocol in Molecular Biology, John Wiley & Son Inc). Once the protein is identified, the corresponding gene can be cloned from the genomic DNA or cDNA library of susceptible insects, and the binding affinity can be directly measured with the PIP-72 peptide. The receptor function that exhibits insecticidal activity by interacting with the PIP-72 peptide can be verified by RNAi-type gene knockout methods (Rajagopal et al., (2002) J. Biol. Chem. 277: 46849-46851).

[0263] Nucleotide constructs, expression cassettes, and vectors

[0264] The use of the term "nucleotide construct" in this document is not intended to limit the embodiments of this disclosure to nucleotide constructs containing DNA. Those skilled in the art will recognize that nucleotide constructs, particularly polynucleotides and oligonucleotides composed of ribonucleotides and combinations of ribonucleotides and deoxyribonucleotides, can also be applied to the methods disclosed herein. The nucleotide constructs, nucleic acids, and nucleotide sequences of the embodiments of this disclosure further encompass all complementary forms of such constructs, molecules, and sequences. Furthermore, the nucleotide constructs, nucleotide molecules, and nucleotide sequences of the embodiments of this disclosure encompass all nucleotide constructs, molecules, and sequences that can be used in the methods of the embodiments of this disclosure to transform plants, including but not limited to those nucleotide constructs, molecules, and sequences composed of deoxyribonucleotides, ribonucleotides, and combinations thereof. Such deoxyribonucleotides and ribonucleotides include both naturally occurring molecules and synthetic analogs. The nucleotide constructs, nucleic acids, and nucleotide sequences of the embodiments also encompass all forms of nucleotide constructs, including but not limited to single-stranded forms, double-stranded forms, hairpin structures, stem-loop structures, etc.

[0265] Another embodiment involves a transformed organism, such as an organism selected from plant and insect cells, bacteria, yeast, baculoviruses, protozoa, nematodes, and algae. The transformed organism comprises a DNA molecule of the present disclosure, an expression cassette containing said DNA molecule, or a vector containing said expression cassette, which can be stably incorporated into the genome of the transformed organism.

[0266] The sequences of this disclosure are provided in a DNA construct for expression in an organism of interest. The construct will include 5′ and 3′ regulatory sequences operatively linked to the sequences of this disclosure. As used herein, the term “operatively linked” refers to a functional link between a promoter and a second sequence, wherein the promoter sequence initiates and mediates transcription of a DNA sequence corresponding to the second sequence. Generally, operatively linked means that the linked nucleic acid sequences are contiguous and that it is necessary to link two protein-coding regions within the same reading frame. The construct may additionally contain at least one additional gene to be co-transformed into the organism. Alternatively, the additional gene may be provided on multiple DNA constructs.

[0267] In some embodiments, the DNA construct comprises a polynucleotide encoding a PIP-72 polypeptide of the present disclosure, said polynucleotide being operatively linked to a heterologous regulatory sequence.

[0268] In some embodiments, the DNA construct comprises a polynucleotide encoding a polypeptide having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with the amino acid sequences shown below: SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 30, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 929, SEQ ID NO: 930, SEQ ID NO: 931, SEQ ID NO: 937, SEQ ID NO: 938, SEQ ID NO: 942, SEQ ID NO: 947 or SEQ ID NO: 948, and is operatively linked to a heterologous regulatory sequence.

[0269] In some embodiments, the DNA construct comprises a polynucleotide encoding a polypeptide, said polynucleotide comprising the amino acid sequence of SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 30, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 929, SEQ ID NO: 930, SEQ ID NO: 931, SEQ ID NO: 937, SEQ ID NO: 938, SEQ ID NO: 942, SEQ ID NO: 947 or SEQ ID NO: 948, and operatively ligated to a heterologous regulatory sequence.

[0270] This DNA construct has multiple restriction sites to ensure that the insertion of the PIP-72 polypeptide gene sequence is under transcriptional regulation by the regulatory region. The DNA construct may also contain a selective marker gene.

[0271] The DNA construct typically comprises, in the 5′ to 3′ transcriptional direction: a transcription and translation initiation region (i.e., a promoter), a DNA sequence of the present disclosure, and a transcription and translation termination region (i.e., a termination region) that functions in the host organism. The transcription initiation region (i.e., the promoter) may be natural, similar, foreign, or heterologous relative to the host organism and / or relative to the sequence of the present disclosure. Additionally, the promoter may be a natural sequence or alternatively a synthetic sequence. The term “foreign” as used herein means that the promoter is not present in the natural organism in which it is introduced. If a promoter is “foreign” or “heterologous” relative to the sequence of the present disclosure, it means that the promoter is not a naturally occurring or operatively linked promoter of the sequence of the present disclosure. As used herein, a chimeric gene contains a coding sequence operatively linked to a transcription initiation region that is heterologous to that coding sequence. If the promoter is a natural or operative sequence, the expression of the operatively linked sequence is altered compared to wild-type expression, resulting in a phenotypic change.

