Insecticide protein

Abstract

Compositions and methods for controlling insect pests are disclosed. In particular, novel insecticidal proteins that are toxic to at least coleopteran insect pests are provided. Also provided are nucleic acid molecules encoding the novel insecticidal proteins. Also disclosed are methods of making the insecticidal proteins and methods of using the insecticidal proteins and nucleic acids encoding the insecticidal proteins of the disclosure, for example, in transgenic plants to confer protection from insect damage.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Application No. 62 / 914,667, filed on October 14, 2019, the contents of which are incorporated herein by reference in their entirety.

[0003] References to sequence listings

[0004] A sequence listing in ASCII text format is provided as an alternative to a paper copy, which is submitted pursuant to 37 CFR §1.821, is named "81942-WO-REG-ORG-P-1_ST25.txt", is approximately 113 kilobytes in size, was generated on September 30, 2020, and was submitted via EFS-Web. This sequence listing is hereby incorporated by reference into this specification for its disclosure. Technical Field

[0005] The present invention relates to the fields of protein engineering, plant molecular biology and pest control. More specifically, the present invention relates to novel proteins and variants thereof having insecticidal activity, nucleic acids (expressed to produce insecticidal proteins), methods for making these insecticidal proteins and corresponding nucleic acids, and methods for controlling insects using these insecticidal proteins and corresponding nucleic acids. Background Art

[0006] Insect pests are a major cause of crop losses. In the United States alone, billions of dollars are lost each year due to infestations of various genera of insects. In addition to losses to field crops, insect pests are a burden to vegetable and fruit growers, to producers of ornamental flowers, and a nuisance to gardeners and homeowners.

[0007] Multiple species of corn rootworm are considered the most destructive corn pests. In the United States alone, three species, Diabrotica virgifera virgifera (also known as the western corn rootworm), D. longicornis barberi (also known as the northern corn rootworm), and D. undecimpunctata howardi (also known as the southern corn rootworm), cause more than $1 billion in losses to corn each year in the U.S. Corn Belt. An important corn rootworm pest in the southern United States is the Mexican corn rootworm (Diabrotica virgifera zeae). In South America, the South American leafworm (Diabrotica Speciosa) is considered an important pest of corn. The western corn rootworm spread to Europe in 1992 and has been causing economic losses throughout major corn-growing regions since 2008. Corn rootworm larvae cause the most substantial plant damage by feeding almost exclusively on corn roots. This damage has been shown to increase plant lodging, reduce grain yield and vegetative yield as well as alter the nutrient content of the grain. Larval feeding also indirectly affects corn by opening access to the roots for bacterial and fungal infections that cause root and stem rot. Adult corn rootworms are active in corn fields in late summer where they feed on ears, silks, and pollen, thereby interfering with normal pollination.

[0008] Corn rootworms are primarily controlled by intensive applications of chemical pesticides, which are active by inhibiting insect growth, preventing insect feeding or reproduction, or causing mortality. Good corn rootworm control can be achieved, but these chemicals can sometimes affect other beneficial organisms as well. Another problem caused by the widespread use of chemical pesticides is the emergence of resistant insect populations. Yet another problem is due to the fact that corn rootworm larvae feed underground, making rescue treatments with insecticides difficult. Therefore, most insecticide applications are made preventively at the time of planting. This practice results in a huge environmental burden. This situation has been partially improved by various farmland management practices, but there is an increasing need for alternative mechanisms of pest control.

[0009] Biological pest control agents, such as Bacillus thuringiensis (Bt) strains expressing pesticidal toxins like delta-endotoxins (Δ-endotoxins; also known as crystalline toxins or Cry proteins), have also been applied to crop plants on a small scale, producing satisfactory results against certain insect pests. These delta-endotoxins are proteins contained within a crystalline matrix, which are known to possess insecticidal activity when ingested by certain insects. Such Cry proteins from Bacillus thuringiensis have been expressed in transgenic crop plants and commercially developed to control certain Lepidoptera and Coleopteran insect pests. For example, starting in 2003, transgenic corn hybrids that control corn rootworms by expressing Cry3Bb1, Cry34Ab1 / Cry35Ab1, or modified Cry3A (mCry3A) or eCry3.1Ab proteins have been commercially available in the United States.

[0010] Although it has been shown that transgenic plants expressing Cry proteins are very effective, insect pests that are resistant to Cry proteins expressed in certain transgenic plants are now known. Therefore, there is still a need to identify new and effective pest control proteins that provide economic benefits to farmers and are environmentally acceptable. Particularly needed are proteins that are toxic to Diabrotica species (a major corn pest) that have different modes of action compared to Cry proteins in existing insect control products to mitigate resistance development. In addition, it is desirable to deliver insect control proteins through these products that minimize the environmental burden (such as through transgenic plants). Summary of the invention

[0011] In view of these needs, in some embodiments, the present disclosure provides insecticidal proteins that are particularly derived from Rhizobium family bacteria, but can also be derived from other types of bacteria. Examples of such insecticidal proteins are illustrated herein and such insecticidal proteins, whether derived from Rhizobium family or other different bacteria, are collectively referred to as Rhizobium family insecticidal proteins (RIPs). The present disclosure also provides variants of RIPs, and proteins substantially identical to RIPs and variants thereof. Examples of the amino acid sequences of the RIPs disclosed herein include, but are not limited to, any one of SEQ ID NOs: 1-21. The RIPs disclosed herein are toxic to insect pests. For example, the proteins disclosed herein can be used to control economically important insect pests, including coleopteran insects, such as western corn rootworms (WCR; corn rootworms), northern corn rootworms (NCR; long-horned bark rootworms), southern corn rootworms (SCR; cucumber eleven-star leafworm rootfeeding subspecies) and / or Mexican corn rootworms (MCR; Mexican corn rootworms).

[0012] The present disclosure also provides nucleic acid molecules comprising one or more nucleotide sequences encoding RIP or variant RIP, their complementary sequences, or nucleotide sequences substantially identical to RIP or RIP variant. Examples of nucleotide sequences encoding RIP or variant RIP disclosed herein include, but are not limited to, any one of SEQ ID NOs: 22-49.

[0013] The present disclosure also provides vectors comprising recombinant nucleic acids encoding the RIP and / or variant RIP of the present disclosure; plants or microorganisms comprising and capable of expressing such nucleic acids; plants transformed with such nucleic acids, such as transgenic corn plants; progeny of such plants, which contain nucleic acids stably incorporated and inherited in a Mendelian manner, and / or seeds of such plants and such progeny. The present disclosure also provides methods of breeding to introduce transgenes comprising nucleic acid molecules of the present disclosure into progeny plants and various germplasms.

[0014] The disclosure also provides compositions and formulations comprising the RIPs and / or variant RIPs of the disclosure, which are capable of inhibiting the ability of pests to survive, grow and / or reproduce, or to limit insect-associated damage or loss to crops, e.g., applying the RIPs or RIP variants as part of a composition or formulation to an area or plant infested by insects, or preventatively treating an area or plant susceptible to insect infestation to confer protection against insect pests.

[0015] The disclosure further provides methods of making RIPs or variants thereof, and methods of using the nucleic acids, e.g., to control insects in microorganisms or to confer protection from insect damage in transgenic plants. Such microorganisms can be, for example, endophytic species that colonize maize roots and deliver the RIPs of the disclosure to the maize rhizosphere, thereby protecting the roots from damage by corn rootworm feeding.

[0016] The RIPs and / or variant RIPs of the present disclosure can be used alone or in combination with other insect control agents and strategies to impart enhanced pest control efficiency against the same insect pests and / or to increase the spectrum of target insects with minimal environmental impact.

[0017] Other aspects and advantages of the present invention will become apparent to those skilled in the art upon study of the following description and non-limiting examples of the invention.

[0018] Brief description of the sequences in the sequence listing

[0019] SEQ ID NO: 1 is the amino acid sequence of Ensif_aridCRW (RIP1Aa).

[0020] SEQ ID NO: 2 is the amino acid sequence of Sinorhiz_GL28CRW (RIP2Aa).

[0021] SEQ ID NO: 3 is the amino acid sequence of Rhizo_bactCRW (RIP3Aa).

[0022] SEQ ID NO:4 is the amino acid sequence of Rhiz_SPYCRW (RIP4Aa).

[0023] SEQ ID NO:5 represents the variant RIP3Aa amino acid sequence.

[0024] SEQ ID NO:6 is the amino acid sequence of RIP3Aa-I50L.

[0025] SEQ ID NO:7 is the amino acid sequence of RIP3Aa-I53L.

[0026] SEQ ID NO: 8 is the amino acid sequence of RIP3Aa-I56L.

[0027] SEQ ID NO:9 is the RIP3Aa-A62C amino acid sequence.

[0028] SEQ ID NO: 10 is the amino acid sequence of RIP3Aa-A62L.

[0029] SEQ ID NO: 11 is the amino acid sequence of RIP3Aa-A64C.

[0030] SEQ ID NO: 12 is the amino acid sequence of RIP3Aa-A64L.

[0031] SEQ ID NO: 13 is the amino acid sequence of RIP3Aa-I81L.

[0032] SEQ ID NO: 14 is the amino acid sequence of RIP3Aa-I126L.

[0033] SEQ ID NO: 15 is the amino acid sequence of RIP3Aa-I153L.

[0034] SEQ ID NO: 16 is the amino acid sequence of RIP3Aa-I169L.

[0035] SEQ ID NO: 17 is the amino acid sequence of RIP3Aa-I185L.

[0036] SEQ ID NO: 18 is the amino acid sequence of RIP3Aa-I207L.

[0037] SEQ ID NO: 19 is the amino acid sequence of RIP3Aa-I219L.

[0038] SEQ ID NO:20 is the amino acid sequence of RIP3Aa-I275L.

[0039] SEQ ID NO:21 is the SUMO-RIP1Aa amino acid sequence.

[0040] SEQ ID NO:22 is the nucleotide sequence of Ensif_aridCRW(rip1Aa).

[0041] SEQ ID NO:23 is the Sinorhiz_GL28CRW (rip2Aa) nucleotide sequence.

[0042] SEQ ID NO:24 is the nucleotide sequence of Rhizo_bactCRW(rip3Aa).

[0043] SEQ ID NO:25 is the nucleotide sequence of Rhiz_SPYCRW (rip4Aa).

[0044] SEQ ID NO: 26 is the nucleotide sequence of rip1Aa optimized for E. coli.

[0045] SEQ ID NO: 27 is the nucleotide sequence of rip2Aa optimized for E. coli.

[0046] SEQ ID NO:28 is the nucleotide sequence of rip3Aa optimized for E. coli.

[0047] SEQ ID NO: 29 is the nucleotide sequence of rip4Aa optimized for E. coli.

[0048] SEQ ID NO: 30 is the nucleotide sequence optimized for rip3Aa-I50L Escherichia coli.

[0049] SEQ ID NO:31 is the nucleotide sequence optimized for rip3Aa-I53L Escherichia coli.

[0050] SEQ ID NO:32 is the nucleotide sequence optimized for rip3Aa-I56L Escherichia coli.

[0051] SEQ ID NO: 33 is the nucleotide sequence optimized for rip3Aa-A62C Escherichia coli.

[0052] SEQ ID NO: 34 is the nucleotide sequence optimized for rip3Aa-A62L Escherichia coli.

[0053] SEQ ID NO:35 is the nucleotide sequence optimized for rip3Aa-A64L Escherichia coli.

[0054] SEQ ID NO:36 is the nucleotide sequence optimized for rip3Aa-A64C Escherichia coli.

[0055] SEQ ID NO: 37 is the nucleotide sequence optimized for rip3Aa-I81L Escherichia coli.

[0056] SEQ ID NO:38 is the nucleotide sequence optimized for rip3Aa-I126L Escherichia coli.

[0057] SEQ ID NO:39 is the nucleotide sequence optimized for rip3Aa-I153L Escherichia coli.

[0058] SEQ ID NO:40 is the nucleotide sequence optimized for rip3Aa-I169L Escherichia coli.

[0059] SEQ ID NO:41 is the nucleotide sequence optimized for rip3Aa-I185L Escherichia coli.

[0060] SEQ ID NO:42 is the nucleotide sequence optimized for rip3Aa-I207L Escherichia coli.

[0061] SEQ ID NO:43 is the nucleotide sequence optimized for rip3Aa-I219L Escherichia coli.

[0062] SEQ ID NO:44 is the nucleotide sequence optimized for rip3Aa-I275L Escherichia coli.

[0063] SEQ ID NO:45 is the SUMO-rip1Aa nucleotide sequence.

[0064] SEQ ID NO:46 is the maize-optimized nucleotide sequence of rip1Aa.

[0065] SEQ ID NO:47 is the maize-optimized nucleotide sequence of rip2Aa.

[0066] SEQ ID NO:48 is the maize-optimized nucleotide sequence of rip3Aa.

[0067] SEQ ID NO:49 is the maize-optimized nucleotide sequence of rip4Aa.

[0068] SEQ ID NO:50 comprises the RIP1Aa cytotoxin domain.

[0069] SEQ ID NO:51 comprises the RIP2Aa cytotoxin domain.

[0070] SEQ ID NO:52 comprises the RIP3Aa cytotoxin domain.

[0071] SEQ ID NO:53 comprises the RIP4Aa cytotoxin domain. DETAILED DESCRIPTION

[0072] This description is not intended to be an exhaustive list of all the different ways in which the present invention may be implemented, or of all the features that may be added to the present invention. For example, features described with respect to one embodiment may be incorporated into other embodiments, and features described with respect to a particular embodiment may be deleted from that embodiment. Therefore, the present invention contemplates that, in some embodiments of the present invention, any feature or combination of features set forth herein may be excluded or omitted. In addition, in view of this disclosure, numerous variations of the various embodiments suggested herein and additions will be apparent to those skilled in the art without departing from the present invention. Therefore, the following description is intended to set forth some specific embodiments of the present invention, and does not exhaustively describe all permutations, combinations, and variations thereof.

[0073] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. The terms used in the description of the invention herein are for the purpose of describing particular embodiments only and are not intended to limit the invention.

[0074] All publications, patent applications, patents, and other references cited herein are incorporated by reference in their entirety for the teachings of the pertinent sentence and / or paragraph to which the reference is addressed.

[0075] The nucleotide sequences provided herein are presented from left to right in the 5' to 3' direction and are represented using the standard code representing nucleotide bases as described in 37 CFR §§1.821-1.825 and World Intellectual Property Organization (WIPO) Standard ST.25, e.g., adenine (A), cytosine (C), thymine (T), and guanine (G).

[0076] Amino acids are also indicated using WIPO Standard ST.25, for example: Alanine (Ala; A), Arginine (Arg; R), Asparagine (Asn; N), Aspartic acid (Asp; D), Cysteine ​​(Cys; C), Glutamine (Gln; Q), Glutamic acid (Glu; E), Glycine (Gly; G), Histidine (His; H), Isoleucine (Ile; 1), Leucine (Leu; L), Lysine (Lys; K), Methionine (Met; M), Phenylalanine (Phe; F), Proline (Pro; P), Serine (Ser; S), Threonine (Thr; T), Tryptophan (Trp; W), Tyrosine (Tyr; Y), and Valine (Val; V). As used herein, "X" or "Xaa" in an amino acid sequence indicates that the amino acid at that position may be any of the 20 known amino acids or may be any of the amino acids listed herein.

[0077] Unless the context indicates otherwise, it is expressly contemplated that the various features of the invention described herein may be used in any combination. Moreover, the present invention also contemplates that in some embodiments of the present invention, any feature or combination of features stated herein may be excluded or omitted. For example, if the specification states that a composition comprises components A, B, and C, it is expressly contemplated that any one or combination of A, B, or C may be omitted and abandoned, singly or in any combination.

[0078] definition

[0079] As used in accordance with this disclosure, unless otherwise indicated, the following terms shall be understood to have the following meanings:

[0080] As used herein and in the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a plant" is a reference to one or more plants and includes equivalents thereof known to those skilled in the art, and so forth.

[0081] As used herein, the term "and / or" refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative ("or").

[0082] The term "about" is used herein to mean approximately, roughly, about, or around. When the term "about" is used in conjunction with a numerical range, it defines the range by extending the boundaries above and below the stated values. Typically, the term "about" is used herein to define a numerical value to a variation of 20%, preferably 10% above and below (higher or lower) above and below the specified value. With respect to temperature, the term "about" means ±1°C, preferably ±0.5°C. When the term "about" is used in the context of the present invention (e.g., in combination with a temperature or molecular weight value), an exact value (i.e., no "about") is preferred.

[0083] As used herein, phrases such as “between about X and Y,” “between about X and about Y,” “from X to Y,” and “from about X to about Y” (and similar phrases) should be interpreted to include X and Y unless the context indicates otherwise.

[0084] As used herein, the term "amplification" means using at least one nucleic acid molecule as a template to construct multiple copies of a nucleic acid molecule or multiple copies complementary to the nucleic acid molecule. Amplification systems include polymerase chain reaction (PCR) systems, ligase chain reaction (LCR) systems, nucleic acid sequence-based amplification (NASBA, Cangene, Mississauga, Ontario), Q-β replicase systems, transcription-based amplification systems (TAS) and strand displacement amplification (SDA). See, for example, Diagnostic Molecular Microbiology: Principles and Applications, PERSING et al., American Society for Microbiology, Washington, DC, (1993). The product of amplification is referred to as "amplicon".

[0085] The "activity" of the insecticidal protein of the present disclosure means that the insecticidal protein acts as an orally active insect control agent, has toxic effects, and / or is able to interfere with or prevent insect feeding, which may or may not cause the death of the insect. When the insecticidal protein of the present disclosure is delivered to an insect, this result is typically the death of the insect, or the insect does not feed on a source from which the insecticidal protein is available to the insect. "Pesticide" is defined as a toxic biological activity that is able to control harmful organisms (such as insects, nematodes, fungi, bacteria or viruses), preferably by killing or destroying them. "Insecticidal" is defined as a toxic biological activity that is able to control insects, preferably by killing them. "Pesticide" is an agent with pesticidal activity. "Insecticide" is an agent with insecticidal activity.

[0086] As used herein, the term "chimeric construct" or "chimeric gene" or "chimeric polynucleotide" or "chimeric nucleic acid" (or similar terms) refers to a construct or molecule comprising two or more polynucleotides from different sources assembled into a single nucleic acid molecule. The term "chimeric construct", "chimeric gene", "chimeric polynucleotide" or "chimeric nucleic acid" refers to any construct or molecule that contains, but is not limited to (1) polynucleotides (e.g., DNA) including regulatory polynucleotides and coding polynucleotides that are not found together in nature (i.e., at least one polynucleotide in the construct is heterologous relative to at least one of its other polynucleotides), or (2) polynucleotides that encode protein portions that are not naturally contiguous, or (3) promoter portions that are not naturally contiguous. In addition, a chimeric construct, chimeric gene, chimeric polynucleotide or chimeric nucleic acid may comprise regulatory polynucleotides and coding polynucleotides derived from different sources, or comprise regulatory polynucleotides and coding polynucleotides derived from the same source but arranged in a manner different from that found in nature. In some embodiments of the present disclosure, a chimeric construct, chimeric gene, chimeric polynucleotide or chimeric nucleic acid comprises an expression cassette comprising a polynucleotide of the present disclosure under the control of a regulatory polynucleotide, particularly a regulatory polynucleotide functional in plants or bacteria.

[0087] A "coding sequence" is a nucleic acid sequence that is transcribed into RNA (such as mRNA, rRNA, tRNA, snRNA, sense RNA or antisense RNA). Preferably, the RNA is then translated in an organism to produce a protein.

[0088] "Controlling" insects means inhibiting the ability of insect pests to survive, grow, feed, and / or reproduce through toxic effects, or limiting insect-related damage or losses in crop plants. "Controlling" insects may or may not mean killing the insects, although it preferably means killing the insects.

[0089] As used herein, "codon optimized" sequence means the following nucleotide sequence, wherein these codons are selected to reflect the specific codon preference that the host cell or organism may have. This is typically accomplished in such a way that the amino acid sequence of the polypeptide encoded by the nucleotide sequence to be optimized is maintained. In certain embodiments, the DNA sequence of the recombinant DNA construct includes a sequence that has been codon optimized for the cell (for example, animal, plant or fungal cell) to be expressed therein for the construct. For example, the construct to be expressed in a plant cell can have its whole or part of its sequence (for example, the first gene suppression element or gene expression element) codon optimized for expression in a plant. See, for example, U.S. Patent number 6,121,014, incorporated herein by reference.

[0090] The terms “comprises” or “comprising” when used in the present specification indicate the presence of stated features, integers, steps, operations, elements, or components, but do not exclude the presence or addition of one or more other features, integers, steps, operations, elements, components, or groups thereof.

[0091] As used herein, the transitional phrase "consisting essentially of (and grammatical variations) means that the scope of the claim is to be interpreted as encompassing the specified materials or steps recited in the claim and those that do not materially alter one or more of the basic and novel characteristics of the claimed invention. Thus, the term "consisting essentially of" when used in the claims of the present invention is not intended to be interpreted as being equivalent to "comprising".

[0092] In the context of the present disclosure, "corresponding to or corresponds to" means that when the amino acid sequences of variant or homolog proteins are aligned with each other, the amino acids "corresponding to" certain enumerated positions in the variant or homolog protein are those aligned with these positions in the reference protein, but not necessarily in these exact numerical positions relative to the specific reference amino acid sequence of the present disclosure. For example, if SEQ ID NO: 1 is the reference sequence and is aligned with SEQ ID NO: 2, then the amino acid Leu (L) at position 153 (L153) of SEQ ID NO: 2 "corresponds to" Leu (L) at position 154 (L154) of SEQ ID NO: 1, or, for example, Asn (N) at position 2 (N2) of SEQ ID NO: 2 "corresponds to" Ala (A) A2 of SEQ ID NO: 1.

[0093] "Delivery" of a composition or toxic protein means that the composition or toxic protein contacts the insect, which promotes oral ingestion of the composition or toxic protein, resulting in toxic effects and control of the insect. The composition or toxic protein can be delivered in many recognized ways, including but not limited to transgenic plant expression, one or more formulated protein compositions, one or more sprayable protein compositions, bait matrix, or any other art-recognized protein delivery system.

[0094] The term "domain" refers to a group of amino acids that are conserved at specific positions along the alignment of the sequences of evolutionarily related proteins. Although amino acids at other positions may differ between homologs, highly conserved amino acids at specific positions indicate amino acids that are likely to be essential in the structure, stability or function of the protein. They are identified by their high conservation in the aligned sequences of a family of protein homologs and can be used as identifiers to determine whether any polypeptide in question belongs to a previously identified polypeptide group.

[0095] "Insect control effective amount" means a concentration of an insecticidal protein that inhibits the ability of insects to survive, grow, feed and / or reproduce through toxic effects, or limits insect-related damage or crop plant losses. An "effective insect control amount" may or may not mean killing insects, although it preferably means killing insects.

[0096] As used herein, "expression cassette" means a nucleic acid sequence capable of directing the expression of a specific nucleotide sequence in an appropriate host cell, comprising a promoter operably connected to a target nucleotide sequence, which is operably connected to a termination signal. It also typically comprises a sequence required for the appropriate translation of the nucleotide sequence. The expression cassette comprising the target nucleotide sequence may have at least one of its components, which is heterologous relative to at least one of its other components. The expression cassette may also be a naturally occurring expression cassette that has been obtained in a recombinant form useful for heterologous expression. However, typically, the expression cassette is heterologous relative to the host, i.e., the specific nucleic acid sequence of the expression cassette is not naturally present in the host cell, and must have been introduced into the host cell or the ancestor of the host cell through a transformation event. The expression of the nucleotide sequence in the expression cassette may be under the control of a constitutive promoter or an inducible promoter, which initiates transcription only when the host cell is exposed to some specific external stimuli. In the case of a multicellular organism (such as a plant), the promoter may also be specific for a specific tissue, or an organ, or a developmental stage.

[0097] The expression cassette comprising the nucleotide sequence of interest may be chimeric, meaning that at least one of its components is heterologous relative to at least one of its other components. The expression cassette may also include a native promoter driving its native gene, but has been obtained in a recombinant form useful for heterologous expression. This use of the expression cassette makes it so that it does not naturally occur in the cell into which it is introduced.

[0098] The expression cassette can also optionally include a transcription and / or translation termination region (i.e., termination region) that plays a role in plants. A variety of transcription terminators are available for use in the expression cassette and are responsible for transcription termination and correct mRNA polyadenylation when exceeding the heterologous nucleotide sequence of interest. The termination region can be natural for the transcription initiation region, can be natural for the nucleotide sequence of interest that is operably connected, can be natural for the plant host, or can be derived from another source (i.e., foreign or heterologous for promoter, nucleotide sequence of interest, plant host, or any combination thereof). Suitable transcription terminators include, but are not limited to, CAMV 35S terminator, tml terminator, nopaline synthase terminator, and / or pea rbcs E9 terminator. These terminators can be used in both monocotyledons and dicotyledons. In addition, the natural transcription terminator of the coding sequence can be used. Any terminator known to play a role in plants that can be used can be used in the context of this disclosure.

[0099] When used with reference to polynucleotides (such as genes, ORFs or parts thereof, or transgenics of plants), the term "expression" refers to the process of converting the genetic information encoded in a gene into RNA (e.g., mRNA, rRNA, tRNA or snRNA) by the "transcription" of a gene (i.e., via the enzymatic action of an RNA polymerase), and into a protein by the "translation" of mRNA in applicable cases (e.g., if the gene encodes a protein). Gene expression can be regulated at many stages of the process. For example, in the case of an antisense construct or a dsRNA construct, expression may refer to only the antisense RNA or only the transcription of the dsRNA, respectively. In an embodiment, "expression" refers to the transcription and stable accumulation of a sense (mRNA) or functional RNA. "Expression" may also refer to the production of a protein.

[0100] "Gene" is a restricted region that is located in the genome and comprises a coding nucleic acid sequence, and typically also comprises other major regulatory nucleic acids that are responsible for controlling the expression (i.e., transcription and translation) of the coding portion. A gene can also comprise other 5' and 3' untranslated sequences and termination sequences. Other possible elements are, for example, introns. As found in nature, the regulatory nucleic acid sequence of a gene may not be operably connected with the associated nucleic acid sequence under normal circumstances, and therefore can not be a mosaic gene.

[0101] "Target gene" refers to any nucleic acid molecule that, when transferred to an organism, such as a bacterium or a plant, confers a desired trait (such as antibiotic resistance, virus resistance, insect resistance, disease resistance, or resistance to other harmful organisms, herbicide tolerance, abiotic stress tolerance, male sterility, modified fatty acid metabolism, modified carbohydrate metabolism, improved nutritional value, improved performance in an industrial process, or altered reproductive capacity) on the bacterium or plant. "Target gene" can also be a gene transferred to a bacterium or a plant for producing a commercially valuable enzyme or metabolite in the plant.

[0102] "Heterologous" nucleic acid sequence or nucleic acid molecule is a nucleic acid sequence or nucleic acid molecule that is not naturally associated with the host cell into which the nucleic acid sequence is introduced, including multiple copies of non-naturally occurring nucleic acid sequences. Heterologous nucleic acid sequence or nucleic acid molecule can comprise chimeric sequences, such as chimeric expression cassettes, in which promoters and coding regions are derived from polygenic organisms. Promoter sequences can be constitutive promoter sequences, tissue-specific promoter sequences, chemically inducible promoter sequences, wound-inducible promoter sequences, stress-inducible promoter sequences, or developmental stage-specific promoter sequences.

[0103] A "homologous" nucleic acid sequence is a nucleic acid sequence naturally associated with a host cell into which it is introduced.

[0104] "Homologous recombination" is the reciprocal exchange of nucleic acid fragments between homologous nucleic acid molecules.

[0105] As used herein, "hypothetical protein" refers to a protein whose existence has been predicted but lacks experimental evidence of its expression in vivo. Sequencing a genome or organism (such as bacteria or plants) usually produces a large number of predicted open reading frames, and the functions of these reading frames are not easy to specify. These proteins, whether isolated or conservative hypothetical proteins, account for about 20% to about 40% of the proteins encoded in each newly sequenced genome. Even if there is enough evidence to show that the product of the gene is expressed, it is difficult to specify its function through technologies such as microarrays and mass spectrometry because it lacks identity with the protein sequence with annotated biochemical functions. Generally, most protein sequences are inferred from computational analysis of genomic DNA sequences. Hypothetical proteins are usually created by gene prediction software during genome analysis. When bioinformatics tools for gene identification find large open reading frames without characteristic homologs in protein databases, such tools usually return the name "hypothetical protein" as an annotation.

[0106] The term "motif" or "consensus sequence" or "signature" refers to a short conserved region in the sequence of evolutionarily related proteins. A motif is often a highly conserved portion of a domain, but may also include only a portion of a domain, or be located outside a conserved domain (if all of the amino acids of the motif are located outside the defined domain).

[0107] In the context of two nucleic acid or amino acid sequences, the term "identity" or "identical" or "substantially identical" refers to two or more sequences or subsequences that have at least 60%, preferably at least 80%, more preferably 90%, even more preferably 95%, and most preferably at least 99% nucleotide or amino acid residue identity when compared and aligned for maximum correspondence, as measured using one of the following sequence comparison algorithms or by visual inspection. Preferably, the basic identity is present in the entire region with a length of at least about 50 residues or bases, more preferably in the entire region of at least about 100 residues or bases, and most preferably these sequences are substantially identical at least about 150 residues or bases. In a particularly preferred embodiment, these sequences are substantially identical throughout the length of the coding region. In addition, substantially identical nucleic acid or amino acid sequences perform substantially the same function.

[0108] For sequence comparison, typically, one sequence serves as a reference sequence for comparison with a test sequence. When using a sequence comparison algorithm, the test sequence and reference sequence are input into a computer (if necessary, subsequence coordinates are specified), and the parameters of the sequence algorithm program are specified. Then, the sequence comparison algorithm calculates the sequence identity percentage of this or these test sequences relative to the reference sequence based on the specified program parameters.

[0109] Optimal alignment of sequences for comparison can be performed, for example, by the local homology algorithm of Smith and Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman and Wunsch, J. Mol. Biol. 48:443 (1970), by the search by similarity method of Pearson and Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Street, Madison, Wisconsin), or by visual inspection (see generally Ausubel et al., below).

[0110] An example of an algorithm suitable for determining percentages of sequence identity and sequence similarity is the BLAST algorithm, which is described in the following literature: Altschul et al., J. Mol. Biol. [Molecular Biology] 215: 403-410 (1990). Software for performing BLAST analysis is available to the public through the National Center for Biotechnology Information (the National Center for Biotechnology Information, US National Library of Medicine, 8600 Rockville Pike, Bethesda, Maryland 20894). This algorithm involves first identifying high-scoring sequence pairs (HSPs) by identifying short words with length W in the query sequence, which match or meet some positive threshold score T when compared with words with the same length in the database sequence. T is referred to as the neighboring word score threshold (Altschul et al., 1990). These initial neighboring word hits serve as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. For nucleotide sequences, the cumulative score is calculated using the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatched residues; always <0). For amino acid sequences, a scoring matrix is ​​used to calculate the cumulative score. Extension of the word hits in each direction is stopped when the cumulative alignment score decreases by the amount X from its maximum achieved value; when the cumulative score approaches 0 or below due to the accumulation of one or more negative-scoring residue alignments; or when the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses a word length (W) of 11, an expectation (E) of 10, a cutoff of 100, M=5, N=-4, and a comparison of both chains as defaults. For amino acid sequences, the BLASTP program uses as defaults a wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff, Proc. Natl. Acad Sci. USA 89:10915 (1989)).

[0111] In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin and Altschul, Proc. Nat'l. Acad. Sci. USA 90:5873-5787 (1993)). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability that a match between two nucleotide or amino acid sequences would occur by chance. For example, a test nucleic acid sequence is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid sequence to the reference nucleic acid sequence is less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.

[0112] Another indication that two nucleic acid sequences are substantially identical is that the two molecules hybridize to each other under stringent conditions. The phrase "specific hybridization" refers to a molecule that binds, doubles or hybridizes only to a specific nucleotide sequence under stringent conditions when the sequence is present in a complex mixture (e.g., total cellular) DNA or RNA. "Substantially binds" refers to complementary hybridization between a probe nucleic acid and a target nucleic acid, and encompasses a small amount of mismatches that can be accommodated by reducing the stringency of the hybridization medium to achieve the desired detection of the target nucleic acid sequence.

[0113] In the context of nucleic acid hybridization experiments (such as DNA hybridization and RNA hybridization), "stringent hybridization conditions" and "stringent hybridization wash conditions" are sequence-dependent and are different under different environmental parameters. Longer sequences hybridize specifically at higher temperatures. A broad guide to nucleic acid hybridization is found in Tijssen (1993) Laboratory Techniques in Biochemistry and Molecular Biology-Hybridization with Nucleic Acid Probes part I chapter 2 "Overview of principles of hybridization and the strategy of nucleic acid probe assays" Elsevier, New York. Generally, highly stringent hybridization and wash conditions are selected to be higher than the thermal melting point (T) for the specific sequence at a defined ionic strength and pH. m ) is about 5° C. Typically, under "stringent conditions," a probe will hybridize to its target sequence, but not to other sequences.

[0114] T mIt is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe. Very stringent conditions are selected to be equal to the T for a specific probe. m . An example of stringent hybridization conditions for hybridization of complementary nucleic acids (which have more than 100 complementary residues on the filter in a Southern or Northern blot) is 50% formamide at 42°C with 1 mg of heparin, and the hybridization is carried out overnight. An example of high stringency washing conditions is 0.15M NaCl at 72°C for about 15 minutes. An example of stringent washing conditions is a 0.2×SSC wash at 65°C for 15 minutes (see, Sambrook, below, for a description of SSC buffer). Typically, a high stringency wash is preceded by a low stringency wash to remove background probe signals. An example of a medium stringency wash for, for example, a duplex of more than 100 nucleotides is 1×SSC at 45°C for 15 minutes. An example of a low stringency wash for, for example, a duplex of more than 100 nucleotides is 4-6×SSC at 40°C for 15 minutes. For short probes (e.g., about 10 to 50 nucleotides), stringent conditions typically involve a salt concentration of less than about 1.0 M Na ions, typically about 0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3, and the temperature is typically at least about 30°C. Stringent conditions can also be achieved by adding destabilizing agents such as formamide. In general, a signal-to-noise ratio of 2-fold (or higher) observed in a particular hybridization assay compared to an unrelated probe indicates that specific hybridization has been detected. If the proteins encoded by nucleic acids that do not hybridize to each other under stringent conditions are substantially identical, they are still substantially identical. For example, this occurs when a copy of a nucleic acid is created using the maximum codon degeneracy allowed by the genetic code.

[0115] The following are examples of settings of hybridization / washing conditions that can be used to clone homologous nucleotide sequences that are substantially identical to the reference nucleotide sequences of the present disclosure: a reference nucleotide sequence preferably hybridizes to the reference nucleotide sequence under the following conditions: in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO4, 1 mM EDTA at 50°C, and washing in 2×SSC, 0.1% SDS at 50°C; more desirably in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO4, 1 mM EDTA at 50°C, and washing in 1×SSC, 0.1% SDS at 50°C; still more desirably in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO4, 1 mM EDTA at 50°C, and washing in 0.5×SSC, 0.1% SDS at 50°C; preferably in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO4, 1 mM EDTA at 50°C, and washing in 0.5×SSC, 0.1% SDS at 50°C. EDTA at 50°C, and washing in 0.1X SSC, 0.1% SDS at 50°C; more preferably in 7% sodium dodecyl sulfate (SDS), 0.5M NaPO4, 1 mM EDTA at 50°C, and washing in 0.1X SSC, 0.1% SDS at 65°C.

[0116] Another indication that two nucleic acid sequences or proteins are substantially identical is that the protein encoded by the first nucleic acid reacts immunologically with or specifically binds to the protein encoded by the second nucleic acid. Thus, a protein is typically substantially identical to a second protein, for example, where the two proteins differ only in conservative substitutions.

[0117] The term "isolated" nucleic acid molecule, polynucleotide or protein is a nucleic acid molecule, polynucleotide or protein that is no longer present in its natural environment. The isolated nucleic acid molecule, polynucleotide or protein of the present disclosure may exist in a purified form, or may be present in a recombinant host, such as a transgenic bacterium or a transgenic plant. Therefore, the claims to "isolated" nucleic acid molecules as listed herein cover nucleic acid molecules when the nucleic acid molecule is contained in a transgenic plant genome.

