Insecticidal polypeptides and uses thereof

Abstract

The present invention relates to insecticidal polypeptides and uses thereof. The present disclosure provides nucleic acids, and variants and fragments thereof, obtained from strains of Bacillus thuringiensis, which encode polypeptides that have pesticidal activity against insect pests, including Lepidoptera and Coleopteran. Particular embodiments of the present disclosure provide isolated nucleic acids encoding pesticidal proteins, pesticidal compositions, DNA constructs, and transformed microorganisms and plants comprising a nucleic acid of the embodiments. These compositions are useful in methods for controlling pests, in particular plant pests.

Description

[0001] This application is a divisional application of Chinese patent application 201880079781.4 “Insecticide polypeptides and uses thereof” filed on November 14, 2018.

[0002] Cross-references

[0003] This application claims the benefit of U.S. Provisional Application No. 62 / 607,372, filed December 19, 2017, which is incorporated herein by reference in its entirety.

[0004] References to sequence listings submitted electronically

[0005] The sequence listing submitted in computer readable form with this specification is named "7492WOPCT_sequencelisting.txt", created on October 24, 2018, and is 1,073 kilobytes in size. The sequence listing is part of this specification and is incorporated herein by reference in its entirety. Technical Field

[0006] The present disclosure relates to naturally occurring and recombinant nucleic acids obtained from novel Bacillus thuringiensis genes encoding pesticidal polypeptides characterized by having pesticidal activity against insect pests. The compositions and methods of the present disclosure utilize the disclosed nucleic acids and the pesticidal polypeptides encoded therein to control plant pests. Background Art

[0007] Insect pests are a major factor in crop losses worldwide. For example, armyworm feeding, black cutworm damage or European corn borer damage can be economically devastating for agricultural producers. Insect pest-related crop losses due to attacks of the European corn borer on field and sweet corn alone amount to approximately one billion dollars in losses and control costs per year.

[0008] Traditionally, the main method for affecting insect pest populations is to use broad-spectrum chemical insecticides. However, consumers and government regulators are also increasingly concerned about the environmental hazards associated with the production and use of chemical synthetic pesticides. Due to such concern, regulators have banned or restricted the use of some of the more dangerous pesticides. Therefore, people have a great interest in developing alternative pesticides.

[0009] The use of microbial agents (such as fungi, bacteria or other insect species) to control insect pests in an agricultural sense provides an environmentally friendly and commercially attractive alternative to chemical synthetic pesticides. In general, the use of biological pesticides has a lower risk of causing pollution and environmental damage, and biological pesticides provide stronger target specificity than the target specificity unique to traditional broad-spectrum chemical insecticides. In addition, biological pesticides often have lower production costs and can therefore increase the economic yield of various crops.

[0010] Certain species of microorganisms of the genus Bacillus are known to have insecticidal activity against a wide range of insect pests, including Lepidoptera, Diptera, Coleoptera, Hemiptera, etc. Bacillus thuringiensis (Bt) and Bacillus papilliae are the most successful biological control agents discovered to date. Insect pathogenicity has also been attributed to strains of B. larvae, B. lentimorbus, B. sphaericus (Harwook, ed., (1989) Bacillus (Plenum Press), 306) and B. cereus (WO96 / 10083). Although pesticidal proteins have also been isolated from the vegetative stages of Bacillus, the pesticidal activity appears to be concentrated in the parasporal crystal protein inclusion bodies. Several genes encoding these pesticidal proteins have been isolated and characterized (see, e.g., U.S. Pat. Nos. 5,366,892 and 5,840,868).

[0011] Microbial insecticides, particularly those obtained from Bacillus strains, play an important role in agriculture as an alternative to chemical control of harmful organisms. Recently, agricultural scientists have developed crop plants with enhanced insect resistance by genetically engineering crop plants to produce pest-killing proteins from Bacillus. For example, corn and cotton plants have been genetically engineered to produce pest-killing proteins isolated from Bt strains (see, for example, Aronson (2002) Cell Mol. Life Sci. [Cell and Molecular Life Sciences] 59 (3): 417-425; Schnepf et al., (1998) Microbiol Mol Biol Rev. [Microbiology and Molecular Biology Review] 62 (3): 775-806). These genetically engineered crops are now widely used in U.S. agriculture and provide farmers with environmentally friendly alternatives to traditional insect control methods. In addition, potatoes genetically engineered to contain Cry toxins that kill pests have been sold to U.S. farmers. Although they have proven to be very successful commercially, these genetically engineered insect-resistant crop plants only provide resistance to a narrow range of economically important insect pests.

[0012] Therefore, there is still a need for a wider range of new Bt toxins with insecticidal activity against insect pests, such as toxins active against a wider variety of insects. In addition, there is still a need for biopesticides active against a variety of insect pests and biopesticides with improved insecticidal activity. Summary of the invention

[0013] Compositions and methods for affecting insect pests are provided. More specifically, embodiments of the present disclosure relate to methods of affecting insects using nucleotide sequences encoding insecticidal peptides to produce transformed microorganisms and plants expressing the insecticidal polypeptides of these embodiments. In some embodiments, these nucleotide sequences encode polypeptides that are insecticides to at least one insect belonging to the order Lepidoptera.

[0014] In some aspects, nucleic acid molecules and fragments and variants thereof are provided that encode polypeptides having pesticidal activity against insect pests. These embodiments further provide fragments and variants of the disclosed nucleotide sequences encoding biologically active (eg, insecticidal) polypeptides.

[0015] In some aspects, isolated pesticidal (e.g., insecticidal) polypeptides encoded by naturally occurring or modified (e.g., mutagenized or manipulated) nucleic acids of the embodiments are provided. In specific aspects, the pesticidal proteins of the embodiments include fragments of full-length proteins and polypeptides produced by mutagenic nucleic acids designed to introduce specific amino acid sequences into the polypeptides of these embodiments. In specific aspects, these polypeptides have enhanced pesticidal activity relative to the activity of the naturally occurring polypeptides derived therefrom.

[0016] In some aspects, the nucleic acids of the embodiments can also be used to produce transgenic (e.g., transformed) monocots or dicots characterized by a genome comprising at least one stably incorporated nucleotide construct containing a coding sequence of the embodiments operably linked to a promoter driving expression of the encoded pesticidal polypeptide. Thus, transformed plant cells, plant tissues, plants, and seeds thereof are also provided.