[0272] In some implementations, the DNA construct may also include a transcriptional enhancer sequence. As used herein, the term "enhancer" refers to a DNA sequence that can stimulate promoter activity and may be an intrinsic element of the promoter or a heterologous element inserted to enhance the promoter's level or tissue specificity. Various enhancers are known in the art, including, for example, introns in plants that have gene expression-enhancing properties (US Patent Application Publication 2009 / 0144863, ubiquitin introns (i.e., maize ubiquitin intron 1 (see, for example, NCBI sequence S94464; Christensen and Quail (1996) Transgenic Res. 5: 213-218; Christensen et al. (1992) Plant Molecular Biology 18: 675-689)), ω-enhancers or ω-prime enhancers (Gallie et al., (1989) Molecular Biology of RNA Cech edited (Liss, New York) 237-256 and Gallie et al., (1987) Gene 60: 217-25), CaMV 35S enhancers (see, for example, Benfey et al., (1990) EMBO) J.9:1685-96), maize AdhI introns (Kyozuka et al. (1991) Mol. Gen. Genet. 228:40-48; Kyozuka et al. (1990) Maydica 35:353-357), enhancers of U.S. Patent 7,803,992, and also enhancers of sugarcane baculovirus (SCBV) of WO2013130813, each cited in this document. The above list of transcriptional enhancers is not intended to be limiting. Any suitable transcriptional enhancer may be used in the described embodiments.

[0273] The termination region may be natural with respect to the transcription start region, natural with respect to the DNA sequence of interest that can be operatively linked, natural with respect to the plant host, or derived from another source (i.e., foreign or heterologous with respect to the promoter, the sequence of interest, the plant host, or any combination thereof).

[0274] The readily available termination regions can be obtained from Ti plasmids of *Agrobacterium tumefaciens*, such as the termination regions of octopaline synthase and carmine synthase. See also Guerineau et al., (1991) Mol. Gen. Genet. 262: 141-144; Proudfoot, (1991) Cell 64: 671-674; Sanfacon et al., (1991) Genes Dev. 5: 141-149; Mogen et al., (1990) Plant Cell 2: 1261-1272; Munroe et al., (1990) Gene 91: 151-158; Ballas et al., (1989) Nucleic Acids Res. 17: 7891-7903 and Joshi et al., (1987) Nucleic Acid Res. 15: 9627-9639.

[0275] If appropriate, nucleic acids can be optimized to improve their expression in a host organism. Therefore, if the host organism is a plant, a synthetic nucleic acid can be synthesized using plant-preferred codons to improve expression. For a discussion of the use of host-preferred codons, see, for example, Campbell and Gowri, (1990) Plant Physiol. 92: 1-11. For example, although the nucleic acid sequences of the embodiments of this disclosure are expressible in both monocotyledonous and dicotyledonous species, the sequences can be modified to address specific codon preferences and GC content preferences for monocotyledonous or dicotyledonous plants, as these preferences have been shown to be different (Murray et al. (1989) Nucleic Acids Res. 17: 477-498). Thus, maize-preferred codons for specific amino acids can be derived from known gene sequences from maize. The use of maize codons for 28 genes from maize plants is listed in Table 4 of Murray et al. (ibid.). Methods for synthesizing plant-preferred genes are readily available in the art. See, for example, U.S. Patents 5,380,831 and 5,436,391, and Murray et al., (1989) Nucleic Acids Res. 17: 477-498, and Liu H et al., Mol Bio Rep37: 677-684, 2010, which are incorporated herein by reference. A codon usage table for maize (Zea maize) is also available at kazusa.or.jp / codon / cgi-bin / showcodon.cgi?species=4577 (accessible with the www prefix). Table 2 shows the optimal codon analysis for maize (modified from Liu H et al., Mol Bio Rep37: 677-684, 2010).