[0118] A "nucleic acid molecule" or "nucleic acid sequence" is a segment of single-stranded or double-stranded DNA or RNA that can be isolated from any source. In the context of the present disclosure, a nucleic acid molecule is typically a segment of DNA. In some embodiments, the nucleic acid molecule of the present disclosure is an isolated nucleic acid molecule.

[0119] "Operably linked" refers to the association of polynucleotides on a single nucleic acid fragment such that the function of one affects the function of the other. For example, when a promoter is able to affect the expression of a coding polynucleotide or a functional RNA (i.e., the coding polynucleotide or the functional RNA is under the transcriptional control of the promoter), the promoter is operably linked to the coding polynucleotide or the functional RNA. A coding polynucleotide in either the sense or antisense orientation can be operably linked to a regulatory polynucleotide.

[0120] As used herein, "killing pests", "killing insects", etc. refers to the ability of the RIP of the present disclosure to control pests or the amount of the RIP that can control pests as defined herein. Thus, the pesticidal RIP can kill or inhibit the ability of pests (e.g., insect pests) to survive, grow, feed, or reproduce.

[0121] The terms "protein," "peptide," and "polypeptide" are used interchangeably herein.

[0122] A "plant" is any plant at any stage of development, especially seed plants. Exemplary plants include, but are not limited to, corn (Zea mays), canola (Brassica napus, Brassica rapa ssp.), alfalfa (Medicago saliva), rice (Oryza sativa, including but not limited to indica and / or japonica), rapeseed (Brassica napus), rye (rye, Secale cereale), sorghum (Sorghum bicolor, Sorghum vulgare), sunflower (Helianthus annus), wheat (Triticum aestivum), soybean (soybean, Glycine max), tobacco (Nicotiana tabacum), potato (Solanum tuberosum), peanut (Arachis hypogaea), cotton (Gossypium hirsutum), sweet potato (Ipomoea candidum), batatas), cassava (Manihot esculenta), coffee (Cofea spp.), coconut (Cocos nucifera), pineapple (Ananas comosus), citrus (Citrus spp.), cocoa (Theobroma cacao), tea (Camellia sinensis), banana (Musas spp.), avocado (Persea americana), fig (Ficus casica), guava (Psidium guajava), mango (Mangifera indica), olive (Olea europaea), papaya (Carica papaya), cashew (Anacardium occidentale), macadamia (macadamia integrifolia), almond (Prunus amygdalus), sugar beet (Beta vulgaris), apple (Malus pumila), blackberry (Rubus), strawberry (Fragaria), walnut (Juglans regia), grape (Vitis vinifera), apricot (Prunus armeniaca), cherry (Prunus), peach (Prunus persica), persica), plum (Prunus domestica), pear (Pyrus communis), watermelon (Citrullus vulgaris), duckweed (Lemna spp.), oats (Avena sativa), barley (Hordium vulgare), vegetables, ornamentals, conifers, and turf grasses (e.g., ornamental, recreational, or forage), and biomass grasses (e.g., switchgrass and miscanthus).

[0123] Vegetables include, but are not limited to, Solanum species (e.g., tomatoes, Lycopersicon esculentum), lettuce (e.g., Lactuea sativa), carrots (Caucus carota), cauliflower (Brassica oleracea), celery (Apium graveolens), eggplant (eggplant, Solanum melongena), asparagus (Asparagus officinalis), okra (Abelmoschus esculentus), green beans (Phaseolus vulgaris), lima beans (Phaseolus limensis), peas (Lathyrus spp.), members of the genus Cucurbita such as C. hubbard, winter squash (C. moschata), zucchini (C. pepo), C. crookneck, C. argyrosperma, C. argyrosperma ssp sororia, C. digitata, C. ecuadorensis, C. foetidissima, C. lundelliana, and C. martinezii, and members of the genus Cucumis such as cucumber (Cucumber, Cucumis sativus), cantaloupe (C. cantalupensis), and muskmelon (C. melo).

[0124] Ornamental plants include, but are not limited to, azaleas (Rhododendron spp.), hydrangeas (Macrophylla hydrangea), hibiscus (Hibiscus rosasanensis), roses (Rosa spp.), tulips (Tulipa spp.), daffodils (Narcissus spp.), petunias (Petunia hybrida), carnations (Dianthus caryophyllus), poinsettias (Euphorbia pulcherima), and chrysanthemums.

[0125] Conifers that can be used to practice the present disclosure include, for example, pines such as loblolly pine (Pinustaeda), slash pine (Pinus elliotii), jack pine (Pinus ponderosa), lodgepole pine (Pinus contorta), and Monterey pine (Pinus radiata); Douglas-fir (Pseudotsuga menziesii); western hemlock (Tsuga canadensis); Sitka spruce (Picea glauca); redwood (Sequoia sempervirens); firs such as silver fir (Abies amabilis) and balsam fir (Abies balsamea); and cedars such as western arborvitae (Thuja plicata) and Alaska yellow-cedar (Chamaecyparis nootkatensis).

[0126] Turf grasses include, but are not limited to, zoysia, bentgrass, fescue, bluegrass, Augustine grass, Bermuda grass, bufallograsses, ryegrass, and orchardgrass.

[0127] Also included are plants that serve primarily as laboratory models, such as Arabidopsis thaliana.

[0128] A "plant cell" is a structural and physiological unit of a plant, comprising a protoplast and a cell wall. A plant cell may be in the form of an isolated single cell or a cultured cell, or as part of a higher organization unit such as, for example, a plant tissue, a plant organ, or a whole plant.

[0129] "Plant cell culture" means a culture of plant units such as, for example, protoplasts, cell culture cells, cells in plant tissue, pollen, pollen tubes, ovules, embryo sacs, zygotes, and embryos at various stages of development.

[0130] "Plant material" refers to leaves, stems, roots, flowers or flower parts, fruits, pollen, egg cells, zygotes, seeds, cuttings, cell or tissue cultures, or any other part or product of a plant.

[0131] A "plant organ" is a distinct and distinct structured and differentiated part of a plant, such as a root, stem, leaf, flower bud or embryo.

[0132] As used herein, "plant tissue" means a group of plant cells organized into structural and functional units. Any plant tissue in a plant or in culture is included. This term includes, but is not limited to, whole plants, plant organs, plant seeds, tissue cultures, and any group of plant cells organized into structural and / or functional units. The use of this term in conjunction with or alone with any specific type of plant tissue as listed above or otherwise covered by this definition is not intended to exclude any other type of plant tissue.

[0133] "Polynucleotide" refers to a polymer composed of many nucleotide monomers covalently bonded in a chain. Such "polynucleotides" include DNA, RNA, modified oligonucleotides (e.g., oligonucleotides containing bases atypical for biological RNA or DNA, such as 2'-O-methylated oligonucleotides), etc. In some embodiments, nucleic acids or polynucleotides can be single-stranded, double-stranded, multi-stranded, or a combination thereof. Unless otherwise indicated, in addition to any explicitly indicated polynucleotides, the specific nucleic acids or polynucleotides of the present disclosure optionally also contain or encode complementary polynucleotides.

[0134] "Polynucleotide of interest" refers to any polynucleotide that, when transferred into an organism (e.g., a plant), confers a desired trait to that organism, such as insect resistance, disease resistance, herbicide tolerance, antibiotic resistance, improved nutritional value, improved performance in an industrial process, production of a commercially valuable enzyme or metabolite, or altered reproductive capacity.

[0135] "Promoter" is a non-translated DNA sequence upstream of the coding region that contains an RNA polymerase binding site and initiates transcription of the DNA. The promoter region may also include other elements that act as regulators of gene expression.

[0136] As used herein, the term "recombination" refers to the following form of a nucleic acid molecule (e.g., DNA or RNA) or protein or organism, which is not usually found in nature and is produced by human intervention for this reason. As used herein, "recombinant nucleic acid molecule" is a nucleic acid molecule comprising a combination of polynucleotides, which do not coexist naturally and are the result of human intervention, for example, a nucleic acid molecule composed of a combination of at least two polynucleotides heterologous to each other, or an artificially synthesized (e.g., a polynucleotide synthesized using an assembled nucleotide sequence) and comprising a nucleic acid molecule of a polynucleotide different from a polynucleotide usually present in nature, or a nucleic acid molecule comprising a transgenic artificially incorporated into the genomic DNA of a host cell and the host cell genome-related flanking DNA. Another example of a recombinant nucleic acid molecule is a DNA molecule produced by inserting a transgenic into the genomic DNA of a plant, which can ultimately lead to the expression of a recombinant RNA / or protein molecule in the organism. As used herein, "recombinant plant" is a plant that is not usually present in nature, is the result of human intervention, and contains transgenic and / or heterologous nucleic acid molecules incorporated into its genome. Due to such genome changes, recombinant plants are significantly different from related wild-type plants.

[0137] "Regulatory elements" refer to sequences involved in controlling the expression of a nucleotide sequence. Regulatory elements include promoters operably linked to a target nucleotide sequence and termination signals. They also typically encompass sequences required for proper translation of the nucleotide sequence.

[0138] Rhizobium family insecticidal protein (RIP) is a protein encoded by a gene found in at least the genome of Rhizobium family bacteria, which contains a cytotoxic domain and is active against insect pests at least in the genus Radix Glechomae. A "variant RIP" is an engineered RIP by replacing and / or deleting at least one amino acid found in a naturally occurring RIP and / or inserting an amino acid into the RIP sequence. As used herein, unless otherwise indicated, when the term "Rhizobium family insecticidal protein" or "RIP" is used alone, it is meant to include both naturally occurring proteins and engineered variant proteins.

[0139] The terms "substitution", "insertion" or "addition" and "deletion" are used herein with reference to amino acid or nucleotide sequences. "Substitution" refers to the replacement of one or more nucleotides or amino acids with different nucleotides or amino acids, respectively. "Insertion" or "addition" is a change in a nucleotide or amino acid sequence that results in the addition of one or more nucleotides or amino acid residues, respectively, compared to a naturally occurring sequence. "Deletion" is defined as a change in a nucleotide or amino acid sequence in which one or more nucleotides or amino acid residues, respectively, are absent. Amino acid substitutions are typically single residue substitutions; insertions will generally be on the order of about 1 to 20 amino acids, although significantly larger insertions can be tolerated. The range of deletions is about 1 to about 20 residues, although in some cases, deletions can be much larger. Substitution, deletion, insertion or any combination thereof can be used to obtain the final variant polypeptide. Typically, some amino acids are changed to minimize changes in the molecule. However, in some cases, greater changes can be tolerated. In certain embodiments, an amino acid substitution can be the result of replacing one amino acid with another amino acid having similar structure and / or chemical properties, such as replacing isoleucine with valine, i.e., a conservative amino acid substitution. Insertion or deletion can optionally be in the range of 1 to 5 amino acids. In an embodiment, substitutions can be made according to known "conservative substitutions". A "conservative substitution" refers to the substitution of an amino acid in one class with an amino acid in the same class, where the class is defined by common physicochemical amino acid side chain properties and high substitution frequencies in homologous proteins found in nature. In contrast, in certain embodiments, the substitution is non-conservative. A "non-conservative substitution" refers to the substitution of an amino acid in one class with an amino acid from another class.

[0140] "Transformation" is a process used to introduce heterologous nucleic acid into a host cell or organism. In specific embodiments, "transformation" means the stable integration of a DNA molecule into the genome (nuclear or plastid) of the organism of interest.

[0141] "Transformed / transgenic / recombinant" refers to a host organism such as a bacterium or plant into which a heterologous nucleic acid molecule has been introduced. The nucleic acid molecule can be stably integrated into the host's genome, or the nucleic acid molecule can also exist as an extrachromosomal molecule. Such extrachromosomal molecules can replicate autonomously. Transformed cells, tissues or plants should be understood to encompass not only the end product of the transformation process, but also its transgenic progeny. "Non-transformed", "non-transgenic" or "non-recombinant" hosts refer to wild-type organisms, such as bacteria or plants, that do not contain the heterologous nucleic acid molecule.

[0142] The present disclosure provides compositions and methods for controlling harmful insect pests. In particular, the present disclosure relates to insecticidal proteins and variants thereof, referred to herein as Rhizobiaceae insecticidal proteins (RIPs), which are active against at least Coleopteran insects, such as corn rootworms (Western corn rootworm; WCR), Diabrotica barbatus (Diabrotica barber) (northern corn rootworm; NCR) and / or cucumber rootworm subspecies eleven-spotted (southern corn rootworm; SCR) and / or other species of the genus Diabrotica (including the Mexican corn rootworm (Mexican corn rootworm; MCR) and the South American rootworm (cucurbit beetle). The inventors of the present invention have discovered that certain proteins described in the art as hypothetical proteins, which are said to be encoded in the genome of at least a gram-negative bacterium in the order Rhizobiales, are surprisingly insecticidal. More specifically, the coding sequences of these hypothetical proteins are present in the genome of bacteria of the family Rhizobiaceae. The family Rhizobiaceae is a family of the phylum Proteobacteria that includes multiple subgroups associated with plants, particularly plant roots. More specifically, the coding sequences of hypothetical proteins with insecticidal effects were found to be present in the genomes of bacteria of the family Rhizobiaceae of the genera Sinorhizobium, Swordworm, Rhizobium, and related genera. Even more specifically, the hypothetical proteins exemplified herein as insecticidal proteins The coding sequences of the white protein include, but are not limited to, those in the genome of Ensiferaridi strain (NCBI: txid1708715), Sinorhizobium species GL28 strain (NCBI: txid1358418), unclassified Rhizobiales bacterial strain (NCBI: txid1909294), and Rhizobium species SPY-1 strain (NCBI: txid2547961). After synthesizing nucleic acid molecules encoding the above-mentioned proteins and expressing the proteins in transgenic Escherichia coli bacteria, the inventors determined that the proteins described in the art as hypothetical proteins surprisingly have insecticidal activity, particularly against rootworm insect pests. Such insecticidal proteins are generally referred to herein as Rhizobium family insecticidal proteins (RIPs), and those specifically exemplified herein are designated as RIP1Aa (Enf_adiCRW; SEQ ID NO: 101). SEQ ID NO: 1), RIP2Aa (Sinorhiz_GL28CRW; SEQ ID NO: 2), RIP3Aa (Rhizo_bactCRW; SEQ ID NO: 3), and RIP4Aa (Rhiz_SPCRW; SEQ ID NO: 4). One skilled in the art will recognize that, using the teachings of the present disclosure, one skilled in the art can identify sequences related to those described above in, but not limited to, bacteria, nucleic acid molecules from environmental samples, and genomic databases, where such sequences may be designated as hypothetical, or they may have some other known function, etc. It is contemplated that such related sequences are included in the present disclosure.The skilled artisan will understand the meaning of the term "related sequences" after reading this disclosure.As described in further detail below, the RIPs of the present disclosure have a unique cytotoxin domain that confers activity against at least coleopteran insect pests.

[0143] The disclosure also relates to nucleic acids (the expression of which produces the RIPs of the disclosure), and to methods of making and using these RIPs to control insect pests. In certain non-limiting embodiments, expression of these nucleic acids produces insecticidal proteins that can be used to control at least coleopteran insects (such as western corn rootworms, northern corn rootworms, and / or southern corn rootworms), particularly when expressed in transgenic plants (such as transgenic corn plants).

[0144] In some non-limiting embodiments, the present disclosure encompasses nucleic acid molecules comprising, consisting essentially of, or consisting of a nucleotide sequence encoding a protein that is toxic to an insect pest (i.e., an insecticidal protein), wherein the nucleotide sequence (a) encodes a protein comprising an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NOs: 1-21, or a toxin fragment thereof; (b) encodes a protein comprising an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NOs: 1-21, or a toxin fragment thereof; NO: 22-49 or a toxin encoding fragment thereof having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity; or (c) is a synthetic sequence of (a) or (b) that has been codon optimized for expression in a transgenic organism. In other embodiments, the insecticidal protein comprises, consists essentially of, or consists of the amino acid sequence of any one of SEQ ID NO: 1-21 or a toxic fragment thereof. In other embodiments, the nucleotide sequence comprises, consists essentially of, or consists of any one of SEQ ID NO: 22-49 or a toxin encoding fragment thereof.

[0145] In some non-limiting embodiments, the present disclosure encompasses chimeric genes comprising a heterologous promoter operably linked to a nucleic acid molecule comprising, consisting essentially of, or consisting of: a nucleotide sequence encoding a protein toxic to an insect pest, wherein the nucleotide sequence (a) encodes a protein comprising an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NOs: 1-21, or a toxin fragment thereof; (b) encodes a protein comprising an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NOs: 1-21, or a toxin fragment thereof; NO: 22-49 or a toxin encoding fragment thereof has at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity; or (c) is a synthetic sequence of (a) or (b) that has been codon optimized for expression in a transgenic organism. In other embodiments, the insecticidal protein comprises the amino acid sequence of any one of SEQ ID NO: 1-21 or a toxic fragment thereof. In other embodiments, the nucleotide sequence comprises any one of SEQ ID NO: 22-49 or a toxin encoding fragment thereof. In some aspects of these embodiments, the chimeric gene is an expression cassette.

[0146] In other non-limiting embodiments, the promoter contained in the chimeric gene or expression cassette of the present disclosure is a plant expressible promoter. In aspects of these embodiments, the plant expressible promoter is selected from the group consisting of: ubiquitin, tuberose yellow virus, corn TrpA, OsMADS 6, maize H3 histone, bacteriophage T3 gene 9 5'UTR, maize sucrose synthase 1, maize alcohol dehydrogenase 1, maize light harvesting complex, maize heat shock protein, maize mtl, pea small subunit RuBP carboxylase, rice actin, rice cyclophilin, Ti plasmid mannopine synthase, Ti plasmid nopaline synthase, petunia chalcone isomerase, legume glycine-rich protein 1, potato glycoprotein, lectin, CaMV 35S and S-E9 small subunit RuBP carboxylase promoter.

[0147] In some non-limiting embodiments, the insecticidal protein encoded by the nucleic acid molecules of the present disclosure or the chimeric genes of the present disclosure or the expression cassettes of the present disclosure is active against coleopteran insect pests. In some aspects of these embodiments, the coleopteran insect pest is in the genus Radix Glechomae. In other aspects, the genus Radix Glechomae insect pest is a corn rootworm (Western corn rootworm; WCR), a barbaric rootworm (Northern corn rootworm; NCR) and / or a cucumber rootworm subspecies of Eleven-spotted Diabrotica (Southern corn rootworm; SCR) and / or other Radix Glechomae species (including the Mexican corn rootworm (Mexican corn rootworm; MCR).

[0148] In some non-limiting embodiments, the chimeric gene or expression cassette of the present disclosure comprises a nucleotide sequence encoding a RIP of the present disclosure, wherein the nucleotide sequence is codon optimized for expression in a transgenic organism. In some aspects of these embodiments, the transgenic organism is a bacterium or a plant.

[0149] In other non-limiting embodiments, the transgenic bacteria are of the genus Bacillus, Clostridium, Xenorhabdus, Photorhabdus, Pasteurella, Escherichia, Pseudomonas, Erwinia, Serratia, Klebsiella, Salmonella, Pasteurella, Xanthomonas, Streptomyces, Rhizobium, Rhodopseudomonas, Sinorhizobium, Sword, Methylophilus, Agrobacterium, Acetobacter, Lactobacillus, Arthrobacter, Azotobacter, Leuconostoc, Sphingolipids, Burkholderia, Candidatus Glomeribacter, Dai's bacteria, Grass Spirillum, Bradyrhizobium, Staphylococcus, Methylophilus, Greedyphage, Streptococcus, Sphingobacteraceae (Chitinophaga) or Alcaligenes. In other embodiments, the transgenic bacteria are Escherichia coli. In other embodiments, the nucleotide sequence comprises, consists essentially of, or consists of any one of SEQ ID NOs: 26-45.

[0150] In other non-limiting embodiments, the transgenic plant is a monocot or a dicot. In other embodiments, the dicot is selected from the group consisting of soybean, sunflower, tomato, Brassica crops, cotton, sugar beet and tobacco. In other aspects, the monocot is selected from the group consisting of barley, maize, oats, rice, sorghum, sugarcane and wheat. In some aspects, the transgenic plant is a maize plant. In other embodiments, the nucleotide sequence comprises codons optimized for expression in maize. In still other embodiments, the nucleotide sequence comprises, consists essentially of, or consists of: any one of SEQ ID NO:46-49.

[0151] In some non-limiting embodiments, the present disclosure encompasses proteins that are toxic to insect pests, and optionally isolated proteins, i.e., insecticidal proteins, wherein the protein or isolated protein comprises, consists essentially of, or consists of: (a) an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NOs: 1-21, or a toxin fragment thereof; (b) an amino acid sequence comprising, consisting essentially of, or consisting of: any one of SEQ ID NOs: 1-21, or a toxin fragment thereof; (c) an amino acid sequence encoded by a nucleotide sequence that has at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NOs: 1-21, or a toxin fragment thereof; NO: 22-49 or a toxin encoding fragment thereof having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity; (d) an amino acid sequence encoded by a nucleotide sequence comprising, consisting essentially of, or consisting of: any one of SEQ ID NO: 22-49 or a toxin encoding fragment thereof; or (e) an amino acid sequence of any one of (a)-(d), comprising a cytotoxin domain selected from the group consisting of: SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, and SEQ ID NO: 53. One skilled in the art will recognize that modifications may be made to the exemplary RIPs encompassed by the present disclosure. Such modifications and substantially identical nucleic acid or amino acid molecules are encompassed by the present disclosure.

[0152] The disclosure also includes engineered Rhizobium family insecticidal proteins, which can be described as mutant RIPs or variant RIPs or modified RIPs disclosed herein. In certain embodiments, modification may include substitution and / or deletion of one or more amino acids in a naturally occurring RIP sequence and / or insertion of one or more other amino acids into a naturally occurring RIP sequence. In other embodiments, modification may include substitution and / or deletion and / or insertion of one or more amino acids in an engineered RIP. Substitution and / or insertion may be with naturally occurring amino acids or non-naturally occurring amino acids. In some non-limiting embodiments, modification comprises, is essentially composed of, or is composed of: substitution and / or insertion and / or deletion of one or more of alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine and / or valine at the amino acid position of the RIP amino acid sequence. Such substitution and / or insertion and / or deletion can be achieved by changing the codons in the nucleotide sequence encoding the RIP, thereby generating a modified rip nucleotide sequence encoding an engineered RIP (which is a mutant RIP or variant RIP or modified RIP of the present disclosure).

[0153] In some non-limiting embodiments, the RIP is modified by the following substitutions and / or insertions: (a) one or more amino acids with aliphatic hydrophobic side chains (e.g., alanine, isoleucine, methionine and / or valine; in embodiments, the amino acid is not alanine); (b) one or more amino acids with aromatic hydrophobic side chains (e.g., phenylalanine, tryptophan and / or tyrosine); (c) one or more amino acids with polar neutral side chains (e.g., asparagine, cysteine, glutamine, serine and / or threonine); (d) one or more amino acids with acidic side chains (e.g., aspartic acid and / or glutamic acid); one or more amino acids with basic side chains (e.g., arginine, histidine and / or lysine); (e) one or more glycine residues; (f) one or more proline residues; or (g) any combination of (a) to (f).

[0154] In other embodiments, amino acids are substituted and / or deleted and / or inserted in any amino acid sequence of the RIP disclosed herein, in particular any amino acid sequence of SEQ ID NO: 1-4. In other embodiments, amino acids are substituted in SEQ ID NO: 3. In other embodiments, the substituted amino acids in SEQ ID NO: 3 are located at positions 50, 52, 56, 62, 64, 81, 126, 153, 169, 185, 207, 219 and / or 275. In other embodiments, the amino acid at position 50 is substituted with L, the amino acid at position 52 is substituted with L, the amino acid at position 56 is substituted with L, the amino acid at position 62 is substituted with C or L, the amino acid at position 64 is substituted with C or L, the amino acid at position 81 is substituted with L, the amino acid at position 126 is substituted with L, the amino acid at position 153 is substituted with L, the amino acid at position 169 is substituted with L, the amino acid at position 185 is substituted with L, the amino acid at position 207 is substituted with L, the amino acid at position 218 is substituted with L, or the amino acid at position 275 is substituted with L.

[0155] In other embodiments, the present disclosure provides a kind of chimeric RIP toxin, it includes the protein fusion tag connected with the complete RIP sequence or a part of the RIP sequence (such as cytotoxin domain).The protein fusion tag can be connected to the N-terminus (for example, at the amino acid 1 or 2 of the RIP sequence), or alternatively, the protein fusion tag can be connected to the C-terminus of the RIP sequence.The protein fusion tag can be polyhistidine, polyarginine, haloalkane dehalogenase, streptavidin binding, glutathione s-transferase (GST), maltose binding protein (MBP), thioredoxin, small ubiquitin-like modifier (SUMO), N-utilizing substance A (NusA), protein disulfide bond isomerase I (DsbA), Mistic, ketosteroid isomerase (KSI) or TrpE, c-myc, hemagglutinin antigen (HA), FLAG, 1D4, calmodulin binding peptide, chitin binding domain, cellulose binding domain, S-tag or Softag3 protein fusion tag. These can be used in methods for producing, isolating or purifying any RIP toxin disclosed herein. The disclosure also provides recombinant polynucleotides, such as constructs, encoding fusion tags connected to the RIP toxins disclosed herein. In some embodiments, a SUMO tag is connected to the N-terminus of a RIP1Aa toxin to produce a SUMO-RIP1Aa toxin (SEQ ID NO: 21) encoded by a SUMO-rip1Aa polynucleotide (SEQ ID NO: 45).

[0156] In some non-limiting embodiments, the disclosure encompasses variant RIPs comprising, consisting essentially of, or consisting of an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to a) SEQ ID NO: 1, and further comprising at least one mutation at a position corresponding to amino acid positions 1-345 of SEQ ID NO: 1; or b) SEQ ID NO: 2 NO:2 having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity, and further comprising at least one mutation at a position corresponding to amino acid position 1-344 of SEQ ID NO:2; or c) SEQ ID NO:2 having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity. NO:3 having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity and further comprising at least one mutation at a position corresponding to amino acid position 1-351 of SEQ ID NO:3; or d) SEQ ID NO:3 having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity. NO:4 has at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity, and further comprises at least one mutation at a position corresponding to amino acid position 1-347 of SEQ ID NO:4. In other non-limiting embodiments, the mutation is at an amino acid position corresponding to amino acid position 50, 52, 56, 62, 64, 81, 126, 153, 169, 185, 207, 219, or 275 of SEQ ID NO:3, or any combination thereof. In other non-limiting embodiments, the mutation is at position 50, 52, 56, 62, 64, 81, 126, 153, 169, 185, 207, 219 or 275 of SEQ ID NO:3.In other embodiments, the mutation at position 50 is I50L, the mutation at position 52 is I52L, the mutation at position 56 is I56L, the mutation at position 62 is A62C or A62L, the mutation at position 64 is A64C or A64L, the mutation at position 81 is I81L, the mutation at position 126 is I126L, the mutation at position 153 is I153L, the mutation at position 169 is I169L, the mutation at position 185 is I185L, the mutation at position 207 is I207L, the mutation at position 2019 is I219L, or the mutation at position 275 is I275L. In yet other embodiments, the variant RIP comprises, consists essentially of, or consists of SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, ID NO:19, or SEQ ID NO:20.

[0157] In some non-limiting embodiments, when tested under the same conditions (e.g., enzyme concentration, protein concentration, pH, temperature and / or time), the variant RIP of the present disclosure has enhanced digestion by mammalian digestive proteases (e.g., pepsin) compared to a suitable control and / or a parent molecule without the modification of the present disclosure. Methods for evaluating protein digestion with pepsin and other digestive proteases are known in the art. For example, digestion using pepsin can be performed under the following conditions: about 37° C. and about pH 1.2, optionally with an enzyme concentration of about 10 units (U) of pepsin / microgram of protein.

[0158] In some embodiments, the RIPs of the present disclosure, including variant RIPs of the present disclosure, are active against coleopteran pests. Coleopteran insects include, but are not limited to, any coleopteran insects currently known or later identified, including those of the suborders Protocoleidae, Myxophagata, Carnivora, and Polyphaga, and any combination thereof.

[0159] In some non-limiting embodiments, the RIP toxins and variant RIP toxins disclosed herein are active against species of the genus Diabrotica. Diabrotica is a genus of beetles in the order Coleoptera, commonly known as "corn rootworms" or "cucumber beetles." Exemplary species of the genus Diabrotica include, but are not limited to, Diabrotica longicornis barberi (northern corn rootworm), Diabrotica nominata subsp. (western corn rootworm), D. eleven-spotted rootworm subsp. (southern corn rootworm), D. balteata (banded cucumber beetle), D. undecimpunctata undecimpunctata (western spotted cucumber beetle), D. significata (3-spotted leaf beetle), D. speciosa (chrysanthemum beetle), Mexican corn rootworm (Mexican corn rootworm), Beni rootworm (D. beiensis), Christa rootworm (D. cristata), Cove rootworm (D. curvipustulata), two-spotted rootworm (D. dissimilis), elegant rootworm (D. elegantula), Emorsitans, Graminea rootworm (D. graminea), Hispanolae rootworm (D. hispanolae), Lemini Root beetle (D.lemniscata), ochre-legged root beetle (D.linsleyi), miller's root beetle (D.milleri), coin-shaped root beetle (D.nummularis), fan-leaved root beetle (D.occlusa), prai root beetle (D.porracea), snail root beetle (D.scutellata), shin root beetle (D.tibialis), three-line root beetle (D.trifasciata) and micro-green root beetle (D.viridula); and any combination thereof.

[0160] Other non-limiting examples of coleopteran insect pests according to the present disclosure include Leptinotarsa ​​spp., such as the potato beetle (Colorado potato beetle); Chrysomelas spp., such as C. scripta (cottonwood leaf beetle); Hypothenemus spp., such as H. hampei (coffee berry borer); Sitophilus spp., such as S. zeamais (maize weevil); Epitrix spp., such as E. hirtipennis (tobacco flea beetle); (tobacco fleabeetle) and E. cucumeris (potato flea beetle): Phyllotreta spp., such as P. cruciferae (crucifer flea beetle) and P. pusilla (western black fleabeetle); Anthonomus spp., such as A. eugenii (pepper weevil); Hemicrepidus spp., such as H. memnonius (wireworms); Melanotus spp., such as M. communis (wireworms); Ceutorhychus spp. spp., such as C. assimilis (cabbage seed pod weevil); Aeolus spp., such as A. mellillus (wireworm); Aeolus spp., such as A. mancus (wheat wireworm); Horistonotus spp., such as H. uhlerii (sand wireworm); Sphenophorus spp., such as S. maidis (maize billbug), S. zeae (timothy billbug), billbug), S. parvulus (bluegrass billbug), and S. callosus (southern corn billbug); Phyllophaga spp. (white grubs); Chaetocnema spp., such as C. pulicaria (corn flea beetle); Popillia spp., such as P. japonensis (Japanese billbug).japonica) (Japanese beetle); Epilaxna spp., such as E. varivestis (Mexican bean beetle); Cerotoma spp., such as C. trifurcate (Bean leaf beetle); Epicauta spp., such as E. pestifera and E. lemniscata (lister beetles); and any combination of the foregoing.

[0161] The RIPs of the present disclosure may also be active against Lepidoptera. Such Lepidoptera include, but are not limited to, any insect classified as Lepidoptera, now known or later identified, including those insect species within the suborders Hypogyria, Proboscis, and Heterotrichoptera, and any combination thereof. Exemplary Lepidoptera include, but are not limited to, Ostrinia spp., such as O. nubilalis (European corn borer); Plutella spp., such as P. xylostella (diamondback moth); Spodoptera spp., such as S. frugiperda (fall armyworm), S. ornithogalli (yellowstriped armyworm), S. praefica (western yellowstriped armyworm), S. eridania (southern armyworm), and S. exigua (beet armyworm); Spodoptera spp., such as A. ipsilon (black cutworm), A. segetum (common cutworm), A. gladiaria (clayback cutworm), and A. sedum (black cutworm). cutworm and A. orthogonia, palewestern cutworm; Striacosta spp., such as S. albicosta (western bean cutworm); Helicoverpa spp., such as H. zea (corn earworm), H. punctigera (native budworm), S. littoralis (Egyptian cotton leafworm), and H. armigera (cotton bollworm); Heliothis spp., such as Heliothis virescens (tobacco budworm); Diatraea spp., such as D. grandiosella, southeastwestern corn borer; borer and D. saccharalis (sugarcane borer); Trichoplusia spp., such as T.ni, cabbage looper; Sesamia spp., such as S. nonagroides, Mediterranean corn borer; Pectinophora spp., such as P. gossypiella, pink bollworm; Cochylis spp., such as C. hospes, banded sunflower moth; Manduca spp., such as M. sexta, tobacco hornworm and M. quinquemaculata, tomato hornworm; Elasmopalpus spp., such as E. lignosellus, lesser cornstalk borer; borer); Pseudoplusia spp., such as P. includens (soybean borer); Anticarsia spp., such as Spodoptera velvet bean caterpillar; Plathypena spp., such as P. scabra (green cloverworm); Pieris spp., such as P. brassicae (cabbage butterfly); Papaipema spp., such as P. nebris (stalk borer); Pseudaletia spp., such as P. unipuncta (common armyworm); Peridroma spp., such as P. saucia (variegated cutworm); cutworm; Keiferia spp., such as K. lycopersicella (tomato pinworm); Artogeia spp., such as A. rapae (imported cabbageworm); Phthorimaea spp., such as P. operculella (potato tuberworm); Crymodes spp., such as C.devastator, glassy cutworm; Feltia spp., such as F. ducens, dingy cutworm; and any combination of the foregoing.

[0162] The RIPs of the disclosure may also be active against Hemiptera, Diptera, Lygus species, and / or other piercing-sucking insects (eg, piercing-sucking insects of the Order Orthoptera or Thysanoptera). Insects of the order Diptera include, but are not limited to, any Diptera insect now known or later identified, including, but not limited to, Liriomyza spp., such as L. trifolii (leaf miner) and L. sativae (vegetable leaf miner); Scrobipalpula spp., such as S. absoluta (tomato leaf miner); Delia spp., such as D. platura (seed corn maggot), D. brassicae (cabbage maggot), and D. radicum (cabbage root fly); Psilia spp., such as P. rosae (carrot rust fly); Tetanops spp., such as P. rosae (carrot rust fly); spp.), such as T. myopaeformis (sugarbeet root maggot); and any combination of the foregoing.

[0163] Insects of the order Orthoptera include, but are not limited to, any Orthoptera insect currently known or later identified, including, but not limited to, Melanoplus spp., such as M. differentialis (Differential grasshopper), M. femurrubrum (Redlegged grasshopper), M. bivittatus (Twostriped grasshopper); and any combination thereof.

[0164] Insects of the order Thysanoptera include, but are not limited to, any Thysanoptera now known or later identified, including, but not limited to, Frankliniella spp., such as F. occidentalis (western flower thrips) and F. fusca (tobacco thrips); and Thrips spp., such as T. tabaci (onion thrips), T. palmi (melon thrips); and any combination of the foregoing.

[0165] The RIPs of the present disclosure may also be active against nematodes. The term "nematode" as used herein encompasses any organism currently known or later identified as being classified as the phylum Nematoda of the animal kingdom, including but not limited to nematodes in the class Adenophora (including, for example, Rhizophora, Isopharyngea, Monodonta, Lanceolata, Trichocephala, Chondriata, Muspa, Lactophaga, Chromatidae, Taeniformes, Chain Rings, and Monogynae) and / or the class Tubulonema (including, for example, Rhabditis, Strongyloides, Ascaridae, Spirulina, Camelidae, Digastricales, Sphaeroidea, and Sphaeroidea).