[0017] In a specific aspect, transformed plants can be produced using nucleic acids that have been optimized to increase expression in host plants. For example, one of the pest-killing polypeptides of the embodiment can be reversely translated to produce the following nucleic acid, which comprises codons optimized for expression in a specific host such as crop plants like corn (corn, Zea mays) plants. The expression of the coding sequence of such transformed plants (such as dicots or monocots) will result in the production of pest-killing polypeptides and give plants increased insect resistance. In some aspects, transgenic plants expressing pest-killing polypeptides are provided, which can be applied to methods affecting various insect pests.

[0018] In some aspects, provided are pesticidal compositions or insecticidal compositions containing the insecticidal polypeptides of the embodiments, and which may optionally include other insecticidal peptides. The embodiments encompass applying these compositions to an environment of insect pests in order to affect the insect pests. DETAILED DESCRIPTION

[0019] The present disclosure relates to compositions and methods for affecting insect pests, particularly plant pests. More specifically, the isolated nucleic acids of the embodiments and fragments and variants thereof comprise nucleotide sequences encoding pesticidal polypeptides (e.g., proteins). The disclosed pesticidal proteins are biologically active (e.g., pesticidal) against insect pests (e.g., but not limited to, insect pests of the order Lepidoptera).

[0020] The compositions of the embodiments include isolated nucleic acids encoding pesticidal polypeptides and fragments and variants thereof, expression cassettes containing the nucleotide sequences of the embodiments, isolated pesticidal proteins, and pesticidal compositions. Some embodiments provide modified pesticidal polypeptides with improved insecticidal activity against Lepidoptera relative to the pesticidal activity of the corresponding wild-type protein. The embodiments further provide plants and microorganisms transformed with these novel nucleic acids, and methods involving the use of such nucleic acids, pesticidal compositions, transformed organisms, and their products to affect insect pests.

[0021] The nucleic acids and nucleotide sequences of the embodiments can be used to transform any organism to produce the encoded pesticidal protein. Methods involving the use of such transformed organisms to affect or control plant pests are provided. The nucleic acids and nucleotide sequences of the embodiments can also be used to transform organelles such as chloroplasts (McBride et al., (1995) Biotechnology 13:362-365; and Kota et al., (1999) Proc. Natl. Acad. Sci. USA 96:1840-1845).

[0022] Embodiments further relate to the identification of fragments and variants of naturally occurring coding sequences encoding biologically active pesticidal proteins. The nucleotide sequences of the embodiments can be directly applied to methods affecting harmful organisms (particularly insect pests, such as lepidopteran pests). Therefore, the embodiments provide novel methods that do not rely on the use of traditional chemically synthesized insecticides to affect insect pests. Embodiments relate to the discovery of naturally occurring biodegradable pesticidal agents and genes encoding them.

[0023] The embodiments further provide fragments and variants of naturally occurring coding sequences that also encode biologically active (e.g., pesticidal) polypeptides. The nucleic acids of the embodiments encompass nucleic acids or nucleotide sequences that have been optimized for expression by cells of a specific organism, such as nucleic acid sequences that are back-translated (i.e., reverse translated) using plant-preferred codons based on the amino acid sequence of a polypeptide with enhanced pesticidal activity. The embodiments further provide mutations that confer improved or altered properties on the polypeptides of the embodiments. See, e.g., U.S. Patent 7,462,760.

[0024] In the following description, a number of terms are used extensively. The following definitions are provided to aid in understanding these embodiments.

[0025] Units, prefixes and symbols may be represented in their SI accepted form. Unless otherwise indicated, nucleic acids are written from left to right in a 5' to 3' direction; amino acid sequences are written from left to right in an amino to carboxyl direction. Numerical ranges include the values ​​defining the ranges. Amino acids may be represented herein by their commonly known three letter symbols or by the single letter symbols recommended by the IUPAC-IUB Biochemical Terminology Commission. Similarly, nucleic acids may be represented by their commonly accepted single letter codes. The above defined terms are more fully defined with reference to the specification as a whole.

[0026] As used herein, "nucleic acid" includes reference to deoxyribonucleotide or ribonucleotide polymers in either single- or double-stranded form, and, unless otherwise limited, encompasses known analogs (e.g., peptide nucleic acids) that have the essential properties of natural nucleotides in that they hybridize to single-stranded nucleic acids in a manner similar to naturally occurring nucleotides.

[0027] As used herein, the terms "encoding" or "encoded" when used in the context of a designated nucleic acid means that the nucleic acid contains the necessary information for translating the nucleotide sequence directly into a designated protein. The information used to encode the protein is specified by codon usage. A protein-encoding nucleic acid may contain non-translated sequences (e.g., introns) within the translated regions of the nucleic acid or may lack such intervening non-translated sequences (e.g., in cDNA).

[0028] As used herein, reference to a particular polynucleotide or its encoded protein "full length sequence" means the entire nucleic acid sequence or the entire amino acid sequence having a natural (non-synthetic) endogenous sequence. A full-length polynucleotide encodes the full-length, catalytically active form of a particular protein.

[0029] As used herein, the term "antisense" used in the context of nucleotide sequence orientation refers to a double-stranded polynucleotide sequence operably linked to a promoter in the direction of antisense strand transcription. The antisense strand is fully complementary to the endogenous transcription product, so that the translation of the endogenous transcription product is often inhibited. Therefore, in the case of using the term "antisense" in the context of a specific nucleotide sequence, the term refers to the complementary strand of the reference transcription product.

[0030] The terms "polypeptide", "peptide" and "protein" are used interchangeably herein to refer to polymers of amino acid residues. These terms apply to amino acid polymers in which one or more amino acid residues are artificial chemical analogs of the corresponding naturally occurring amino acids, as well as to naturally occurring amino acid polymers.

[0031] The terms "residue" or "amino acid residue" or "amino acid" are used interchangeably herein to refer to an amino acid that is incorporated into a protein, polypeptide, or peptide (collectively referred to as a "protein"). The amino acid may be a naturally occurring amino acid and, unless otherwise limited, may encompass known analogs of natural amino acids that can function in a similar manner to the naturally occurring amino acids.

[0032] The polypeptides of the embodiments can be produced from the nucleic acids disclosed herein or by using standard molecular biology techniques. For example, the proteins of the embodiments can be produced by expressing the recombinant nucleic acids of the embodiments in appropriate host cells or alternatively by a combination of in vitro procedures.

[0033] As used herein, the terms "isolated" and "purified" are used interchangeably and refer to a nucleic acid or polypeptide, or biologically active portion thereof, that is substantially or essentially free of components that normally accompany or interact with the nucleic acid or polypeptide as found in its naturally occurring environment. Thus, an isolated or purified nucleic acid or polypeptide is substantially free of other cellular material or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized.