[0276] Table 2

[0277] amino codon high RSCU Low RSCU amino codon high RSCU Low RSCU acid count count acid count count Phe UUU 115 0.04 2,301 1.22 Ala GCU 629 0.17 3,063 1.59 UUC* 5,269 1.96 1,485 0.78 GCC* 8,057 2.16 1,136 0.59 Ser UCU 176 0.13 2,498 1.48 GCA 369 0.1 2,872 1.49 UCC* 3,489 2.48 1,074 0.63 GCG* 5,835 1.57 630 0.33 UCA 104 0.07 2,610 1.54 Tyr UAU 71 0.04 1,632 1.22 UCG* 1,975 1.4 670 0.4 UAC* 3,841 1.96 1,041 0.78 AGU 77 0.05 1,788 1.06 His CAU 131 0.09 1,902 1.36 AGC* 2,617 1.86 1,514 0.89 CAC* 2,800 1.91 897 0.64 Leu UUA 10 0.01 1,326 0.79 Cys UGU 52 0.04 1,233 1.12 UUG 174 0.09 2,306 1.37 UGC* 2,291 1.96 963 0.88 CUU 223 0.11 2,396 1.43 Gln CAA 99 0.05 2,312 1.04 CUC* 5,979 3.08 1,109 0.66 CAG* 3,557 1.95 2,130 0.96 CUA 106 0.05 1,280 0.76 Arg CGU 153 0.12 751 0.74 CUG* 5,161 2.66 1,646 0.98 CGC* 4,278 3.25 466 0.46 Pro CCU 427 0.22 1,900 1.47 CGA 92 0.07 659 0.65 CCC* 3,035 1.59 601 0.47 CGG* 1,793 1.36 631 0.62 CCA 311 0.16 2,140 1.66 AGA 83 0.06 1,948 1.91 CCG* 3,846 2.02 513 0.4 AGG* 1,493 1.14 1,652 1.62 Ile AUU 138 0.09 2,388 1.3 Asn AAU 131 0.07 3,074 1.26 AUC* 4,380 2.85 1,353 0.74 AAC* 3,814 1.93 1,807 0.74 AUA 88 0.06 1,756 0.96 Lys AAA 130 0.05 3,215 0.98 Thr ACU 136 0.09 1,990 1.43 AAG* 5,047 1.95 3,340 1.02 ACC* 3,398 2.25 991 0.71 Asp GAU 312 0.09 4,217 1.38 ACA 133 0.09 2,075 1.5 GAC* 6,729 1.91 1,891 0.62 ACG* 2,378 1.57 495 0.36 Gly GGU 363 0.13 2,301 1.35 Val GUU 182 0.07 2,595 1.51 GGC* 7,842 2.91 1,282 0.75 GUC* 4,584 1.82 1,096 0.64 GGA 397 0.15 2,044 1.19 GUA 74 0.03 1,325 0.77 GGG* 2,186 0.81 1,215 0.71 GUG* 5,257 2.08 1,842 1.07 Glu GAA 193 0.06 4,080 1.1 GAG* 6,010 1.94 3,307 0.9

[0278] Chi-square contingency tests are used to compare codon usage to identify the optimal codon. Codons that appear significantly more frequently (P ≤ 0.01) are indicated by an asterisk.

[0279] The codon usage table for soybean (Glycine max) is shown in Table 3 and can also be found at kazusa.or.jp / codon / cgi-bin / showcodon.cgi?species=3847&aa=1&style=N (accessible with the www prefix).

[0280] Table 3

[0281] TTT F 21.2 (10493) TCT S 18.4 (9107) TTC F 21.2 (10487) TCC S 12.9 (6409) TTA L 9.2 (4545) TCA S 15.6 (7712) TTG L 22.9 (11340) TCG S 4.8 (2397) CTT L 23.9 (11829) CCT P 18.9 (9358) CTC L 17.1 (8479) CCC P 10.1 (5010) CTA L 8.5 (4216) CCA P 19.1 (9461) CTG L 12.7 (6304) CCG P 4.7 (2312) ATT I 25.1 (12411) ACT T 17.1 (8490) ATC I 16.3 (8071) ACC T 14.3 (7100) ATA I 12.9 (6386) ACA T 14.9 (7391) ATG M 22.7 (11218) ACG T 4.3 (2147) GTT V 26.1 (12911) GCT A 26.7 (13201) GTC V 11.9 (5894) GCC A 16.2 (8026) GTA V 7.7 (3803) GCA A 21.4 (10577) GTG V 21.4 (10610) GCG A 6.3 (3123) TAT Y 15.7 (7779) TGT C 8.1 (3995) TAC Y 14.9 (7367) TGC C 8.0 (3980) TAA * 0.9 (463) TGA * 1.0 (480) TAG * 0.5 (263) TGG W 13.0 (6412) CAT H 14.0 (6930) CGT R 6.6 (3291) CAC H 11.6 (5759) CGC R 6.2 (3093) CAA Q 20.5 (10162) CGA R 4.1 (2018) CAG Q 16.2 (8038) CGG R 3.1 (1510) AAT N 22.4 (11088) AGT S 12.6 (6237) AAC N 22.8 (11284) AGC S 11.3 (5594) AAA K 26.9 (13334) AGA R 14.8 (7337) AAG K 35.9 (17797) AGG R 13.3 (6574) GAT D 32.4 (16040) GGT G 20.9 (10353) GAC D 20.4 (10097) GGC G 13.4 (6650) GAA E 33.2 (16438) GGA G 22.3 (11022) GAG E 33.2 (16426) GGG G 13.0 (6431)

[0282] In some implementations, the recombinant nucleic acid molecule encoding the PIP-72 polypeptide has a maize-optimized codon.