[0166] Nematodes include, but are not limited to, parasitic nematodes, such as root-knot nematodes, cyst nematodes, and / or decay nematodes. Exemplary genera of nematodes according to the present disclosure include, but are not limited to: Root-knot nematodes (root-knot nematodes), Heterodera (cyst nematodes), Globodera (cyst nematodes), Penetrate nematodes (piercing nematodes), Renoides (kidney-shaped kidney-shaped nematodes), Pratylenchus (rot nematodes), Aphelenchus (leaf nematodes), Heliconematodes (helical nematodes), Nematodes (lance nematodes), Pseudo-burrs (short and thick root nematodes), Long-nematodes, Pearl Nematodes (pseudo-root-knot nematodes), Subanguina, Stinging Nematodes (thorn nematodes), Microcircle Nematodes (microcircle nematodes), Angiospermum (angiosperm nematodes), Ditylenchus (dendrum nematodes), Conynematode (conynematodes), Hemicyclenematode (hemicycle nematodes), Sheathnematode (sheath nematodes), Subroot Nematode (subroot nematodes), Hypsoperine, Ditylenchus (large stem nematodes), Melinius, Punctodera, Quinisulcius, Shield Nematode (shield nematodes), Xiphinema (dagger nematodes), Dwarf Nematode (dwarf nematodes), Penelopenetes (piercing nematodes), Bursaphelenchus (roundworms), and any combination thereof.

[0167] Exemplary plant parasitic nematodes according to the present disclosure include, but are not limited to: Belonolaimus gracilis, Belonolaimus longicaudatus, Bursaphelenchus xylophilus, pine wood nematode, Criconemoides ornata, Ditylenchus destructor, potato rot nematode, Ditylenchus dipsaci, stem and bulb nematode, Globodera pallida, potato cyst nematode, Globodera rostochiensis (golden nematode), Heterodera glycines, soybean cyst nematode, Heteroderaschachtii, sugar beet cyst nematode. nematode); Heterodera zeae, corn cyst nematode; Heterodera avenae,cereal cyst nematode), Heterodera carotae, Heterodera trifolii, Hoplolaimus columbus, Hoplolaimus galeatus, Hoplolaimusmagnistylus, Longidorus breviannulatus, Meloidogynearenaria, Meloidogyne chitwoodi, Meloidogyne hapla, Meloidogyne incognita, Meloidogyne javanica, Mesocriconema xenoplax, Nacobbus aberrans, Naccobus dorsalis, Paratrichodorus christiei, Paratrichodorus minor), Pratylenchus brachyurus, Pratylenchus crenatus, Pratylenchus hexincisus, Pratylenchus negletus, Pratylenchus penetrans, Pratylenchus projectus, Pratylenchus scribneri, Pratylenchus tenuicaudatus, Pratylenchus thornei, Pratylenchus zeae, Punctodera chaccoensis, Quinisulciusacutus, Radopholus similis, Rotylenchulus reniformis, Tylenchorhynchus dubius), Tylenchulus semipenetrans, Siphinema americanum, X. Mediterraneum, and any combination of the foregoing.

[0168] The present disclosure also encompasses recombinant vectors and / or recombinant constructs, which may also be referred to as vectors or constructs, and which include expression cassettes and / or nucleic acid molecules of the present disclosure. In such vectors, these nucleic acids are preferably in expression cassettes, which include regulatory elements for expressing nucleotide molecules in host cells capable of expressing nucleotide molecules. Such regulatory elements typically include promoters and termination signals and preferably also include elements, which allow effective translation of polypeptides encoded by nucleic acids of the present disclosure. The vectors comprising nucleic acids can replicate (preferably as extrachromosomal molecules) in specific host cells and can therefore be used to amplify nucleic acids of the present disclosure in these host cells.

[0169] The disclosure also encompasses host cells comprising the recombinant vectors, expression cassettes or nucleic acid molecules of the disclosure. In other embodiments, such vectors are viral vectors and are used to replicate nucleotide sequences in specific host cells (e.g., insect cells or plant cells). Recombinant vectors are also used to transform the nucleic acid molecules of the disclosure into host cells, whereby these nucleic acid molecules are stably integrated into the DNA of a transgenic host. In certain embodiments, the host cell is a bacterial cell or a plant cell. In some aspects of these embodiments, the bacterial cell is in Bacillus, Clostridium, Xenorhabdus, Photorhabdus, Pasteurella, Escherichia, Pseudomonas, Erwinia, Serratia, Klebsiella, Salmonella, Pasteurella, Xanthomonas, Streptomyces, Rhizobium, Sinorhizobium, Sword, Rhodopseudomonas, Methylophilus, Agrobacterium, Acetobacter, Lactobacillus, Arthrobacter, Azotobacter, Leuconostoc, Sphingolipids, Burkholderia, Candidatus Glomeribacter, Dai Shi, Grass Spirillum, Bradyrhizobium, Staphylococcus, Methylophilus, Greedy Phage, Streptococcus, Sphingobacteraceae or Alcaligenes. In other aspects of these embodiments, the host cell for such recombinant vectors is an endophyte or epiphyte. In some other aspects of these embodiments, the host cell is a plant cell, such as a dicot cell or a monocot cell. In other aspects, the dicot cell is selected from the group consisting of soybean cells, sunflower cells, tomato cells, Brassica crop cells, cotton cells, sugar beet cells, and tobacco cells. In still other aspects, the monocot cell is selected from the group consisting of barley cells, maize cells, oat cells, rice cells, sorghum cells, sugar cane cells, and wheat cells.

[0170] In some non-limiting embodiments of the present disclosure, at least one of the nucleic acid molecules of the present disclosure is inserted into an appropriate expression cassette (comprising a promoter and a termination signal). The expression of the nucleic acid may be constitutive, or an inducible promoter that responds to various types of stimuli to initiate transcription may be used. In another embodiment, the cell in which the insecticidal protein of the present disclosure is expressed is a microorganism, such as a virus, a bacterium, or a fungus. In yet another embodiment, a virus (such as a baculovirus) contains the nucleic acid of the present disclosure in its genome and expresses a large amount of the corresponding insecticidal protein after infecting an appropriate eukaryotic cell (suitable for viral replication and expression of the nucleic acid). The insecticidal protein thus produced is used as an insecticide. Alternatively, a baculovirus engineered to include the nucleic acid is used to infect insects in vivo and kill them by expression of the insecticidal toxin or by a combination of viral infection and expression of the insecticidal toxin. In a further embodiment, the present disclosure also encompasses a method for producing a polypeptide having insecticidal activity, the method comprising culturing a host cell under conditions where a nucleic acid molecule encoding the polypeptide is expressed.

[0171] Bacterial cells are also hosts for expressing nucleic acids disclosed herein. In one embodiment, non-pathogenic symbiotic bacteria (so-called endophytes) that can live and replicate in plant tissues are used, or non-pathogenic symbiotic bacteria (so-called epiphytes) that can settle in the leaf or rhizosphere are used. Such bacteria include bacteria of the following genus: Agrobacterium, Alcaligenes, Azospirillum, Azotobacter, Bacillus, Corynebacterium, Enterobacter, Erwinia, Flavobacterium, Klebsiella, Pseudomonas, Rhizobium, Sinorhizobium, Sword Pseudomonas, Serratia, Streptomyces, Sphingomonas, Burkholderia, Candidatus Glomeribacter, Dai Shi, Grass Spirillum, Bradyrhizobium, Staphylococcus, Methylophilus, Greedy Phage, Streptococcus, Sphingobacteraceae and Xanthomonas. Symbiotic fungi such as Trichoderma and Glucobra are also possible hosts for expressing the nucleic acids of the invention for the same purpose.

[0172] These gene manipulation techniques are specific and known in the art for different available hosts. For example, expression vectors pKK223-3 and pKK223-2 can be used to express heterologous genes after tac or trc promoters in Escherichia coli (in transcription or translation fusion). In order to express the operon encoding multiple ORFs, the simplest method is to insert the operon into a vector (such as pKK223-3) in transcriptional fusion, allowing the homologous ribosome binding site of the heterologous gene to be utilized. Overexpression techniques in gram-positive species (such as Bacillus) are also known in the art, and can be used in the context of the present disclosure (Quax et al., in: Industrial Microorganisms: Basic and Applied Molecular Genetics [industrial microorganisms: basic and applied molecular genetics], editor Baltz et al., American Society for Microbiology [American Society for Microbiology], Washington (1993)). Alternative systems for overexpression rely on, for example, yeast vectors, and include the use of Pichia, Saccharomyces, and Kluyveromyces (Sreekrishna, In: Industrial microorganisms: basic and applied molecular genetics, Baltz, Hegeman, and Skatrud, eds., American Society for Microbiology, Washington (1993); Dequin and Barre, Biotechnology L2:173-177 (1994); van den Berg et al., Biotechnology 8:135-139 (1990)).

[0173] In yet other embodiments, the disclosure encompasses methods for controlling insect pests, the method comprising delivering an insecticidal protein of an insect control effective amount of the disclosure to the insect pests. In some aspects of these embodiments, the insecticidal protein is delivered by a transgenic plant or by topical application of an insecticidal composition comprising the insecticidal protein. In other aspects, the transgenic plant or the insecticidal composition comprises a second insecticide different from the RIP of the disclosure. In still other aspects, the second insecticide is a protein, a dsRNA, or a chemical. In still other aspects, the protein is selected from the group consisting of: Cry protein, VIP toxin, potato glycoprotein, protease, protease inhibitor, urease, α-amylase inhibitor, pore-forming protein, lectin, engineered antibody or antibody fragment, or chitinase; or the chemical is a carbamate, pyrethroid, organophosphate, friprole, neonicotinoid, organochloride, nereis toxin or a combination thereof; or the chemical comprises an active ingredient selected from the group consisting of: carbofuran, carbaryl, methomyl, bifenthrin, tefluthrin, permethrin, cypermethrin, λ-cypermethrin, cypermethrin, deltamethrin, chlorpyrifos, chloroxyfos, dimethoate, anthracene, malathion, methyl parathion, phorate, terbufos, tert-butylpyrimidophos, fipronil, acetamiprid, imidacloprid, thiacloprid, thiamethoxam, endosulfan, sulfamethoxam and a combination thereof.

[0174] In some embodiments of the present disclosure, at least one of the RIP toxins of the present disclosure is expressed in a higher organism such as a plant. Transgenic plants expressing insecticidal proteins in an effective amount to control insects protect themselves from damage by insect pests. When insect pests begin to feed on such transgenic plants, they also ingest such expressed insecticidal proteins. This can prevent insects from further feeding on plant tissues and / or can even injure or kill insects. The nucleic acid molecules of the present disclosure are inserted into an expression cassette, and then the nucleic acid can be stably integrated into the genome of the plant. In other embodiments, the nucleic acid molecules are included in non-pathogenic self-replicating viruses. Plants transformed according to the present disclosure can be monocots or dicots, and include, but are not limited to, corn, wheat, oats, turf grass, pasture grass, flax, barley, rye, sweet potato, bean, pea, chicory, lettuce, cabbage, cauliflower, broccoli, turnip, radish, spinach, asparagus, onion, garlic, pepper, celery, squash, pumpkin, hemp, zucchini, apple, pear, quince, melon, plum, cherry, peach, nectarine, apricot, strawberry, grape, raspberry, blackberry, pineapple, avocado, papaya, mango, banana, soybean, tomato, sorghum, sugarcane, sugar beet, sunflower, rapeseed, clover, tobacco, carrot, cotton, alfalfa, rice, potato, eggplant, cucumber, Arabidopsis species, and woody plants, such as coniferous and deciduous trees.

[0175] In some embodiments, the present disclosure encompasses a method of producing a protein that is toxic to an insect pest, i.e., an insecticidal protein, comprising: (a) obtaining a host cell comprising a gene, which gene itself comprises an expression cassette and / or a nucleic acid molecule of the present disclosure; and (b) growing the host cell or a transgenic host comprising the host cell under conditions where the transgenic host cell produces the protein that is toxic to an insect pest.

[0176] In other embodiments, the disclosure encompasses methods of producing a transgenic plant or plant part having enhanced insect resistance compared to a control plant or plant part, the method comprising: (a) introducing a chimeric gene or expression cassette or vector comprising a nucleic acid molecule encoding an insecticidal protein of the disclosure into a plant or plant part, wherein the insecticidal protein is expressed in the plant or plant part, thereby producing a plant or plant part having enhanced insect resistance. In other embodiments, the chimeric gene, expression cassette or vector may encode a RIP toxin of the disclosure, the RIP toxin comprising, consisting essentially of, or consisting of an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical or similar to any one of SEQ ID NOs: 1-21. "Enhanced" insect resistance can be measured by any toxic effect of the transgenic plant on insect pests that feed on the transgenic plant. The enhanced insect resistance can be 0%, at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 125%, at least 150%, at least 200%, at least 300%, at least 400%, at least 500%, at least 600%, at least 700%, at least 800%, at least 900%, or at least 1000% insecticidal activity compared to a control plant that does not express the insecticidal protein. Plants or plant parts with enhanced insect resistance compared to control plants or plant parts can be produced by plant transformation, plant tissue culture or breeding methods. Plants or plant parts can be produced by sexual or asexual reproduction methods. Any suitable control plant or plant part can be used, such as a plant with the same or similar genetic background grown in the same environment. In an embodiment, the control plant or plant part has the same genetic background and is grown in the same environment as the described plant, but does not contain the molecules of the present disclosure, while the described plant contains the nucleic acid molecules of the present disclosure.

[0177] In other embodiments, the disclosure encompasses a method of enhancing insect resistance in a plant or plant part compared to a control plant or plant part, the method comprising expressing a nucleic acid molecule or expression cassette of the disclosure in the plant or plant part, wherein expression of the heterologous nucleic acid of the expression cassette results in the plant or plant part having enhanced insect resistance compared to the control plant or plant part. In some embodiments, the expression cassette or nucleic acid molecule comprises a promoter operably linked to a heterologous nucleic acid molecule comprising a nucleotide sequence comprising, consisting essentially of, or consisting of: (a) a nucleotide sequence of any one of SEQ ID NOs: 22-49; (b) a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a nucleotide sequence of any one of SEQ ID NOs: 22-49; (c) a nucleotide sequence encoding a protein, wherein the amino acid sequence of the protein comprises, consists essentially of, or consists of: any one of SEQ ID NOs: 1-21; (d) a nucleotide sequence encoding a protein, wherein the amino acid sequence of the protein is identical to that of SEQ ID NOs: 1-22. The amino acid sequence of any one of IDNO: 1-21 is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical; (e) the nucleotide sequence of any one of the above (a) to (d), which is codon-optimized for expression in a transgenic host organism; or (f) a nucleotide sequence complementary to the nucleotide sequence of any one of the above (a) to (e). Nucleic acid molecules or expression cassettes can be introduced into plants. In some embodiments, nucleic acid molecules or expression cassettes can be introduced into plant parts, and plants comprising nucleic acid molecules or expression cassettes can be produced from the plant parts.

[0178] In certain embodiments, the disclosure encompasses methods for producing plants with enhanced insect resistance compared to control plants, the method comprising detecting heterologous nucleic acids comprising nucleic acid molecules or expression cassettes of the disclosure in plant parts, and producing plants from the plant parts, thereby producing plants with enhanced insect resistance compared to control plants. In a further embodiment, the disclosure encompasses methods for identifying plants or plant parts with enhanced insect resistance compared to control plants or plant parts, the method comprising detecting nucleic acid molecules or expression cassettes of the disclosure in the plant or plant parts, thereby identifying plants or plant parts with enhanced insect resistance. In a further embodiment, the expression cassette or its diagnostic fragment is detected in an amplified product from a nucleic acid sample of the plant or plant part. The diagnostic fragment can be a nucleic acid molecule of at least 10 consecutive nucleotides long, which is unique to the expression cassette of the disclosure.

[0179] In other embodiments, the present disclosure encompasses a method of producing a plant having enhanced insect resistance compared to a control plant or plant part, the method comprising crossing a first parent plant with a second parent plant, wherein at least the first parent plant comprises in its genome a heterologous nucleic acid comprising a nucleic acid molecule or expression cassette of the present disclosure; and producing progeny generations, wherein the progeny generations comprise at least one plant having in its genome the heterologous nucleic acid and exhibiting enhanced insect resistance compared to a control plant.

[0180] In some aspects of the above-described embodiments, the method of the present disclosure gives the insect resistance of the enhancement of plant or plant part for coleopteran insect pests. In the example, the insect control of coleopteran insect pests was demonstrated. In other aspects, the method of the present disclosure gives the insect resistance of the enhancement of plant or plant part for root firefly species (including corn root firefly, Bar's root firefly, cucumber eleven star leaf beetle root feeding subspecies, Mexican corn root firefly and / or South American leaf beetle (Diabrotica peciosa)) and / or related species. In other embodiments, the method of the present disclosure gives the insect resistance of the enhancement of plant or plant part for western corn root firefly, Bar's root firefly and / or cucumber eleven star leaf beetle root feeding subspecies.

[0181] In some embodiments, the disclosure encompasses transgenic plants comprising heterologous nucleic acid molecules or expression cassettes of the disclosure, which confer enhanced insect resistance to the transgenic plants when transcribed or translated. In some aspects of these embodiments, the heterologous nucleic acid molecule or expression cassette comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 91% at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% at least 99% or 100% identical to any one of SEQ ID NO: 21-49. In other embodiments, the transgenic plant is a dicot or a monocot. In another aspect, the transgenic plant is alfalfa, apple, apricot, artichoke, arugula, asparagus, avocado, banana, bean, beet, blackberry, blueberry, broccoli, Brussels sprouts, cabbage, canola, cantaloupe, carrot, cassava, cauliflower, celery, cherry, cilantro, citrus, clementine, coffee bean, corn, cotton, cucumber, Douglas fir, eggplant, endive, kohlrabi, eucalyptus, fennel, fig, gourd, grape, grapefruit, honeydew melon, jicama, kiwi, lettuce, leek, Lemon, lime, loblolly pine, mango, melon, mushroom, nut, okra, onion, orange, ornamental plant, papaya, parsley, pea, peach, peanut, pear, pepper, persimmon, pine, pineapple, plantain, plum, pomegranate, poplar, potato, pumpkin, quince, radiata pine, red chicory, radish, raspberry, rice, rye, sorghum, southern pine, soybean, spinach, small pumpkin, strawberry, beet, sunflower, sweet potato, liquidambar, citrus, tea, tobacco, tomato, turf, vine, watermelon, potato or green zucchini. In still other aspects, transgenic plant is millet, switchgrass, maize, sorghum, wheat, oat, lawn grass, pasture grass, flax, rice, sugarcane, rape or barley. In other embodiments, transgenic plant is transgenic maize (corn) plant, it comprises rip coding sequence (wherein codon optimized for expression in maize), for example any one of SEQ ID NO:46-49.

[0182] In some embodiments, the present disclosure encompasses nucleic acid molecules encoding the insecticidal proteins of the present disclosure that are modified and optimized for expression in transgenic plants. Although in many cases genes from microbial organisms can be expressed at high levels in plants without modification, low expression in transgenic plants may be due to microbial nucleic acids having codons that are not preferred in plants. It is known in the art that all organisms have specific preferences for codon usage, and the codons of the nucleic acids described in the present disclosure can be altered to conform to the preferences of plants while maintaining the amino acids encoded thereby, or making certain amino acid changes to the encoded insecticidal proteins. In addition, high expression in plants is best achieved by coding sequences having a GC content of at least about 35%, preferably greater than about 45%, more preferably greater than about 50%, and most preferably greater than about 60%. Microbial nucleic acids with low GC content may be poorly expressed in plants due to the presence of ATTTA motifs, which can destabilize the message, and AATAAA motifs, which can cause inappropriate polyadenylation. In an embodiment, the sequence can be modified to cater to the specific codon preference and GC content preference of monocots or dicots, as these preferences have been shown to be different (Murray et al. Nucl. Acids Res. [Nucleic Acids Research] 17: 477-498 (1989)). In addition, the nucleic acids are screened for the presence of irrational splice sites that may cause message shortening. All changes that need to be made within these nucleic acids (such as those described above) can be changed using the well-known techniques of site-directed mutagenesis, PCR, and synthetic gene construction, for example, using the methods described in published patent applications EP 0 385 962, EP 0 359 472, and WO93 / 07278.

[0183] In some embodiments of the present disclosure, the coding sequence of the insecticidal protein of the present disclosure is manufactured according to the program disclosed in U.S. Patent No. 5,625,136 (incorporated herein by reference). In this program, the preferred codon of maize is used, i.e., the single codon of the amino acid in maize that is most frequently encoded. The preferred codon of maize for specific amino acid can be for example derived from the known gene sequence of maize. The maize codon usage for 28 genes from maize plant is found in Murray et al., Nucleic Acids Research [nucleic acid sequence] 17:477-498 (1989), and its disclosure is incorporated herein by reference. In this way, these nucleotide sequences can be optimized for expression in any plant. It is recognized that all or any part of the gene sequence can be optimized or synthetic. That is, synthetic or partially optimized sequences can also be used.

[0184] For more efficient translation initiation, sequences adjacent to the initiator methionine can be modified. For example, they can be modified by including sequences known to be effective in plants. Joshi has proposed a suitable consensus sequence for plants (NAR 15:6643-6653 (1987)), and Clontech has proposed another consensus translation initiator (1993 / 1994 catalog, page 210). These consensus sequences are suitable for use with the nucleic acids disclosed herein. In an embodiment, these sequences are incorporated into a construct comprising a nucleic acid, up to and including ATG (without modifying the second amino acid), or alternatively up to and including GTC after ATG (with the possibility of modifying the second amino acid of the transgenic).

[0185] The expression of these nucleic acids in transgenic plants is driven by promoters that work in plants. The selection of promoters will vary according to the time and space requirements of expression, and also according to the target species. Therefore, the expression of nucleic acids disclosed herein in leaves, stalks or stems, spikes, inflorescences (e.g., spikes, panicles, cobs, etc.), roots, and / or seedlings is preferred. However, in many cases, protection is sought for more than one type of insect pest, and therefore expression in multiple tissues is desirable. Although it has been shown that many promoters from dicots are operable in monocots and vice versa, it is desirable to select dicot promoters for expression in dicots, and select monocot promoters for expression in monocots. However, there is no restriction on the origin of the selected promoter; it is sufficient as long as they can effectively drive nucleic acids to be expressed in desired cells.

[0186] In certain embodiments, promoters for constitutive expression are used, including actin or ubiquitin or cmp promoters, or CaMV35S and 19S promoters. The nucleic acid of the present disclosure can also be expressed under the regulation of promoters regulated by chemical methods. The preferred technology for chemical induction of genetic expression is described in detail in open application EP 0 332 104 (Ciba-Geigy) and U.S. Patent No. 5,614,395. The preferred promoter for chemical induction is the tobacco PR-1a promoter.

[0187] In other embodiments, a class of wound-inducible promoters can be used. Numerous promoters have been described that express at the site of wounding and also at the site of infection by plant pathogens. Ideally, such promoters should be locally active only at the site of infection, and in this way, the insecticidal proteins of the present disclosure accumulate only in cells that need to synthesize these proteins to kill invading insect pests. Such preferred promoters include those described by Stanford et al. Mol. Gen. Genet. 215:200-208 (1989), Xu et al. Plant Molec. Biol. 22:573-588 (1993), Logemann et al. Plant Cell 1:151-158 (1989), Rohrmeier and Lehle, Plant Molec. Biol. 22:783-792 (1993), Firek et al. Plant Molec. Biol. 22:129-142 (1993), and Warner et al. Plant J. 3:191-201 (1993).

[0188] Tissue-specific or tissue-preferred promoters for expressing genes encoding the insecticidal proteins of the present disclosure in plants, particularly corn, are those that are expressed directly in roots, pith, leaves, or pollen, particularly roots. Such promoters, such as those isolated from PEPC or trpA, are disclosed in U.S. Pat. No. 5,625,136, or those isolated from MTL are disclosed in U.S. Pat. No. 5,466,785. Both U.S. patents are incorporated herein by reference in their entirety.

[0189] In addition, promoters that function in plastids can be used. Non-limiting examples of such promoters include the phage T3 gene 9 5'UTR and other promoters disclosed in U.S. Patent No. 7,579,516. Other promoters suitable for use in the present disclosure include, but are not limited to, the S-E9 small subunit RuBP carboxylase promoter and the Kunitz trypsin inhibitor gene promoter (Kti3).

[0190] In some embodiments of the present disclosure, an inducible promoter can be used. Thus, for example, a chemically regulated promoter can be used to regulate the expression of the nucleotide sequence of the present disclosure by applying an exogenous chemical regulator. The expression of the nucleotide sequence of the present disclosure is regulated by a chemically regulated promoter so that the polypeptide of the present disclosure can be synthesized only when the crop plant is treated with an induced chemical. Depending on the purpose, when the expression of the nucleotide sequence of the present disclosure is induced by the application of chemicals, the promoter can be a chemically inducible promoter, or when the application of chemicals suppresses the expression of the nucleotide sequence of the present disclosure, the promoter can be a chemically repressible promoter.

[0191] Chemically inducible promoters are known in the art and include, but are not limited to, the maize In2-2 promoter (which is activated by benzenesulfonamide herbicide safeners), the maize GST promoter (which is activated by hydrophobic electrophilic compounds used as pre-emergence herbicides), and the tobacco PR-1a promoter (which is activated by salicylic acid) (e.g., the PR1a system), steroid-responsive promoters (see, e.g., Schena et al. (1991) Proc. Natl. Acad. Sci. USA 88, 10421-10425 and McNellis et al. (1998) Plant Genetics, Inc., 1996). J. [Plant Journal] 14, 247-257) as well as tetracycline-inducible promoters and tetracycline-repressible promoters (see, for example, Gatz et al. (1991) Mol. Gen. Genet. [Molecular Genetics] 227, 229-237 and U.S. Pat. Nos. 5,814,618 and 5,789,156), Lac repressor system promoters, copper-inducible system promoters, salicylic acid-inducible system promoters (e.g., PR1a system), glucocorticoid-inducible promoters (Aoyama et al. (1997) Plant J. [Plant Journal] 11: 605-612) and ecdysone-inducible system promoters.

[0192] Other non-limiting examples of inducible promoters include ABA-inducible and cell swelling-inducible promoters, the auxin binding protein gene promoter (Schwob et al. (1993) Plant J. 4:423-432), the UDP-glucose flavonoid glycosyltransferase promoter (Ralston et al. (1988) Genetics 119:185-197), the MPI proteinase inhibitor promoter (Cordero et al. (1994) Plant J. 6:141-150), and the glyceraldehyde-3-phosphate dehydrogenase promoter (Kohler et al. (1995) Plant J. 10:114-115). Mol. Biol. [Plant Molecular Biology] 29: 1293-1298; Martinez et al. (1989) J. Mol. Biol. [Journal of Molecular Biology] 208: 551-565; and Quigley et al. (1989) J. Mol. Evol. [Journal of Molecular Evolution] 29: 412-421). Also included are benzenesulfonamide-inducible (U.S. Pat. No. 5,364,780) and ethanol-inducible (International Patent Application Publication Nos. WO 97 / 06269 and WO 97 / 06268) systems and glutathione S-transferase promoters. Likewise, any of the inducible promoters described in Gatz (1996) Current Opinion Biotechnol. 7: 168-172 and Gatz (1997) Annu. Rev. Plant Physiol. Plant Mol. Biol. 48: 89-108 may be used. Other chemically inducible promoters suitable for directing the expression of the disclosed nucleotide sequences in plants are disclosed in U.S. Pat. No. 5,614,395, which is incorporated herein by reference in its entirety. Chemical induction of gene expression is also described in detail in published application EP 0 332 104 (granted to Ciba-Geigy) and U.S. Pat. No. 5,614,395. In some embodiments, the promoter for chemical induction may be the tobacco PR-1a promoter.

[0193] In further embodiments, the nucleotide sequences of the present disclosure may be operably associated with a promoter that is inducible by wounding or by infestation with a pest or pathogen (e.g., an insect or nematode plant pest). Numerous promoters have been described that express at sites of wounding and / or at sites of pest attack (e.g., insect / nematode feeding) or plant pathogen infection. Ideally, such promoters should be locally active only at or adjacent to the site of attack, and in this way, expression of the nucleotide sequences of the present disclosure will be concentrated in the cells invaded or fed. Such promoters include, but are not limited to, those described by Stanford et al., Mol. Gen. Genet. [Molecular and General Genetics] 215:200-208 (1989); Xu et al., Plant Molec. Biol. [Plant Molecular Biology] 22:573-588 (1993); Logemann et al., Plant Cell [Plant Cell] 1:151-158 (1989); Rohrmeier and Lehle, Plant Molec. Biol. [Plant Molecular Biology] 22:783-792 (1993); Firek ...1:151-158 (1993); Firek et al., Plant Cell [Plant Cell] 1:151-158 (1989); Rohrmeier and Lehle, Plant Molec. Biol. [Plant Molecular Biology] 1:151-158 (1993); Firek et al., Plant Cell [Plant Cell] 1:151-158 (1993); Molec. Biol. 22: 129-142 (1993); Warner et al., Plant J. 3: 191-201 (1993); U.S. Patent No. 5,750,386; U.S. Patent No. 5,955,646; U.S. Patent No. 6,262,344; U.S. Patent No. 6,395,963; U.S. Patent No. 6,703,541; U.S. Patent No. 7,078,589; U.S. Patent No. 7,196,247; U.S. Patent No. 7,223,901; and U.S. Patent Application Publication No. 2010043102.

[0194] In some embodiments of the present disclosure, a "minimal promoter" or "basic promoter" is used. Minimal promoters can recruit and bind RNA polymerase II complexes and their accessory proteins to allow transcription initiation and extension. In some embodiments, minimal promoters are constructed to contain only the nucleotides / nucleotide sequences of selected promoters necessary for the binding of transcription factors and the transcription of target nucleotide sequences, and this target nucleotide sequence is operably associated with a minimal promoter including but not limited to a TATA box sequence. In other embodiments, minimal promoters lack cis sequences that recruit and bind transcription factors, and these transcription factors regulate (e.g., enhance, repress, confer tissue specificity, confer induction or repression) transcription. Minimal promoters are usually placed upstream (i.e., 5') of the nucleotide sequence to be expressed. Therefore, nucleotides / nucleotide sequences from any promoter available with the present disclosure can be selected to be used as minimal promoters.

[0195] A large number of other sequences can be incorporated into the expression cassettes described in the present disclosure. These sequences include sequences that have been shown to enhance expression, such as intron sequences (e.g., from Adhl and bronzel) and viral leader sequences (e.g., from TMV, MCMV, and AMV).

[0196] The nucleic acid of the present disclosure may be more preferably targeted for expression in plants for different cell localizations. In some cases, localization in the cytosol may be desirable, while in other cases, localization in a certain subcellular organelle may be preferred. Subcellular localization of transgenics encoding enzymes is performed using techniques well known in the art. Typically, DNA encoding a target peptide from a gene product of known organelle targeting is operated and fused upstream of the nucleic acid. Many such target sequences for chloroplasts are known and have demonstrated their functions in heterologous constructs. The expression of the nucleic acid of the present disclosure is also targeted to the endoplasmic reticulum or vacuole of the host cell. The technology for realizing it is well known in the art.

[0197] Vectors suitable for plant transformation are described elsewhere in this specification. For Agrobacterium-mediated transformation, binary vectors or vectors carrying at least one T-DNA border sequence are suitable, and for direct gene transfer, any vector is suitable, and it is perhaps preferred that the linear DNA containing only the purpose construct is used. In the case of direct gene transfer, conversion or co-transformation with a single DNA species can be used (Schocher et al., Biotechnology [biotechnology] 4: 1093-1096 (1986)). For both direct gene transfer and Agrobacterium-mediated transformation, conversion is usually (but not necessarily) carried out with a selective marker, which can provide resistance to antibiotics (kanamycin, hygromycin or methotrexate) or herbicides (basta). The plant transformation vector comprising the nucleic acid molecules of the present disclosure may also include the following genes (e.g., phosphomannose isomerase; PMI), which provide positive selection of transgenic plants, as disclosed in U.S. Patents 5,767,378 and 5,994,629 (incorporated herein by reference). However, the choice of selectable marker is not critical to the present disclosure.

[0198] In some embodiments, nucleic acid can be transformed into nuclear genome. In another embodiment, nucleic acid of the present disclosure is directly transformed into plastid genome. The main advantage of plastid transformation is that plastids are usually able to express bacterial genes without substantial codon optimization, and plastids can express multiple open reading frames under the control of a single promoter. In U.S. Patent Nos. 5,451,513, 5,545,817 and 5,545,818, in PCT Application No. WO 95 / 16783, and in McBride et al., (1994), Proc. Nati. Acad. Sci. USA [Proceedings of the National Academy of Sciences of the United States] 91, 7301-7305, plastid transformation technology is widely described. Basic chloroplast transformation technology involves, for example, using biolistic or protoplast transformation (e.g., calcium chloride or PEG-mediated transformation), the cloned plastid DNA region flanking the selective marker is introduced into a suitable target tissue together with the target gene. These 1 to 1.5 kb flanking regions (designated targeting sequences) promote homologous recombination with the plastid genome and thus allow replacement or modification of specific regions of the plastid. Initially, point mutations in the chloroplast 16S rRNA and rps12 genes (conferring resistance to spectinomycin and / or streptomycin) were used as selective markers for transformation (Svab, Z., Hajdukiewicz, P., and Maliga, P. (1990) Proc. Nati. Acad. Sci. USA [Proceedings of the National Academy of Sciences of the United States] 87, 8526-8530; Staub, JM, and Maliga, P. (1992) Plant Cell [Plant Cell] 4, 39-45). This produces stable homoplasmic transformants at a frequency of about 1 per 100 target leaf bombardments. The presence of a cloning site between these markers allows the establishment of plastid targeting vectors for the introduction of foreign genes (Staub, JM and Maliga, P. (1993) EMBO J. [European Journal of Molecular Biology] 12, 601-606). A substantial increase in transformation frequency is obtained by replacing a recessive rRNA or r-protein antibiotic resistance gene with a dominant selectable marker (the bacterial aadA gene, which encodes the spectinomycin-detoxifying enzyme aminoglycoside-3'-adenylyltransferase) (Svab, Z. and Maliga, P. (1993) Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences of the United States of America] 90, 913-917).Previously, this marker has been successfully used for high-frequency transformation of the plastid genome of the green alga Chlamydomonas reinhardtii (Goldschmidt-Clermont, M. (1991) Nucl. Acids Res. [Nucleic Acids Research] 19: 4083-4089). Other selective markers for plastid transformation are known in the art and are included in the scope of the present disclosure. Typically, about 15-20 cell division cycles are required after transformation to reach a homoplasmic state. Plastid expression (wherein the gene is inserted into all of the thousands of copies of the circular plastid genome present in each plant cell by homologous recombination) takes advantage of the huge copy number of genes over nuclear expression to allow expression levels that can easily exceed 10% of total soluble plant proteins. In a preferred embodiment, the nucleic acid of the present disclosure is inserted into a plastid-targeted vector and transformed into the plastid genome of the desired plant host. Plants homozygous for a plastid genome comprising a nucleic acid of the present disclosure are obtained and these plants are preferentially capable of overexpressing the nucleic acid.

[0199] In other embodiments, the transgenic plant of the present disclosure may include a heterologous nucleic acid molecule encoding at least one other desired proterties. Other proterties may be encoded on a heterologous nucleic acid molecule identical to the nucleic acid molecule of the present disclosure, or may be encoded on a second heterologous nucleic acid molecule. Other desired proterties may confer insect resistance to a second insect pest, insect resistance to the same insect pest, abiotic stress tolerance, male sterility, herbicide resistance, bacterial disease resistance, fungal disease resistance, viral disease resistance, nematode resistance, modified fatty acid metabolism, modified carbohydrate metabolism, improved nutritional value, improved performance in an industrial process, or the reproductive capacity of a change. Other desired proterties may also induce commercially valuable enzymes or metabolites to be produced in plants.