[0034] An "isolated" nucleic acid is generally free of sequences that naturally flank the nucleic acid (i.e., sequences located at the 5' and 3' ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived (such as, for example, protein coding sequences). For example, in various embodiments, an isolated nucleic acid can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb, or 0.1 kb of nucleotide sequences that naturally flank the nucleic acid in the genomic DNA of the cell from which the nucleic acid is derived.

[0035] As used herein, the term "isolated" or "purified", as used in reference to the polypeptides of the embodiments, means that the isolated protein is substantially free of cellular material and includes protein preparations having less than about 30%, 20%, 10% or 5% (by dry weight) of contaminated protein. When the protein of the embodiments or its biologically active portion is recombinantly produced, the culture medium contains less than about 30%, 20%, 10% or 5% (by dry weight) of chemical precursors or non-target protein chemicals.

[0036] As used herein, "recombinant" nucleic acid molecules (or DNA) refer to nucleic acid sequences (or DNA) in recombinant bacteria or plant host cells. In some embodiments, an "isolated" or "recombinant" nucleic acid does not contain sequences (preferably protein-encoding sequences) that naturally flank the nucleic acid in the genomic DNA of the organism from which the nucleic acid is derived (i.e., sequences located at the 5' and 3' ends of the nucleic acid). For the purposes of this disclosure, "isolated" or "recombinant" when used to refer to nucleic acid molecules excludes isolated chromosomes.

[0037] As used herein, "non-genomic nucleic acid sequence" or "non-genomic nucleic acid molecule" refers to a nucleic acid molecule having one or more changes in the nucleic acid sequence compared to a native or genomic nucleic acid sequence. In some embodiments, changes in a native or genomic nucleic acid molecule include, but are not limited to: changes in the nucleic acid sequence due to the degeneracy of the genetic code; codon optimization of a nucleic acid sequence for expression in a plant; changes in the nucleic acid sequence that introduce at least one amino acid substitution, insertion, deletion, and / or addition compared to a native or genomic sequence; removal of one or more introns associated with the genomic nucleic acid sequence; insertion of one or more heterologous introns; deletion of one or more upstream or downstream regulatory regions associated with the genomic nucleic acid sequence; insertion of one or more heterologous upstream or downstream regulatory regions; deletion of 5' and / or 3' untranslated regions associated with the genomic nucleic acid sequence; insertion of heterologous 5' and / or 3' untranslated regions; and modification of polyadenylation sites. In some embodiments, the non-genomic nucleic acid molecule is a cDNA. In some embodiments, the non-genomic nucleic acid molecule is a synthetic nucleic acid sequence.

[0038] Throughout this specification, the word "comprising" or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or groups of multiple elements, integers or steps, but not the exclusion of any other element, integer or step, or groups of multiple elements, integers or steps.

[0039] As used herein, the term "affecting insect pests" refers to changes that affect insect feeding, growth and / or behavior at any developmental stage, including but not limited to: killing the insect; retarding growth; preventing reproductive capacity; repelling feeding activity; and the like.

[0040] As used herein, the terms "pesticide activity" and "insecticide activity" are used synonymously to refer to the activity of an organism or substance (e.g., such as a protein) that can be measured by, but not limited to, pest mortality, pest weight loss, pest resistance, and other behavioral and physical changes in pests after ingestion and exposure for an appropriate length of time. Thus, an organism or substance with pesticide activity adversely affects at least one measurable parameter of pest fitness. For example, a "pesticidal protein" is a protein that exhibits pesticide activity by itself or in combination with other proteins.

[0041] As used herein, the term "insecticide effective amount" means an amount of a substance or organism that has pesticidal activity when present in a pest environment. For each substance or organism, the pesticidal effective amount is determined empirically for each pest affected in a particular environment. Similarly, when the pest is an insect pest, "insecticide effective amount" can be used to mean "pesticidal effective amount".

[0042] As used herein, the term "recombinant engineering" or "engineering" means the use of recombinant DNA technology to introduce (e.g., engineer) changes in protein structure based on an understanding of the protein's mechanism of action and consideration of the amino acids that are introduced, deleted, or substituted.

[0043] As used herein, the term "mutant nucleotide sequence" or "mutation" or "mutagenized nucleotide sequence" means a nucleotide sequence that has been mutated or altered to contain one or more nucleotide residues (e.g., base pairs) that are not present in the corresponding wild-type sequence. Such mutagenesis or alteration consists of one or more additions, deletions, substitutions or replacements of nucleic acid residues. When mutation is performed by adding, removing or replacing amino acids at a proteolytic site, such additions, removals or replacements may be within or adjacent to the proteolytic site motif as long as the purpose of the mutation is achieved (i.e., as long as proteolysis at the site is altered).

[0044] The mutant nucleotide sequence may encode a mutant insecticidal toxin showing improved or reduced insecticidal activity, or encode an amino acid sequence that confers improved or reduced insecticidal activity to a polypeptide containing it. As used herein, the term "mutant" or "mutation" in the context of a protein or polypeptide or amino acid sequence refers to a sequence that has been mutated or altered to contain one or more amino acid residues that are not present in the corresponding wild-type sequence. Such a mutagenesis or change consists of one or more additions, deletions, or substitutions or replacements of amino acid residues. The mutant polypeptide shows improved or reduced insecticidal activity, or represents an amino acid sequence that confers improved insecticidal activity to a polypeptide containing it. Therefore, the term "mutant" or "mutation" refers to one or both of the mutant nucleotide sequence and the encoded amino acid. The mutant can be used alone or in any compatible combination with other mutants of the embodiment or with other mutants. "Mutant polypeptides" may instead show a reduction in insecticidal activity. In the case of adding more than one mutation to a particular nucleic acid or protein, the mutations may be added simultaneously or sequentially; if sequentially, the mutations can be added in any suitable order.

[0045] As used herein, the term "improved insecticidal activity" or "improved pesticidal activity" refers to an insecticidal polypeptide of the embodiments having enhanced insecticidal activity relative to the activity of its corresponding wild-type protein, and / or an insecticidal polypeptide that is effective against a wider range of insects, and / or an insecticidal polypeptide that is specific to insects that are not sensitive to the toxicity of the wild-type protein. The discovery of improved or enhanced pesticidal activity requires demonstration of an increase in pesticidal activity of at least 10% against an insect target, or an increase in pesticidal activity of at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 100%, 150%, 200% or 300% or more relative to the pesticidal activity of the wild-type insecticidal polypeptide determined against the same insect.