[0283] Other sequence modifications are known to enhance gene expression in the host cell. These include the removal of sequences encoding pseudopolyadenylation signals, exon-intron splicing site signals, transposon-like repeat sequences, and other well-characterized sequences that may be detrimental to gene expression. The GC content of a sequence can be adjusted to the average level for a given host cell, calculated by referencing known genes expressed in the host cell. As used herein, the term "host cell" refers to a cell containing a vector and supporting the replication and / or expression of that expression vector. Host cells can be prokaryotic cells (e.g., *E. coli*) or eukaryotic cells (e.g., yeast, insect, amphibian, or mammalian cells, or monocot or dicotyledonous plant cells). An example of a monocotyledonous plant host cell is a maize host cell. When possible, sequences are modified to avoid predictable hairpin secondary mRNA structures.

[0284] The expression cassette may also contain a 5′ leader sequence. Such leader sequences can enhance translation. Translation leader sequences are known in the art and include: microRNA virus leader sequences, such as the EMCV leader sequence (5′ untranslated region of encephalomyocarditis) (Elroy-Stein et al., (1989) Proc. Natl. Acad. Sci. USA 86: 6126-6130); potato Y virus group leader sequences, such as the TEV leader sequence (tobacco etch virus) (Gallie et al., (1995) Gene 165(2): 233-238), the MDMV leader sequence (maize dwarf mosaic virus), and human immunoglobulin heavy chain binding protein (BiP) (Macejak et al., (1991) Nature 353: 90-94); and untranslated leader sequences of the coat protein mRNA (AMV RNA 4) from alfalfa mosaic virus (Jobling et al., (1987) Nature). 325:622-625); Tobacco mosaic virus leader sequence (TMV) (Gallie et al., (1989), Molecular Biology of RNA, edited by Cech (Liss, New York), pp. 237-256); and Maize chlorotic mottle virus leader sequence (MCMV) (Lommel et al., (1991) Virology 81:382-385). See also, Della-Cioppa et al., (1987) Plant Physiol. 84:965-968. Such constructs may also contain a “signal sequence” or “leader sequence” to facilitate co-translational or post-translational transport of peptides to specific intracellular structures, such as chloroplasts (or other plastids), endoplasmic reticulum, or Golgi apparatus.

[0285] As used herein, a “signal sequence” refers to a sequence known or suspected of inducing co-translational or post-translational peptide transport across the cell membrane. In eukaryotes, this typically involves secretion into the Golgi apparatus with some degree of glycosylation. Bacterial insecticidal toxins are often synthesized as prototoxins, which are activated by proton transfer in the gut of the target pest (Chang, (1987) Methods Enzymol. 153: 507-516). In some embodiments, the signal sequence is located within the native sequence or may be derived from the sequence of the described embodiment. As used herein, a “leader sequence” refers to any sequence whose translation produces an amino acid sequence sufficient to trigger co-translational transport of the peptide chain to subcellular organelles. Thus, this includes leader sequences that are targeted for transport and / or glycosylated via entry into the endoplasmic reticulum, through vacuoles, plastids (including chloroplasts), mitochondria, etc. Nuclear-encoded proteins targeting the thylakoid cavity of chloroplasts possess characteristic bipartite transit peptides, composed of a matrix-targeting signal peptide and a lumen-targeting signal peptide. The matrix-targeting information is located at the proximal amino group of this transit peptide. The lumen-targeting signal peptide, located at the proximal carboxyl group of this transit peptide, contains all the information for targeting the lumen. In recent years, proteomics studies of higher plant chloroplasts have successfully identified many nuclear-encoded lumen proteins (Kieselbach et al. FEBS LETT 480:271-276, 2000; Peltier et al. PlantCell 12:319-341, 2000; Bricker et al. Biochim. Biophys Acta 1503:350-356, 2001), whose lumen-targeting signal peptides have the potential for use according to this disclosure. Kieselbach et al., Photosynthesis Research, 78:249-264, 2003, reported approximately 80 proteins from Arabidopsis, as well as homologous proteins from spinach and peas. Specifically, Table 2 of that publication, which is incorporated herein by reference, discloses 85 chloroplast lumen proteins identified by their accession numbers (see also U.S. Patent Application Publication 2009 / 09044298). Furthermore, the recently published draft rice genome (Goff et al., Science 296:92-100, 2002) is a suitable source of lumen-targeting signal peptides that can be used according to this disclosure.