[0200] In some embodiments, the desired additional trait is a second pesticide.The second pesticide can be active against any plant pest, including insects, nematodes, fungi, viruses, or bacteria. Examples of insect plant pests include, but are not limited to, Nilaparvata spp. (e.g., N. lugens (brown planthopper)); Laodelphax spp. (e.g., L. striatellus (small brown planthopper)); Nephotettix spp. (e.g., N. virescens or N. cincticeps (green leafhopper), or N. nigropictus (rice leafhopper)); Sogatella spp. (e.g., S. furcifera, white-backed planthopper); Blissus spp. (e.g., N. virescens or N. cincticeps (green leafhopper), or N. nigropictus (rice leafhopper); spp.) (e.g., B. leucopterus (chinch bug)); Scotinophora spp. (e.g., S. vermidulate, rice blackbug); Acrosternum spp. (e.g., A. hilare (green stink bug)); Parnara spp. (e.g., P. guttata, riceskipper); Chilo spp. (e.g., C. suppressalis (rice striped stem borer), C. auricilius (gold-fringed stemborer), or C. polychrysus (dark-headed stem borer); Chilotraea spp. (e.g. C. polychrysa (rice stalk borer); Sesamia spp. (e.g. S. inferens (pink riceborer); Tryporyza spp.) (e.g., T. innotata (white rice borer), or T. incertulas (yellow rice borer)); Cnaphalocrocis spp. (e.g., C. medinalis, rice leafroller); Agromyza spp. (e.g., A. oryzae (leafminer), or A. parvicornis (corn blot leafminer)); Conyza spp. (e.g., D. grandiosella, southwestern corn borer); Narnaga spp. (e.g., N. aenescens, green rice borer); caterpillar); Xanthodes spp. (e.g., X. transversa, green caterpillar); Spodoptera spp. (e.g., Spodoptera frugiperda (fall armyworm), Spodoptera exigua, beet armyworm, Spodoptera littoralis, climbing cutworm, or S. praefica, western yellow striped armyworm); Mythimna spp. (e.g., Mythmnaseperata, Pseudaletia seperata); Spodoptera spp. (e.g., corn earworm (corn ear moth)); Colaspis spp. (e.g., C. brunnea, grape colaspis); Lissorhoptrus spp. (e.g., L. oryzophilus (rice water weevil); Echinocnemus spp. (e.g., E. squamos (rice plant weevil)); Diclodispa spp. (e.g., D. armigera, rice hispa); Oulema spp. (e.g., O. oryzophilus (rice water weevil));oryzae (leaf beetle); Sitophilus spp. (e.g., S. oryzae, rice weevil); Pachydiplosis spp. (e.g., P. oryzae, rice gall midge); Hydrellia spp. (e.g., H. griseola (small rice leaf miner), or H. sasakii, rice stem maggot); Chlorops spp. (e.g., C. oryzae, stem maggot); Rootworm species (e.g., O. nubilalis (Western corn rootworm), O. barnetta (Northern corn rootworm), O. eleven-spotted (Southern corn rootworm), O. mexicana (Mexican corn rootworm); Cucumis sativus (banded cucumber beetle); Ostrinia spp. (e.g., O. nubilalis, European corn borer); Spodoptera species (e.g., Agrotissima (black cutworm); Elasmopalpus spp. (e.g., E. lignosellus (lesser cornstalk borer); Cyclocephalus spp. (wireworm); Cyclocephala spp. (e.g., C. borealis, northern masked rhinoceros beetle); chafer or southern masked chafer (C. immaculata); Popillia spp. (e.g., P. japonica, Japanese beetle); Chaetocnema spp. (e.g., C. pulicaria, corn flea beetle); Sphenophorus spp. (e.g., S. maidis, maize billbug); Rhopalosiphum spp. (e.g., R. maidis (corn leaf aphid); Anuraphis spp. (e.g., A.maidiradicis, corn root aphid); Melanoplus spp. (e.g. M. femurrubrum (redlegged grasshopper), M. differentialis, differential grasshopper or M. sanguinipes (migratory grasshopper); Hylemya spp. (e.g. H. platura, seed corn maggot); Anaphothrips spp. (e.g. A. obscrurus, grass thrips); Solenopsis spp. (e.g. S. milesta, thief ant); Tetranychus spp. (e.g. T. urticae, twospotted spider mite); mite, T. cinnabarinus, carmine spider mite; Spodoptera spp. (e.g., corn earworm (cotton bollworm), or H. armigera (American bollworm)); Pectinophora spp. (e.g., P. gossypiella, pink bollworm); Earias spp. (e.g., E. vittella, spotted bollworm); Heliothis spp. (e.g., H. virescens (tobacco budworm)); Heliothis spp. (e.g., A. grandis (bollworm); weevil); Pseudatomoscelis spp. (e.g., P. seriatus, cotton fleahopper); Trialeurodes spp. (e.g., T. abutiloneus (banded-winged whitefly), T. vaporariorum (greenhouse whitefly); Bemisia spp. (e.g., B.argentifolii (silverleaf whitefly); Aphis spp. (e.g., A. gossypii (cotton aphid); Lygus spp. (e.g., L. lineolaris (tarnished plant bug) or L. hesperus (western tarnished plant bug)); Euschistus spp. (e.g., E. conspersus (consperse stinkbug)); Chlorochroa spp. (e.g., C. sayi (Say stinkbug)); Nezara spp. (e.g., spp.) (e.g. N. viridula (southern green stink bug)); Thrips species (e.g. T. tabaci (onion thrips)); Frankliniella spp. (e.g. F. fusca (tobacco thrips) or F. occidentalis (western flower thrips); Leptinotarsa ​​spp. (e.g. L. decemlineata (Colorado potato beetle), L. juncta (false potato beetle) or L. texana (Texan false potato beetle)). potato beetle); Lema species (e.g. three-lined potato beetle (L. trilineata); Epitrix spp. (e.g. potato flea beetle (E. cucumeris), tobacco flea beetle (E. hirtipennis) (flea beetle), or tuber flea beetle (E. tuberis); Meloidogyne species (e.g. E.vittata, striped blister beetle); Phaedon spp. (e.g., P. cochleariae (mustard leaf beetle); Epilaxna spp. (e.g., E. varivetis (Mexican bean beetle); Acheta spp. (e.g., A. domesticus (house cricket); Empoasca spp. (e.g., E. fabae (potato leafhopper); Myzus spp. (e.g., M. persicae (green peach aphid); Paratrioza spp. (e.g., spp.) (e.g., P. cockerelli (psyllid)); Conoderus spp. (e.g., C. falli, southern potato wireworm or C. vespertinus, tobacco wireworm); Conoderus spp. (e.g., P. operculella, potato tuberworm); Macrosiphum spp. (e.g., M. euphorbiae (potato aphid); Thyanta spp. (e.g., T. Pallidovirens (red shouldered stinkbug); Conoderus spp. (e.g., P. operculella, potato tuberworm); Spodoptera spp. (e.g., P. operculella, potato tuberworm); fruitworm); Lycopodiella spp. (e.g., Mylotrichum solani (tomato pinworm); Limonius spp. (wireworms); Manduca spp. (e.g., M. sexta (tobacco hornworm) and M. quinquemaculata (tomato hornworm); Liriomyza spp. (e.g., L. sativae, L. trifoliata, L. quinquemacul ...trifolli or L. huidobrensis (leaf miners); Drosophilla spp. (e.g. Drosophila melanogaster, D. yakuba, D. Pseudoobscura or D. simulans); Carabus spp. (e.g. C. granulatus); Chironomus spp. (e.g. C. tentanus); Ctenocephalus spp. (C. felis, cat flea); Diaprepes spp. (e.g. D. abbreviation, root weevil); Ips spp. (e.g. I. pini (pine engraver); Tribolium spp. (e.g. T. innotata, red floor beetle); beetle); Glossina spp. (e.g., G. Morsitans, tsetse fly); Anopheles spp. (e.g., A. gambiae (malaria mosquito); Helicoverpa spp. (e.g., H. armigera (African Bollworm); Acyrthosiphon spp. (e.g., A. pisum, pea aphid); Apis spp. (e.g., A. melifera (honey bee); Homalodisca spp. (e.g., H. coagulate (glassy-winged sharpshooter); Aedes spp. (e.g., Ae. aegypti (yellow fever mosquito)); fever mosquito); Bombyx spp. (e.g., B. mori (silkworm); Locusta spp. (e.g., L. migratoria (migratory locust); Boophilus spp. (e.g., B. microplus (cattle tick); Acanthoscurria spp. (e.g., A.Gomesiana (red-haired chololate bird eater); Diploptera spp. (e.g., D. punctata, pacific beetle cockroach); Heliconius spp. (e.g., H. erato (red passion flower butterfly) or H. melpomene (postman butterfly); Curculio spp. (e.g., C. glandium, acorn weevil); Plutella xylostella (e.g., P. xylostella, diamondback moth); Amblyomma spp. (e.g., A. variegatum, cattle tick); tick); Anteraea species (e.g. A. Yamamai (silkmoth); and Armigeress pp. (e.g. A. subalbatus).

[0201] The RIP toxins disclosed herein can be used in combination with other insecticides to increase the target range of pests. In addition, the RIP toxins disclosed herein are used in combination with a second insecticide (which has a different mode of action or targets a different receptor in the insect gut) for prevention and / or management of insect resistance. In some embodiments, the RIP toxins disclosed herein are combined with a second insecticide protein selected from the group consisting of Cry1A, Cry1Ab, Cry1Ac, Cry1A.105, Cry1Ae, Cry1B, Cry1B variants, Cry1C, Cry1C variants, Cry1D, Cry1D variants, Cry1E, Cry1F, Cry1A / F chimeras, Cry1G, Cry1H, Cry1I, Cry1J, Cry1K, Cry1L, Cry2A, Cry2Ab, Cry2Ae, Cry3, Cry3A variants body, Cry3B, Cry4B, Cry6, Cry7, Cry8, Cry9, Cry15, Cry34, Cry35, Cry43A, Cry43B, Cry46A, Cry51Aa1, PtIP-96, PtIP-83, PHI-4, M P467, MP81, PS149B1, DIG-3, DIG-5, DIG-10, DIG-11, DIG-17, DIG-657, IRDIG28688.1, IRDIG28688.1, IRDIG28684.1, IRDIG28 682.1, IRDIG28680.1, IRDIG28674.1, IRDIG28672.1, IRDIG27642, IRDIG28688.1, IRDIG28686.1, IRDIG28684.1, IRDIG28682 .1, IRDIG28680.1, IRDIG28674.1, IRDIG28672.1, IRDIG27642, IRDIG28678.2, IRDIG28678.1, IRDIG31125.1, IRDIG28696.1, IRDIG29781.1, IRDIG29779.1, IRDIG30844.1, IRDIG30850.1, IRDIG30852.1, IRDIG30854.1, IRDIG30856.1, IRDIG30858.1, IRDIG30862.1, IRDIG30860.1, IRDIG30848.1, RETIRE2021, VIP3A, VIP3B, VIP3Ab, binary VIP1 and VIP2, or other vegetative insecticidal proteins, mCry3A, eCry3.1Ab, AXMI-001, AXMI-002, AXMI-030, AXMI-035, AXMI-036, AXMI-045, AXMI52, AXMI58, AXMI88, AXMI97, AX MI102, AXMI112, AXMI113, AXMI115, AXMI117, AXMI100, AXMI-115, AXMI-113, and AXMI-005, AXMI134, AXMI-15 0, AXMI171, AXMI-184, AXMI196, AXMI204, AXMI207, AXMI209, AXMI205, AXMI218, AXMI220, AXMI221z, AXMI222z, AXMI223z, AXMI224z and AXMI225z, AXMI238, AXMI270, AXMI279, AXMI345, AXMI-R1 and its variants, IP3 and its variants, ET29, E T33, ET34, ET35, ET66, ET70, TIC400, TIC407, TIC417, TIC431, TIC800, TIC807, TIC834, TIC836, TIC844, TIC853, TIC860 or variants thereof, TIC867 or variants thereof, TIC868 or variants thereof, TIC869, TIC900 or related proteins, TIC901, TIC1100, TIC1201, TIC1362, TIC1414, TIC1415, TIC1422, TIC1497, TIC1498, TIC1885, TIC1886, TIC1922, TIC1925, TIC1974, TIC2032, TIC2120, TIC2160, TIC3131, TIC3244, TIC6757, TIC7243, TIC7472, and TIC7473 proteins, or hybrid proteins or chimeras made from any of the foregoing insecticidal proteins. The second insecticide can also be an agent selected from the group comprising: alpha amylase, peroxidase, cholesterol oxidase, potato glycoprotein, protease, protease inhibitor, urease, alpha-amylase inhibitor, pore-forming protein, chitinase, lectin, engineered antibody or antibody fragment, Bacillus cereus insecticidal protein, Xenorhabdus species (such as X. nematophila or Xenorhabdus bovienii) insecticidal protein, Photorhabdus species (such as P. luminescens or P. asymobiotica) insecticidal protein, Brevibacillus species (such as B. laterosporous) insecticidal protein, Lysinibacillus spp.In some embodiments, the second agent may be an insecticidal protein derived from at least one insecticide toxin complex (Tc) from Photorhabdus, Xenorhabus, Serratia or Yersinia. In still other embodiments, the second insecticidal protein may be a binary toxin derived from insecticide bacteria, such as ISP1A and ISP2A from Brevibacillus laterosporus or BinA and BinB from Bacillus sphaericus. The combination of the RIP of the present disclosure and the second pesticide can be expressed by both in a transgenic plant. In some embodiments, the transgenic plant is a transgenic corn plant. In other embodiments, the combination in the transgenic corn plant is a RIP of the present disclosure and mCry3A and / or eCry3.1Ab and / or Cry3Bb1 and / or Cry34 / Cry35. .

[0202] In some embodiments, the transgenic plants disclosed herein may include at least one non-protein second pesticidal agent. In a preferred embodiment, the second pesticidal agent is an interfering RNA molecule. Interfering RNA molecules typically include at least one RNA fragment for a target gene, a spacer sequence, and a second RNA fragment complementary to the first RNA fragment, thereby forming a double-stranded RNA structure. When an organism recognizes double-stranded RNA (dsRNA) molecules and hydrolyzes them, RNA interference (RNAi) occurs. The resulting hydrolyzate is a small RNA fragment of about 19-24 nucleotides in length, which is referred to as small interfering RNA (siRNA). These siRNAs then diffuse or are carried to the entire organism, including across the cell membrane, where they hybridize with mRNA (or other RNA) and cause the hydrolysis of RNA. Interfering RNA is recognized by the RNA interference silencing complex (RISC), and the effector strand (or "guide strand") of RNA is located in the complex. This guide strand serves as a recognition and destruction template for the duplex sequence. Each time siRNA hybridizes with its complementary RNA target, this process is repeated, effectively preventing those mRNAs from being translated, and thus "silencing" the expression of specific genes transcribed therein. Interfering RNA is known in the art to be used for insect control (see, for example, Publication WO 2013 / 192256, which is incorporated herein by reference). Interfering RNA designed for insect control produces non-natural double-stranded RNA that utilizes the natural RNAi pathway in insects to trigger the downregulation of target genes, which may result in cessation of feeding and / or growth and may result in death of insect pests. Interfering RNA molecules may confer insect resistance to target pests identical to the proteins disclosed herein or may target different pests. Target insect plant pests may be fed by chewing, sucking or piercing. Interfering RNA is known in the art to be used for insect control. In other embodiments, interfering RNA may confer resistance to non-insect plant pests (such as nematode pests or viral pests).

[0203] Co-expression of more than one pesticide in the same transgenic plant can be achieved by making a single recombinant vector (comprising the coding sequence of more than one pesticide in a so-called molecular stack) and genetically engineering the plant so as to contain and express all the pesticides in the transgenic plant. Such molecular stacking can also be prepared by using mini-chromosomes, as described in, for example, U.S. Patent 7,235,716. Alternatively, a transgenic plant comprising a nucleic acid encoding a first pesticide can be re-transformed with a different nucleic acid encoding a second pesticidal agent, etc. Alternatively, a plant (parent 1) can be genetically engineered for expression of the genes disclosed herein. A second plant (parent 2) can be genetically engineered for expression of a second pesticide. By hybridizing parent 1 with parent 2, a progeny plant expressing all the genes introduced into parent 1 and parent 2 is obtained.

[0204] Transgenic plants or seeds including the insecticidal proteins of the present disclosure may also be treated with insecticides or insecticidal seed coatings, as described in U.S. Pat. Nos. 5,849,320 and 5,876,739 (incorporated herein by reference). Where the insecticides or insecticidal seed coatings of the present disclosure and the transgenic plants or seeds are active against the same target insects (e.g., coleopteran pests or rootworm target pests), the combination (i) is useful in methods for further enhancing the activity of the compositions of the present disclosure against the target insects and (ii) in methods for preventing resistance to the compositions of the present disclosure by providing yet another mechanism of action against the target insects. Thus, the present disclosure provides methods for enhancing control of rootworm insect populations, the methods comprising providing transgenic plants or seeds of the present disclosure and applying the insecticides or insecticidal seed coatings of the present disclosure to the plants or the seeds.

[0205] Even where the insecticidal seed coating is active against different insects, the insecticidal seed coating is useful for expanding the spectrum of insect control, for example by adding an insecticidal seed coating active against Lepidoptera insects to transgenic seeds of the present disclosure (active against Coleoptera and some Lepidoptera in some embodiments), the resulting coated transgenic seeds control both Lepidoptera and Coleoptera pests.

[0206] Examples of such insecticides and / or insecticidal seed coatings include, but are not limited to, carbamates, pyrethroids, organophosphates, friprole, neonicotinoids, organochlorides, nereistoxins, or combinations thereof. In another embodiment, the insecticide or insecticidal seed coating is selected from the group consisting of carbofuran, carbaryl, methomyl, bifenthrin, tefluthrin, permethrin, cyfluthrin, lambda-cyhalothrin, cypermethrin, deltamethrin, chlorpyrifos, chloroxyfos, dimethoate, ethoxyprothion, malathion, methyl parathion, phorate, terbufos, tebupirimiphos, fipronil, acetamiprid, imidacloprid, thiacloprid, thiamethoxam, endosulfan, sulfamic acid, and combinations thereof. Commercial products containing such insecticides and insecticidal seed coatings include, but are not limited to (Carbofuran), (Methomyl, Methomyl, Nanaid), (aminonaphthalene), (Bifenthrin), (Tefluthrin), (Cypermethrin), (Cypermethrin), Delta (Deltamethrin), (λ-cyhalothrin), (Permethrin), (Permethrin), (Bifenthrin), (Bifenthrin), (tefluthrin)), (λ-cyhalothrin), (Chlorpyrifos), (Chlorophene Oxygen), (Methoate), (Phorate), (Phorate, flucythinate), (Phorate), (terbufos), (Dimethoate), isochlorophos, (fipronil)), (Thiamethoxam), (Imidacloprid), (Imidacloprid), (thiamethoxam) and (Cyfluthrin, Cypermethrin).

[0207] In some embodiments, the present disclosure also encompasses a composition comprising an insecticidal protein disclosed herein in an effective amount for controlling insects. In a further embodiment, the composition comprises a suitable agricultural carrier and the RIP disclosed herein. The agricultural carrier may include an adjuvant, a mixture, an enhancer, etc. that are beneficial to the application of the active ingredient (such as the protein disclosed herein, including a protein comprising, consisting essentially of, or consisting of: an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to any one of SEQ ID NO: 1-21). Suitable carriers should not be phytotoxic to valuable crops (particularly at the concentration used when the composition is applied in the presence of crops), and should not react chemically with the compounds of the active ingredients herein (i.e., polypeptides or other composition components disclosed herein). Such mixtures may be designed for direct application to crops, or may be concentrates or formulations that are typically diluted with additional carriers and adjuvants before application. They may include inert or active ingredients and may be solid (such as, for example, dusts, powders, granules, water-dispersible granules or wettable powders) or liquid (such as emulsifiable concentrates, solutions, emulsions or suspensions). Suitable agricultural carriers may include liquid carriers such as water, toluene, xylene, naphtha, crop oils, acetone, methyl ethyl ketone, cyclohexanone, trichloroethylene, perchloroethylene, ethyl acetate, amyl acetate, butyl acetate, propylene glycol monomethyl ether and diethylene glycol methyl ether, methanol, ethanol, isopropanol, amyl alcohol, ethylene glycol, propylene glycol, glycerol, and the like. Water is generally the carrier of choice for diluting concentrates. Suitable solid carriers may include talc, pyrophyllite clay, silica, attapulgite clay, kieselguhr, chalk, diatomaxeous earth, lime, calcium carbonate, bentonite, Fuller's earth, cottonseed hulls, wheat flour, soy flour, pumice, wood flour, walnut shell flour, lignin, and the like. In other embodiments, the proteins of the present disclosure can be encapsulated in a synthetic matrix (such as a polymer) and applied to the surface of a host (such as a plant). Insect uptake of host cells allows the insect control agent to be delivered to the insect and results in a toxic effect on the insect pest.

[0208] In other embodiments, the composition of the present disclosure can be a powder, dust, pill, granule, spray, emulsion, colloid or solution. The composition of the present disclosure can be prepared by dehydrating, freeze-drying, homogenizing, extracting, filtering, centrifuging, sedimenting or concentrating the culture of bacterial cells. The composition of the present disclosure may contain at least 1%, about 5%, at least 10%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 97% or at least 99% of the polypeptide of the present disclosure by weight. The composition of the present disclosure may include at least a second insecticide (which can be insecticidal, nematicidal, fungicidal, bactericidal). At least the second insecticide can have an insecticidal effect on the same insect or a different insect as the polypeptide of the present disclosure. The second insecticide can be a polypeptide. The insecticide can be an interfering RNA. The second pesticide can be a microorganism (such as bacteria) comprising a nucleic acid molecule encoding a pesticide and / or comprising a pesticide (such as a polypeptide or interfering RNA). The microorganism can be attenuated, heat-inactivated or freeze-dried. The microorganism may die or be unable to reproduce. The second pesticide can be an insecticide, such as carbofuran, carbaryl, methomyl, bifenthrin, tefluthrin, permethrin, cyfluthrin, lambda-cyhalothrin, cypermethrin, deltamethrin, chlorpyrifos, chloroxyfos, dimethoate, ethoxyproline, malathion, methyl parathion, phorate, terbufos, tert-butyl pyrimidine phosphorus (tebupirimiphos), fipronil, acetamiprid, imidacloprid, thiacloprid, thiamethoxam, endosulfan, sulfamic acid, and a combination thereof, or a commercial product containing an insecticide and an insecticide seed coating as described above.

[0209] The compositions of the present disclosure (e.g., compositions comprising the protein of the present disclosure and agriculturally acceptable carriers) can be used in traditional agricultural methods. Agriculturally acceptable carriers are formulations that can be used to apply the compositions comprising the polypeptide of the present disclosure to plants or seeds. For example, the compositions of the present disclosure can be mixed with water and / or fertilizers, and can be applied to desired places before and / or after emergence by any means, such as aircraft spray barrels, irrigation equipment, direct injection spray equipment, backpack spray barrels, livestock dip tanks, farm equipment used in ground spraying (e.g., spray tube sprayers, hand sprayers), etc. The desired place can be soil, plants, etc.

[0210] The compositions of the present disclosure can be applied to seeds or plant propagules in any physiological state at the following time: any time between seed harvest and sowing; or during or after sowing; and / or after germination. Preferably, the seeds or plant propagules are in a sufficiently durable state so that no damage or minimal damage, including physical damage or biological damage, is caused during the treatment process. The formulation can be applied to seeds or plant propagules using conventional coating techniques and machines (such as fluidized bed technology, drum grinding method, static rotation (rotostatic) seed processor and drum coater).

[0211] In some embodiments, the disclosure also includes methods of controlling a population of coleopteran pests, the methods comprising contacting the pest population with an insect-controlling effective amount of a RIP of the disclosure, wherein the protein comprises, consists essentially of, or consists of an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 1-21. The contacting includes ingestion or ingestion of the insecticidal protein by members of the pest population. The insecticidal protein may be incorporated into an insect dietary food, or may be expressed or present in plant tissues that are then ingested by the insect population. In further embodiments, controlling a population of coleopteran pests includes killing the insects by contacting the insects with an insect-controlling effective amount of an insecticidal protein of the disclosure.

[0212] The disclosure further encompasses a method for increasing plant yield, the method comprising growing a plant or its seeds having stably incorporated into its genome a nucleic acid molecule of the expression cassette of the disclosure in a field, and wherein the field is infested with a pest against which the polypeptide has insecticidal activity.

[0213] Once a desired nucleic acid has been transformed into a particular plant species, it can be propagated within that species or moved into other varieties of the same species (particularly including commercial varieties) using conventional breeding techniques.

[0214] In some embodiments, the present disclosure encompasses a method of providing a corn grower with a means of controlling a rootworm pest population in a corn crop, the method comprising (a) selling or providing to the grower a transgenic corn seed comprising a nucleic acid molecule, expression cassette, vector or chimeric gene of the present disclosure; and (b) advertising to the grower that the transgenic corn seed produces a transgenic corn plant that controls a rootworm pest population.

[0215] In some embodiments, the disclosure also encompasses methods of identifying an insecticidal protein comprising, consisting essentially of, or consisting of a nucleotide sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NOs: 1-21, or a toxin fragment thereof, the method comprising the steps of: (a) generating a primer pair that will amplify the SEQ ID NOs from a nucleic acid sample; NO:22-25 or its complementary sequence, (b) amplifying the orthologous polynucleotide from the nucleic acid sample, (c) identifying the nucleotide sequence of the orthologous polynucleotide, (d) producing a protein encoded by the orthologous polynucleotide, and (e) determining that the protein of step (d) has insecticidal activity against insect pests.

[0216] Examples

[0217] Embodiments of the present invention can be better understood by reference to the following examples. The foregoing and following embodiments of the present invention and the description of various embodiments are not intended to limit the claims, but are illustrative thereof. Therefore, it should be understood that the claims are not intended to be limited to the specific details of these examples. It should be understood by those skilled in the art that other embodiments of the present invention can be practiced without departing from the spirit and scope of the present disclosure, and the scope of the present disclosure is defined by the appended claims. Recombinant DNA and molecular cloning techniques recognized in the art can be found in, for example, J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press (2001); T. J. Silhavy, M. L. Berman, and L. W. Enquist, Experiments with Gene Fusions, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1984) and Ausubel, FM et al., Current Protocols in Molecular Biology, New York, John Wiley and Sons Inc., (1988), Reiter et al., Methods in Arabidopsis Research, World Scientific Publishing Co., Ltd., (1989). Press [World Scientific Press] (1992), and Schultz et al., Plant Molecular Biology Manual [Plant Molecular Biology Manual], Kluwer Academic Publishers [Kluwer Academic Publishers] (1998).

[0218] Example 1: Identification of sequences encoding insecticidal proteins from the family Rhizobiaceae.

[0219] Based on a proprietary algorithm, candidate nucleotide sequences encoding proteins described as hypothetical proteins in the art were identified in the genomes of Gram-negative bacteria belonging to the genus Sinorhizobium, Sword Pseudomonas, Rhizobium, and related genera belonging to the Rhizobiaceae family. Four candidate sequences were selected for expression and testing against insect pests. Four candidate nucleotide sequences were identified in the genomes of the Sword Pseudomonas alidi strain (NCBI: txid1708715) (SEQ ID NO: 22), Sinorhizobium species GL28 strain (NCBI: txid1358418) (SEQ ID NO: 23), unclassified Rhizobiales bacterial strain (NCBI: txid1909294) (SEQ ID NO: 24), and Rhizobium species strain SPY-1 (NCBI: txid2547961) (SEQ ID NO: 25). The codon optimized version of each candidate coding sequence was generated, SEQ ID NO: 26-29, and introduced into the pET29a bacterial expression vector separately, designated as pET29a-26, pET29a-27, pET29a-28 and pET29a-29, to produce protein. Each pET29a expression vector was transformed into E. coli BL21*(DE3) and lysate was prepared from isopropyl β-D-1-thiogalactopyranoside (IPTG)-induced culture (producing protein overnight at about 18°C). In the feed incorporation bioassay experiment, the insecticidal activity of lysate to western corn rootworm (WCR) was detected. In brief, the E. coli lysate was mixed with an equal volume of heated artificial insect feed (Bioserv, Frenchtown, New Jersey (NJ)) in a 1.5 mL centrifuge tube and then applied to a small culture dish. After the diet-sample mixture cooled and solidified, 12 WCR larvae were added to each plate. The plates were sealed and maintained under ambient laboratory conditions with respect to temperature, light, and relative humidity. Buffer without lysate, lysate from E. coli BL21*(DE3) culture (containing an empty pET29a vector), and artificial insect diet alone served as negative controls. Percent mortality and growth inhibition observations were obtained at 4 and 6 days post-infection, designated as s=small larvae, m=medium larvae, and l=large larvae.

[0220] The results shown in Table 1 indicate that lysates from E. coli cultures expressing proteins encoded in the genome of Rhizobiaceae bacteria surprisingly have insecticidal activity against rootworm insect pests. These Rhizobiaceae insecticidal proteins (RIPs) were named Enf_adiCRW (RIP1Aa; SEQ ID NO: 1), which was expressed by the pET29a-26 vector; Sinorhiz_GL28CRW (RIP2Aa; SEQ ID NO: 2), which was expressed by the pET29a-27 vector; Rhizo_bactCRW (RIP3Aa; SEQ ID NO: 3), which was expressed by the pET29a-28 vector; and Rhiz_SPYCRW (RIP4Aa; SEQ ID NO: 4), which was expressed by the pET29a-29 vector.

[0221] Table 1: Insecticidal activity of RIPs against WCR

[0222]

[0223] Table 2 shows the alignment and sequence identity comparison of active RIPs against WCR. National Institutes of Health (NIH) gene sequence database (U.S. National Library of Medicine) searches indicate that the disclosed RIPs are not significantly identical to any other sequences. However, the disclosed RIPs appear to have cytotoxic domains. Enf_adiCRW protein (RIP1Aa; SEQ ID NO: 1) has 345 amino acids, is 38 kDa, and contains a cytotoxic domain from about amino acid position 40 to about amino acid position 234. Sinorhiz_GL28CRW protein (RIP2Aa; SEQ ID NO: 2) has 344 amino acids, is 37.7 kDa, and contains a cytotoxic domain from about amino acid position 39 to about amino acid position 233. Rhizo_bactCRW protein (RIP3Aa; SEQ ID NO: 3) has 351 amino acids, is 38.5 kDa, and contains a cytotoxic domain from about amino acid position 46 to about amino acid position 240. The Rhiz_SPYCRW protein (RIP4Aa; SEQ ID NO: 4) has 347 amino acids, is 38.2 kDa, and contains a cytotoxin domain from about amino acid 40 to about amino acid 234. Other bacteria, such as Bacillus thuringiensis, are known to produce proteins with cytotoxin domains that are primarily active against Diptera and not Coleoptera, particularly not rootworm pests. However, the cytotoxin domains of the RIPs of the present disclosure appear to be unique, and without being bound by theory, it is believed that these unique cytotoxin domains of the RIPs of the present disclosure are responsible for their unexpected activity against coleoptera pests, particularly against rootworm pests.

[0224] Table 2. Alignment and percent identity comparison of WCR-active RIPs.

[0225]

[0226]

[0227] Under amino acids, "." indicates identical amino acids

[0228] Table 3. Alignment of RIP cytotoxic domains.

[0229]

[0230]

[0231] Example 2. Efficacy of RIP against corn rootworm.

[0232] To determine the efficacy of the RIPs of the present disclosure against western corn rootworm (WCR; corn rootworm), lysates containing the RIPs of the present disclosure were tested against WCR larvae over a range of concentrations in a dietary incorporation assay essentially as described in Example 1. Twelve newborn larvae were tested at each concentration. Mortality and percent growth were determined on days 4 and 6 for RIP2Aa and on days 3 and 6 for RIP3Aa.

[0233] As shown in Table 4, lysates containing RIP2Aa (SEQ ID NO: 2) were effective against WCR even at a 1:100 dilution, producing at least 25% mortality on day 6, compared to no mortality in the control treatment. RIP3Aa was active at the lowest concentration of 1:64, producing 100% mortality by day 6.

[0234] Table 4. Efficacy of RIP lysates against western corn rootworm.

[0235]

[0236]

[0237] The insecticidal properties of the RIP2Aa protein were further characterized. Two liters of E. coli BL21*(DE3) cells carrying pET-rip2Aa were grown in LB medium at 37°C. IPTG (1 mM) was added to the culture when the OD reached 0.8-1.0, and the culture was then moved to 18°C ​​for 18 hours. The cell pellet was harvested and resuspended in 20 mM Tris (pH 8.5) containing 10% glycerol. The cells were lysed using a French press; the lysate was then spun at 100k xg in an ultracentrifuge. The supernatant was collected, filtered, and then loaded onto a HiPrepQ anion exchange column, which was pre-equilibrated in 20 mM Tris (pH 8.5) containing 10% glycerol. The HiPrepQ column efficiently binds RIP2Aa; the protein was eluted from the column using a linear NaCl gradient. High salt buffer consisted of 20 mM Tris (pH 8.5), 0.5 M NaCl containing 10% glycerol. The purest fractions were combined and then concentrated to approximately 2 mL. The RIP2Aa protein was loaded onto a Sephadex 200 gel filtration column that had been pre-equilibrated in 1X PBS. The purity of the fractions from the Sephadex 200 column was analyzed by SDS-PAGE (RIP2Aa (SEQ ID NO: 2) has a predicted molecular weight of 37.7 kDa). The purest fractions were combined and then concentrated to approximately 7 mg / mL and then stored at -80°C. A series of concentrations of pure RIP2Aa protein were then tested against 12 WCR larvae in a diet incorporation assay essentially as described above. As shown in Table 5, RIP2Aa is effective against WCR; 75 μg / mL of RIP2Aa produced at least 75% mortality on the 6th day.

[0238] Table 5. Activity of RIP2Aa against western corn rootworm.

[0239]

[0240] Example 3. Biological activities of RIP variants.

[0241] Variant RIPs are generated by making single or double amino acid substitutions in the native RIP3Aa sequence (SEQ ID NO: 3). The variants made included the following: the I50 residue was changed to L (RIP3Aa-I150L; Xaa50 of SEQ ID NO:5 is L; SEQ ID NO:6), the I53 residue was changed to L (RIP3Aa-I53L; Xaa53 of SEQ ID NO:5 is L; SEQ ID NO:7), the I56 residue was changed to L (RIP3Aa-I56L; Xaa56 of SEQ ID NO:5 is L; SEQ ID NO:8), the A62 residue was changed to C or L (RIP3Aa-A62C or RIP3Aa-A62L; Xaa62 of SEQ ID NO:5 is C or L; SEQ ID NO:9 or SEQ ID NO:10), the A64 residue was changed to C or L (RIP3Aa-A64C or RIP3Aa-A64L; Xaa64 of SEQ ID NO:5 is C or L; SEQ ID NO:11). NO:11 or SEQ ID NO:12), the I81 residue was changed to L (RIP3Aa-I81L; Xaa81 of SEQ ID NO:5 is L; SEQ ID NO:13), the I126 residue was changed to L (RIP3Aa-I126L; Xaa126 of SEQ ID NO:5 is L; SEQ ID NO:14), the I153 residue was changed to L (RIP3Aa-I153L; Xaa153 of SEQ ID NO:5 is L; SEQ ID NO:15), the I169 residue was changed to L (RIP3Aa-I169L; Xaa169 of SEQ ID NO:5 is L; SEQ ID NO:16), the I185 residue was changed to L (RIP3Aa-I185L; Xaa185 of SEQ ID NO:5 is L; SEQ ID NO:17). NO:17), the I207 residue was changed to L (RIP3Aa-I207L; Xaa207 of SEQ ID NO:5 is L; SEQ ID NO:18), the I219 residue was changed to L (RIP3Aa-I219L; Xaa219 of SEQ ID NO:5 is L; SEQ ID NO:19), and the I275 residue was changed to L (RIP3Aa-I275L; Xaa275 of SEQ ID NO:5 is L; SEQ ID NO:20).

[0242] Each variant was tested in a diet incorporation assay against newborn WCR larvae essentially as described in Example 1. The results are shown in Table 6. The results indicate that certain amino acid substitutions can be made in the RIP and still retain insecticidal activity against Radix Gnaphalidae. Certain mutations do appear to delay full toxicity, suggesting that these positions may be important in the mode of action of the Rhizobiaceae insecticidal proteins.

[0243] Table 6. Insecticidal activity of variant RIPs.

[0244]

[0245] Example 4. Transformation of maize with a RIP coding sequence.

[0246] A nucleotide sequence encoding RIP1Aa, RIP2Aa, RIP3Aa, RIP4Aa or a variant thereof of the present disclosure, such as any one of SEQ ID NOs: 1-5, or a maize optimized nucleotide sequence, such as any one of SEQ ID NOs: 42-45, which can be generated as described, for example, in U.S. Pat. No. 6,051,760, is transformed into corn to control corn rootworm.

[0247] Two plant expression cassettes were constructed to introduce the RIP coding sequence into maize. The first cassette contained the maize ubiquitin 1 (Ubil) promoter operably connected to the rip coding sequence, which was operably connected to the maize Ubi361 terminator. The second cassette contained the maize Ubil promoter operably connected to the pmi coding sequence encoding the selective marker phosphomannose isomerase (PMI), which was operably connected to the maize Ubil terminator. A recombinant plant transformation binary vector containing the two expression cassettes was generated for maize transformation experiments.