[0046] For example, improved pesticidal or insecticidal activity is provided, wherein a greater or narrower range of insects is affected by the polypeptide relative to the range of insects affected by the wild-type Bt toxin. Where multifunctionality is desired, a wider range of effects may be desired, whereas where, for example, beneficial insects may otherwise be affected by the use or presence of the toxin, a narrower range of effects may be desired. Although the embodiments are not bound by any particular mechanism of action, improved pesticidal activity may also be provided by alterations in one or more characteristics of the polypeptide; for example, the stability or longevity of the polypeptide in the insect gut may be increased relative to the stability or longevity of the corresponding wild-type protein.

[0047] As used herein, the term "toxin" refers to a polypeptide that exhibits pesticidal activity or insecticidal activity or improved pesticidal activity or improved insecticidal activity. "Bt" or "Bacillus thuringiensis" toxins are intended to include the broader class of Cry toxins found in various Bt strains, including, for example, Cry1, Cry2 or Cry3 toxins.

[0048] The term "proteolytic site" or "cleavage site" refers to an amino acid sequence that confers sensitivity to a class of proteases or a specific protease, such that a polypeptide containing the amino acid sequence is digested by the class of proteases or a specific protease. It is considered that a proteolytic site is "sensitive" to one or more proteases that recognize the site. It should be understood in the art that the efficiency of digestion will vary, and a reduction in digestion efficiency can result in an increase in the stability or lifespan of the polypeptide in the insect gut. Therefore, a proteolytic site can confer sensitivity to more than one protease or a class of proteases, but the efficiency of digestion of various proteases at the site may be different. Proteolytic sites include, for example, trypsin sites, chymotrypsin sites, and elastase sites.

[0049] Studies have shown that the insect gut proteases of Lepidoptera include trypsin, chymotrypsin, and elastase. See, for example, Lenz et al., (1991) Arch. Insect Biochem. Physiol. 16:201-212; and Hedegus et al., (2003) Arch. Insect Biochem. Physiol. 53:30-47. For example, about 18 different trypsin enzymes have been found in the midgut of Helicoverpa armigera larvae (see Gatehouse et al., (1997) Insect Biochem. Mol. Biol. 27:929-944). The preferred proteolytic substrate sites of these proteases have been studied. See, e.g., Peterson et al. (1995) Insect Biochem. Mol. Biol. 25: 765-774.

[0050] Efforts have been made to understand the mechanism of action of Bt toxins and to engineer toxins with improved properties. It has been shown that insect gut proteases can affect the effects of Bt Cry proteins on insects. Some proteases activate Cry proteins by processing them from a "protoxin" form into a toxic form or "toxin". See, Oppert (1999) Arch. Insect Biochem. Phys. [Archives of Insect Biochemistry and Physiology] 42: 1-12; and Carroll et al., (1997) J. Invertebrate Pathology [Journal of Invertebrate Pathology] 70: 41-49. This activation of the toxin can include the removal of the N-terminal peptide and the C-terminal peptide from the protein, and can also include internal cleavage of the protein. Other proteases can degrade Cry proteins. See Oppert, supra.

[0051] Comparison of the amino acid sequences of Cry toxins of different specificities reveals five highly conserved sequence blocks. Structurally, the toxin contains three different domains, from N-terminal to C-terminal they are: a cluster of seven α helices involved in pore formation (called "domain 1"), three antiparallel β sheets involved in cell binding (called "domain 2") and a β sandwich (called "domain 3"). The location and properties of these domains are known to those skilled in the art. See, for example, Li et al., (1991) Nature [Nature], 305: 815-821 and Morse et al., (2001) Structure [Structure], 9: 409-417. When referring to a specific domain such as domain 1, it should be understood that the exact endpoints of the domain are not critical for a specific sequence, as long as the sequence or a portion thereof includes a sequence that provides at least some functions attributed to the specific domain. Therefore, for example, when referring to "domain 1", it means that a specific sequence includes a cluster of seven α helices, but the exact endpoints of the sequence used or quoted about the cluster are not critical. Those skilled in the art are familiar with the determination of such endpoints and the assessment of such functions.

[0052] In order to better characterize and improve Bt toxins, strains of the bacterium Bt were studied. Crystal preparations prepared from cultures of Bt strains were found to have pesticidal activity against a large number of lepidopteran pests (see, e.g., Example 1). Efforts were made to identify nucleotide sequences encoding crystalline proteins from selected strains, and to isolate the wild-type (i.e., naturally occurring) nucleic acids of the embodiments from these bacterial strains, clone into expression vectors, and transform into Escherichia coli. Depending on the characteristics of a given preparation, it is recognized that demonstration of pesticidal activity sometimes requires trypsin pretreatment to activate the pesticidal protein. Therefore, it should be understood that some pesticidal proteins require protease digestion (e.g., by trypsin, chymotrypsin, and the like) for activation, while other proteins are biologically active (e.g., pesticidal) without activation.

[0053] Such molecules can be altered by methods such as those described in U.S. Patent No. 7,462,760. In addition, nucleic acid sequences can be engineered to encode polypeptides containing additional mutations that confer improved or altered pesticidal activity relative to the pesticidal activity of naturally occurring polypeptides. The nucleotide sequence of such engineered nucleic acids contains mutations not found in the wild-type sequence.

[0054] The mutant polypeptides of the embodiments are generally prepared by a method comprising the following steps: obtaining a nucleic acid sequence encoding a Cry family polypeptide; analyzing the structure of the polypeptide to identify specific "target" sites for mutagenizing potential gene sequences based on considerations of the target domain function proposed in the toxin mode of action; introducing one or more mutations into the nucleic acid sequence to produce the desired changes in one or more amino acid residues of the encoded polypeptide sequence; and determining the pesticidal activity of the produced polypeptide.

[0055] Many Bt insecticidal toxins are related to various degrees by the similarity of their amino acid sequences and tertiary structures, and methods for obtaining the crystal structures of Bt toxins are well known. Exemplary high-resolution crystal structure analyses of both Cry3A and Cry3B polypeptides are available in the literature. The resolved structure of the Cry3A gene (Li et al., (1991) Nature [Nature] 353: 815-821) provides insights into the relationship between the structure and function of the toxin. A comprehensive consideration of the disclosed structural analysis of Bt toxins and reported functions associated with specific structures, motifs, etc. indicates that specific regions of the toxin are associated with specific functions and discrete steps of the protein's mode of action. For example, many toxins isolated from Bt are generally described as including three domains: a seven-helix bundle involving pore formation, a three-lamellar domain associated with receptor binding, and a β-sandwich motif (Li et al., (1991) Nature [Nature] 305: 815-821).