[0286] Suitable chloroplast transport peptides (CTPs) are well known to those skilled in the art, including chimeric CTPs, which include, but are not limited to, N-terminal, central, or C-terminal domains of CTPs derived from the following enzymes: rice (Oryza sativa) 1-deoxy-D-xylitol-5-phosphate synthase, rice superoxide dismutase, rice soluble starch synthase, rice NADP-dependent malate kinase, rice phosphate-2-dehydro-3-deoxyheptanol aldolase 2, rice L-ascorbic acid peroxidase 5 or rice phosphoglucan aqueous 2-kinase, maize (Zea Mays) ssRUBISCO, maize β-glucosidase, maize malate dehydrogenase, and maize M-type thioredoxin (US Patent Application Publication 2012 / 0304336). US patents US20130205440A1, US20130205441A1, and US20130210114A1 disclose chloroplast transport peptides.

[0287] The PIP-72 polypeptide gene targeting chloroplasts can be optimized for expression in chloroplasts to account for differences in codon usage between the plant cell nucleus and this organelle. In this way, nucleic acids of interest can be synthesized using chloroplast-preferred codons. See, for example, U.S. Patent 5,380,831, which is incorporated herein by reference.

[0288] In preparing expression cassettes, multiple DNA fragments can be manipulated to provide DNA sequences in the correct orientation, and, where appropriate, in the correct reading frame. For this purpose, adaptors or linkers can be used to ligate the DNA fragments together, or other manipulations can be involved to provide convenient restriction sites, remove redundant DNA, remove restriction sites, etc. This may involve in vitro mutagenesis, primer repair, restriction enzyme digestion, annealing, and substitution (e.g., conversion and transversion).