[0248] The binary vector was transformed into Agrobacterium tumefaciens using standard molecular biology techniques.To prepare Agrobacterium for transformation, cells were grown overnight in liquid YPC medium at 28°C and 220 rpm.

[0249] Agrobacterium transformation of immature maize embryos was performed substantially as described in Negrotto et al., 2000, Plant Cell Reports 19:798-803. For this example, all culture medium components were substantially as described in Negrotto et al. (as above). However, various culture medium components known in the art may be substituted.

[0250] Briefly, Agrobacterium strain LBA4404 (pSB1) containing a binary vector plant transformation vector was grown on YEP (yeast extract (5 g / L), peptone (10 g / L), NaCl (5 g / L), 15 g / l agar, pH 6.8) solid medium at 28°C for 2-4 days. Approximately 0.8×10 9 Agrobacterium was suspended in LS-inf medium (Negrotto et al., supra) supplemented with 100 μM As. The bacteria were pre-induced in this medium for 30 to 60 minutes.

[0251] The immature embryos from the genotype that is suitable for are excised in the liquid LS-inf+100 μM As from the big ear of 8-12 days. Rinse these embryos with fresh infection culture medium. Add agrobacterium solution then, and these embryos are vortexed for 30 seconds and allowed to settle 5 minutes with bacteria. Then these embryo scutels are transferred upwards in the LSA culture medium, and cultivated two to three days in the dark. Subsequently, embryos between 20 and 25 of every petri plate (petri plate) are transferred to the LSDc culture medium that is supplemented with cefotaxime (250mg / l) and silver nitrate (1.6mg / l), and cultivated 10 days at 28 ℃ in the dark.

[0252] The immature embryos that produce embryonic callus are transferred to the LSD1M0.5S culture medium. On this culture medium, the selection that culture continues for about 6 weeks is carried out, and there is the subculture step of about 3 weeks. The callus that survives is transferred to the Reg1 culture medium that is supplemented with mannose. Afterwards in illumination (16 hours illumination / 8 hours dark scheme) cultivation, green tissue is transferred to the Reg2 culture medium that does not have growth regulator, hatches about 1 to 2 weeks. These plantlets are transferred to the Magenta GA-7 box (Magenta Corp, Chicago, Illinois) that contains Reg3 culture medium and it is grown in illumination.

[0253] After transformation, selection and regeneration, use The analysis was used to determine whether the pmi gene and the rip maize codon-optimized coding sequence were present in the plants. The presence of the vector backbone was also tested in the plants. Plants that were negative for the vector backbone and contained a copy of the transgenic from the binary vector were transferred to the greenhouse and tested for insecticidal activity against WCR.

[0254] Example 5. Rhizobium insecticidal protein in combination with a second insecticide.

[0255] The RIP of the present disclosure as described above is purified as described in Example 2. dsRNAs directed to essential targets and known to have insecticidal activity are prepared. In non-limiting examples, dsRNAs can target genes encoding vacuolar ATP synthase, β-tubulin, 26S proteosome subunit p28 protein, EF1α48D, troponin I, transmembrane tetraprotein, clathrin heavy chain, γ-coat body, β-coat body and / or juvenile hormone epoxide hydrolase (PCT Patent Application No. PCT / US 17 / 044825; PCT / US 17 / 044831; PCT / US 17 / 044832; U.S. Patent No. 7,812,219; each incorporated herein by reference). The efficacy of dsRNA and purified proteins against WCR was tested in a dietary incorporation assay performed essentially as described in Example 1.

[0256] Example 6. In situ genome editing in plant cells to generate modified RIPs.

[0257] The following examples illustrate the use of genome editing of plant cell genomes in situ to incorporate mutations described herein (including but not limited to the mutations described in Example 3) into the coding sequence of native RIPs, including RIP3Aa (SEQ ID NO: 3), or into the coding sequence of an already modified RIP3Aa protein.

[0258] Directed genome modification (also known as genome editing) can be used to introduce mutations in a specific DNA sequence. These genome editing technologies, including zinc finger nucleases (ZNFs), transcription activator-like effector nucleases (TALENs), meganucleases, and clustered regularly interspaced short palindromic repeats (CRISPRs), have been successfully applied to more than 50 different organisms including crop plants. See, for example, Belhaj, K., et al., Plant Methods [Plant Methods] 9, 39 (2013); Jiang, W., et al., Nucleic Acids Res [Nucleic Acids Research], 41, e188 (2013)). The CRISPR / Cas system for genome editing is based on the transient expression of an engineered single guide RNA (sgRNA) of the Cas9 nuclease and a specified target polynucleotide sequence.

[0259] Cas9 is a large monomeric DNA nuclease that is guided to a DNA target sequence by a complex of two 20-nucleotide (nt) noncoding RNAs: CRIPSR RNA (crRNA) and transactivating crRNA (tracrRNA), which are functionally available as a single synthetic RNA chimera. The Cas9 protein contains two nuclease domains homologous to RuvC and HNH nucleases. The HNH nuclease domain cuts the complementary DNA strand, while the RuvC-like domain cuts the non-complementary strand, thus introducing a blunt cut in the target DNA.

[0260] When Cas9 and sgRNA are transiently expressed in living maize cells, double-strand breaks (DSBs) in specific target DNA are generated in transgenic maize cells. Mutations at the break sites are introduced by non-homologous end joining and homology-directed DNA repair pathways.

[0261] By using a recombinant plasmid expressing Cas9 nuclease and sgRNA target (maize codon optimized for rip3Aa or mutant rip3Aa sequence) in transgenic maize, specific mutations (such as the mutations described in Example 3 above) are introduced into the coding sequence of the native RIP3Aa insecticidal protein (SEQ ID NO: 3) or mutant RIP3Aa protein. The method is implemented by the Agrobacterium infiltration method of Agrobacterium tumefaciens, which carries a binary plasmid containing a specified target sequence of interest. After the sgRNA binds to the target rip3Aa or mutant rip3Aa coding sequence, the Cas9 nuclease specifically cuts the coding sequence and introduces the desired one or more mutations during the DNA repair process. Thus, the now mutated rip3Aa coding sequence will encode a modified variant RIP3Aa protein, such as the variant described in Example 3, for example, wherein the mutation at residue I50 is changed to L, then residue I53 is changed to L, residue I56 is changed to L, residue A62 is changed to C or L, residue A64 is changed to C or L, residue I81 is changed to L, residue I126 is changed to L, residue I153 is changed to L, residue I169 is changed to L, residue I185 is changed to L, residue I207 is changed to L, residue I219 is changed to L, residue I275 is changed to L, or any combination thereof.

[0262] Plant cells containing genome-edited rip coding sequences were screened by PCR and sequencing. Calli with genome-edited mutations in rip or modified rip coding sequences were induced and regenerated into plants for phenotypic evaluation to evaluate the insecticidal activity of the expressed RIP against the following: western corn rootworm (Diplodia zeae), northern corn rootworm (Diplodia barnetta), southern corn rootworm (Diplodia elevenspotted cucumber root feeding subspecies) and / or Mexican corn rootworm (Diplodia mexicana).

[0263] Example 7. Testing of RIPs for insecticidal activity against lepidopteran pests.

[0264] The pET-6His-SUMO construct containing rip1Aa (SEQ ID NO:21) was made to express tagged RIP1Aa (SEQ ID NO:49). The pET-6His-SUMO-rip1Aa construct was transformed into E. coli BL21*(DE3) for protein production. Lysates from bacterial cultures expressing 6his-SUMO-RIP1Aa were tested for biological activity against a group of lepidopteran insect pests including corn ear moth (CEW), fall armyworm (FAW) and soybean looper (SBL) using a diet overlay bioassay. For each experiment, newborn WCR larvae were tested using lysates from BL21* bacterial cultures containing the gene encoding SUMO-RIP1Aa (SEQ ID NO:1) containing a hexa-histidine tag at the N-terminus. The positive control consisted of larvae exposed to lysates of E. coli BL21* expressing the Vip3 protein. Buffer alone and lysate from a BL21*(DE3) bacterial culture carrying an empty pET29 vector were used as negative controls. Mortality was assessed on day 7. The results of the bioassays showed that the labeled RIP1Aa was active against WCR but not against any of the lepidopteran pests tested.

[0265] As described above, bacterial lysates containing RIP2Aa (SEQ ID NO: 2) were also tested against lepidopteran pests including corn ear moth, European corn borer, black cutworm, and fall armyworm. The results of the bioassays showed that RIP2Aa had no insecticidal activity against any lepidopteran pest insects at the concentrations tested.

[0266] It should be understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes based on the description thereof will suggest themselves to those skilled in the art and will be included within the spirit and scope of the present application and the scope of the appended claims.