[0056] As reported in U.S. Pat. Nos. 7,105,332 and 7,462,760, the toxicity of Cry proteins can be improved by targeting the region located between alpha helices 3 and 4 of domain 1 of the toxin. This theory is premised on a body of knowledge about insecticidal toxins, including: 1) alpha helices 4 and 5 of domain 1 of Cry3A toxin are reported to be inserted into the lipid bilayer of cells lining the midgut of susceptible insects (Gazit et al. (1998) Proc. Natl. Acad. Sci. USA 95: 12289-12294); 2) the inventors' knowledge of the locations of trypsin and chymotrypsin cleavage sites within the amino acid sequence of the wild-type protein; 3) the observation that the wild-type protein is more active against certain insects after in vitro activation by trypsin or chymotrypsin treatment; and 4) digestion of the toxin from the 3' end is reported to result in reduced toxicity to insects.

[0057] A series of mutations can be generated and placed in various background sequences to produce novel polypeptides with enhanced or altered pesticidal activity. See, for example, U.S. Patent 7,462,760. These mutants include, but are not limited to: adding at least one more protease sensitive site (e.g., trypsin cleavage site) in the region between helices 3 and 4 of domain 1; replacing the original protease sensitive site in the wild-type sequence with a different protease sensitive site; adding multiple protease sensitive sites at specific positions; adding amino acid residues near one or more protease sensitive sites to change the folding of the polypeptide and thereby enhance the digestion of the polypeptide at the one or more protease sensitive sites; and adding mutations to protect the polypeptide from the effects of degradative digestion that reduces toxicity (e.g., making a series of mutations in which the wild-type amino acid is replaced by valine to protect the polypeptide from digestion). Mutations can be used alone or in any combination to provide the polypeptides of the embodiments.

[0058] Homologous sequences were identified by similarity search on the non-redundant database (nr) of the National Center for Bioinformatics Information (NCBI) using BLAST and PSI-BLAST. Homologous proteins are mainly composed of Cry toxins of Bacillus thuringiensis.

[0059] Mutations that are additional or alternative protease sensitive sites may be sensitive to several classes of proteases, such as serine proteases (including trypsin and chymotrypsin), or enzymes such as elastase. Thus, mutations that are additional or alternative protease sensitive sites can be designed so that the site is easily recognized and / or cleaved by a class of proteases (e.g., mammalian proteases or insect proteases). Protease sensitive sites can also be designed to be cleaved by a specific class of enzymes or a specific enzyme known to be produced in an organism, such as, for example, chymotrypsin produced by corn earworm (Heliothis zea) (Lenz et al., (1991) Arch. Insect Biochem. Physiol. [Insect Biochemistry and Physiology Archives] 16: 201-212). Mutations can also confer resistance to proteolytic digestion, for example, resistance to digestion by chymotrypsin at the C-terminus of the peptide.

[0060] The presence of additional and / or alternative protease sensitive sites in the amino acid sequence of the encoded polypeptide can improve the pesticidal activity and / or specificity of the polypeptide encoded by the nucleic acid of the embodiment. Therefore, the nucleotide sequence of the embodiment can be recombinantly engineered or manipulated to produce a polypeptide with improved or altered insecticidal activity and / or specificity compared to an unmodified wild-type toxin. In addition, the mutations disclosed herein can be placed in other nucleotide sequences or used in combination with other nucleotide sequences to provide improved properties. For example, a protease sensitive site that is susceptible to cleavage by insect chymosin (e.g., chymosin found in bertha armyworm or corn ear moth) (Hegedus et al. (2003) Arch. Insect Biochem. Physiol. [Insect Biochemistry and Physiology Archives] 53: 30-47; and Lenz et al., (1991) Arch. Insect Biochem. Physiol. [Insect Biochemistry and Physiology Archives] 16: 201-212) can be placed in a Cry background sequence to provide improved toxicity to the sequence. In this manner, the embodiments provide toxicity polypeptides with improved properties.

[0061] For example, the mutagenic Cry nucleotide sequence can include other mutants, which include other codons that introduce a second trypsin-sensitive amino acid sequence (except the naturally occurring trypsin site) into the encoded polypeptide. The alternative addition mutants of the embodiment include other codons that are designed to introduce at least one other different protease-sensitive site into the polypeptide, such as the chymotrypsin-sensitive site that is directly located at the 5' or 3' of the naturally occurring trypsin site. Alternatively, substitution mutants can be produced, in which at least one codon of the nucleic acid encoding the naturally occurring protease-sensitive site is destroyed, and alternative codons are introduced into the nucleic acid sequence to provide different (e.g., replacement) protease-sensitive sites. Replacement mutants can also be added to the Cry sequence, in which the naturally occurring trypsin cleavage site present in the encoded polypeptide is destroyed, and a chymotrypsin or elastase cleavage site is introduced in its position.

[0062] It is recognized that any nucleotide sequence encoding an amino acid sequence that is a proteolytic site or a putative proteolytic site can be used (e.g., a sequence such as RR or LKM), and the exact identity of the codons used to introduce any of these cleavage sites into a variant polypeptide can vary depending on the use (i.e., expression in a specific plant species). It should also be recognized that any disclosed mutation can be introduced into any polynucleotide sequence of the embodiments, which comprises codons for amino acid residues that provide a modified targeted native trypsin cleavage site. Therefore, variants of full-length toxins or fragments thereof can be modified to include additional or alternative cleavage sites, and these embodiments are intended to be encompassed by the scope of the embodiments disclosed herein.

[0063] Those skilled in the art will appreciate that any useful mutation can be added to the sequence of the embodiment as long as the encoded polypeptide retains pesticidal activity. Therefore, the sequence can also be mutated so that the encoded polypeptide is resistant to proteolytic digestion of chymosin. More than one recognition site can be added to a specific position in any combination, and multiple recognition sites can be added to or removed from the toxin. Therefore, additional mutations can include three, four or more recognition sites. It should be recognized that multiple mutations can be engineered in any suitable polynucleotide sequence; therefore, the full-length sequence or its fragments can be modified to include additional or alternative cleavage sites and to make it resistant to proteolytic digestion. In this way, the embodiment provides Cry toxins containing mutations that improve pesticidal activity and improved compositions and methods for using other Bt toxins to affect pests.