[0289] A variety of promoters are available for implementation of the embodiments disclosed herein. Promoters can be selected based on the desired results. Nucleic acids can be combined with constitutive promoters, tissue-preferred promoters, inducible promoters, or other promoters for expression in a host organism. The promoters of this invention include homologs of cis-elements known to influence gene regulation and exhibit homology with the promoter sequences of this invention. These cis-elements include, but are not limited to: oxygen-responsive cis-elements (Cowen et al., J Biol. Chem. 268(36): 26904-26910(1993)), light-regulating elements (Bruce and Quaill, Plant Cell 2(11): 1081-1089(1990); Bruce et al., EMBO J. 10: 3015-3024(1991); Rocholl et al. ... Sci. 97: 189-198 (1994); Block et al., Proc. Natl. Acad. Sci. USA, 87: 5387-5391 (1990); Giuliano et al., Proc. Natl. Acad. Sci. USA, 85: 7089-7093 (1988); Staiger et al., Proc. Natl. Acad. Sci. USA, 86: 6930-6934 (1989); Izawa et al., Plant Cell 6: 1277-1287 (1994); Menkens et al., Trends in Biochemistry 20: 506-510 (1995); Foster et al., FASEBJ. 8: 192-200 (1994); Plessel et al., Mol Gen Gene 254:258-266 (1997); Green et al., EMBO J. 6:2543-2549 (1987); Kuhlemeier et al., Ann. Rev. Plant Physiol. 38:221-257 (1987); Villain et al., J. Biol. Chem. 271:32593-32598 (1996); Lam et al., Plant Cell 2:857-866 (1990); Gilmartin et al., Plant Cell 2:369-378 (1990); Datta et al., Plant Cell 1:1069-1077 (1989); Gilmartin et al., Plant Cell 2:369-378 (1990); Castresana et al., EMBO J.7: 1929–1936 (1988); Ueda et al., Plant Cell 1: 217–227 (1989); Terzaghi et al., Annu.Rev. Plant Physiol. Plant Mol. Biol. 46: 445-474 (1995); Green et al., EMBO J. 6: 2543-2549 (1987); Villain et al., J. Biol. Chem. 271: 32593-32598 (1996); Tjaden et al., Plant Cell 6: 107-118 (1994); Tjaden et al., Plant Physiol. 108: 1109-1117 (1995); Ngai et al., Plant J. 12: 1021-1234 (1997); Bruce et al., EMBO J. 10: 3015-3024 (1991); Ngai et al., Plant J. 12: 1021-1034 (1997)), gibberellin response elements (Muller et al., J. Plant Physiol. 145: 606-613 (1995); Croissant et al., Plant Science 116: 27-35 (1996); Lohmer et al., EMBO J. 10: 617-624 (1991); Rogers et al., Plant Cell 4: 1443-1451 (1992); Lanahan et al., Plant Cell 4: 203-211 (1992); Skriver et al., Proc. Natl. Acad. Sci. USA, 88: 7266-7270 (1991); Gilmartin et al., Plant Cell 2: 369-378 (1990); Huang et al., Plant Mol. Biol. 14: 655-668 (1990); Gubler et al., Plant Cell 7: 1879-1891 (1995)), abscisic acid response element (Busk et al., Plant Cell 9: 2261-2270 (1997); Guiltinan et al., Science 250: 267-270 (1990); Shen et al., Plant Cell 7: 295-307 (1995); Shen et al., Plant Cell 8: 1107-1119 (1996); Seo et al., Plant Mol. Biol. 27: 1119-1131 (1995); Marcotte et al., Plant Cell 1: 969-976 (1989); Shen et al., Plant Cell 7:295-307 (1995); Iwasaki et al., Mol Gen Genet 247:391-398 (1995); Hattori et al., Genes Dev.6:609-618 (1992); Thomas et al., Plant Cell 5:1401-1410 (1993)), elements similar to abscisic acid-responsive elements (Ellerstrom et al., Plant Mol. Biol. 32:1019-1027 (1996)), auxin-responsive elements (Liu et al., Plant Cell 6:645-657 (1994); Liu et al., Plant Physiol. 115:397-407 (1997); Kosugi et al., Plant J. 7:877-886 (1995); Kosugi et al., Plant Cell 9:1607-1619 (1997); Ballas et al., J. Mol. Biol. 233:580-596 (1993)), cis elements responsive to methyl jasmonate treatment (Beaudoin and Rothstein, Plant Mol. Biol. 33:835-846 (1997)), cis elements responsive to abscisic acid and stress response (Straub et al., Plant Mol. Biol. 26: 617-630 (1994)), ethylene-responsive cis-electrode (Itzhaki et al., Proc. Natl. Acad. Sci. USA, 91: 8925-8929 (1994); Montgomery et al., Proc. Natl. Acad. Sci. USA, 90: 5939-5943 (1993); Sessa et al., Plant Mol. Biol. 28: 145-153 (1995); Shinshi et al., Plant Mol. Biol. 27: 923-932 (1995)), salicylic acid-responsive cis-electrode (Strange et al., Plant J. 11: 1315-1324 (1997); Qin et al., Plant Cell 6:863-874 (1994)), cis elements responding to water stress and abscisic acid (Lam et al., J. Biol. Chem. 266:17131-17135 (1991); Thomas et al., Plant Cell 5:1401-1410 (1993); Pla et al., Plant Mol Biol 21:259-266 (1993)), cis elements essential for M-phase specific expression (Ito et al., Plant Cell 10:331-341 (1998)), sucrose-responsive elements (Huang et al., Plant Mol. Biol. 14:655-668 (1990); Hwang et al., Plant Mol Biol 36:331-341 (1998); Grierson et al., Plant J. Biol. 266:863-874 (1994)), cis elements responding to water stress and abscisic acid (Lam et al., J. Biol. Chem. 266:17131-17135 (1991); Thomas et al., Plant Cell 5:1401-1410 (1993); Pla et al., Plant Mol Biol 21:259-266 (1993)), cis elements essential for M-phase specific expression (Ito et al., Plant Cell 10:331-341 (1998)), sucrose-responsive elements (Huang et al., Plant Mol. Biol. 14:655-668 (1990); Hwang et al., Plant Mol Biol 36:331-341 (1998); Grierson et al., Plant J. Biol. Chem. 266:863-874 (1994)), cis elements responding to water stress and absci5:815-826 (1994)), heat shock responsive elements (Pelham et al., Trends Genet. 1:31-35 (1985)), elements responsive to auxin and / or salicylic acid (also reported for photoregulation) (Lam et al., Proc. Natl. Acad. Sci. USA, 86:7890-7897 (1989); Benfey et al., Science 250:959-966 (1990)), elements responsive to ethylene and salicylic acid (Ohme-Takagi et al., Plant Mol. Biol. 15:941-946 (1990)), elements responsive to trauma and abiotic stress (Loake et al., Proc. Natl. Acad. Sci. USA, 89:9230-9234 (1992); Miri et al., Plant Mol. Biol ...). Mol. Biol. 33: 257-266 (1997)), antioxidant-responsive elements (Rushmore et al., J. Biol. Chem. 266: 11632-11639; Dalton et al., Nucleic Acids Res. 22: 5016-5023 (1994)), Sph elements (Suzuki et al., Plant Cell 9: 799-807 (1997)), inducer-responsive elements (Fukuda et al., Plant Mol. Biol. 34: 81-87 (1997); Rushton et al., EMBO J. 15: 5690-5700 (1996)), metal-responsive elements (Stuart et al., Nature 317: 828-831 (1985); Westin et al., EMBO J. 7: 3763-3770 (1988); Thiele et al., Nucleic Acids Res. 20: 1183-1191 (1992); Faisst et al., Nucleic Acids Res. 20: 3-26 (1992)), low temperature response elements (Baker et al., Plant Mol. Biol. 24: 701-713 (1994); Jiang et al., Plant Mol. Biol. 30: 679-684 (1996); Nordin et al., Plant Mol. Biol. 21: 641-653 (1993); Zhou et al., J. Biol. Chem. 267: 23515-23519 (1992)), drought response elements (Yamaguchi et al., Plant Cell 6: 251-264 (1994); Wang et al., Plant Mol. Biol.28:605-617 (1995); Bray EA, Trends in Plant Science 2:48-54 (1997)), glutenin enhancer elements (Colot et al., EMBO J.6:3559-3564 (1987); Thomas et al., Plant Cell 2:1171-1180 (1990); Kreis et al., Philos.Trans.R.Soc.Lond., B314:355-365 (1986)), light-independent regulatory elements (Lagrange et al., Plant Cell 9:1469-1479 (1997); Villain et al., J.Biol.Chem.271:32593-32598 (1996)), OCS enhancer elements (Bouchez ...3559-3564 (1987); Thomas et al., Plant Cell 2:3559-3564 (1987); Thomas et al., Plant Cell 2:3559-3564 (1987); Thomas et al., Plant Cell 2:3559-356 J.8:4197-4204 (1989); Foley et al., Plant J.3:669-679 (1993)), ACGT elements (Foster et al., FASEB J.8:192-200 (1994); Izawa et al., Plant Cell 6:1277-1287 (1994); Izawa et al., J.Mol.Biol.230:1131-1144 (1993)), negative cis elements in plastid-related genes (Zhou et al., J.Biol.Chem.267:23515-23519 (1992); Lagrange et al., Mol.Cell Biol.13:2614-2622 (1993); Lagrange et al., Plant Cell 9:1469-1479 (1997); Zhou et al., J. Biol. Chem. 267:23515-23519 (1992)), alcohol-soluble gluten box elements (Forde et al., Nucleic Acids Res. 13:7327-7339 (1985); Colott et al., EMBO J. 6:3559-3564 (1987); Thomas et al., Plant Cell 2:1171-1180 (1990); Thompson et al., Plant Mol. Biol. 15:755-764 (1990); Vicente et al., Proc. Natl. Acad. Sci. USA 94:7685-7690 (1997))...