[0267] All publications and patent applications mentioned in this specification are indicative of the technical levels of those skilled in the art to which the invention pertains. All publications and patent applications are incorporated herein by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. Sequence Listing <110> Syngenta Crop Protection AG REYNOLDS, Clarance M. <120> Insecticide protein <130> 81942-US-REG-ORG-NAT-1 <160> 53 <170> PatentIn version 3.5 <210> 1 <211> 345 <212> PRT <213> Alibaba <400> 1 Met Ala Asp Ile Leu Ala Pro Asp Glu Val Arg Leu Lys Asn Ile Ser 1 5 10 15 Ala Val Arg Arg Leu Arg Ser Arg Gly Gly Pro Phe Leu Phe Ile Gly 20 25 30 Ala Thr Ala Asp Val Ser Glu Gln Phe Gln Glu Ile Val Ala Ile Asp 35 40 45 Asn Ile Asp Tyr Leu Thr Gln Ala Val Gln Leu Thr Ala Leu Phe Asn 50 55 60 Gly Ala Ile Asn Asn Glu Thr Gly Arg Phe Glu Ser Asn Thr Ala Arg 65 70 75 80 Gln Leu Ile Ala Asp Phe Asn Ala Ser Leu Pro Glu Ser Asp Arg Ala 85 90 95 Tyr Lys Ile Gly Ile Phe Lys Ser Tyr Gln Thr Thr Leu Thr Gln Thr 100 105 110 His Ser Val Val Ser Gly Met Ile Asp Lys Ile Val Glu Ala Leu Lys 115 120 125 Gln Val Leu Gly Val Ala Leu Gly Thr Ser Thr Val Ala Gln Leu Thr 130 135 140 Ala Ala Val Thr Asp Ala Phe Thr Asp Leu Lys Ser Gln Glu Gly Asp 145 150 155 160 Ala Trp Ile Phe Trp Glu Lys Lys Thr Ala Glu Lys Thr Thr Tyr Ser 165 170 175 Tyr Ala Ile Leu Phe Ala Ile Gln Asp Ser Ser Thr Gly Met Met Met 180 185 190 Phe Ala Met Pro Met Ser Leu Leu Ile Glu Val Asn Val Ser Tyr Glu 195 200 205 Lys Val Leu Trp Ile Thr Ile Asp Asp Thr Glu Thr Tyr Ser Val Thr 210 215 220 Leu Asp Thr Met Lys Val Gly Gln Ile Leu Phe Pro Pro Ser Pro Gly 225 230 235 240 Ser Ser Val Leu Arg Gln Ala Leu Ala Pro Pro Pro Arg Lys Ala Glu 245 250 255 Leu Gly Lys Glu Leu Glu Phe Ser Asp Ile Thr Asp Ile Gln Val Thr 260 265 270 Asn Trp Ser Lys Thr Lys Thr Phe Ala Thr Ala Lys His Gly Ser Tyr 275 280 285 Val Lys Glu Phe His Leu Glu Gln Val Met Ala Phe Gln Pro Glu Val 290 295 300 Leu His Pro Leu Gln Asp Glu Asp Lys Cys Leu Val Ser Phe Thr Arg 305 310 315 320 Ser Gly Glu Arg Lys Ser Val Gly Val Ile Leu Asn Gly Thr Leu Pro 325 330 335 Asp Gly Thr Leu Trp Phe Val Ser Gln 340 345 <210> 2 <211> 344 <212> PRT <213> Sinorhizobium species <400> 2 Met Asn Glu Val Leu Arg Asn Ser Val Gly Gln His Ser His Ile Ser 1 5 10 15 Lys Arg Ala Leu Arg Pro Arg Ser Ser Pro Val Val Phe Ile Asn Thr 20 25 30 Thr Gln Glu Glu Ala Glu Gln Phe Gln Glu Ile Val Ala Ile Asp Asp 35 40 45 Ile Asp Tyr Leu Thr Gln Ala Ile Ala Leu Thr Ala Leu Phe Asn Gly 50 55 60 Ala Ile Asp Asn Thr Thr Gly Arg Phe Ile Pro Gly Lys Ala Arg Glu 65 70 75 80 Leu Ile Ala Asn Tyr Asn Glu Ser Leu Asp Ala Asn Ser Gln Pro Tyr 85 90 95 Lys Ile Gly Ile Phe Asn Thr His Gln Thr Thr Leu Thr Gln Gln Asn 100 105 110 Ser Ala Val Ser Ala Met Ile Ser Gln Ile Leu Glu Thr Leu Lys Arg 115 120 125 Val Met Gly Val Ala Leu Gly Ala Ser Ser Val Ala Gln Met Thr Ala 130 135 140 Ala Val Thr Asp Ala Phe Thr Asn Leu Asp Glu Gln Ser Gly Asp Ala 145 150 155 160 Trp Ile Phe Trp Glu Lys Lys Thr Ser Asn Lys Thr Thr Tyr Ser Tyr 165 170 175 Ala Ile Leu Phe Ala Phe Gln Asp Ser Thr Thr Gly Lys Leu Met Phe 180 185 190 Ala Leu Pro Met Ser Leu Glu Ile Glu Val Asp Val Ser Tyr Glu Arg 195 200 205 Val Leu Phe Ile Thr Val Glu Asp Lys Glu Thr Tyr Ser Val Lys Leu 210 215 220 Asp Thr Met Lys Val Gly Gln Leu Leu Phe Pro Lys Ser Pro Gly Ala 225 230 235 240 Asn Ala Leu Gln Ser Ala Arg Arg Leu Gly Thr Arg Ser Gly Ser Ala 245 250 255 Asp Leu Leu Ala Glu Pro Arg Pro Ile Thr Asp Ile Val Val Thr Asn 260 265 270 Trp Ala Lys Thr Lys Thr Phe Ala Thr Ala Ala Thr Gly Leu Tyr Thr 275 280 285 Asn Asp His Pro Leu Val Gln Val Met Ala Ser Glu Pro Asn Val Val 290 295 300 Asn Pro Leu Tyr Asp Gly Asn Gln Tyr Leu Val Ser Phe Ala Leu Asp 305 310 315 320 Gly Val Arg Gln Thr Leu Gly Phe Leu Leu Asn Cys Thr Leu Pro Asp 325 330 335 Gly Glu Leu Trp Phe Val Ser Ile 340 <210> 3 <211> 351 <212> PRT <213> Rhizobiales species <400> 3 Met Lys Gln Gly Glu Ala Ala Met Asn Glu Val Leu Ala Asn Asn Ile 1 5 10 15 Gly Gln His Pro Gln Val Ser Arg Arg Ala Leu Arg Ser Pro Arg Ser 20 25 30 Pro Val Val Phe Ile Asn Thr Thr Ser Glu Glu Ala Glu Gln Phe Gln 35 40 45 Glu Ile Val Ala Ile Asp Asp Ile Asp Tyr Leu Thr Gln Ala Val Ala 50 55 60 Leu Thr Ala Leu Phe Asn Gly Ala Ile Asp Asn Thr Thr Gly Arg Phe 65 70 75 80 Ile Pro Gly Arg Ala Arg Glu Leu Ile Ala Asn Tyr Asn Glu Ser Leu 85 90 95 Asp Pro Glu Ser Gln Arg Tyr Lys Ile Gly Ile Phe Asn Thr His Gln 100 105 110 Thr Thr Leu Thr Gln Gln Asn Ser Ala Val Ser Ala Met Ile Asn Gln 115 120 125 Ile Leu Glu Thr Leu Lys Asn Val Met Gly Val Ala Leu Gly Thr Thr 130 135 140 Ser Val Thr Gln Met Thr Ala Ala Ile Thr Asp Ala Phe Thr Asn Leu 145 150 155 160 Asn Glu Gln Ser Gly Asp Ala Trp Ile Phe Trp Glu Lys Lys Thr Ser 165 170 175 Asn Lys Thr Thr Tyr Ser Tyr Ala Ile Leu Phe Ala Phe Gln Asp Ser 180 185 190 Ser Thr Gly Lys Leu Met Phe Ala Leu Pro Met Ser Leu Glu Ile Glu 195 200 205 Val Asp Val Ser Tyr Glu Arg Val Leu Phe Ile Thr Val Glu Asp Lys 210 215 220 Glu Thr Tyr Ser Val Lys Leu Asp Thr Met Lys Val Gly Gln Leu Leu 225 230 235 240 Phe Pro Lys Ser Pro Gly Ala Asn Ala Leu Gln Thr Val Arg Arg Leu 245 250 255 Arg Thr Arg Ser Gly Ser Ser Gly Leu Leu Thr Ala Pro Ser Pro Val 260 265 270 Thr Asn Ile Val Val Thr Asn Trp Ala Lys Thr Thr Thr Phe Ala Thr 275 280 285 Ser Ala Asn Gly Phe Tyr Thr Glu Asn His Pro Leu Val Gln Leu Met 290 295 300 Ala Val Gly Pro Thr Val Val Asn Pro Leu Tyr Asp Gly Asn Gln Tyr 305 310 315 320 Leu Val Ser Phe Asp Leu Asn Gly Gly Arg Gln Thr Arg Gly Leu Ile 325 330 335 Leu Asn Gly Thr Leu Pro Asp Gly Glu Leu Trp Phe Val Ser Met 340 345 350 <210> 4 <211> 347 <212> PRT <213> species <400> 4 Met Asn Glu Ile Val Thr Asn Pro Ala Pro Ala His Ala Ser Arg Pro 1 5 10 15 Gln Leu Arg Ser Leu Thr Gly Lys Arg Ser Pro Ile Val Phe Leu Gln 20 25 30 Ala Ala Val Asp Gln Lys Glu Gln Tyr Gln Glu Ile Met Ala Leu Asp 35 40 45 Asp Ile Asp Tyr Val Thr Gln Ala Leu Gly Ile Gly Ala Leu Leu Asn 50 55 60 Gly Ala Ile Asp Thr Ala Thr Gly Arg Phe Asp Ala Gly Arg Ala Arg 65 70 75 80 Gln Leu Ile Val Asp Phe Asn Gln Ala Leu Pro Pro Ala Asp Ser Lys 85 90 95 Tyr Lys Leu Ala Ile Met Asn Ser Tyr Gln Ser Thr Val Ser Gln Glu 100 105 110 Asn Ser Val Val Ser Gly Met Ile Asp Lys Ile Leu Asp Val Leu Lys 115 120 125 Thr Ala Ile Gly Val Ala Leu Gly Gln Lys Ser Ile Asp Gln Ile Thr 130 135 140 Ala Ala Val Thr Asp Ala Phe Thr Asn Leu Lys Ser Gln Asp Gly Asp 145 150 155 160 Ala Trp Ile Phe Trp Gln Lys Arg Glu Ala His Lys Thr Val Tyr Ser 165 170 175 Tyr Ala Ile Leu Phe Ala Val Gln Asp Glu Ser Thr Gly Arg Val Met 180 185 190 Leu Ala Phe Pro Met Ser Leu Glu Ile Glu Val Asn Val Glu Phe Glu 195 200 205 Lys Val Leu Trp Ile Thr Val Lys Asp Ser His Asn Tyr Ser Val Lys 210 215 220 Val Asp Ala Met Lys Ile Ala Gln Leu Leu Phe Pro Lys Ala Pro Gly 225 230 235 240 Ser Gln Thr Leu Gln Ser Ile Ala Ser Ala Pro Arg Leu Arg Gly Leu 245 250 255 Ala Asp Val Glu Tyr Gln Thr Arg Ala Ser Asp Ile Thr Asp Ile Thr 260 265 270 Val Thr Asn Trp Ser Gln Ser Thr Leu Phe Ala Arg Ala Ala Lys Gly 275 280 285 Ser Leu Val Thr Ala Gly Ser Leu Gln Gln Ile Met Ala Phe Glu Pro 290 295 300 Ala Ile Asp Ile Pro Leu Glu Pro Glu Asn His Tyr Leu Val His Tyr 305 310 315 320 Lys Leu Asn Gly Glu Ala Lys Gln Ile Gly Met Ile Phe Asn Asp Tyr 325 330 335 Leu Pro Asp Ser Thr Leu Trp Phe Val Ser Arg 340 345 <210> 5 <211> 351 <212> PRT <213> Artificial sequence <220> <223> Engineered RIP3Aa variants. <220> <221> Features not yet classified <222> (50)..(50) <223> X=I or L <220> <221> Features not yet classified <222> (53)..(53) <223> X=I or L <220> <221> Features not yet classified <222> (56)..(56) <223> X=I or L <220> <221> Features not yet classified <222> (62)..(62) <223> X = A, C or L <220> <221> Features not yet classified <222> (64)..(64) <223> X = A, C or L <220> <221> Features not yet classified <222> (81)..(81) <223> X=I or L <220> <221> Features not yet classified <222> (126)..(126) <223> X=I or L <220> <221> Features not yet classified <222> (153)..(153) <223> X=I or L <220> <221> Features not yet classified <222> (169)..(169) <223> X=I or L <220> <221> Features not yet classified <222> (185)..(185) <223> X=I or L <220> <221> Features not yet classified <222> (207)..(207) <223> X=I or L <220> <221> Features not yet classified <222> (219)..(219) <223> X=I or L <220> <221> Features not yet classified <222> (275)..(275) <223> X=I or L <400> 5 Met Lys Gln Gly Glu Ala Ala Met Asn Glu Val Leu Ala Asn Asn Ile 1 5 10 15 Gly Gln His Pro Gln Val Ser Arg Arg Ala Leu Arg Ser Pro Arg Ser 20 25 30 Pro Val Val Phe Ile Asn Thr Thr Ser Glu Glu Ala Glu Gln Phe Gln 35 40 45 Glu Xaa Val Ala Xaa Asp Asp Xaa Asp Tyr Leu Thr Gln Xaa Val Xaa 50 55 60 Leu Thr Ala Leu Phe Asn Gly Ala Ile Asp Asn Thr Thr Gly Arg Phe 65 70 75 80 Xaa Pro Gly Arg Ala Arg Glu Leu Ile Ala Asn Tyr Asn Glu Ser Leu 85 90 95 Asp Pro Glu Ser Gln Arg Tyr Lys Ile Gly Ile Phe Asn Thr His Gln 100 105 110 Thr Thr Leu Thr Gln Gln Asn Ser Ala Val Ser Ala Met Xaa Asn Gln 115 120 125 Ile Leu Glu Thr Leu Lys Asn Val Met Gly Val Ala Leu Gly Thr Thr 130 135 140 Ser Val Thr Gln Met Thr Ala Ala Xaa Thr Asp Ala Phe Thr Asn Leu 145 150 155 160 Asn Glu Gln Ser Gly Asp Ala Trp Xaa Phe Trp Glu Lys Lys Thr Ser 165 170 175 Asn Lys Thr Thr Tyr Ser Tyr Ala Xaa Leu Phe Ala Phe Gln Asp Ser 180 185 190 Ser Thr Gly Lys Leu Met Phe Ala Leu Pro Met Ser Leu Glu Xaa Glu 195 200 205 Val Asp Val Ser Tyr Glu Arg Val Leu Phe Xaa Thr Val Glu Asp Lys 210 215 220 Glu Thr Tyr Ser Val Lys Leu Asp Thr Met Lys Val Gly Gln Leu Leu 225 230 235 240 Phe Pro Lys Ser Pro Gly Ala Asn Ala Leu Gln Thr Val Arg Arg Leu 245 250 255 Arg Thr Arg Ser Gly Ser Ser Gly Leu Leu Thr Ala Pro Ser Pro Val 260 265 270 Thr Asn Xaa Val Val Thr Asn Trp Ala Lys Thr Thr Thr Phe Ala Thr 275 280 285 Ser Ala Asn Gly Phe Tyr Thr Glu Asn His Pro Leu Val Gln Leu Met 290 295 300 Ala Val Gly Pro Thr Val Val Asn Pro Leu Tyr Asp Gly Asn Gln Tyr 305 310 315 320 Leu Val Ser Phe Asp Leu Asn Gly Gly Arg Gln Thr Arg Gly Leu Ile 325 330 335 Leu Asn Gly Thr Leu Pro Asp Gly Glu Leu Trp Phe Val Ser Met 340 345 350 <210> 6 <211> 351 <212> PRT <213> Artificial Sequence <220> <223> RIP3Aa- I50L <400> 6 Met Lys Gln Gly Glu Ala Ala Met Asn Glu Val Leu Ala Asn Asn Ile 1 5 10 15 Gly Gln His Pro Gln Val Ser Arg Arg Ala Leu Arg Ser Pro Arg Ser 20 25 30 Pro Val Val Phe Ile Asn Thr Thr Ser Glu Glu Ala Glu Gln Phe Gln 35 40 45 Glu Leu Val Ala Ile Asp Asp Ile Asp Tyr Leu Thr Gln Ala Val Ala 50 55 60 Leu Thr Ala Leu Phe Asn Gly Ala Ile Asp Asn Thr Thr Gly Arg Phe 65 70 75 80 Ile Pro Gly Arg Ala Arg Glu Leu Ile Ala Asn Tyr Asn Glu Ser Leu 85 90 95 Asp Pro Glu Ser Gln Arg Tyr Lys Ile Gly Ile Phe Asn Thr His Gln 100 105 110 Thr Thr Leu Thr Gln Gln Asn Ser Ala Val Ser Ala Met Ile Asn Gln 115 120 125 Ile Leu Glu Thr Leu Lys Asn Val Met Gly Val Ala Leu Gly Thr Thr 130 135 140 Ser Val Thr Gln Met Thr Ala Ala Ile Thr Asp Ala Phe Thr Asn Leu 145 150 155 160 Asn Glu Gln Ser Gly Asp Ala Trp Ile Phe Trp Glu Lys Lys Thr Ser 165 170 175 Asn Lys Thr Thr Tyr Ser Tyr Ala Ile Leu Phe Ala Phe Gln Asp Ser 180 185 190 Ser Thr Gly Lys Leu Met Phe Ala Leu Pro Met Ser Leu Glu Ile Glu 195 200 205 Val Asp Val Ser Tyr Glu Arg Val Leu Phe Ile Thr Val Glu Asp Lys 210 215 220 Glu Thr Tyr Ser Val Lys Leu Asp Thr Met Lys Val Gly Gln Leu Leu 225 230 235 240 Phe Pro Lys Ser Pro Gly Ala Asn Ala Leu Gln Thr Val Arg Arg Leu 245 250 255 Arg Thr Arg Ser Gly Ser Ser Gly Leu Leu Thr Ala Pro Ser Pro Val 260 265 270 Thr Asn Ile Val Val Thr Asn Trp Ala Lys Thr Thr Thr Phe Ala Thr 275 280 285 Ser Ala Asn Gly Phe Tyr Thr Glu Asn His Pro Leu Val Gln Leu Met 290 295 300 Ala Val Gly Pro Thr Val Val Asn Pro Leu Tyr Asp Gly Asn Gln Tyr 305 310 315 320 Leu Val Ser Phe Asp Leu Asn Gly Gly Arg Gln Thr Arg Gly Leu Ile 325 330 335 Leu Asn Gly Thr Leu Pro Asp Gly Glu Leu Trp Phe Val Ser Met 340 345 350 <210> 7 <211> 351 <212> PRT <213> Artificial Sequence <220> <223> RIP3Aa-I53L <400> 7 Met Lys Gln Gly Glu Ala Ala Met Asn Glu Val Leu Ala Asn Asn Ile 1 5 10 15 Gly Gln His Pro Gln Val Ser Arg Arg Ala Leu Arg Ser Pro Arg Ser 20 25 30 Pro Val Val Phe Ile Asn Thr Thr Ser Glu Glu Ala Glu Gln Phe Gln 35 40 45 Glu Ile Val Ala Leu Asp Asp Ile Asp Tyr Leu Thr Gln Ala Val Ala 50 55 60 Leu Thr Ala Leu Phe Asn Gly Ala Ile Asp Asn Thr Thr Gly Arg Phe 65 70 75 80 Ile Pro Gly Arg Ala Arg Glu Leu Ile Ala Asn Tyr Asn Glu Ser Leu 85 90 95 Asp Pro Glu Ser Gln Arg Tyr Lys Ile Gly Ile Phe Asn Thr His Gln 100 105 110 Thr Thr Leu Thr Gln Gln Asn Ser Ala Val Ser Ala Met Ile Asn Gln 115 120 125 Ile Leu Glu Thr Leu Lys Asn Val Met Gly Val Ala Leu Gly Thr Thr 130 135 140 Ser Val Thr Gln Met Thr Ala Ala Ile Thr Asp Ala Phe Thr Asn Leu 145 150 155 160 Asn Glu Gln Ser Gly Asp Ala Trp Ile Phe Trp Glu Lys Lys Thr Ser 165 170 175 Asn Lys Thr Thr Tyr Ser Tyr Ala Ile Leu Phe Ala Phe Gln Asp Ser 180 185 190 Ser Thr Gly Lys Leu Met Phe Ala Leu Pro Met Ser Leu Glu Ile Glu 195 200 205 Val Asp Val Ser Tyr Glu Arg Val Leu Phe Ile Thr Val Glu Asp Lys 210 215 220 Glu Thr Tyr Ser Val Lys Leu Asp Thr Met Lys Val Gly Gln Leu Leu 225 230 235 240 Phe Pro Lys Ser Pro Gly Ala Asn Ala Leu Gln Thr Val Arg Arg Leu 245 250 255 Arg Thr Arg Ser Gly Ser Ser Gly Leu Leu Thr Ala Pro Ser Pro Val 260 265 270 Thr Asn Ile Val Val Thr Asn Trp Ala Lys Thr Thr Thr Phe Ala Thr 275 280 285 Ser Ala Asn Gly Phe Tyr Thr Glu Asn His Pro Leu Val Gln Leu Met 290 295 300 Ala Val Gly Pro Thr Val Val Asn Pro Leu Tyr Asp Gly Asn Gln Tyr 305 310 315 320 Leu Val Ser Phe Asp Leu Asn Gly Gly Arg Gln Thr Arg Gly Leu Ile 325 330 335 Leu Asn Gly Thr Leu Pro Asp Gly Glu Leu Trp Phe Val Ser Met 340 345 350 <210> 8 <211> 351 <212> PRT <213> Artificial Sequence <220> <223> RIP3Aa-I56L <400> 8 Met Lys Gln Gly Glu Ala Ala Met Asn Glu Val Leu Ala Asn Asn Ile 1 5 10 15 Gly Gln His Pro Gln Val Ser Arg Arg Ala Leu Arg Ser Pro Arg Ser 20 25 30 Pro Val Val Phe Ile Asn Thr Thr Ser Glu Glu Ala Glu Gln Phe Gln 35 40 45 Glu Ile Val Ala Ile Asp Asp Leu Asp Tyr Leu Thr Gln Ala Val Ala 50 55 60 Leu Thr Ala Leu Phe Asn Gly Ala Ile Asp Asn Thr Thr Gly Arg Phe 65 70 75 80 Ile Pro Gly Arg Ala Arg Glu Leu Ile Ala Asn Tyr Asn Glu Ser Leu 85 90 95 Asp Pro Glu Ser Gln Arg Tyr Lys Ile Gly Ile Phe Asn Thr His Gln 100 105 110 Thr Thr Leu Thr Gln Gln Asn Ser Ala Val Ser Ala Met Ile Asn Gln 115 120 125 Ile Leu Glu Thr Leu Lys Asn Val Met Gly Val Ala Leu Gly Thr Thr 130 135 140 Ser Val Thr Gln Met Thr Ala Ala Ile Thr Asp Ala Phe Thr Asn Leu 145 150 155 160 Asn Glu Gln Ser Gly Asp Ala Trp Ile Phe Trp Glu Lys Lys Thr Ser 165 170 175 Asn Lys Thr Thr Tyr Ser Tyr Ala Ile Leu Phe Ala Phe Gln Asp Ser 180 185 190 Ser Thr Gly Lys Leu Met Phe Ala Leu Pro Met Ser Leu Glu Ile Glu 195 200 205 Val Asp Val Ser Tyr Glu Arg Val Leu Phe Ile Thr Val Glu Asp Lys 210 215 220 Glu Thr Tyr Ser Val Lys Leu Asp Thr Met Lys Val Gly Gln Leu Leu 225 230 235 240 Phe Pro Lys Ser Pro Gly Ala Asn Ala Leu Gln Thr Val Arg Arg Leu 245 250 255 Arg Thr Arg Ser Gly Ser Ser Gly Leu Leu Thr Ala Pro Ser Pro Val 260 265 270 Thr Asn Ile Val Val Thr Asn Trp Ala Lys Thr Thr Thr Phe Ala Thr 275 280 285 Ser Ala Asn Gly Phe Tyr Thr Glu Asn His Pro Leu Val Gln Leu Met 290 295 300 Ala Val Gly Pro Thr Val Val Asn Pro Leu Tyr Asp Gly Asn Gln Tyr 305 310 315 320 Leu Val Ser Phe Asp Leu Asn Gly Gly Arg Gln Thr Arg Gly Leu Ile 325 330 335 Leu Asn Gly Thr Leu Pro Asp Gly Glu Leu Trp Phe Val Ser Met 340 345 350 <210> 9 <211> 351 <212> PRT <213> Artificial Sequence <220> <223> RIP3Aa - A62C <400> 9 Met Lys Gln Gly Glu Ala Ala Met Asn Glu Val Leu Ala Asn Asn Ile 1 5 10 15 Gly Gln His Pro Gln Val Ser Arg Arg Ala Leu Arg Ser Pro Arg Ser 20 25 30 Pro Val Val Phe Ile Asn Thr Thr Ser Glu Glu Ala Glu Gln Phe Gln 35 40 45 Glu Ile Val Ala Ile Asp Asp Ile Asp Tyr Leu Thr Gln Cys Val Ala 50 55 60 Leu Thr Ala Leu Phe Asn Gly Ala Ile Asp Asn Thr Thr Gly Arg Phe 65 70 75 80 Ile Pro Gly Arg Ala Arg Glu Leu Ile Ala Asn Tyr Asn Glu Ser Leu 85 90 95 Asp Pro Glu Ser Gln Arg Tyr Lys Ile Gly Ile Phe Asn Thr His Gln 100 105 110 Thr Thr Leu Thr Gln Gln Asn Ser Ala Val Ser Ala Met Ile Asn Gln 115 120 125 Ile Leu Glu Thr Leu Lys Asn Val Met Gly Val Ala Leu Gly Thr Thr 130 135 140 Ser Val Thr Gln Met Thr Ala Ala Ile Thr Asp Ala Phe Thr Asn Leu 145 150 155 160 Asn Glu Gln Ser Gly Asp Ala Trp Ile Phe Trp Glu Lys Lys Thr Ser 165 170 175 Asn Lys Thr Thr Tyr Tyr Ser Tyr Ala Ile Leu Phe Ala Phe Gln Asp Ser 180 185 190 Ser Thr Gly Lys Leu Met Phe Ala Leu Pro Met Ser Leu Glu Ile Glu 195 200 205 Val Asp Val Ser Tyr Glu Arg Val Leu Phe Ile Thr Val Glu Asp Lys 210 215 220 Glu Thr Tyr Ser Val Lys Leu Asp Thr Met Lys Val Gly Gln Leu Leu 225 230 235 240 Phe Pro Lys Ser Pro Gly Ala Asn Ala Leu Gln Thr Val Arg Arg Leu 245 250 255 Arg Thr Arg Ser Gly Ser Ser Gly Leu Leu Thr Ala Pro Ser Pro Val 260 265 270 Thr Asn Ile Val Val Thr Asn Trp Ala Lys Thr Thr Thr Phe Ala Thr 275 280 285 Ser Ala Asn Gly Phe Tyr Thr Glu Asn His Pro Leu Val Gln Leu Met 290 295 300 Ala Val Gly Pro Thr Val Val Asn Pro Leu Tyr Asp Gly Asn Gln Tyr 305 310 315 320 Leu Val Ser Phe Asp Leu Asn Gly Gly Arg Gln Thr Arg Gly Leu Ile 325 330 335 Leu Asn Gly Thr Leu Pro Asp Gly Glu Leu Trp Phe Val Ser Met 340 345 350 <210> 10 <211> 351 <212> PRT <213> Artificial Sequence <220> <223> RIP3Aa - A62L <400> 10 Met Lys Gln Gly Glu Ala Ala Met Asn Glu Val Leu Ala Asn Asn Ile 1 5 10 15 Gly Gln His Pro Gln Val Ser Arg Arg Ala Leu Arg Ser Pro Arg Ser 20 25 30 Pro Val Val Phe Ile Asn Thr Thr Ser Glu Glu Ala Glu Gln Phe Gln 35 40 45 Glu Ile Val Ala Ile Asp Asp Ile Asp Tyr Leu Thr Gln Leu Val Ala 50 55 60 Leu Thr Ala Leu Phe Asn Gly Ala Ile Asp Asn Thr Thr Gly Arg Phe 65 70 75 80 Ile Pro Gly Arg Ala Arg Glu Leu Ile Ala Asn Tyr Asn Glu Ser Leu 85 90 95 Asp Pro Glu Ser Gln Arg Tyr Lys Ile Gly Ile Phe Asn Thr His Gln 100 105 110 Thr Thr Leu Thr Gln Gln Asn Ser Ala Val Ser Ala Met Ile Asn Gln 115 120 125 Ile Leu Glu Thr Leu Lys Asn Val Met Gly Val Ala Leu Gly Thr Thr 130 135 140 Ser Val Thr Gln Met Thr Ala Ala Ile Thr Asp Ala Phe Thr Asn Leu 145 150 155 160 Asn Glu Gln Ser Gly Asp Ala Trp Ile Phe Trp Glu Lys Lys Thr Ser 165 170 175 Asn Lys Thr Thr Tyr Ser Tyr Ala Ile Leu Phe Ala Phe Gln Asp Ser 180 185 190 Ser Thr Gly Lys Leu Met Phe Ala Leu Pro Met Ser Leu Glu Ile Glu 195 200 205 Val Asp Val Ser Tyr Glu Arg Val Leu Phe Ile Thr Val Glu Asp Lys 210 215 220 Glu Thr Tyr Ser Val Lys Leu Asp Thr Met Lys Val Gly Gln Leu Leu 225 230 235 240 Phe Pro Lys Ser Pro Gly Ala Asn Ala Leu Gln Thr Val Arg Arg Leu 245 250 255 Arg Thr Arg Ser Gly Ser Ser Gly Leu Leu Thr Ala Pro Ser Pro Val 260 265 270 Thr Asn Ile Val Val Thr Asn Trp Ala Lys Thr Thr Thr Phe Ala Thr 275 280 285 Ser Ala Asn Gly Phe Tyr Thr Glu Asn His Pro Leu Val Gln Leu Met 290 295 300 Ala Val Gly Pro Thr Val Val Asn Pro Leu Tyr Asp Gly Asn Gln Tyr 305 310 315 320 Leu Val Ser Phe Asp Leu Asn Gly Gly Arg Gln Thr Arg Gly Leu Ile 325 330 335 Leu Asn Gly Thr Leu Pro Asp Gly Glu Leu Trp Phe Val Ser Met 340 345 350 <210> 11 <211> 351 <212> PRT <213> Artificial Sequence <220> <223> RIP3Aa-A64C <400> 11 Met Lys Gln Gly Glu Ala Ala Met Asn Glu Val Leu Ala Asn Asn Ile 1 5 10 15 Gly Gln His Pro Gln Val Ser Arg Arg Ala Leu Arg Ser Pro Arg Ser 20 25 30 Pro Val Val Phe Ile Asn Thr Thr Ser Glu Glu Ala Glu Gln Phe Gln 35 40 45 Glu Ile Val Ala Ile Asp Asp Ile Asp Tyr Leu Thr Gln Ala Val Cys 50 55 60 Leu Thr Ala Leu Phe Asn Gly Ala Ile Asp Asn Thr Thr Gly Arg Phe 65 70 75 80 Ile Pro Gly Arg Ala Arg Glu Leu Ile Ala Asn Tyr Asn Glu Ser Leu 85 90 95 Asp Pro Glu Ser Gln Arg Tyr Lys Ile Gly Ile Phe Asn Thr His Gln 100 105 110 Thr Thr Leu Thr Gln Gln Asn Ser Ala Val Ser Ala Met Ile Asn Gln 115 120 125 Ile Leu Glu Thr Leu Lys Asn Val Met Gly Val Ala Leu Gly Thr Thr 130 135 140 Ser Val Thr Gln Met Thr Ala Ala Ile Thr Asp Ala Phe Thr Asn Leu 145 150 155 160 Asn Glu Gln Ser Gly Asp Ala Trp Ile Phe Trp Glu Lys Lys Thr Ser 165 170 175 Asn Lys Thr Thr Tyr Ser Tyr Ala Ile Leu Phe Ala Phe Gln Asp Ser 180 185 190 Ser Thr Gly Lys Leu Met Phe Ala Leu Pro Met Ser Leu Glu Ile Glu 195 200 205 Val Asp Val Ser Tyr Glu Arg Val Leu Phe Ile Thr Val Glu Asp Lys 210 215 220 Glu Thr Tyr Ser Val Lys Leu Asp Thr Met Lys Val Gly Gln Leu Leu 225 230 235 240 Phe Pro Lys Ser Pro Gly Ala Asn Ala Leu Gln Thr Val Arg Arg Leu 245 250 255 Arg Thr Arg Ser Gly Ser Ser Gly Leu Leu Thr Ala Pro Ser Pro Val 260 265 270 Thr Asn Ile Val Val Thr Asn Trp Ala Lys Thr Thr Thr Phe Ala Thr 275 280 285 Ser Ala Asn Gly Phe Tyr Thr Glu Asn His Pro Leu Val Gln Leu Met 290 295 300 Ala Val Gly Pro Thr Val Val Asn Pro Leu Tyr Asp Gly Asn Gln Tyr 305 310 315 320 Leu Val Ser Phe Asp Leu Asn Gly Gly Arg Gln Thr Arg Gly Leu Ile 325 330 335 Leu Asn Gly Thr Leu Pro Asp Gly Glu Leu Trp Phe Val Ser Met 340 345 350 <210> 12 <211> 351 <212> PRT <213> Artificial Sequence <220> <223> RIP3Aa - A64L <400> 12 Met Lys Gln Gly Glu Ala Ala Met Asn Glu Val Leu Ala Asn Asn Ile 1 5 10 15 Gly Gln His Pro Gln Val Ser Arg Arg Ala Leu Arg Ser Pro Arg Ser 20 25 30 Pro Val Val Phe Ile Asn Thr Thr Ser Glu Glu Ala Glu Gln Phe Gln 35 40 45 Glu Ile Val Ala Ile Asp Asp Ile Asp Tyr Leu Thr Gln Ala Val Leu 50 55 60 Leu Thr Ala Leu Phe Asn Gly Ala Ile Asp Asn Thr Thr Gly Arg Phe 65 70 75 80 Ile Pro Gly Arg Ala Arg Glu Leu Ile Ala Asn Tyr Asn Glu Ser Leu 85 90 95 Asp Pro Glu Ser Gln Arg Tyr Lys Ile Gly Ile Phe Asn Thr His Gln 100 105 110 Thr Thr Leu Thr Gln Gln Asn Ser Ala Val Ser Ala Met Ile Asn Gln 115 120 125 Ile Leu Glu Thr Leu Lys Asn Val Met Gly Val Ala Leu Gly Thr Thr 130 135 140 Ser Val Thr Gln Met Thr Ala Ala Ile Thr Asp Ala Phe Thr Asn Leu 145 150 155 160 Asn Glu Gln Ser Gly Asp Ala Trp Ile Phe Trp Glu Lys Lys Thr Ser 165 170 175 Asn Lys Thr Thr Tyr Ser Tyr Ala Ile Leu Phe Ala Phe Gln Asp Ser 180 185 190 Ser Thr Gly Lys Leu Met Phe Ala Leu Pro Met Ser Leu Glu Ile Glu 195 200 205 Val Asp Val Ser Tyr Glu Arg Val Leu Phe Ile Thr Val Glu Asp Lys 210 215 220 Glu Thr Tyr Ser Val Lys Leu Asp Thr Met Lys Val Gly Gln Leu Leu 225 230 235 240 Phe Pro Lys Ser Pro Gly Ala Asn Ala Leu Gln Thr Val Arg Arg Leu 245 250 255 Arg Thr Arg Ser Gly Ser Ser Gly Leu Leu Thr Ala Pro Ser Pro Val 260 265 270 Thr Asn Ile Val Val Thr Asn Trp Ala Lys Thr Thr Thr Phe Ala Thr 275 280 285 Ser Ala Asn Gly Phe Tyr Thr Glu Asn His Pro Leu Val Gln Leu Met 290 295 300 Ala Val Gly Pro Thr Val Val Asn Pro Leu Tyr Asp Gly Asn Gln Tyr 305 310 315 320 Leu Val Ser Phe Asp Leu Asn Gly Gly Arg Gln Thr Arg Gly Leu Ile 325 330 335 Leu Asn Gly Thr Leu Pro Asp Gly Glu Leu Trp Phe Val Ser Met 340 345 350 <210> 13 <211> 351 <212> PRT <213> Artificial Sequence <220> <223> RIP3Aa-I81L <400> 13 Met Lys Gln Gly Glu Ala Ala Met Asn Glu Val Leu Ala Asn Asn Ile 1 5 10 15 Gly Gln His Pro Gln Val Ser Arg Arg Ala Leu Arg Ser Pro Arg Ser 20 25 30 Pro Val Val Phe Ile Asn Thr Thr Ser Glu Glu Ala Glu Gln Phe Gln 35 40 45 Glu Ile Val Ala Ile Asp Asp Ile Asp Tyr Leu Thr Gln Ala Val Ala 50 55 60 Leu Thr Ala Leu Phe Asn Gly Ala Ile Asp Asn Thr Thr Gly Arg Phe 65 70 75 80 Leu Pro Gly Arg Ala Arg Glu Leu Ile Ala Asn Tyr Asn Glu Ser Leu 85 90 95 Asp Pro Glu Ser Gln Arg Tyr Lys Ile Gly Ile Phe Asn Thr His Gln 100 105 110 Thr Thr Leu Thr Gln Gln Asn Ser Ala Val Ser Ala Met Ile Asn Gln 115 120 125 Ile Leu Glu Thr Leu Lys Asn Val Met Gly Val Ala Leu Gly Thr Thr 130 135 140 Ser Val Thr Gln Met Thr Ala Ala Ile Thr Asp Ala Phe Thr Asn Leu 145 150 155 160 Asn Glu Gln Ser Gly Asp Ala Trp Ile Phe Trp Glu Lys Lys Thr Ser 165 170 175 Asn Lys Thr Thr Tyr Ser Tyr Ala Ile Leu Phe Ala Phe Gln Asp Ser 180 185 190 Ser Thr Gly Lys Leu Met Phe Ala Leu Pro Met Ser Leu Glu Ile Glu 195 200 205 Val Asp Val Ser Tyr Glu Arg Val Leu Phe Ile Thr Val Glu Asp Lys 210 215 220 Glu Thr Tyr Ser Val Lys Leu Asp Thr Met Lys Val Gly Gln Leu Leu 225 230 235 240 Phe Pro Lys Ser Pro Gly Ala Asn Ala Leu Gln Thr Val Arg Arg Leu 245 250 255 Arg Thr Arg Ser Gly Ser Ser Gly Leu Leu Thr Ala Pro Ser Pro Val 260 265 270 Thr Asn Ile Val Val Thr Asn Trp Ala Lys Thr Thr Thr Phe Ala Thr 275 280 285 Ser Ala Asn Gly Phe Tyr Thr Glu Asn His Pro Leu Val Gln Leu Met 290 295 300 Ala Val Gly Pro Thr Val Val Asn Pro Leu Tyr Asp Gly Asn Gln Tyr 305 310 315 320 Leu Val Ser Phe Asp Leu Asn Gly Gly Arg Gln Thr Arg Gly Leu Ile 325 330 335 Leu Asn Gly Thr Leu Pro Asp Gly Glu Leu Trp Phe Val Ser Met 340 345 350 <210> 14 <211> 351 <212> PRT <213> Artificial Sequence <220> <223> RIP3Aa-I126L <400> 14 Met Lys Gln Gly Glu Ala Ala Met Asn Glu Val Leu Ala Asn Asn Ile 1 5 10 15 Gly Gln His Pro Gln Val Ser Arg Arg Ala Leu Arg Ser Pro Arg Ser 20 25 30 Pro Val Val Phe Ile Asn Thr Thr Ser Glu Glu Ala Glu Gln Phe Gln 35 40 45 Glu Ile Val Ala Ile Asp Asp Ile Asp Tyr Leu Thr Gln Ala Val Ala 50 55 60 Leu Thr Ala Leu Phe Asn Gly Ala Ile Asp Asn Thr Thr Gly Arg Phe 65 70 75 80 Ile Pro Gly Arg Ala Arg Glu Leu Ile Ala Asn Tyr Asn Glu Ser Leu 85 90 95 Asp Pro Glu Ser Gln Arg Tyr Lys Ile Gly Ile Phe Asn Thr His Gln 100 105 110 Thr Thr Leu Thr Gln Gln Asn Ser Ala Val Ser Ala Met Leu Asn Gln 115 120 125 Ile Leu Glu Thr Leu Lys Asn Val Met Gly Val Ala Leu Gly Thr Thr 130 135 140 Ser Val Thr Gln Met Thr Ala Ala Ile Thr Asp Ala Phe Thr Asn Leu 145 150 155 160 Asn Glu Gln Ser Gly Asp Ala Trp Ile Phe Trp Glu Lys Lys Thr Ser 165 170 175 Asn Lys Thr Thr Tyr Tyr Ser Tyr Ala Ile Leu Phe Ala Phe Gln Asp Ser 180 185 190 Ser Thr Gly Lys Leu Met Phe Ala Leu Pro Met Ser Leu Glu Ile Glu 195 200 205 Val Asp Val Ser Tyr Glu Arg Val Leu Phe Ile Thr Val Glu Asp Lys 210 215 220 Glu Thr Tyr Ser Val Lys Leu Asp Thr Met Lys Val Gly Gln Leu Leu 225 230 235 240 Phe Pro Lys Ser Pro Gly Ala Asn Ala Leu Gln Thr Val Arg Arg Leu 245 250 255 Arg Thr Arg Ser Gly Ser Ser Gly Leu Leu Thr Ala Pro Ser Pro Val 260 265 270 Thr Asn Ile Val Val Thr Asn Trp Ala Lys Thr Thr Thr Phe Ala Thr 275 280 285 Ser Ala Asn Gly Phe Tyr Thr Glu Asn His Pro Leu Val Gln Leu Met 290 295 300 Ala Val Gly Pro Thr Val Val Asn Pro Leu Tyr Asp Gly Asn Gln Tyr 305 310 315 320 Leu Val Ser Phe Asp Leu Asn Gly Gly Arg Gln Thr Arg Gly Leu Ile 325 330 335 Leu Asn Gly Thr Leu Pro Asp Gly Glu Leu Trp Phe Val Ser Met 340 345 350 <210> 15 <211> 351 <212> PRT <213> Artificial Sequence <220> <223> RIP3Aa-I153L <400> 15 Met Lys Gln Gly Glu Ala Ala Met Asn Glu Val Leu Ala Asn Asn Ile 1 5 10 15 Gly Gln His Pro Gln Val Ser Arg Arg Ala Leu Arg Ser Pro Arg Ser 20 25 30 Pro Val Val Phe Ile Asn Thr Thr Ser Glu Glu Ala Glu Gln Phe Gln 35 40 45 Glu Ile Val Ala Ile Asp Asp Ile Asp Tyr Leu Thr Gln Ala Val Ala 50 55 60 Leu Thr Ala Leu Phe Asn Gly Ala Ile Asp Asn Thr Thr Gly Arg Phe 65 70 75 80 Ile Pro Gly Arg Ala Arg Glu Leu Ile Ala Asn Tyr Asn Glu Ser Leu 85 90 95 Asp Pro Glu Ser Gln Arg Tyr Lys Ile Gly Ile Phe Asn Thr His Gln 100 105 110 Thr Thr Leu Thr Gln Gln Asn Ser Ala Val Ser Ala Met Ile Asn Gln 115 120 125 Ile Leu Glu Thr Leu Lys Asn Val Met Gly Val Ala Leu Gly Thr Thr 130 135 140 Ser Val Thr Gln Met Thr Ala Ala Leu Thr Asp Ala Phe Thr Asn Leu 145 150 155 160 Asn Glu Gln Ser Gly Asp Ala Trp Ile Phe Trp Glu Lys Lys Thr Ser 165 170 175 Asn Lys Thr Thr Tyr Ser Tyr Ala Ile Leu Phe Ala Phe Gln Asp Ser 180 185 190 Ser Thr Gly Lys Leu Met Phe Ala Leu Pro Met Ser Leu Glu Ile Glu 195 200 205 Val Asp Val Ser Tyr Glu Arg Val Leu Phe Ile Thr Val Glu Asp Lys 210 215 220 Glu Thr Tyr Ser Val Lys Leu Asp Thr Met Lys Val Gly Gln Leu Leu 225 230 235 240 Phe Pro Lys Ser Pro Gly Ala Asn Ala Leu Gln Thr Val Arg Arg Leu 245 250 255 Arg Thr Arg Ser Gly Ser Ser Gly Leu Leu Thr Ala Pro Ser Pro Val 260 265 270 Thr Asn Ile Val Val Thr Asn Trp Ala Lys Thr Thr Thr Phe Ala Thr 275 280 285 Ser Ala Asn Gly Phe Tyr Thr Glu Asn His Pro Leu Val Gln Leu Met 290 295 300 Ala Val Gly Pro Thr Val Val Asn Pro Leu Tyr Asp Gly Asn Gln Tyr 305 310 315 320 Leu Val Ser Phe Asp Leu Asn Gly Gly Arg Gln Thr Arg Gly Leu Ile 325 330 335 Leu Asn Gly Thr Leu Pro Asp Gly Glu Leu Trp Phe Val Ser Met 340 345 350 <210> 16 <211> 351 <212> PRT <213> Artificial Sequence <220> <223> RIP3Aa-I169L <400> 16 Met Lys Gln Gly Glu Ala Ala Met Asn Glu Val Leu Ala Asn Asn Ile 1 5 10 15 Gly Gln His Pro Gln Val Ser Arg Arg Ala Leu Arg Ser Pro Arg Ser 20 25 30 Pro Val Val Phe Ile Asn Thr Thr Ser Glu Glu Ala Glu Gln Phe Gln 35 40 45 Glu Ile Val Ala Ile Asp Asp Ile Asp Tyr Leu Thr Gln Ala Val Ala 50 55 60 Leu Thr Ala Leu Phe Asn Gly Ala Ile Asp Asn Thr Thr Gly Arg Phe 65 70 75 80 Ile Pro Gly Arg Ala Arg Glu Leu Ile Ala Asn Tyr Asn Glu Ser Leu 85 90 95 Asp Pro Glu Ser Gln Arg Tyr Lys Ile Gly Ile Phe Asn Thr His Gln 100 105 110 Thr Thr Leu Thr Gln Gln Asn Ser Ala Val Ser Ala Met Ile Asn Gln 115 120 125 Ile Leu Glu Thr Leu Lys Asn Val Met Gly Val Ala Leu Gly Thr Thr 130 135 140 Ser Val Thr Gln Met Thr Ala Ala Ile Thr Asp Ala Phe Thr Asn Leu 145 150 155 160 Asn Glu Gln Ser Gly Asp Ala Trp Leu Phe Trp Glu Lys Lys Thr Ser 165 170 175 Asn Lys Thr Thr Tyr Ser Tyr Ala Ile Leu Phe Ala Phe Gln Asp Ser 180 185 190 Ser Thr Gly Lys Leu Met Phe Ala Leu Pro Met Ser Leu Glu Ile Glu 195 200 205 Val Asp Val Ser Tyr Glu Arg Val Leu Phe Ile Thr Val Glu Asp Lys 210 215 220 Glu Thr Tyr Ser Val Lys Leu Asp Thr Met Lys Val Gly Gln Leu Leu 225 230 235 240 Phe Pro Lys Ser Pro Gly Ala Asn Ala Leu Gln Thr Val Arg Arg Leu 245 250 255 Arg Thr Arg Ser Gly Ser Ser Gly Leu Leu Thr Ala Pro Ser Pro Val 260 265 270 Thr Asn Ile Val Val Thr Asn Trp Ala Lys Thr Thr Thr Phe Ala Thr 275 280 285 Ser Ala Asn Gly Phe Tyr Thr Glu Asn His Pro Leu Val Gln Leu Met 290 295 300 Ala Val Gly Pro Thr Val Val Asn Pro Leu Tyr Asp Gly Asn Gln Tyr 305 310 315 320 Leu Val Ser Phe Asp Leu Asn Gly Gly Arg Gln Thr Arg Gly Leu Ile 325 330 335 Leu Asn Gly Thr Leu Pro Asp Gly Glu Leu Trp Phe Val Ser Met 340 345 350 <210> 17 <211> 351 <212> PRT <213> Artificial Sequence <220> <223> RIP3Aa-I185L <400> 17 Met Lys Gln Gly Glu Ala Ala Met Asn Glu Val Leu Ala Asn Asn Ile 1 5 10 15 Gly Gln His Pro Gln Val Ser Arg Arg Ala Leu Arg Ser Pro Arg Ser 20 25 30 Pro Val Val Phe Ile Asn Thr Thr Ser Glu Glu Ala Glu Gln Phe Gln 35 40 45 Glu Ile Val Ala Ile Asp Asp Ile Asp Tyr Leu Thr Gln Ala Val Ala 50 55 60 Leu Thr Ala Leu Phe Asn Gly Ala Ile Asp Asn Thr Thr Gly Arg Phe 65 70 75 80 Ile Pro Gly Arg Ala Arg Glu Leu Ile Ala Asn Tyr Asn Glu Ser Leu 85 90 95 Asp Pro Glu Ser Gln Arg Tyr Lys Ile Gly Ile Phe Asn Thr His Gln 100 105 110 Thr Thr Leu Thr Gln Gln Asn Ser Ala Val Ser Ala Met Ile Asn Gln 115 120 125 Ile Leu Glu Thr Leu Lys Asn Val Met Gly Val Ala Leu Gly Thr Thr 130 135 140 Ser Val Thr Gln Met Thr Ala Ala Ile Thr Asp Ala Phe Thr Asn Leu 145 150 155 160 Asn Glu Gln Ser Gly Asp Ala Trp Ile Phe Trp Glu Lys Lys Thr Ser 165 170 175 Asn Lys Thr Thr Tyr Ser Tyr Ala Leu Leu Phe Ala Phe Gln Asp Ser 180 185 190 Ser Thr Gly Lys Leu Met Phe Ala Leu Pro Met Ser Leu Glu Ile Glu 195 200 205 Val Asp Val Ser Tyr Glu Arg Val Leu Phe Ile Thr Val Glu Asp Lys 210 215 220 Glu Thr Tyr Ser Val Lys Leu Asp Thr Met Lys Val Gly Gln Leu Leu 225 230 235 240 Phe Pro Lys Ser Pro Gly Ala Asn Ala Leu Gln Thr Val Arg Arg Leu 245 250 255 Arg Thr Arg Ser Gly Ser Ser Gly Leu Leu Thr Ala Pro Ser Pro Val 260 265 270 Thr Asn Ile Val Val Thr Asn Trp Ala Lys Thr Thr Thr Phe Ala Thr 275 280 285 Ser Ala Asn Gly Phe Tyr Thr Glu Asn His Pro Leu Val Gln Leu Met 290 295 300 Ala Val Gly Pro Thr Val Val Asn Pro Leu Tyr Asp Gly Asn Gln Tyr 305 310 315 320 Leu Val Ser Phe Asp Leu Asn Gly Gly Arg Gln Thr Arg Gly Leu Ile 325 330 335 Leu Asn Gly Thr Leu Pro Asp Gly Glu Leu Trp Phe Val Ser Met 340 345 350 <210> 18 <211> 351 <212> PRT <213> Artificial Sequence <220> <223> RIP3Aa-I207L <400> 18 Met Lys Gln Gly Glu Ala Ala Met Asn Glu Val Leu Ala Asn Asn Ile 1 5 10 15 Gly Gln His Pro Gln Val Ser Arg Arg Ala Leu Arg Ser Pro Arg Ser 20 25 30 Pro Val Val Phe Ile Asn Thr Thr Ser Glu Glu Ala Glu Gln Phe Gln 35 40 45 Glu Ile Val Ala Ile Asp Asp Ile Asp Tyr Leu Thr Gln Ala Val Ala 50 55 60 Leu Thr Ala Leu Phe Asn Gly Ala Ile Asp Asn Thr Thr Gly Arg Phe 65 70 75 80 Ile Pro Gly Arg Ala Arg Glu Leu Ile Ala Asn Tyr Asn Glu Ser Leu 85 90 95 Asp Pro Glu Ser Gln Arg Tyr Lys Ile Gly Ile Phe Asn Thr His Gln 100 105 110 Thr Thr Leu Thr Gln Gln Asn Ser Ala Val Ser Ala Met Ile Asn Gln 115 120 125 Ile Leu Glu Thr Leu Lys Asn Val Met Gly Val Ala Leu Gly Thr Thr 130 135 140 Ser Val Thr Gln Met Thr Ala Ala Ile Thr Asp Ala Phe Thr Asn Leu 145 150 155 160 Asn Glu Gln Ser Gly Asp Ala Trp Ile Phe Trp Glu Lys Lys Thr Ser 165 170 175 Asn Lys Thr Thr Tyr Ser Tyr Ala Ile Leu Phe Ala Phe Gln Asp Ser 180 185 190 Ser Thr Gly Lys Leu Met Phe Ala Leu Pro Met Ser Leu Glu Leu Glu 195 200 205 Val Asp Val Ser Tyr Glu Arg Val Leu Phe Ile Thr Val Glu Asp Lys 210 215 220 Glu Thr Tyr Ser Val Lys Leu Asp Thr Met Lys Val Gly Gln Leu Leu 225 230 235 240 Phe Pro Lys Ser Pro Gly Ala Asn Ala Leu Gln Thr Val Arg Arg Leu 245 250 255 Arg Thr Arg Ser Gly Ser Ser Gly Leu Leu Thr Ala Pro Ser Pro Val 260 265 270 Thr Asn Ile Val Val Thr Asn Trp Ala Lys Thr Thr Thr Phe Ala Thr 275 280 285 Ser Ala Asn Gly Phe Tyr Thr Glu Asn His Pro Leu Val Gln Leu Met 290 295 300 Ala Val Gly Pro Thr Val Val Asn Pro Leu Tyr Asp Gly Asn Gln Tyr 305 310 315 320 Leu Val Ser Phe Asp Leu Asn Gly Gly Arg Gln Thr Arg Gly Leu Ile 325 330 335 Leu Asn Gly Thr Leu Pro Asp Gly Glu Leu Trp Phe Val Ser Met 340 345 350 <210> 19 <211> 351 <212> PRT <213> Artificial Sequence <220> <223> RIP3Aa-I219L <400> 19 Met Lys Gln Gly Glu Ala Ala Met Asn Glu Val Leu Ala Asn Asn Ile 1 5 10 15 Gly Gln His Pro Gln Val Ser Arg Arg Ala Leu Arg Ser Pro Arg Ser 20 25 30 Pro Val Val Phe Ile Asn Thr Thr Ser Glu Glu Ala Glu Gln Phe Gln 35 40 45 Glu Ile Val Ala Ile Asp Asp Ile Asp Tyr Leu Thr Gln Ala Val Ala 50 55 60 Leu Thr Ala Leu Phe Asn Gly Ala Ile Asp Asn Thr Thr Gly Arg Phe 65 70 75 80 Ile Pro Gly Arg Ala Arg Glu Leu Ile Ala Asn Tyr Asn Glu Ser Leu 85 90 95 Asp Pro Glu Ser Gln Arg Tyr Lys Ile Gly Ile Phe Asn Thr His Gln 100 105 110 Thr Thr Leu Thr Gln Gln Asn Ser Ala Val Ser Ala Met Ile Asn Gln 115 120 125 Ile Leu Glu Thr Leu Lys Asn Val Met Gly Val Ala Leu Gly Thr Thr 130 135 140 Ser Val Thr Gln Met Thr Ala Ala Ile Thr Asp Ala Phe Thr Asn Leu 145 150 155 160 Asn Glu Gln Ser Gly Asp Ala Trp Ile Phe Trp Glu Lys Lys Thr Ser 165 170 175 Asn Lys Thr Thr Tyr Tyr Ser Tyr Ala Ile Leu Phe Ala Phe Gln Asp Ser 180 185 190 Ser Thr Gly Lys Leu Met Phe Ala Leu Pro Met Ser Leu Glu Ile Glu 195 200 205 Val Asp Val Ser Tyr Glu Arg Val Leu Phe Leu Thr Val Glu Asp Lys 210 215 220 Glu Thr Tyr Ser Val Lys Leu Asp Thr Met Lys Val Gly Gln Leu Leu 225 230 235 240 Phe Pro Lys Ser Pro Gly Ala Asn Ala Leu Gln Thr Val Arg Arg Leu 245 250 255 Arg Thr Arg Ser Gly Ser Ser Gly Leu Leu Thr Ala Pro Ser Pro Val 260 265 270 Thr Asn Ile Val Val Thr Asn Trp Ala Lys Thr Thr Thr Phe Ala Thr 275 280 285 Ser Ala Asn Gly Phe Tyr Thr Glu Asn His Pro Leu Val Gln Leu Met 290 295 300 Ala Val Gly Pro Thr Val Val Asn Pro Leu Tyr Asp Gly Asn Gln Tyr 305 310 315 320 Leu Val Ser Phe Asp Leu Asn Gly Gly Arg Gln Thr Arg Gly Leu Ile 325 330 335 Leu Asn Gly Thr Leu Pro Asp Gly Glu Leu Trp Phe Val Ser Met 340 345 350 <210> 20 <211> 351 <212> PRT <213> Artificial Sequence <220> <223> RIP3Aa-I275L <400> 20 Met Lys Gln Gly Glu Ala Ala Met Asn Glu Val Leu Ala Asn Asn Ile 1 5 10 15 Gly Gln His Pro Gln Val Ser Arg Arg Ala Leu Arg Ser Pro Arg Ser 20 25 30 Pro Val Val Phe Ile Asn Thr Thr Ser Glu Glu Ala Glu Gln Phe Gln 35 40 45 Glu Ile Val Ala Ile Asp Asp Ile Asp Tyr Leu Thr Gln Ala Val Ala 50 55 60 Leu Thr Ala Leu Phe Asn Gly Ala Ile Asp Asn Thr Thr Gly Arg Phe 65 70 75 80 Ile Pro Gly Arg Ala Arg Glu Leu Ile Ala Asn Tyr Asn Glu Ser Leu 85 90 95 Asp Pro Glu Ser Gln Arg Tyr Lys Ile Gly Ile Phe Asn Thr His Gln 100 105 110 Thr Thr Leu Thr Gln Gln Asn Ser Ala Val Ser Ala Met Ile Asn Gln 115 120 125 Ile Leu Glu Thr Leu Lys Asn Val Met Gly Val Ala Leu Gly Thr Thr 130 135 140 Ser Val Thr Gln Met Thr Ala Ala Ile Thr Asp Ala Phe Thr Asn Leu 145 150 155 160 Asn Glu Gln Ser Gly Asp Ala Trp Ile Phe Trp Glu Lys Lys Thr Ser 165 170 175 Asn Lys Thr Thr Tyr Ser Tyr Ala Ile Leu Phe Ala Phe Gln Asp Ser 180 185 190 Ser Thr Gly Lys Leu Met Phe Ala Leu Pro Met Ser Leu Glu Ile Glu 195 200 205 Val Asp Val Ser Tyr Glu Arg Val Leu Phe Ile Thr Val Glu Asp Lys 210 215 220 Glu Thr Tyr Ser Val Lys Leu Asp Thr Met Lys Val Gly Gln Leu Leu 225 230 235 240 Phe Pro Lys Ser Pro Gly Ala Asn Ala Leu Gln Thr Val Arg Arg Leu 245 250 255 Arg Thr Arg Ser Gly Ser Ser Gly Leu Leu Thr Ala Pro Ser Pro Val 260 265 270 Thr Asn Leu Val Val Thr Asn Trp Ala Lys Thr Thr Thr Phe Ala Thr 275 280 285 Ser Ala Asn Gly Phe Tyr Thr Glu Asn His Pro Leu Val Gln Leu Met 290 295 300 Ala Val Gly Pro Thr Val Val Asn Pro Leu Tyr Asp Gly Asn Gln Tyr 305 310 315 320 Leu Val Ser Phe Asp Leu Asn Gly Gly Arg Gln Thr Arg Gly Leu Ile 325 330 335 Leu Asn Gly Thr Leu Pro Asp Gly Glu Leu Trp Phe Val Ser Met 340 345 350 <210> 21 <211> 464 <212> PRT <213> Artificial Sequence <220> <223> SUMO-RIP1Aa <220> <221> Unclassified <222> (1)..