[0064] Mutation can protect polypeptide from protease degradation, for example, by removing putative proteolytic sites from different regions, such as putative serine protease sites and elastase recognition sites. Some or all of such putative sites can be removed or changed to reduce proteolysis at the original site position. Proteolytic changes can be assessed by comparing mutant polypeptides with wild-type toxins or by comparing mutant toxins with different amino acid sequences. Putative proteolytic sites and proteolytic sites include but are not limited to the following sequences: RR, trypsin cleavage site; LKM, chymotrypsin site; and trypsin site. As long as the pesticidal activity of the polypeptide increases, these sites can be changed by adding or deleting any number and type of amino acid residues. , 240, 250, 260, 270, or 280 or more amino acid changes or additions relative to the native or background sequence.

[0065] The compositions of the embodiments include nucleic acids encoding pesticidal polypeptides and fragments and variants thereof. In particular, the embodiments provide an isolated nucleic acid molecule comprising a nucleotide sequence encoding the amino acid sequence of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66 NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 92, SEQ ID NO:94, SEQ ID NO:96, SEQ ID NO:98, SEQ ID NO:100, SEQ ID NO:102, SEQ ID NO:104, SEQ ID NO:106, SEQ ID NO:108, SEQ ID NO:110, SEQ ID NO:112, SEQ ID NO:114, SEQ ID NO:116, SEQ ID NO:118, SEQ ID NO:120, SEQ ID NO:122, SEQ ID NO:124, SEQ ID NO:126, SEQ ID NO:128, SEQ ID NO:130, SEQ ID NO:132, SEQ ID NO:134, SEQ ID NO:136, SEQ ID NO:138, SEQ ID NO:140, SEQ ID NO:142, SEQID NO: 144, SEQ ID NO: 146, SEQ ID NO: 148, SEQ ID NO: 150, SEQ ID NO: 152, SEQ ID NO: 154, SEQ ID NO: 156, SEQ ID NO: 158, SEQ ID NO: 160, SEQ ID NO: 162, SEQ ID NO: 164, SEQ ID NO: 166, SEQ ID NO: 168, SEQ ID NO: 170, SEQ ID NO: 172, SEQ ID NO: 174, SEQ ID NO: 176, SEQ ID NO: 178, SEQ ID NO: 180, SEQ ID NO: 182, SEQ ID NO: 184, SEQ ID NO: 186, SEQ ID NO: 188, SEQ ID NO: 190, SEQ ID NO: 192, SEQ ID NO: 194, SEQ ID NO: 196, SEQ ID NO: 198, SEQ ID NO: 200, SEQ ID NO: 202, SEQ ID NO: 204, SEQ ID NO: 206, SEQ ID NO: 208, SEQ ID NO: 210, SEQ ID NO: 212, SEQ ID NO: 214, SEQ ID NO: 216, SEQ ID NO: 218, SEQ ID NO: 220, SEQ ID NO: 222, SEQ ID NO: 224, SEQ ID NO: 226, SEQ ID NO: 228, SEQ ID NO: 230, SEQ ID NO: 232, SEQ ID NO: 234, SEQ ID NO: 236, SEQ ID NO: 238, SEQ ID NO: 240, SEQ ID NO: 242, SEQ ID NO: 244, SEQ ID NO: 246, SEQ ID NO: 248, SEQ ID NO: 250, SEQ ID NO: 252, SEQ ID NO: 254, SEQ ID NO: 256, SEQ ID NO: 258, SEQ ID NO: 260, SEQ ID NO: 262, SEQ ID NO: 264, SEQ ID NO: 266, SEQ ID NO: 268, SEQ ID NO: 270, SEQ ID NO: 272, SEQ ID NO: 274, SEQ ID NO: 276, SEQ ID NO: 278, SEQ ID NO: 280, SEQ ID NO: 282, and SEQ IDNO: 284, and the nucleotide sequences encoding said amino acid sequences, such as the nucleotide sequences shown below: SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID NO: 91, SEQ ID NO: 93, SEQ ID NO: 95, SEQ ID NO: 97, SEQ ID NO: 99, SEQ ID NO: 101, SEQ ID NO: 103, SEQ ID NO: 105, SEQ ID NO: 107, SEQ ID NO: 109, SEQ ID NO: 111, SEQ ID NO: 113, SEQ ID NO: 115, SEQ ID NO: 117, SEQ ID NO: 119, SEQ ID NO: 121, SEQ ID NO: 123, SEQ ID NO: 125, SEQ ID NO: 127, SEQ ID NO: 129, SEQ ID NO: 131, SEQ ID NO: 133, SEQ ID NO: 135, SEQ ID NO: 137, SEQ ID NO: 139, SEQ ID NO: 141, SEQ ID NO: 143, SEQ ID NO: 145, SEQ IDNO: 147, SEQ ID NO: 149, SEQ ID NO: 151, SEQ ID NO: 153, SEQ ID NO: 155, SEQ ID NO: 157, SEQ ID NO: 159, SEQ ID NO: 161, SEQ ID NO: 163, SEQ ID NO: 165, SEQ ID NO: 167, SEQ ID NO: 169, SEQ ID NO: 171, SEQ ID NO: 173, SEQ ID NO: 175, SEQ ID NO: 177, SEQ ID NO: 179, SEQ ID NO: 181, SEQ ID NO: 183, SEQ ID NO: 185, SEQ ID NO: 187, SEQ ID NO: 189, SEQ ID NO: 191, SEQ ID NO: 193, SEQ ID NO: 195, SEQ ID NO: 197, SEQ ID NO: 199, SEQ ID NO: 201, SEQ ID NO: 203, SEQ ID NO: 205, SEQ ID NO: 207, SEQ ID NO: 209, SEQ ID NO: 211, SEQ ID NO: 213, SEQ ID NO: 215, SEQ ID NO: 217, SEQ ID NO: 219, SEQ ID NO: 221, SEQ ID NO: 223, SEQ ID NO: 225, SEQ ID NO: 227, SEQ ID NO: 229, SEQ ID NO: 231, SEQ ID NO: 233, SEQ ID NO: 235, SEQ ID NO: 237, SEQ ID NO: 239, SEQ ID NO: 241, SEQ ID NO: 243, SEQ ID NO: 245, SEQ ID NO: 247, SEQ ID NO: 249, SEQ ID NO: 251, SEQ ID NO: 253, SEQ ID NO: 255, SEQ ID NO: 257, SEQ ID NO: 259, SEQ ID NO: 261, SEQ ID NO: 263, SEQ ID NO: 265, SEQ ID NO: 267, and SEQ ID NO: 269, and their fragments and variants.