Claims

1. A recombinant PIP-72 polypeptide, said recombinant PIP-72 polypeptide having insecticidal activity against the western maize root borer (Diabrotica virgifera virgifera), wherein said PIP-72 polypeptide is selected from SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:927, SEQ ID NO:928, SEQ ID NO:608, SEQ ID NO:609, SEQ ID NO:610, SEQ ID NO:611, SEQ ID NO:612, SEQ ID NO:613, SEQ ID NO:614, SEQ ID NO:615, SEQ ID NO:616, SEQ ID NO:617, SEQ ID NO:618, SEQ ID NO:619, SEQ ID NO:620, SEQ ID NO:621, SEQ ID NO:622, SEQ ID NO:623, SEQ ID NO:624, SEQ ID NO:625, SEQ ID NO:626, SEQ ID NO:627, SEQ ID NO:628, SEQ ID NO:62 ...9, SEQ ID NO:620, SEQ ID NO:621 NO: 628, SEQ ID NO: 629, SEQ ID NO: 630, SEQ ID NO: 631, SEQ ID NO: 632, SEQ ID NO: 633, SEQ ID NO: 634, SEQ ID NO: 635, SEQ ID NO: 636, SEQ ID NO: 637, SEQ ID NO: 638, SEQ ID NO: 639, SEQ ID NO: 640, SEQ ID NO:641, SEQ ID NO:642, SEQ ID NO:643, SEQ ID NO:644, SEQ ID NO:645, SEQ ID NO:646, SEQ ID NO:647, SEQ ID NO:648, SEQ ID NO:649, SEQ ID NO:650, SEQ ID NO:651, SEQ ID NO:652, SEQ ID NO:653, SEQ ID NO:654, SEQ ID NO:655, SEQ ID NO:656, SEQ ID NO:657, SEQ ID NO:658, SEQ ID NO:659, SEQ ID NO:660, SEQ ID NO:661, SEQ ID NO:662, SEQ ID NO:663, SEQ ID NO:664, SEQ ID NO:665, SEQ ID NO:666, SEQ ID NO:667, SEQ ID NO:668, SEQ IDNO:669, SEQ ID NO:670, SEQ ID NO:671, SEQ ID NO:672, SEQ ID NO:673, SEQ ID NO:674, SEQ ID NO:675, SEQ ID NO:676, SEQ ID NO:677, SEQ ID NO:678, SEQ ID NO:679, SEQ ID NO:680, SEQ ID NO:681, SEQ ID NO:682, SEQ ID NO:683, SEQ ID NO:684, SEQ ID NO:685, SEQ ID NO:686, SEQ ID NO:687, SEQ ID NO:688, SEQ ID NO:689, SEQ ID NO:690, SEQ ID NO:691, SEQ ID NO:692, SEQ ID NO:693, SEQ ID NO:694, SEQ ID NO:695, SEQ ID NO:696, SEQ ID NO:697, SEQ ID NO:698, SEQ ID NO:699, SEQ ID NO:700, SEQ ID NO:701, SEQ ID NO:702, SEQ ID NO:703, SEQ ID NO:704, SEQ ID NO:705, SEQ ID NO:706, SEQ ID NO:707, SEQ ID NO:708, SEQ ID NO:771, SEQ ID NO:825, SEQ ID NO:826, SEQ ID NO:827, SEQ ID NO:828, SEQ ID NO:829, SEQ ID NO:830, SEQ ID NO:831, SEQ ID NO:832, SEQ ID NO:833, SEQ ID NO:834, SEQ ID NO:835, SEQ ID NO:836, SEQ ID NO:837, SEQ ID NO:838, SEQ ID NO:839, SEQ ID NO:840, SEQ ID NO:841, SEQ ID NO:842, SEQ ID NO:843, SEQ ID NO:844, SEQ ID NO:903, SEQ ID NO:904, SEQ ID NO:905, SEQ ID NO:906, SEQ ID NO:907, SEQ ID NO:908, SEQ ID NO:909, SEQ ID NO:910, SEQ ID NO:911, SEQ ID NO:912, SEQ ID NO:913 and SEQ IDNO:914。 2. The recombinant PIP-72 polypeptide according to claim 1, wherein the PIP-72 polypeptide is selected from SEQ ID NO:903, SEQ ID NO:904, SEQ ID NO:905, SEQ ID NO:906, SEQ ID NO:907, SEQ ID NO:908, SEQ ID NO:909, SEQ ID NO:910, SEQ ID NO:911, SEQ ID NO:912, SEQ ID NO:913 and SEQ ID NO:

914.

3. A DNA construct comprising a heterologous nucleic acid molecule, said heterologous nucleic acid molecule encoding the PIP-72 polypeptide according to claim 1 or 2.

4. An isolated polynucleotide comprising a nucleic acid molecule, said nucleic acid molecule encoding the PIP-72 polypeptide according to claim 1 or 2.

5. An expression cassette comprising the isolated polynucleotide of claim 4 operably linked to a heterologous regulatory element.

6. The expression cassette of claim 5, wherein the regulatory element is a promoter capable of expressing a protein in a plant.

7. A method for producing transgenic plants, comprising introducing the DNA construct of claim 3 into a plant.

8. A method for producing transgenic plants, comprising stabilizing the plants using the DNA construct of claim 3.

9. A method of producing seeds, comprising producing seeds from a plant produced by the method according to claim 7 or 8, wherein the seeds contain the nucleic acid molecule.

10. A method for producing offspring plants, comprising producing offspring plants from seeds produced by the method according to claim 9.

11. A method of producing cells, comprising transforming host cells with the DNA construct of claim 3.

12. The method of claim 11, wherein the host cell is a bacterial cell or a plant cell.

13. The method according to claim 12, wherein the plant cell is a monocotyledonous plant cell or a dicotyledonous plant cell.

14. A composition comprising the recombinant PIP-72 polypeptide of claim 1 or 2.

15. A fusion protein comprising the PIP-72 polypeptide of claim 1 or 2.

16. A method for controlling the western maize rootworm (Diabrotica virgifera virgifera), the method comprising contacting the western maize rootworm (Diabrotica virgifera virgifera) with an insecticidal effective amount of the PIP-72 polypeptide of claim 1 or 2.

17. A method for inhibiting or killing the western corn rootworm (Diabrotica virgifera virgifera), the method comprising contacting the western corn rootworm (Diabrotica virgifera virgifera) with a composition comprising an insecticidally effective amount of the PIP-72 polypeptide of claim 1 or 2.

18. A method for controlling the western corn rootworm (Diabrotica virgiferavirgifera) resistant to insecticidal proteins, the method comprising contacting the western corn rootworm (Diabrotica virgiferavirgifera) with an insecticidal effective amount of the PIP-72 polypeptide of claim 1 or 2.

19. A method for controlling the infection of transgenic plants by the western maize rootworm (Diabrotica virgifera virgifera) and providing resistance management against the western maize rootworm (Diabrotica virgifera virgifera), the method comprising expressing the PIP-72 polypeptide of claim 1 or 2 in the plant.

20. A method for identifying a nucleotide sequence encoding the PIP-72 polypeptide of claim 1 or 2 in a biological sample, the method comprising contacting the sample with a polynucleotide hybridized to the nucleotide sequence under stringent hybridization conditions, and detecting the binding of the polynucleotide to the nucleotide sequence, wherein the binding diagnoses the nucleotide sequence in the sample.

21. A method for identifying the PIP-72 polypeptide of claim 1 or 2 in a sample, the method comprising contacting the sample with an antibody specifically bound to the polypeptide and detecting the binding, wherein the binding diagnoses the presence of the polypeptide in the sample.

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