(119) <223> SUMO tag <400> 21 Met Gly Ser Ser His His His His His His Gly Ser Gly Leu Val Pro 1 5 10 15 Arg Gly Ser Ala Ser Met Ser Asp Ser Glu Val Asn Gln Glu Ala Lys 20 25 30 Pro Glu Val Lys Pro Glu Val Lys Pro Glu Thr His Ile Asn Leu Lys 35 40 45 Val Ser Asp Gly Ser Ser Glu Ile Phe Phe Lys Ile Lys Lys Thr Thr 50 55 60 Pro Leu Arg Arg Leu Met Glu Ala Phe Ala Lys Arg Gln Gly Lys Glu 65 70 75 80 Met Asp Ser Leu Arg Phe Leu Tyr Asp Gly Ile Arg Ile Gln Ala Asp 85 90 95 Gln Thr Pro Glu Asp Leu Asp Met Glu Asp Asn Asp Ile Ile Glu Ala 100 105 110 His Arg Glu Gln Ile Gly Gly Met Ala Asp Ile Leu Ala Pro Asp Glu 115 120 125 Val Arg Leu Lys Asn Ile Ser Ala Val Arg Arg Leu Arg Ser Arg Gly 130 135 140 Gly Pro Phe Leu Phe Ile Gly Ala Thr Ala Asp Val Ser Glu Gln Phe 145 150 155 160 Gln Glu Ile Val Ala Ile Asp Asn Ile Asp Tyr Leu Thr Gln Ala Val 165 170 175 Gln Leu Thr Ala Leu Phe Asn Gly Ala Ile Asn Asn Glu Thr Gly Arg 180 185 190 Phe Glu Ser Asn Thr Ala Arg Gln Leu Ile Ala Asp Phe Asn Ala Ser 195 200 205 Leu Pro Glu Ser Asp Arg Ala Tyr Lys Ile Gly Ile Phe Lys Ser Tyr 210 215 220 Gln Thr Thr Leu Thr Gln Thr His Ser Val Val Ser Gly Met Ile Asp 225 230 235 240 Lys Ile Val Glu Ala Leu Lys Gln Val Leu Gly Val Ala Leu Gly Thr 245 250 255 Ser Thr Val Ala Gln Leu Thr Ala Ala Val Thr Asp Ala Phe Thr Asp 260 265 270 Leu Lys Ser Gln Glu Gly Asp Ala Trp Ile Phe Trp Glu Lys Lys Thr 275 280 285 Ala Glu Lys Thr Thr Tyr Ser Tyr Ala Ile Leu Phe Ala Ile Gln Asp 290 295 300 Ser Ser Thr Gly Met Met Met Phe Ala Met Pro Met Ser Leu Leu Ile 305 310 315 320 Glu Val Asn Val Ser Tyr Glu Lys Val Leu Trp Ile Thr Ile Asp Asp 325 330 335 Thr Glu Thr Tyr Ser Val Thr Leu Asp Thr Met Lys Val Gly Gln Ile 340 345 350 Leu Phe Pro Pro Ser Pro Gly Ser Ser Val Leu Arg Gln Ala Leu Ala 355 360 365 Pro Pro Pro Arg Lys Ala Glu Leu Gly Lys Glu Leu Glu Phe Ser Asp 370 375 380 Ile Thr Asp Ile Gln Val Thr Asn Trp Ser Lys Thr Lys Thr Phe Ala 385 390 395 400 Thr Ala Lys His Gly Ser Tyr Val Lys Glu Phe His Leu Glu Gln Val 405 410 415 Met Ala Phe Gln Pro Glu Val Leu His Pro Leu Gln Asp Glu Asp Lys 420 425 430 Cys Leu Val Ser Phe Thr Arg Ser Gly Glu Arg Lys Ser Val Gly Val 435 440 445 Ile Leu Asn Gly Thr Leu Pro Asp Gly Thr Leu Trp Phe Val Ser Gln 450 455 460 <210> 22 <211> 1038 <212> DNA <213> Aliidiomarina <400> 22 atggctgata ttcttgcacc tgatgaggtt cgactgaaaa acatctctgc tgtcaggaga 60 cttaggtctc gcggtggacc cttcctgttt attggtgcca cggcagacgt ctccgagcaa 120 tttcaagaaa ttgttgcaat cgataacatc gattatctca cccaggccgt tcagttaacg 180 gctctgttca atggtgccat taacaacgag accggaaggt tcgagtctaa tacagcacgc 240 cagctgatag cagatttcaa tgcgagtttg cctgaaagcg accgagcata caaaattggc 300 attttcaaaa gctaccaaac aacactcact cagacgcaca gcgtcgtctc cggcatgatc 360 gacaaaatcg ttgaggcgct gaaacaggtc ctcggcgtgg ctttgggcac gagcacggtt 420 gctcagctga ctgccgcagt cactgacgcg ttcacggatc tgaagtcgca ggagggcgac 480 gcatggattt tttgggagaa aaagaccgcg gaaaagacca catattccta cgctattctc 540 ttcgcgatcc aagacagcag cactggcatg atgatgttcg cgatgccgat gtctttgctg 600 atcgaggtga acgtcagcta tgagaaggtg ctgtggatta caattgatga cacagaaacc 660 tattccgtta cgctcgacac aatgaaggtc ggacaaatcc tgtttccacc gagtcccggt 720 tcgagtgtac tccggcaagc tctcgccccg ccgccccgca aagcggagct cggaaaggaa 780 ttggagtttt cagatatcac tgacatccag gtcacgaact ggtcgaagac aaagaccttt 840 gcaacggcaa agcacgggtc atatgtcaag gagttccatc ttgagcaggt gatggcgttt 900 caacccgaag ttcttcatcc cctccaggat gaagacaaat gccttgtgtc attcacacga 960 agcggtgagc gtaagagtgt gggagtcatt ctgaacggca cgttgccgga cggaacccta 1020 tggttcgtgt cgcaatag 1038 <210> 23 <211> 1035 <212> DNA <213> Sinorhizobium species <400> 23 atgaatgaag tgctgaggaa ctccgttggg caacattctc acatctccaa gcgggctttg 60 cgcccgagaa gctcgcccgt cgtattcata aacaccactc aagaagaggc ggagcagttt 120 caggaaatcg tcgccatcga tgatatcgac tatctgaccc aggcgatcgc gttgacggcg 240. ctgtttaacg gcgccattga caatacaacc ggtcgcttca ttcccggcaa ggcccgcgag ttgatcgcca actacaacga aagcctcgac gctaacagcc agccttacaa gatcggcatt ttcaacaccc accagaccac gctgacccag caaaacagtg ctgtgtcggc gatgatcagc cagattctcg aaaccttgaa gcgcgtgatg ggggtcgccc tcggcgcttc ttcggtcgcg 420 cagatgaccg cagcagtac tgatgcgttc accaacctcg acgagcagtc gggggatgcc 480. tggatcttct gggaaaaga gacgagcaac aagactacct acagctacgc gatccttttc gcgtttcagg acagcacgac cgggaagctc atgtttgctc tgcccatgtc actggagatc gaggtggacg tcagctacga gcgcgttctc ttcatcaccg tcgaagacaa ggaaacctat tcggtcaaac tcgatacgat gaaggttggc cagcttctct tccccaagtc acctggtgcg 720 aacgcacttc aatcggcgcg ccggctcggt acgcgctcgg gcagcgccga tctgctggct 780 gaccccgtc cgatcaccga catcgtcgtg acgaactggg caaaacga gactttcgca acggccgcaa ccggcctgta cactaacgat cacccgctcg ttcaggtgat ggcctcggaa 900 cccaatgtcg tcaatccgct gtacgacggc aaccagtacc tggtctcctt cgccctcgat 960 ggcgtacggc aaacactcgg gttccttctg aattgcacct tgcccgatgg tgaactgtgg 1020 ttcgtgtcga tctag 1035 <210> 24 <211> 1056 <212> DNA <213> Rhizobiales species <400> 24 atgaagcaag gagaagctgc aatgaatgaa gttctggcaa acaacattgg ccaacatcct 60 caggtatcca ggcgagcctt gcgttcgcca aggtcacccg tcgtattcat caacaccaca 120 tcggaagaag ctgaacagtt ccaggaaatc gtcgcgatcg atgacatcga ctatttgacc 180 caagcggtcg ctctgacggc attattcaat ggcgcgatcg acaatacgac cgggcgcttc 240 atccccggga gggctcgaga gctgattgcc aattacaatg aaagccttga tccggaaagc 300 cagcggtaca agatcggcat ctttaatacc catcaaacga cgctcactca gcaaaacagt 360 gccgtttccg caatgatcaa ccagatcctc gaaacgctga agaacgttat gggggtggcg 420 ctcggcacaa cttcggtcac ccagatgacc gctgcgataa ccgacgcttt caccaatctc 480 aatgagcagt caggcgacgc gtggatcttc tgggagaaaa agaccagcaa taagacgacc 540 tacagctacg cgatcctttt cgcgttccag gatagctcga ccgggaagct gatgttcgca 600 cttcccatgt cgttggaaat cgaagtcgac gtcagttacg aacgcgtcct cttcattaca 660 gtcgaagaca aggagaccta ttccgtgaag ctcgatacca tgaaggtcgg acagcttctc 720 tttccgaagt ctccgggcgc gaatgccctg cagacggtgc ggaggcttcg tacgcgttcg 780 gggagctccg gcttgctcac tgcgccgagt ccggtcacaa atatcgtcgt tacgaactgg 840 gcaaaaacga cgaccttcgc aacctccgca aatggttttt atacggagaa ccacccgcta 900 gttcagctga tggcggttgg ccccactgtg gtcaaccccc tgtacgacgg caaccagtac 960 ctggtctcct tcgatctcaa tggcggtcgg caaacacgcg gtctcatctt gaacggcacc 1020 ttgcctgatg gcgaactgtg gttcgtgtcg atgtag 1056 <210> 25 <211> 1044 <212> DNA <213> Rhizobium species <400> 25 atgaatgaaa tcgtcaccaa tcccgcccca gcccatgctt cacgccctca gcttcgcagt 60 ctgaccggca aacgcagccc gattgtcttt cttcaggctg cggtcgacca gaaggagcag 120 taccaggaga tcatggcgct cgacgacatc gactatgtca cacaggcact cggtatcgga 180 gccttgctca atggcgccat cgacacggcg accggccgtt tcgatgcggg cagggcgcgc 240 cagctgatcg tcgatttcaa ccaggcgctg ccgccggccg acagcaaata caagctggcc 300 atcatgaaca gctaccagtc gaccgtatcg caagaaaact cggtcgtctc cggcatgatc 360 gacaagatcc tcgatgttct caagacggca atcggcgtgg cgctcggcca gaagtcgatc 420 gaccagatca ctgccgccgt gaccgatgcc ttcaccaatt tgaaatcgca ggacggcgat 480 gcctggatct tctggcaaaa gcgggaggcc cataagaccg tgtattctta cgccatcctc 540 tttgccgtcc aggacgagtc gaccggtcgc gtcatgctcg cctttcccat gtcactggaa 600 atcgaagtca atgtggaatt cgagaaggtc ctctggatca ccgtcaagga cagccacaat 660 tattcggtca aggtggatgc gatgaagatc gcccagctgc ttttccccaa ggcgccaggc 720 agtcagacgc tgcagtcgat cgcctccgcc cctcggcttc ggggcctggc agacgtcgag 780 taccagaccc gagctagcga catcacggat atcacggtca ccaactggtc gcagtccacg 840 ctcttcgccc gggccgccaa gggcagcctt gtgaccgctg gctcgttgca gcagatcatg 900 gcctttgaac cagccatcga tattccgctc gaaccggaaa accattatct cgttcactac 960 aagctaaatg gggaggccaa gcagatcggg atgatcttca atgattatct ccctgacagc 1020 acactctggt tcgtcagccg ataa 1044 <210> 26 <211> 1038 <212> DNA <213> Artificial Sequence <220> <223> Escherichia coli codon-optimized <400> 26 atggccgaca tattagcgcc agacgaagtc agactgaaaa acatctccgc ggttcgcaga 60 cttagatcgc gcggcggccc ctttttgttt attggtgcga ccgccgatgt ttcggaacag 120 ttccaggaga tagtcgcaat agacaacatc gactatttga cccaggctgt gcaacttacc 180 gctttattca acggagctat caataatgaa actggacgtt ttgagagtaa taccgcgcgt 240 caattaatag ccgacttcaa tgcctcgtta cccgaatctg atcgtgccta taaaattggg 300 atattcaaat cataccagac aacactgact caaacacact cagtcgtctc cggcatgata 360 gacaaaattg tcgaagcatt aaagcaggtc ctgggtgtag ctttagggac gtcaactgtg 420 gcgcaattaa ccgcggccgt cacggatgcg ttcaccgact taaaatccca agagggggac 480 gcgtggattt tctgggagaa aaaaaccgcg gagaaaacta cttattccta cgctattctg 540 ttcgcgatcc aagacagctc tacaggcatg atgatgttcg caatgcctat gtcccttttg 600 atagaagtaa acgtatcata cgaaaaagtc ttgtggataa cgatagatga cactgagaca 660 tacagcgtca cattagacac tatgaaggta ggtcaaattt tatttcctcc ctccccaggc 720 tccagcgttt tgcggcaagc acttgcccca cctccccgta aagccgagct tggcaaagag 780 cttgagtttt cagacataac agatatacag gtgaccaact ggagtaaaac caagactttc 840 gcgaccgcca aacatggttc gtacgttaaa gagtttcatt tggagcaggt aatggccttc 900 cagcccgagg tattacatcc cttgcaagac gaggacaagt gtttagtctc attcacccgc 960 agcggagaac gcaagtcggt aggtgtcatc ttaaacggaa cgcttcccga tggcacgctg 1020 tggtttgtaa gccaataa 1038 <210> 27 <211> 1035 <212> DNA <213> Artificial Sequence <220> <223> Escherichia coli codon-optimized <400> 27 atgaacgagg ttttacggaa ctcagtgggc caacattcgc acatctccaa acgggccctt 60 cggcctagat cgtcgcctgt ggtatttatc aacacaaccc aagaggaggc ggagcaattc 120 caggaaattg ttgcaatcga cgacatcgat tacttgaccc aggcaattgc tctgaccgct 180 ttattcaatg gggctatcga taataccact ggtcgcttta tcccaggcaa agctcgggaa 240 cttatagcca actacaacga atcgcttgat gctaacagtc aaccatataa aattggcata 300 tttaacacac accagaccac gcttactcaa caaaattcag cggttagtgc gatgattagc 360 cagattcttg agactcttaa gcgcgtaatg ggtgttgcgt tgggtgcatc aagtgtggcg 420 caaatgacag cggccgtaac cgacgcgttc acaaacctgg atgagcagtc gggcgatgct 480 tggatcttct gggagaagaa gacaagtaat aaaaccacct actcttacgc tatacttttc 540 gccttccaag acagcactac cggcaaattg atgttcgcct taccgatgtc cttagagatc 600 gccttccaag acagcactac cggcaaattg atgttcgcct taccgatgtc cttagagatc 600 gaagtcgatg tgtcctatga acgcgttctt tttataacgg tggaggataa ggagacgtat 660 gaagtcgatg tgtcctatga acgcgttctt tttataacgg tggaggataa ggagacgtat 660 tcggttaaac ttgatacaat gaaggtgggg cagttactgt ttccaaagtc ccctggcgcg 720 tcggttaaac ttgatacaat gaaggtgggg cagttactgt ttccaaagtc ccctggcgcg 720 aatgcgttac aaagtgcaag acgtttgggg actagaagtg gatccgcaga ccttttagcc 780 aatgcgttac aaagtgcaag acgtttgggg actagaagtg gatccgcaga ccttttagcc 780 gagccacgtc cgataacgga catagtcgta actaattggg caaagacaaa gacatttgct 840 gagccacgtc cgataacgga catagtcgta actaattggg caaagacaaa gacatttgct 840 acagcggcga ctggcctgta cacaaatgac cacccgctgg tacaagtgat ggcatcggag 900 acagcggcga ctggcctgta cacaaatgac cacccgctgg tacaagtgat ggcatcggag 900 cctaatgtgg ttaatccgtt atatgacggc aatcagtatt tagtttcttt cgccttggac 960 cctaatgtgg ttaatccgtt atatgacggc aatcagtatt tagtttcttt cgccttggac 960 ggtgtgcggc agacgcttgg cttcttatta aactgcacat taccagatgg tgagctttgg 1020 ggtgtgcggc agacgcttgg cttcttatta aactgcacat taccagatgg tgagctttgg 1020 ttcgtcagta tataa 1035 ttcgtcagta tataa 1035 <210> 28<210> 28 <211> 1056<211> 1056 <212> DNA<212> DNA <213> 人工序列<213> Artificial sequence <220><220> <223> 大肠杆菌密码子优化的 <223> Escherichia coli codon-optimized <400> 28 <400> 28 atgaaacagg gagaggctgc gatgaacgaa gtattggcca acaatatcgg gcaacatcca 60 atgaaacagg gagaggctgc gatgaacgaa gtattggcca acaatatcgg gcaacatcca 60 caggtatctc gccgtgcgtt gcgctcgccc cgcagccccg tggtgttcat taacacaaca 120 tccgaggaag ccgaacaatt tcaagagatc gttgcaatcg atgacattga ttatcttact 180 caggcagtgg ccctgacagc tttatttaac ggtgcaattg acaatactac tggtcgtttc 240 attccaggac gcgcacgtga attaattgct aattacaacg aatcattaga cccagaatcg 300 cagcgttaca agatcggaat ctttaatacc caccaaacga ccttgactca acaaaattcg 360 gctgtttccg cgatgattaa ccaaatttta gaaacactga agaatgtgat gggggtagct 420 ctggggacta cgtcggtcac gcagatgact gccgcaatca ccgatgcatt cactaacctg 480 aatgagcaat cgggggacgc ttggatcttt tgggaaaaaa agacctccaa taagacaaca 540 tattcctacg caatcttatt tgcttttcag gattcgtcaa caggtaaact gatgtttgca 600 ttgccaatgt cacttgaaat cgaggtagac gtatcatacg aacgtgtcct gtttattacg 660 gtcgaggaca aagaaactta ctcggtcaag cttgatacca tgaaagttgg ccagttgttg 720 ttcccgaaaa gtccgggggc aaatgctttg cagacagtgc gtcgcttgcg tacgcgctca 780 ggttcatcgg ggttgttgac ggccccgtca ccagtaacta atatcgttgt aacgaactgg 840 gctaagacga ccacatttgc gacctcggca aatgggtttt atacggaaaa tcatcccttg 900 gttcagttga tggcggttgg ccctacggtc gtaaaccccc tttacgacgg caaccagtac 960 ttagtgagct tcgacttgaa tggtggtcgc cagacacgcg ggcttatcct gaatgggacg 1020 cttccagacg gagagttgtg gtttgtctct atgtaa 1056 <210> 29 <211> 1044 <212> DNA <213> Artificial Sequence <220> <223> Escherichia coli codon-optimized <400> 29 atgaatgaga ttgtaactaa tcctgctccg gctcacgcgt cacgcccgca actgcgcagt 60 ttgacgggga aacgtagtcc tattgttttt ttacaagccg ccgttgacca aaaagaacaa 120 tatcaggaga tcatggcttt ggatgacatc gattatgtta cgcaagcact ggggattggc 180 gcgttactga atggagcgat tgacaccgct acgggacgtt tcgatgccgg gcgcgcacgt 240 cagctgatcg ttgactttaa ccaagccttg cctcccgctg actcgaagta caaactggcg 300 attatgaact cttaccaatc gacagtgtca caagaaaact cagtggtgtc cggaatgatt 420. gacaaaatct tagatgtttt gaaaacggcg atcggggtag cattgggtca aaagtctatt gatcaaatca cagcggccgt cacggacgcg tttacgaatt tgaatccca ggacggtgat gcgtggattt tctggcaaaa acgtgaggct cataaaacag tttatagcta cgcaattctt 540 ttcgccgttc aggattc gaccggccgt gttatgttag catttccgat gtctcttgaa attgaggtta atgtggagtt tgaaaaggtt ctgtggatca cggttaagga ctcacacaat 720. tactccgtca aagttgacgc tatgaaaatt gctcaattgc tgttccccaa ggcccccggt tctcagactc ttcagtccat tgcgagcgcg cctcgtctgc gcggattagc cgacgttgaa 780 tatcagactc gcgcctcgga catcacagac atcacggtca cgaactggtc gcaaagcact ttgttcgcgc gcgctgccaa gggaagtctg gttacagctg gatcgttaca acagatcatg 960. gcgttcgac ctgccatcga catcccgctg gaaccagaga atcactatct ggtccattac aaactgaatg gagaagcgaa acagatcgga atgatcttta acgattactt gccagacagc acgctgtggt ttgtgtctcg ttaa 1044 <210> 30 <211> 1056 <212> DNA <213> Artificial Sequence <220> <223> Escherichia coli codon-optimized <400> 30 atgaaacagg gagaggctgc gatgaacgaa gtattggcca acaatatcgg gcaacatcca 60 caggtatctc gccgtgcgtt gcgctcgccc cgcagccccg tggtgttcat taacacaaca 120 tccgaggaag ccgaacaatt tcaagagctt gttgcaatcg atgacattga ttatcttact 180 caggcagtgg ccctgacagc tttatttaac ggtgcaattg acaatactac tggtcgtttc 240 attccaggac gcgcacgtga attaattgct aattacaacg aatcattaga cccagaatcg 300 cagcgttaca agatcggaat ctttaatacc caccaaacga ccttgactca acaaaattcg 360 gctgtttccg cgatgattaa ccaaatttta gaaacactga agaatgtgat gggggtagct 420 ctggggacta cgtcggtcac gcagatgact gccgcaatca ccgatgcatt cactaacctg 480 aatgagcaat cgggggacgc ttggatcttt tgggaaaaaa agacctccaa taagacaaca 540 tattcctacg caatcttatt tgcttttcag gattcgtcaa caggtaaact gatgtttgca 600 ttgccaatgt cacttgaaat cgaggtagac gtatcatacg aacgtgtcct gtttattacg 660 gtcgaggaca aagaaactta ctcggtcaag cttgatacca tgaaagttgg ccagttgttg 720 ttcccgaaaa gtccgggggc aaatgctttg cagacagtgc gtcgcttgcg tacgcgctca 780 ggttcatcgg ggttgttgac ggccccgtca ccagtaacta atatcgttgt aacgaactgg 840 gctaagacga ccacatttgc gacctcggca aatgggtttt atacggaaaa tcatcccttg 900 gttcagttga tggcggttgg ccctacggtc gtaaaccccc tttacgacgg caaccagtac 960 ttagtgagct tcgacttgaa tggtggtcgc cagacacgcg ggcttatcct gaatgggacg 1020 cttccagacg gagagttgtg gtttgtctct atgtaa 1056 <210> 31 <211> 1056 <212> DNA <213> Artificial sequence <220> <223> Escherichia coli codon-optimized <400> 31 atgaaacagg gagaggctgc gatgaacgaa gtattggcca acaatatcgg gcaacatcca 60 caggtatctc gccgtgcgtt gcgctcgccc cgcagccccg tggtgttcat taacacaaca 120 tccgaggaag ccgaacaatt tcaagagatc gttgcacttg atgacattga ttatcttact 180 caggcagtgg ccctgacagc tttatttaac ggtgcaattg acaatactac tggtcgtttc 240 attccaggac gcgcacgtga attaattgct aattacaacg aatcattaga cccagaatcg 300 cagcgttaca agatcggaat ctttaatacc caccaaacga ccttgactca acaaaattcg 360 gctgtttccg cgatgattaa ccaaatttta gaaacactga agaatgtgat gggggtagct 420 ctggggacta cgtcggtcac gcagatgact gccgcaatca ccgatgcatt cactaacctg 480 aatgagcaat cgggggacgc ttggatcttt tgggaaaaaa agacctccaa taagacaaca 540 tattcctacg caatcttatt tgcttttcag gattcgtcaa caggtaaact gatgtttgca 600 ttgccaatgt cacttgaaat cgaggtagac gtatcatacg aacgtgtcct gtttattacg 660 gtcgaggaca aagaaactta ctcggtcaag cttgatacca tgaaagttgg ccagttgttg 720 ttcccgaaaa gtccgggggc aaatgctttg cagacagtgc gtcgcttgcg tacgcgctca 780 ggttcatcgg ggttgttgac ggccccgtca ccagtaacta atatcgttgt aacgaactgg 840 gctaagacga ccacatttgc gacctcggca aatgggtttt atacggaaaa tcatcccttg 900 gttcagttga tggcggttgg ccctacggtc gtaaaccccc tttacgacgg caaccagtac 960 ttagtgagct tcgacttgaa tggtggtcgc cagacacgcg ggcttatcct gaatgggacg 1020 cttccagacg gagagttgtg gtttgtctct atgtaa 1056 <210> 32 <211> 1056 <212> DNA <213> Artificial Sequence <220> <223> Escherichia coli codon-optimized <400> 32 atgaaacagg gagaggctgc gatgaacgaa gtattggcca acaatatcgg gcaacatcca 60 caggtatctc gccgtgcgtt gcgctcgccc cgcagccccg tggtgttcat taacacaaca 120 tccgaggaag ccgaacaatt tcaagagatc gttgcaatcg atgaccttga ttatcttact 180 caggcagtgg ccctgacagc tttatttaac ggtgcaattg acaatactac tggtcgtttc 240 attccaggac gcgcacgtga attaattgct aattacaacg aatcattaga cccagaatcg 300 cagcgttaca agatcggaat ctttaatacc caccaaacga ccttgactca acaaaattcg 360 gctgtttccg cgatgattaa ccaaatttta gaaacactga agaatgtgat gggggtagct 420 ctggggacta cgtcggtcac gcagatgact gccgcaatca ccgatgcatt cactaacctg 480 aatgagcaat cgggggacgc ttggatcttt tgggaaaaaa agacctccaa taagacaaca 540 tattcctacg caatcttatt tgcttttcag gattcgtcaa caggtaaact gatgtttgca 600 ttgccaatgt cacttgaaat cgaggtagac gtatcatacg aacgtgtcct gtttattacg 660 gtcgaggaca aagaaactta ctcggtcaag cttgatacca tgaaagttgg ccagttgttg 720 ttcccgaaaa gtccgggggc aaatgctttg cagacagtgc gtcgcttgcg tacgcgctca 780 ggttcatcgg ggttgttgac ggccccgtca ccagtaacta atatcgttgt aacgaactgg 840 gctaagacga ccacatttgc gacctcggca aatgggtttt atacggaaaa tcatcccttg 900 gttcagttga tggcggttgg ccctacggtc gtaaaccccc tttacgacgg caaccagtac 960 ttagtgagct tcgacttgaa tggtggtcgc cagacacgcg ggcttatcct gaatgggacg 1020 cttccagacg gagagttgtg gtttgtctct atgtaa 1056 <210> 33 <211> 1056 <212> DNA <213> Artificial Sequence <220> <223> Escherichia coli codon-optimized <400> 33 atgaaacagg gagaggctgc gatgaacgaa gtattggcca acaatatcgg gcaacatcca 60 caggtatctc gccgtgcgtt gcgctcgccc cgcagccccg tggtgttcat taacacaaca 120 tccgaggaag ccgaacaatt tcaagagatc gttgcaatcg atgacattga ttatcttact 180 cagtgtgtgg ccctgacagc tttatttaac ggtgcaattg acaatactac tggtcgtttc 240 attccaggac gcgcacgtga attaattgct aattacaacg aatcattaga cccagaatcg 300 cagcgttaca agatcggaat ctttaatacc caccaaacga ccttgactca acaaaattcg 360 gctgtttccg cgatgattaa ccaaatttta gaaacactga agaatgtgat gggggtagct 420 ctggggacta cgtcggtcac gcagatgact gccgcaatca ccgatgcatt cactaacctg 480 aatgagcaat cgggggacgc ttggatcttt tgggaaaaaa agacctccaa taagacaaca 540 tattcctacg caatcttatt tgcttttcag gattcgtcaa caggtaaact gatgtttgca 600 ttgccaatgt cacttgaaat cgaggtagac gtatcatacg aacgtgtcct gtttattacg 660 gtcgaggaca aagaaactta ctcggtcaag cttgatacca tgaaagttgg ccagttgttg 720 ttcccgaaaa gtccgggggc aaatgctttg cagacagtgc gtcgcttgcg tacgcgctca 780 ggttcatcgg ggttgttgac ggccccgtca ccagtaacta atatcgttgt aacgaactgg 840 gctaagacga ccacatttgc gacctcggca aatgggtttt atacggaaaa tcatcccttg 900 gttcagttga tggcggttgg ccctacggtc gtaaaccccc tttacgacgg caaccagtac 960 ttagtgagct tcgacttgaa tggtggtcgc cagacacgcg ggcttatcct gaatgggacg 1020 cttccagacg gagagttgtg gtttgtctct atgtaa 1056 <210> 34 <211> 1056 <212> DNA <213> Artificial Sequence <220> <223> Escherichia coli codon-optimized <400> 34 atgaaacagg gagaggctgc gatgaacgaa gtattggcca acaatatcgg gcaacatcca 60 caggtatctc gccgtgcgtt gcgctcgccc cgcagccccg tggtgttcat taacacaaca 120 tccgaggaag ccgaacaatt tcaagagatc gttgcaatcg atgacattga ttatcttact 180 cagcttgtgg ccctgacagc tttatttaac ggtgcaattg acaatactac tggtcgtttc 240 attccaggac gcgcacgtga attaattgct aattacaacg aatcattaga cccagaatcg 300 cagcgttaca agatcggaat ctttaatacc caccaaacga ccttgactca acaaaattcg 360 gctgtttccg cgatgattaa ccaaatttta gaaacactga agaatgtgat gggggtagct 420 ctggggacta cgtcggtcac gcagatgact gccgcaatca ccgatgcatt cactaacctg 480 aatgagcaat cgggggacgc ttggatcttt tgggaaaaaa agacctccaa taagacaaca 540 tattcctacg caatcttatt tgcttttcag gattcgtcaa caggtaaact gatgtttgca 600 ttgccaatgt cacttgaaat cgaggtagac gtatcatacg aacgtgtcct gtttattacg 660 gtcgaggaca aagaaactta ctcggtcaag cttgatacca tgaaagttgg ccagttgttg 720 ttcccgaaaa gtccgggggc aaatgctttg cagacagtgc gtcgcttgcg tacgcgctca 780 ggttcatcgg ggttgttgac ggccccgtca ccagtaacta atatcgttgt aacgaactgg 840 gctaagacga ccacatttgc gacctcggca aatgggtttt atacggaaaa tcatcccttg 900 gttcagttga tggcggttgg ccctacggtc gtaaaccccc tttacgacgg caaccagtac 960 ttagtgagct tcgacttgaa tggtggtcgc cagacacgcg ggcttatcct gaatgggacg 1020 cttccagacg gagagttgtg gtttgtctct atgtaa 1056 <210> 35 <211> 1056 <212> DNA <213> Artificial Sequence <220> <223> Escherichia coli codon-optimized <400> 35 atgaaacagg gagaggctgc gatgaacgaa gtattggcca acaatatcgg gcaacatcca 60 caggtatctc gccgtgcgtt gcgctcgccc cgcagccccg tggtgttcat taacacaaca 120 tccgaggaag ccgaacaatt tcaagagatc gttgcaatcg atgacattga ttatcttact 180 caggcagtgt gtctgacagc tttatttaac ggtgcaattg acaatactac tggtcgtttc 240 attccaggac gcgcacgtga attaattgct aattacaacg aatcattaga cccagaatcg 300 cagcgttaca agatcggaat ctttaatacc caccaaacga ccttgactca acaaaattcg 360 gctgtttccg cgatgattaa ccaaatttta gaaacactga agaatgtgat gggggtagct 420 ctggggacta cgtcggtcac gcagatgact gccgcaatca ccgatgcatt cactaacctg 480 aatgagcaat cgggggacgc ttggatcttt tgggaaaaaa agacctccaa taagacaaca 540 tattcctacg caatcttatt tgcttttcag gattcgtcaa caggtaaact gatgtttgca 600 ttgccaatgt cacttgaaat cgaggtagac gtatcatacg aacgtgtcct gtttattacg 660 gtcgaggaca aagaaactta ctcggtcaag cttgatacca tgaaagttgg ccagttgttg 720 ttcccgaaaa gtccgggggc aaatgctttg cagacagtgc gtcgcttgcg tacgcgctca 780 ggttcatcgg ggttgttgac ggccccgtca ccagtaacta atatcgttgt aacgaactgg 840 gctaagacga ccacatttgc gacctcggca aatgggtttt atacggaaaa tcatcccttg 900 gttcagttga tggcggttgg ccctacggtc gtaaaccccc tttacgacgg caaccagtac 960 ttagtgagct tcgacttgaa tggtggtcgc cagacacgcg ggcttatcct gaatgggacg 1020 cttccagacg gagagttgtg gtttgtctct atgtaa 1056 <210> 36 <211> 1056 <212> DNA <213> Artificial Sequence <220> <223> Escherichia coli codon-optimized <400> 36 atgaaacagg gagaggctgc gatgaacgaa gtattggcca acaatatcgg gcaacatcca 60 caggtatctc gccgtgcgtt gcgctcgccc cgcagccccg tggtgttcat taacacaaca 120 tccgaggaag ccgaacaatt tcaagagatc gttgcaatcg atgacattga ttatcttact 180 caggcagtgc ttctgacagc tttatttaac ggtgcaattg acaatactac tggtcgtttc 240 attccaggac gcgcacgtga attaattgct aattacaacg aatcattaga cccagaatcg 300 cagcgttaca agatcggaat ctttaatacc caccaaacga ccttgactca acaaaattcg 360 gctgtttccg cgatgattaa ccaaatttta gaaacactga agaatgtgat gggggtagct 420 ctggggacta cgtcggtcac gcagatgact gccgcaatca ccgatgcatt cactaacctg 480 aatgagcaat cgggggacgc ttggatcttt tgggaaaaaa agacctccaa taagacaaca 540 tattcctacg caatcttatt tgcttttcag gattcgtcaa caggtaaact gatgtttgca 600 ttgccaatgt cacttgaaat cgaggtagac gtatcatacg aacgtgtcct gtttattacg 660 gtcgaggaca aagaaactta ctcggtcaag cttgatacca tgaaagttgg ccagttgttg 720 ttcccgaaaa gtccgggggc aaatgctttg cagacagtgc gtcgcttgcg tacgcgctca 780 ggttcatcgg ggttgttgac ggccccgtca ccagtaacta atatcgttgt aacgaactgg 840 gctaagacga ccacatttgc gacctcggca aatgggtttt atacggaaaa tcatcccttg 900 gttcagttga tggcggttgg ccctacggtc gtaaaccccc tttacgacgg caaccagtac 960 ttagtgagct tcgacttgaa tggtggtcgc cagacacgcg ggcttatcct gaatgggacg 1020 cttccagacg gagagttgtg gtttgtctct atgtaa 1056 <210> 37 <211> 1056 <212> DNA <213> Artificial sequence <220> <223> Escherichia coli codon-optimized <400> 37 atgaaacagg gagaggctgc gatgaacgaa gtattggcca acaatatcgg gcaacatcca 60 caggtatctc gccgtgcgtt gcgctcgccc cgcagccccg tggtgttcat taacacaaca 120 tccgaggaag ccgaacaatt tcaagagatc gttgcaatcg atgacattga ttatcttact 180 caggcagtgg ccctgacagc tttatttaac ggtgcaattg acaatactac tggtcgtttc 240 cttccaggac gcgcacgtga attaattgct aattacaacg aatcattaga cccagaatcg 300 cagcgttaca agatcggaat ctttaatacc caccaaacga ccttgactca acaaaattcg 360 gctgtttccg cgatgattaa ccaaatttta gaaacactga agaatgtgat gggggtagct 420 ctggggacta cgtcggtcac gcagatgact gccgcaatca ccgatgcatt cactaacctg 480 aatgagcaat cgggggacgc ttggatcttt tgggaaaaaa agacctccaa taagacaaca 540 tattcctacg caatcttatt tgcttttcag gattcgtcaa caggtaaact gatgtttgca 600 ttgccaatgt cacttgaaat cgaggtagac gtatcatacg aacgtgtcct gtttattacg 660 gtcgaggaca aagaaactta ctcggtcaag cttgatacca tgaaagttgg ccagttgttg 720 ttcccgaaaa gtccgggggc aaatgctttg cagacagtgc gtcgcttgcg tacgcgctca 780 ggttcatcgg ggttgttgac ggccccgtca ccagtaacta atatcgttgt aacgaactgg 840 gctaagacga ccacatttgc gacctcggca aatgggtttt atacggaaaa tcatcccttg 900 gttcagttga tggcggttgg ccctacggtc gtaaaccccc tttacgacgg caaccagtac 960 ttagtgagct tcgacttgaa tggtggtcgc cagacacgcg ggcttatcct gaatgggacg 1020 cttccagacg gagagttgtg gtttgtctct atgtaa 1056 <210> 38 <211> 1056 <212> DNA <213> Artificial sequence <220> <223> Escherichia coli codon-optimized <400> 38 atgaaacagg gagaggctgc gatgaacgaa gtattggcca acaatatcgg gcaacatcca 60 caggtatctc gccgtgcgtt gcgctcgccc cgcagccccg tggtgttcat taacacaaca 120 tccgaggaag ccgaacaatt tcaagagatc gttgcaatcg atgacattga ttatcttact 180 caggcagtgg ccctgacagc tttatttaac ggtgcaattg acaatactac tggtcgtttc 240 attccaggac gcgcacgtga attaattgct aattacaacg aatcattaga cccagaatcg 300 cagcgttaca agatcggaat ctttaatacc caccaaacga ccttgactca acaaaattcg 360 gctgtttccg cgatgcttaa ccaaatttta gaaacactga agaatgtgat gggggtagct 420 ctggggacta cgtcggtcac gcagatgact gccgcaatca ccgatgcatt cactaacctg 480 aatgagcaat cgggggacgc ttggatcttt tgggaaaaaa agacctccaa taagacaaca 540 tattcctacg caatcttatt tgcttttcag gattcgtcaa caggtaaact gatgtttgca 600 ttgccaatgt cacttgaaat cgaggtagac gtatcatacg aacgtgtcct gtttattacg 660 gtcgaggaca aagaaactta ctcggtcaag cttgatacca tgaaagttgg ccagttgttg 720 ttcccgaaaa gtccgggggc aaatgctttg cagacagtgc gtcgcttgcg tacgcgctca 780 ggttcatcgg ggttgttgac ggccccgtca ccagtaacta atatcgttgt aacgaactgg 840 gctaagacga ccacatttgc gacctcggca aatgggtttt atacggaaaa tcatcccttg 900 gttcagttga tggcggttgg ccctacggtc gtaaaccccc tttacgacgg caaccagtac 960 ttagtgagct tcgacttgaa tggtggtcgc cagacacgcg ggcttatcct gaatgggacg 1020 cttccagacg gagagttgtg gtttgtctct atgtaa 1056 <210> 39 <211> 1056 <212> DNA <213> Artificial Sequence <220> <223> Escherichia coli codon-optimized <400> 39 atgaaacagg gagaggctgc gatgaacgaa gtattggcca acaatatcgg gcaacatcca 60 caggtatctc gccgtgcgtt gcgctcgccc cgcagccccg tggtgttcat taacacaaca 120 tccgaggaag ccgaacaatt tcaagagatc gttgcaatcg atgacattga ttatcttact 180 caggcagtgg ccctgacagc tttatttaac ggtgcaattg acaatactac tggtcgtttc 240 attccaggac gcgcacgtga attaattgct aattacaacg aatcattaga cccagaatcg 300 cagcgttaca agatcggaat ctttaatacc caccaaacga ccttgactca acaaaattcg 360 gctgtttccg cgatgattaa ccaaatttta gaaacactga agaatgtgat gggggtagct 420 ctggggacta cgtcggtcac gcagatgact gccgcactta ccgatgcatt cactaacctg 480 aatgagcaat cgggggacgc ttggatcttt tgggaaaaaa agacctccaa taagacaaca 540 tattcctacg caatcttatt tgcttttcag gattcgtcaa caggtaaact gatgtttgca 600 ttgccaatgt cacttgaaat cgaggtagac gtatcatacg aacgtgtcct gtttattacg 660 gtcgaggaca aagaaactta ctcggtcaag cttgatacca tgaaagttgg ccagttgttg 720 ttcccgaaaa gtccgggggc aaatgctttg cagacagtgc gtcgcttgcg tacgcgctca 780 ggttcatcgg ggttgttgac ggccccgtca ccagtaacta atatcgttgt aacgaactgg 840 gctaagacga ccacatttgc gacctcggca aatgggtttt atacggaaaa tcatcccttg 900 gttcagttga tggcggttgg ccctacggtc gtaaaccccc tttacgacgg caaccagtac 960 ttagtgagct tcgacttgaa tggtggtcgc cagacacgcg ggcttatcct gaatgggacg 1020 cttccagacg gagagttgtg gtttgtctct atgtaa 1056 <210> 40 <211> 1056 <212> DNA <213> Artificial Sequence <220> <223> Escherichia coli codon-optimized <400> 40 atgaaacagg gagaggctgc gatgaacgaa gtattggcca acaatatcgg gcaacatcca 60 caggtatctc gccgtgcgtt gcgctcgccc cgcagccccg tggtgttcat taacacaaca 120 tccgaggaag ccgaacaatt tcaagagatc gttgcaatcg atgacattga ttatcttact 180 caggcagtgg ccctgacagc tttatttaac ggtgcaattg acaatactac tggtcgtttc 240 attccaggac gcgcacgtga attaattgct aattacaacg aatcattaga cccagaatcg 300 cagcgttaca agatcggaat ctttaatacc caccaaacga ccttgactca acaaaattcg 360 gctgtttccg cgatgattaa ccaaatttta gaaacactga agaatgtgat gggggtagct 420 ctggggacta cgtcggtcac gcagatgact gccgcaatca ccgatgcatt cactaacctg 480 aatgagcaat cgggggacgc ttggcttttt tgggaaaaaa agacctccaa taagacaaca 540 tattcctacg caatcttatt tgcttttcag gattcgtcaa caggtaaact gatgtttgca 600 ttgccaatgt cacttgaaat cgaggtagac gtatcatacg aacgtgtcct gtttattacg 660 gtcgaggaca aagaaactta ctcggtcaag cttgatacca tgaaagttgg ccagttgttg 720 ttcccgaaaa gtccgggggc aaatgctttg cagacagtgc gtcgcttgcg tacgcgctca 780 ggttcatcgg ggttgttgac ggccccgtca ccagtaacta atatcgttgt aacgaactgg 840 gctaagacga ccacatttgc gacctcggca aatgggtttt atacggaaaa tcatcccttg 900 gttcagttga tggcggttgg ccctacggtc gtaaaccccc tttacgacgg caaccagtac 960 ttagtgagct tcgacttgaa tggtggtcgc cagacacgcg ggcttatcct gaatgggacg 1020 cttccagacg gagagttgtg gtttgtctct atgtaa 1056 <210> 41 <211> 1056 <212> DNA <213> Artificial Sequence <220> <223> Escherichia coli codon-optimized <400> 41 atgaaacagg gagaggctgc gatgaacgaa gtattggcca acaatatcgg gcaacatcca 60 caggtatctc gccgtgcgtt gcgctcgccc cgcagccccg tggtgttcat taacacaaca 120 tccgaggaag ccgaacaatt tcaagagatc gttgcaatcg atgacattga ttatcttact 180 caggcagtgg ccctgacagc tttatttaac ggtgcaattg acaatactac tggtcgtttc 240 attccaggac gcgcacgtga attaattgct aattacaacg aatcattaga cccagaatcg 300 cagcgttaca agatcggaat ctttaatacc caccaaacga ccttgactca acaaaattcg 360 gctgtttccg cgatgattaa ccaaatttta gaaacactga agaatgtgat gggggtagct 420 ctggggacta cgtcggtcac gcagatgact gccgcaatca ccgatgcatt cactaacctg 480 aatgagcaat cgggggacgc ttggatcttt tgggaaaaaa agacctccaa taagacaaca 540 tattcctacg cacttttatt tgcttttcag gattcgtcaa caggtaaact gatgtttgca 600 ttgccaatgt cacttgaaat cgaggtagac gtatcatacg aacgtgtcct gtttattacg 660 gtcgaggaca aagaaactta ctcggtcaag cttgatacca tgaaagttgg ccagttgttg 720 ttcccgaaaa gtccgggggc aaatgctttg cagacagtgc gtcgcttgcg tacgcgctca 780 ggttcatcgg ggttgttgac ggccccgtca ccagtaacta atatcgttgt aacgaactgg 840 gctaagacga ccacatttgc gacctcggca aatgggtttt atacggaaaa tcatcccttg 900 gttcagttga tggcggttgg ccctacggtc gtaaaccccc tttacgacgg caaccagtac 960 ttagtgagct tcgacttgaa tggtggtcgc cagacacgcg ggcttatcct gaatgggacg 1020 cttccagacg gagagttgtg gtttgtctct atgtaa 1056 <210> 42 <211> 1056 <212> DNA <213> Artificial Sequence <220> <223> Escherichia coli codon-optimized <400> 42 atgaaacagg gagaggctgc gatgaacgaa gtattggcca acaatatcgg gcaacatcca 60 caggtatctc gccgtgcgtt gcgctcgccc cgcagccccg tggtgttcat taacacaaca 120 tccgaggaag ccgaacaatt tcaagagatc gttgcaatcg atgacattga ttatcttact 180 caggcagtgg ccctgacagc tttatttaac ggtgcaattg acaatactac tggtcgtttc 240 attccaggac gcgcacgtga attaattgct aattacaacg aatcattaga cccagaatcg 300 cagcgttaca agatcggaat ctttaatacc caccaaacga ccttgactca acaaaattcg 360 gctgtttccg cgatgattaa ccaaatttta gaaacactga agaatgtgat gggggtagct 420 ctggggacta cgtcggtcac gcagatgact gccgcaatca ccgatgcatt cactaacctg 480 aatgagcaat cgggggacgc ttggatcttt tgggaaaaaa agacctccaa taagacaaca 540 tattcctacg caatcttatt tgcttttcag gattcgtcaa caggtaaact gatgtttgca 600 ttgccaatgt cacttgaact tgaggtagac gtatcatacg aacgtgtcct gtttattacg 660 gtcgaggaca aagaaactta ctcggtcaag cttgatacca tgaaagttgg ccagttgttg 720 ttcccgaaaa gtccgggggc aaatgctttg cagacagtgc gtcgcttgcg tacgcgctca 780 ggttcatcgg ggttgttgac ggccccgtca ccagtaacta atatcgttgt aacgaactgg 840 gctaagacga ccacatttgc gacctcggca aatgggtttt atacggaaaa tcatcccttg 900 gttcagttga tggcggttgg ccctacggtc gtaaaccccc tttacgacgg caaccagtac 960 ttagtgagct tcgacttgaa tggtggtcgc cagacacgcg ggcttatcct gaatgggacg 1020 cttccagacg gagagttgtg gtttgtctct atgtaa 1056 <210> 43 <211> 1056 <212> DNA <213> Artificial Sequence <220> <223> Escherichia coli codon-optimized <400> 43 atgaaacagg gagaggctgc gatgaacgaa gtattggcca acaatatcgg gcaacatcca 60 caggtatctc gccgtgcgtt gcgctcgccc cgcagccccg tggtgttcat taacacaaca 120 tccgaggaag ccgaacaatt tcaagagatc gttgcaatcg atgacattga ttatcttact 180 caggcagtgg ccctgacagc tttatttaac ggtgcaattg acaatactac tggtcgtttc 240 attccaggac gcgcacgtga attaattgct aattacaacg aatcattaga cccagaatcg 300 cagcgttaca agatcggaat ctttaatacc caccaaacga ccttgactca acaaaattcg 360 gctgtttccg cgatgattaa ccaaatttta gaaacactga agaatgtgat gggggtagct 420 ctggggacta cgtcggtcac gcagatgact gccgcaatca ccgatgcatt cactaacctg 480 aatgagcaat cgggggacgc ttggatcttt tgggaaaaaa agacctccaa taagacaaca 540 tattcctacg caatcttatt tgcttttcag gattcgtcaa caggtaaact gatgtttgca 600 ttgccaatgt cacttgaaat cgaggtagac gtatcatacg aacgtgtcct gtttcttacg 660 gtcgaggaca aagaaactta ctcggtcaag cttgatacca tgaaagttgg ccagttgttg 720 ttcccgaaaa gtccgggggc aaatgctttg cagacagtgc gtcgcttgcg tacgcgctca 780 ggttcatcgg ggttgttgac ggccccgtca ccagtaacta atatcgttgt aacgaactgg 840 gctaagacga ccacatttgc gacctcggca aatgggtttt atacggaaaa tcatcccttg 900 gttcagttga tggcggttgg ccctacggtc gtaaaccccc tttacgacgg caaccagtac 960 ttagtgagct tcgacttgaa tggtggtcgc cagacacgcg ggcttatcct gaatgggacg 1020 cttccagacg gagagttgtg gtttgtctct atgtaa 1056 <210> 44 <211> 1056 <212> DNA <213> Artificial Sequence <220> <223> Escherichia coli codon-optimized <400> 44 atgaaacagg gagaggctgc gatgaacgaa gtattggcca acaatatcgg gcaacatcca 60 caggtatctc gccgtgcgtt gcgctcgccc cgcagccccg tggtgttcat taacacaaca 120 tccgaggaag ccgaacaatt tcaagagatc gttgcaatcg atgacattga ttatcttact 180 caggcagtgg ccctgacagc tttatttaac ggtgcaattg acaatactac tggtcgtttc 240 attccaggac gcgcacgtga attaattgct aattacaacg aatcattaga cccagaatcg 300 cagcgttaca agatcggaat ctttaatacc caccaaacga ccttgactca acaaaattcg 360 gctgtttccg cgatgattaa ccaaatttta gaaacactga agaatgtgat gggggtagct 420 ctggggacta cgtcggtcac gcagatgact gccgcaatca ccgatgcatt cactaacctg 480 aatgagcaat cgggggacgc ttggatcttt tgggaaaaaa agacctccaa taagacaaca 540 tattcctacg caatcttatt tgcttttcag gattcgtcaa caggtaaact gatgtttgca 600 ttgccaatgt cacttgaaat cgaggtagac gtatcatacg aacgtgtcct gtttattacg 660 gtcgaggaca aagaaactta ctcggtcaag cttgatacca tgaaagttgg ccagttgttg 720 ttcccgaaaa gtccgggggc aaatgctttg cagacagtgc gtcgcttgcg tacgcgctca 780 ggttcatcgg ggttgttgac ggccccgtca ccagtaacta atcttgttgt aacgaactgg 840 gctaagacga ccacatttgc gacctcggca aatgggtttt atacggaaaa tcatcccttg 900 gttcagttga tggcggttgg ccctacggtc gtaaaccccc tttacgacgg caaccagtac 960 ttagtgagct tcgacttgaa tggtggtcgc cagacacgcg ggcttatcct gaatgggacg 1020 cttccagacg gagagttgtg gtttgtctct atgtaa 1056 <210> 45 <211> 1395 <212> DNA <213> Artificial sequence <220> <223> SUMO-rip1Aa <220> <221> Unclassified <222> (1)..(357) <223> SUMO tag <400> 45 atgggcagca gccatcatca tcatcatcac ggcagcggcc tggtgccgcg cggcagcgct 60 agcatgtcgg actcagaagt caatcaagaa gctaagccag aggtcaagcc agaagtcaag 120 cctgagactc acatcaattt aaaggtgtcc gatggatctt cagagatctt cttcaagatc 180 aaaaagacca ctcctttaag aaggctgatg gaagcgttcg ctaaaagaca gggtaaggaa 240 atggactcct taagattctt gtacgacggt attagaattc aagctgatca gacccctgaa 300 gatttggaca tggaggataa cgatattatt gaggctcaca gagaacagat tggtggtatg 360 gccgacatat tagcgccaga cgaagtcaga ctgaaaaaca tctccgcggt tcgcagactt 420 agatcgcgcg gcggcccctt tttgtttatt ggtgcgaccg ccgatgtttc ggaacagttc 480 caggagatag tcgcaataga caacatcgac tatttgaccc aggctgtgca acttaccgct 540 ttattcaacg gagctatca taatgaact ggacgttttg agagtaac cgcgcgtca 600 ttaatagccg acttcaatgc ctcgttaccc gatctgatc gtgcctataa aattgggata 660 ttcaatcat accagacac actgactca accactcag tcgtctccgg catgatagac 720 aaaattgtcg aagcattaaa gcaggtcctg ggtgtagctt tagggacgtc aactgtggcg 780 cattaaccg cggccgtcac ggatgcgttc accgactta aatcccaaga gggggacgcg 840 tggattttct gggagaaaa aaccggcggag aaactactt attcctacgc tattctgttc 900 gcgatccaag acagctctac agcatgatg atgttcgcaa tgcctatgtc ccttttgata 960 gagtaacg tatcatacga aaagtcttg tggataacga taggacac tgagacatac 1020 agcgtcacat tagacactt gaaggtaggt caattttat ttccccctc cccaggctcc 1080 agcgttttgc ggcaagcact tgcccacct cccgtaag ccgagcttgg gcgagctt 1140 gagttttcag acatacaga tatacaggtg accaactgga gtaaaaccaa gactttcgcg 1200 accgccaaac atggttcgta cgttaaagag tttcatttgg agcaggtaat ggccttccag 1260 cccgaggtat tacatccctt gcaagacgag gacaagtgtt tagtctcatt cacccgcagc 1320 ggagaacgca agtcggtagg tgtcatctta aacggaacgc ttcccgatgg cacgctgtgg 1380 tttgtaagcc aataa 1395 <210> 46 <211> 1035 <212> DNA <213> Artificial Sequence <220> <223> Maize-optimized rip1Aa <400> 46 atggctgaca tactcgcccc ggacgaggtg cggctgaaaa atatatccgc tgttaggcgt 60 ttgaggagca ggggagggcc ttttttgttc atcggcgcga ccgctgacgt gtctgagcag 120 tttcaagaga tagtggctat agacaacatc gactacctca cgcaggccgt ccagttgacc 180 gccctgttca acggcgcgat taacaacgag acgggccgtt tcgagtcgaa taccgccaga 240 cagctgatcg cggacttcaa tgcctccctg cccgagagtg acagagctta caagatcggc 300 atcttcaagt cttaccaaac tacactcact cagacgcata gcgttgtgtc cgggatgatc 360 gacaagatag tggaagcgct taaacaggtt ctgggggtgg ccctcggcac tagtacggtg 420 gctcagctca ccgccgccgt cacagacgct ttcacggacc tcaagagcca agagggggat 480 gcgtggattt tctgggagaa gaagactgcc gagaagacca catactcata cgccatcctg 540 ttcgctattc aagactcctc aactggcatg atgatgttcg caatgccgat gtctctcctg 600 attgaggtta atgtgtcata cgaaaaggtt ctctggatca ctatcgacga taccgagacc 660 tactctgtta ctctcgacac gatgaaggtg ggacagatac tcttccctcc gtcgcctggg 720 tcttccgtgc tgaggcaagc cctcgcgccc ccgccacgta aggccgagct gggcaaagag 780 cttgagttct cagatatcac ggacatccag gttaccaact ggagtaagac gaagaccttc 840 gctactgcta agcacggatc ttatgtcaag gagttccacc tcgagcaggt tatggctttt 900 caaccagagg ttctgcaccc ccttcaagat gaagacaaat gcctggtctc ttttacgcgg 960 tcgggcgagc ggaagagtgt cggggtgatc ctgaatggta cccttccaga cggtacgctc 1020 tggtttgtta gccag 1035 <210> 47 <211> 1032 <212> DNA <213> Artificial Sequence <220> <223> Zea mays-optimized RIP2Aa <400> 47 atgaacgaag ttctcaggaa ctctgtcggc cagcactcgc atattagcaa gagggccctc 60 cggcccagaa gtagcccggt tgttttcata aatacgacgc aagaggaagc cgagcaattc 120 caagagatcg tcgctatcga tgacattgat taccttacgc aggcgatcgc tctcactgct 180 ctgttcaacg gtgctataga caacacgacg gggaggttta tccctgggaa ggcccgcgag 240 ctcatagcga attataacga gtccctcgat gcgaactcgc agccttacaa gatcggtata 300 ttcaacaccc accagacgac gctgacccag caaaattctg cagtctcagc catgatcagc 360 cagatccttg agaccctcaa gcgcgtcatg ggtgtcgcac tgggggcctc atcggtggct 420 caaatgaccg ctgctgtgac cgatgcgttt actaatctcg acgagcagtc aggcgacgcc 480 tggatcttct gggagaagaa gacctctaat aagaccacct actcttatgc catcctcttc 540 gcatttcaag acagtacgac ggggaaattg atgtttgcac tcccgatgtc gctcgaaatt 600 gaggtcgacg tcagctacga acgtgttctg ttcattacag tggaggataa ggagacatac 660 tcagtgaagc tggacaccat gaaggtgggt cagcttttgt tcccgaagtc tcctggggct 720 aatgcgctcc aaagcgcccg gcgcctgggc actcggagtg gcagcgccga cttgctggcc 780 gaaccgcgcc ccataaccga tatcgtggtg accaactggg ccaagacgaa aacattcgct 840 actgcagcta ccggacttta cacaaacgac cacccgctcg ttcaggtcat ggcaagtgag 900 cccaatgtcg ttaacccgtt gtatgacggc aaccagtatt tggtgagctt tgcactcgac 960 ggggtgcggc agacgttggg ctttttgctc aactgcaccc tccccgacgg ggagttgtgg 1020 ttcgtttcta tt 1032 <210> 48 <211> 1053 <212> DNA <213> Artificial Sequence <220> <223> Maize - optimized RIP3Aa <400> 48 atgaagcagg gtgaggcagc catgaacgaa gttcttgcaa acaacatagg acagcacccc 60 caagtgtccc gccgggcgtt gagatcacca aggagccctg ttgtgttcat aaacacgacc 120 tccgaggagg cggagcaatt ccaggagatc gttgctattg acgacataga ctacctcacc 180 caggcggtcg ctttgacagc gctgtttaac ggcgccatcg ataacaccac cgggcggttc 240 atcccgggaa gggcgaga gctgatcgcc aactacaatg agtcgttgga cccggaatct 300 cagagatata aaatcggaat ctttaatacc caccagacta ccctgaccca gcaaaattct 360 gcagtgagcg ccatgattaa tcaaattctc gagacactga agaatgtgat gggtgtggca 420 cttggaacca cgtccgtgac tcagatgaca gccgcgataa ccgatgcgtt caccaacctg 480 aacgagcaga gtggtgatgc ttggatttc tgggagaaga agacccttaa caagaccacg 540 tactcttacg ccatcctgtt tgcttttcag gactccagca ccggcaagct gatgttcgcc 600 ctgccaatgt ctcttgagat tgaggtggat gtctcctacg agagggtgct cttcatcacg 660 gtggaggaca aagacata ctctgtgaag ctggatacga tgaaagtggg gcagctgttg 720 ttcccaaagt ctcccggggc aaatgcactc cagacagtca gaaggctcag gactaggtcg 780 ggtagctcgg gtttgctcac tgcccctagc cctgtgacaa atatcgttgt caaaactgg 840 gccaagacca ctaccttgc gacatcggcg aatggattct acacggagagaa tcatccgctt 900 gtccagctca tggctgtggg cccgaccgtt gtgaaccccc tttatgatgg aaatcaatat 960 ctggtgtcat ttgacctcaa cggtggccgg cagacgcgtg gccttatcct gaacggcact 1020 cttcccgacg gtgagctgtg gtttgtctct atg 1053 <210> 49 <211> 1041 <212> DNA <213> Artificial Sequence <220> <223> Maize-Optimized RIP4Aa <400> 49 atgaatgaga ttgtgacgaa tcccgctcct gctcacgcca gtagacccca actccggtcg 60 cttacgggta agaggtcccc tatcgtgttt ctccaggcgg cagtcgacca gaaagagcaa 120 taccaggaga ttatggcgct ggacgacata gattatgtga ctcaggcgct ggggatcggc 180 gctcttttga atggagctat cgacacggcg acaggtagat tcgatgctgg ccgcgcacgg 240 caactcatag tcgattttaa tcaggcgctg cctcctgctg acagcaagta caagttggcg 300 atcatgaatt cttaccagtc gacagtgtcg caagagaaca gcgttgtgag cggtatgatt 360 gataagatcc tcgatgtcct gaagaccgca attggtgttg cgttgggaca gaagtcaata 420 gaccaaatta cggcagcggt gactgatgcc ttcacgaacc tcaaaagtca agacggcgat 480 gcgtggatct tttggcagaa aagggaggca cataaaaccg tgtactccta cgcgatactc 540 tttgctgttc aagatgagtc cacgggacgc gtgatgctgg cgttccctat gagcctggag 600 atcgaagtga acgtggagtt cgagaaggtc ctttggatca cggttaagga ttcacataac 660 tattcagtta aggtggacgc catgaaaatc gctcagcttt tgttccctaa agctccgggt 720 agtcagactc ttcaaagcat agcatcagct cctcgcctca gaggccttgc agacgttgag 780 taccagacca gagccagtga tatcacagac ataaccgtca cgaactggag ccagtctacg 840 ctgtttgcta gagctgctaa ggggtcactg gtgacagccg gctcgctcca acaaattatg 900 gctttcgagc ccgccatcga tatcccactt gaacccgaga atcactacct ggtgcactac 960 aagttgaacg gtgaggccaa gcaaatcggg atgattttca atgactatct gccggactcg 1020 acgctttggt ttgtttcgag a 1041 <210> 50 <211> 195 <212> PRT <213> Pseudomonas alldii <400> 50 Gln Phe Gln Glu Ile Val Ala Ile Asp Asn Ile Asp Tyr Leu Thr Gln 1 5 10 15 Ala Val Gln Leu Thr Ala Leu Phe Asn Gly Ala Ile Asn Asn Glu Thr 20 25 30 Gly Arg Phe Glu Ser Asn Thr Ala Arg Gln Leu Ile Ala Asp Phe Asn 35 40 45 Ala Ser Leu Pro Glu Ser Asp Arg Ala Tyr Lys Ile Gly Ile Phe Lys 50 55 60 Ser Tyr Gln Thr Thr Leu Thr Gln Thr His Ser Val Val Ser Gly Met 65 70 75 80 Ile Asp Lys Ile Val Glu Ala Leu Lys Gln Val Leu Gly Val Ala Leu 85 90 95 Gly Thr Ser Thr Val Ala Gln Leu Thr Ala Ala Val Thr Asp Ala Phe 100 105 110 Thr Asp Leu Lys Ser Gln Glu Gly Asp Ala Trp Ile Phe Trp Glu Lys 115 120 125 Lys Thr Ala Glu Lys Thr Thr Tyr Ser Tyr Ala Ile Leu Phe Ala Ile 130 135 140 Gln Asp Ser Ser Thr Gly Met Met Met Phe Ala Met Pro Met Ser Leu 145 150 155 160 Leu Ile Glu Val Asn Val Ser Tyr Glu Lys Val Leu Trp Ile Thr Ile 165 170 175 Asp Asp Thr Glu Thr Tyr Ser Val Thr Leu Asp Thr Met Lys Val Gly 180 185 190 Gln Ile Leu 195 <210> 51 <211> 195 <212> PRT <213> Chinese species of <400> 51 Phe Gln Glu Ile Val Ala Ile Asp Asp Ile Asp Tyr Leu Thr Gln Ala 1 5 10 15 Ile Ala Leu Thr Ala Leu Phe Asn Gly Ala Ile Asp Asn Thr Thr Gly 20 25 30 Arg Phe Ile Pro Gly Lys Ala Arg Glu Leu Ile Ala Asn Tyr Asn Glu 35 40 45 Ser Leu Asp Ala Asn Ser Gln Pro Tyr Lys Ile Gly Ile Phe Asn Thr 50 55 60 His Gln Thr Thr Leu Thr Gln Gln Asn Ser Ala Val Ser Ala Met Ile 65 70 75 80 Ser Gln Ile Leu Glu Thr Leu Lys Arg Val Met Gly Val Ala Leu Gly 85 90 95 Ala Ser Ser Val Ala Gln Met Thr Ala Ala Val Thr Asp Ala Phe Thr 100 105 110 Asn Leu Asp Glu Gln Ser Gly Asp Ala Trp Ile Phe Trp Glu Lys Lys 115 120 125 Thr Ser Asn Lys Thr Thr Tyr Ser Tyr Ala Ile Leu Phe Ala Phe Gln 130 135 140 Asp Ser Thr Thr Gly Lys Leu Met Phe Ala Leu Pro Met Ser Leu Glu 145 150 155 160 Ile Glu Val Asp Val Ser Tyr Glu Arg Val Leu Phe Ile Thr Val Glu 165 170 175 Asp Lys Glu Thr Tyr Ser Val Lys Leu Asp Thr Met Lys Val Gly Gln 180 185 190 Leu Leu Phe 195 <210> 52 <211> 195 <212> PRT <213> Rhizobiales species <400> 52 Gln Phe Gln Glu Ile Val Ala Ile Asp Asp Ile Asp Tyr Leu Thr Gln 1 5 10 15 Ala Val Ala Leu Thr Ala Leu Phe Asn Gly Ala Ile Asp Asn Thr Thr 20 25 30 Gly Arg Phe Ile Pro Gly Arg Ala Arg Glu Leu Ile Ala Asn Tyr Asn 35 40 45 Glu Ser Leu Asp Pro Glu Ser Gln Arg Tyr Lys Ile Gly Ile Phe Asn 50 55 60 Thr His Gln Thr Thr Leu Thr Gln Gln Asn Ser Ala Val Ser Ala Met 65 70 75 80 Ile Asn Gln Ile Leu Glu Thr Leu Lys Asn Val Met Gly Val Ala Leu 85 90 95 Gly Thr Thr Ser Val Thr Gln Met Thr Ala Ala Ile Thr Asp Ala Phe 100 105 110 Thr Asn Leu Asn Glu Gln Ser Gly Asp Ala Trp Ile Phe Trp Glu Lys 115 120 125 Lys Thr Ser Asn Lys Thr Thr Tyr Ser Tyr Ala Ile Leu Phe Ala Phe 130 135 140 Gln Asp Ser Ser Thr Gly Lys Leu Met Phe Ala Leu Pro Met Ser Leu 145 150 155 160 Glu Ile Glu Val Asp Val Ser Tyr Glu Arg Val Leu Phe Ile Thr Val 165 170 175 Glu Asp Lys Glu Thr Tyr Ser Val Lys Leu Asp Thr Met Lys Val Gly 180 185 190 Gln Leu Leu 195 <210> 53 <211> 195 <212> PRT <213> species <400> 53 Gln Tyr Gln Glu Ile Met Ala Leu Asp Asp Ile Asp Tyr Val Thr Gln 1 5 10 15 Ala Leu Gly Ile Gly Ala Leu Leu Asn Gly Ala Ile Asp Thr Ala Thr 20 25 30 Gly Arg Phe Asp Ala Gly Arg Ala Arg Gln Leu Ile Val Asp Phe Asn 35 40 45 Gln Ala Leu Pro Pro Ala Asp Ser Lys Tyr Lys Leu Ala Ile Met Asn 50 55 60 Ser Tyr Gln Ser Thr Val Ser Gln Glu Asn Ser Val Val Ser Gly Met 65 70 75 80 Ile Asp Lys Ile Leu Asp Val Leu Lys Thr Ala Ile Gly Val Ala Leu 85 90 95 Gly Gln Lys Ser Ile Asp Gln Ile Thr Ala Ala Val Thr Asp Ala Phe 100 105 110 Thr Asn Leu Lys Ser Gln Asp Gly Asp Ala Trp Ile Phe Trp Gln Lys 115 120 125 Arg Glu Ala His Lys Thr Val Tyr Ser Tyr Ala Ile Leu Phe Ala Val 130 135 140 Gln Asp Glu Ser Thr Gly Arg Val Met Leu Ala Phe Pro Met Ser Leu 145 150 155 160 Glu Ile Glu Val Asn Val Glu Phe Glu Lys Val Leu Trp Ile Thr Val 165 170 175 Lys Asp Ser His Asn Tyr Ser Val Lys Val Asp Ala Met Lys Ile Ala 180 185 190 Gln Leu Leu 195