[0066] In particular, the embodiments provide isolated nucleic acid molecules encoding the amino acid sequence shown in SEQ ID NO:64, SEQ ID NO:152, SEQ ID NO:170 or SEQ ID NO:184, or nucleotide sequences encoding the amino acid sequence (e.g., the nucleotide sequence shown in SEQ ID NO:63, SEQ ID NO:151, SEQ ID NO:169 or SEQ ID NO:183) and fragments and variants thereof.

[0067] Also of interest is the optimized nucleotide sequence of the pest-killing protein of the coding embodiment. As used herein, the phrase "optimized nucleotide sequence" refers to a nucleic acid optimized for expression in a specific organism such as a plant. Methods known in the art can be used to prepare optimized nucleotide sequences for any purpose organism. See, for example, U.S. Patent No. 7,462,760, which describes the optimized nucleotide sequence of the disclosed pest-killing protein of the coding. In this example, the nucleotide sequence is prepared by reverse translating the amino acid sequence of the protein and changing the nucleotide sequence to include maize preference codons while still encoding the same amino acid sequence. Murray et al., (1989) Nucleic Acids Res. [Nucleic Acids Research] 17: 477-498 describes the program in more detail. The best nucleotide sequence can be applied to increase the expression of the pest-killing protein in plants, such as monocots of Gramineae or Poaceae families, such as maize or corn plants.

[0068] In a specific embodiment, the pesticidal proteins of the embodiments provide full-length insecticidal polypeptides, fragments of full-length insecticidal polypeptides and variant polypeptides, which are produced from mutagenized nucleic acids designed to introduce specific amino acid sequences into the polypeptides of the embodiments. In a specific embodiment, the amino acid sequence introduced into the polypeptide comprises a sequence that provides a cleavage site for an enzyme such as a protease.

[0069] It is known in the art that the pesticidal activity of Bt toxins is usually activated by various proteases cleaving peptides in the insect gut. Because peptides may not always be completely and effectively cleaved in the insect gut, fragments of full-length toxins may have enhanced pesticidal activity compared to the full-length toxin itself. Therefore, some polypeptides of the embodiments include fragments of full-length insecticidal polypeptides, and some polypeptide fragments, variants and mutations will have enhanced pesticidal activity relative to the activity of the naturally occurring insecticidal polypeptides from which they are derived, especially if the naturally occurring insecticidal polypeptides are not activated in vitro with proteases before activity screening. Therefore, the present application covers truncated versions or fragments of the sequences.

[0070] As long as the polypeptide retains pesticidal activity, mutations can be placed in any background sequence (including such truncated polypeptides). Those skilled in the art can easily compare two or more proteins about pesticidal activity using tests known in the art or described elsewhere herein. It should be understood that the polypeptide of the embodiment can be produced by expression of nucleic acids disclosed herein or by using standard molecular biology techniques.

[0071] It has been recognized that pesticidal proteins may be oligomeric and will differ in molecular weight, number of residues, component peptides, activity against specific pests and other characteristics. However, by the methods described herein, proteins active against various pests can be isolated and characterized. The pesticidal proteins of the embodiments can be used in combination with other Bt toxins or other insecticidal proteins to increase the insect target range. In addition, the pesticidal proteins of the embodiments are used in combination with other Bt toxins or other insecticidal principles with different properties to prevent and / or manage insect resistance. Other insecticides include protease inhibitors (both types of serine and cysteine), alpha-amylase and peroxidase.

[0072] The fragment and variant of nucleotide and amino acid sequence and the polypeptide encoded thereby are also contained in the embodiment.As used herein, the term " fragment " refers to a part of the nucleotide sequence of polynucleotide or a part of the amino acid sequence of the polypeptide of embodiment.The fragment of nucleotide sequence can encode the protein fragment that retains the biological activity of natural or corresponding full-length protein, therefore possesses the activity of killing harmful organisms.Therefore, it is generally acknowledged that some polynucleotide and amino acid sequences of embodiment can be correctly referred to as fragment and mutant.

[0073] It should be understood that the term "fragment", as used to refer to the nucleic acid sequences of the embodiments, also encompasses sequences used as hybridization probes. Such nucleotide sequences do not usually encode fragment proteins that retain biological activity. Therefore, the fragment range of the nucleotide sequence can be from at least about 20 nucleotides, about 50 nucleotides, about 100 nucleotides, up to the full-length nucleotide sequence of the protein of the encoding embodiment.

[0074] Fragments of the nucleotide sequences of the embodiments encoding the biologically active portion of the pesticidal protein of the embodiments will encode at least 15, 25, 30, 50, 100, 200, 250 or 300 consecutive amino acids, or up to the total number of amino acids present in the pesticidal polypeptide of the embodiments. Fragments of the nucleotide sequences of the embodiments that can be used as hybridization probes or PCR primers generally do not need to encode the biologically active portion of the pesticidal protein. Therefore, fragments of the nucleic acids of the embodiments can encode the biologically active portion of the pesticidal protein, or can be fragments that can be used as hybridization probes or PCR primers using the methods disclosed herein. The biologically active portion of the pesticidal protein can be prepared as follows: isolate a portion of one of the nucleotide sequences of the embodiments, express the coding portion of the pesticidal protein (e.g., by in vitro recombinant expression), and evaluate the activity of the coding portion of the pesticidal protein.

[0075] The nucleic acid as a fragment of the nucleotide sequence of the embodiment comprises at least 16, 20, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 850, 900 or 950 nucleotides, or up to the number of nucleotides in the nucleotide sequence disclosed herein. Specific embodiments contemplate a fragment derived from (e.g., produced from) the first nucleic acid of the embodiment, wherein the fragment encodes a truncated toxin having pesticidal activity. The truncated polypeptide encoded by the polynucleotide fragment of the embodiment has comparable or improved pesticidal activity relative to the activity of the corresponding full-length polypeptide encoded by the first nucleic acid from which the fragment is derived. It is conceivable that such nucleic acid fragments of the embodiment can be truncated at the 3' end of the natural or corresponding full-length coding sequence. The nucleic acid fragment can also be truncated at both the 5' and 3' ends of the natural or corresponding full-length coding sequence.