Claims

1. A nucleic acid molecule consisting of a nucleotide sequence encoding a protein toxic to insect pests, wherein the nucleotide sequence (a) encodes a protein consisting of SEQ ID NO: 3; or (b) consists of any one of SEQ ID NOs: 24, 28, 30-44 and 48.

2. The nucleic acid molecule of claim 1, wherein the protein toxic to insect pests consists of SEQ ID NO:

3.

3. The nucleic acid molecule of claim 1, wherein the nucleotide sequence consists of any one of SEQ ID NOs: 24, 28, 30-44 and 48.

4. A chimeric gene comprising a heterologous promoter, and the nucleic acid molecule according to any one of claims 1 to 3 operably linked to the heterologous promoter. The chimeric gene of claim 4 , wherein the heterologous promoter is a plant-expressible promoter.

6. The chimeric gene of claim 5, wherein the plant expressible promoter is selected from the group consisting of: ubiquitin, tuberose yellow virus, maize TrpA, OsMADS 6, maize H3 histone, bacteriophage T3 gene 9 5'UTR, maize sucrose synthase 1, maize alcohol dehydrogenase 1, maize light harvesting complex, maize heat shock protein, maize mtl, pea small subunit RuBP carboxylase, rice actin, rice cyclophilin, Ti plasmid mannopine synthase, Ti plasmid nopaline synthase, petunia chalcone isomerase, legume glycine-rich protein 1, potato glycoprotein, lectin, CaMV 35S, and S-E9 small subunit RuBP carboxylase promoter.

7. The chimeric gene of claim 4, wherein the insect pest is a coleopteran insect pest.

8. The chimeric gene of claim 7, wherein the coleopteran insect pest is a Diabrotica insect pest.

9. The chimeric gene of claim 8, wherein the Diabrotica insect pest is the corn rootworm.

10. A protein toxic to insect pests, wherein the protein consists of the following amino acid sequence: (a) an amino acid sequence consisting of SEQ ID NO: 3; or (b) an amino acid sequence encoded by a nucleotide sequence consisting of any one of SEQ ID NO: 28, 30-44 and 48.

11. The protein of claim 10, wherein the insect pest is a coleopteran insect pest.

12. The protein of claim 11, wherein the coleopteran insect pest is a Diabrotica insect pest.

13. The protein of claim 12, wherein the Diabrotica insect pest is a corn rootworm. A recombinant vector comprising the chimeric gene according to claim 4 .

15. A host cell comprising the recombinant vector according to claim 14, wherein the host cell is a bacterial cell.

16. The host cell of claim 15, wherein the bacterial cell is in the genus Escherichia.

17. The host cell of claim 16, wherein the Escherichia cell is an Escherichia coli cell.

18. An insecticidal composition comprising the protein of claim 10 and an agriculturally acceptable carrier.

19. The composition of claim 18, wherein the agriculturally acceptable carrier is selected from the group consisting of dusts, pellets, granules, sprays, emulsions, colloids and solutions.

20. The composition of claim 18 or 19, wherein the composition further comprises a second pesticide.

21. The composition of claim 20, wherein the second pesticide is a biological agent or a chemical agent.

22. The composition of claim 21, wherein (a) the biological agent is or is derived from a Bacillus thuringiensis insecticidal protein, a Bacillus cereus insecticidal protein, a Xenorhabdus species insecticidal protein, a Photorhabdus species insecticidal protein, a Bacillus laterosporus insecticidal protein, a Bacillus sphaericus insecticidal protein, a Chromobacterium species insecticidal protein, a Yersinia entomophaga insecticidal protein, a Paenibacillus popiliae insecticidal protein, or a Clostridium species insecticidal protein; (b) the biological agent is or is derived from a dsRNA, a Cry protein, a Vip protein, a potato glycoprotein, a protease, a protease inhibitor, a urease, an α-amylase inhibitor, a pore-forming protein, a lectin, an engineered antibody or antibody fragment, or a chitinase; (c) the chemical agent is a carbamate, a pyrethroid, an organophosphate, a friprole, a neonicotinoid, an organochloride , nereis toxins or a combination thereof; or (d) the chemical agent comprises an active ingredient selected from the group consisting of: carbofuran, carbaryl, methomyl, cypermethrin, tefluthrin, permethrin, cyfluthrin, λ-cyfluthrin, cypermethrin, deltamethrin, chlorpyrifos, chloroxyfos, dimethoate, anthracene, malathion, methyl parathion, phorate, terbufos, tert-butylpyrimidophos, fipronil, acetamiprid, imidacloprid, thiacloprid, thiamethoxam, thiosulfuron, sulfamethoxam and a combination thereof.

23. A method for producing an insecticidal protein, the method comprising culturing the host cell of claim 15 under conditions whereby the host cell produces the insecticidal protein.

24. A method of producing a transgenic maize plant or maize plant part having enhanced insect resistance compared to a control maize plant or maize plant part, the method comprising: (a) introducing into a maize plant or maize plant part The chimeric gene of claim 4, wherein the protein toxic to an insect pest is expressed in the maize plant or maize plant part, thereby producing a maize plant or maize plant part having enhanced insect resistance.

25. The method of claim 24, wherein the introducing is accomplished by: (a) transforming the maize plant or maize plant part; or (b) crossing a first maize plant comprising the mosaic gene with a second, different maize plant.

26. The method of claim 25, wherein the chimeric gene encodes an insecticidal protein consisting of the amino acid sequence of SEQ ID NO:

3.

27. A method of controlling insect pests, the method comprising delivering an effective amount of the protein toxic to insect pests of claim 10 to the insect pests or their environment.

28. The method of claim 27, wherein the protein toxic to insect pests is delivered via a transgenic plant or by topical application of an insecticidal composition comprising the protein.

29. The method of claim 28, wherein the transgenic plant or the insecticidal composition comprises a second insecticide different from the protein that is toxic to an insect pest.

30. The method of claim 29, wherein the second insecticide is a protein, a dsRNA, or a chemical.

31. The method of claim 30, wherein (a) the protein is selected from the group consisting of Cry protein, Vip protein, potato glycoprotein, protease, protease inhibitor, urease, α-amylase inhibitor, pore-forming protein, lectin, engineered antibody or antibody fragment, or chitinase; (b) the chemical is a carbamate, pyrethroid, organophosphate, friprole, neonicotinoid, organochloride, nereistoxin or a combination thereof; or (c) the chemical comprises an active ingredient selected from the group consisting of carbofuran, carbaryl, methomyl, bifenthrin, tefluthrin, permethrin, cyfluthrin, λ-cyfluthrin, cypermethrin, deltamethrin, chlorpyrifos, chloroxyfos, dimethoate, anthiophos, malathion, methyl parathion, phorate, terbufos, tert-butylpyrimidophos, fipronil, acetamiprid, imidacloprid, thiacloprid, thiamethoxam, endosulfan, sulfamethoxam and a combination thereof.

32. The method of any one of claims 27-31, wherein the insect pest is a coleopteran insect pest.

33. The method of claim 32, wherein the coleopteran insect pest is a species of the genus Diabrotica.

34. The method of claim 33, wherein the Diabrotica species is Diabrotica zea.

35. A method for identifying an insecticidal protein encoded by the nucleotide sequence of SEQ ID NO: 24, the method comprising the steps of: (a) generating a primer pair that will amplify the polynucleotide of SEQ ID NO: 24 or its complement from a nucleic acid sample, (b) amplifying an orthologous gene from the nucleic acid sample, (c) identifying the polynucleotide sequence of the orthologous gene, (d) producing a protein encoded by the orthologous gene, and (e) determining that the protein of step (d) has insecticidal activity against insect pests.

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