[0076] The term "variant" as used herein refers to substantially similar sequences. For nucleotide sequences, conservative variants include sequences that, due to the degeneracy of the genetic code, encode the amino acid sequence of one of the pesticidal polypeptides of the embodiments. One of ordinary skill in the art will readily appreciate that, due to the degeneracy of the genetic code, there are many nucleotide sequences encoding the present disclosure.

[0077] In some embodiments, the nucleic acid molecule encoding the polypeptide is a non-genomic nucleic acid sequence. As used herein, a "non-genomic nucleic acid sequence" or "non-genomic nucleic acid molecule" or "non-genomic polynucleotide" refers to a nucleic acid molecule having one or more changes in a nucleic acid sequence compared to a natural or genomic nucleic acid sequence. In some embodiments, changes in natural or genomic nucleic acid molecules include, but are not limited to: changes in nucleic acid sequences due to the degeneracy of the genetic code; codon optimization of nucleic acid sequences for expression in plants; changes in nucleic acid sequences introducing at least one amino acid substitution, insertion, deletion and / or addition compared to a natural or genomic sequence; removal of one or more introns associated with the genomic nucleic acid sequence; insertion of one or more heterologous introns; deletion of one or more upstream or downstream regulatory regions associated with the genomic nucleic acid sequence; insertion of one or more heterologous upstream or downstream regulatory regions; deletion of 5' and / or 3' untranslated regions associated with the genomic nucleic acid sequence; insertion of heterologous 5' and / or 3' untranslated regions; and modification of polyadenylation sites. In some embodiments, the non-genomic nucleic acid molecule is a cDNA. In some embodiments, the non-genomic nucleic acid molecule is a synthetic nucleic acid sequence.

[0078] When appropriate, nucleic acid can be optimized to increase expression in the host organism. Therefore, when the host organism is a plant, synthetic nucleic acids can be synthesized using plant preference codons to improve expression. For discussion of the use of host preference codons, see, for example, Campbell and Gowri, (1990) Plant Physiol. [Plant Physiology] 92: 1-11. For example, although the nucleotide sequence of the embodiment can be expressed in monocotyledonous and dicotyledonous plant species, the sequence can be modified to explain the codon preference and GC content preference specific to monocotyledonous or dicotyledonous plants, because these preferences have shown differences (Murray et al. (1989) Nucleic Acids Res. [Nucleic Acids Research] 17: 477-498). Thus, the maize preference codons of specific amino acids can be derived from the known gene sequences of maize. The maize codons from 28 kinds of genes of maize plants are listed in Table 4 of Murray et al. (ibid). Methods for synthesizing plant-preferred genes are available in the art. See, for example, U.S. Patent Nos. 5,380,831, and 5,436,391, and Murray et al., (1989), Nucleic Acids Res., 17:477-498, and Liu H et al., Mol Bio Rep, 37:677-684, 2010, incorporated herein by reference. A codon usage table for corn (Zea maize) can also be found at kazusa.or.jp / codon / cgi-bin / showcodon.cgi?species=4577, which can be accessed using the www prefix.

[0079] A soybean (Glycine max) codon usage table is shown in Table 3 and can also be found at kazusa.or.jp / codon / cgi-bin / showcodon.cgi?species=3847&aa=1&style=N, which can be accessed using the www prefix.

[0080] The technician will further understand that changes can be introduced by the sudden change of nucleotide sequence, resulting in changes in the amino acid sequence of the encoded polypeptide, without changing the biological activity of these proteins. Therefore, variant nucleic acid molecules can be produced in the following manner: one or more nucleotides are replaced, added and / or deleted and introduced into the corresponding nucleotide sequence disclosed herein, so that one or more amino acid substitutions, additions or deletions are introduced into the encoded protein. Sudden changes can be introduced by standard techniques, such as directed mutagenesis and PCR-mediated mutagenesis. This type of variant nucleic acid sequence is also encompassed by the disclosure.

[0081] For example, these naturally occurring allelic variants can be identified using well-known molecular biology techniques such as polymerase chain reaction (PCR) and hybridization techniques as outlined herein.

[0082] In some embodiments, the polynucleotides encoding the following polypeptides are non-genomic nucleic acid sequences: SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:74, SEQ ID NO:76, SEQ ID NO:78, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:84, SEQ ID NO:86, SEQ ID NO:88, SEQ ID NO:90, SEQ ID NO:92, SEQ ID NO:94, SEQ ID NO:96, SEQ ID NO:98, SEQ ID NO:100, SEQ ID NO:102, SEQ ID NO:104, SEQ ID NO:106, SEQ ID NO:108, SEQ ID NO:110, SEQ ID NO:112, SEQ ID NO:114, SEQ ID NO:116, SEQ ID NO:118, SEQ ID NO:120, SEQ ID NO:122, SEQ ID NO:124, SEQ ID NO:126, SEQ ID NO:128, SEQ ID NO:130, SEQ ID NO:132, SEQ ID NO:134, SEQ ID NO:136, SEQ ID NO:138, SEQ ID NO:140, SEQ ID NO:142, SEQ ID NO:144, SEQ ID NO:146, SEQ IDNO: 148, SEQ ID NO: 150, SEQ ID NO: 152, SEQ ID NO: 154, SEQ ID NO: 156, SEQ ID NO: 158, SEQ ID NO: 160, SEQ ID NO: 162, SEQ ID NO: 164, SEQ ID NO: 166, SEQ ID NO: 168, SEQ ID NO: 170, SEQ ID NO: 172, SEQ ID NO: 174, SEQ ID NO: 176, SEQ ID NO: 178, SEQ ID NO: 180, SEQ ID NO: 182, SEQ ID NO: 184, SEQ ID NO: 186, SEQ ID NO: 188, SEQ ID NO: 190, SEQ ID NO: 192, SEQ ID NO: 194, SEQ ID NO: 196, SEQ ID NO: 198, SEQ ID NO: 200, SEQ ID NO: 202, SEQ ID NO: 204, SEQ ID NO: 206, SEQ ID NO: 208, SEQ ID NO: 210, SEQ ID NO: 212, SEQ ID NO: 214, SEQ ID NO: 216, SEQ ID NO: 218, SEQ ID NO: 220, SEQ ID NO: 222, SEQ ID NO: 224, SEQ ID NO: 226, SEQ ID NO: 228, SEQ ID NO: 230, SEQ ID NO: 232, SEQ ID NO: 234, SEQ ID NO: 236, SEQ ID NO: 238, SEQ ID NO: 240, SEQ ID NO: 242, SEQ ID NO: 244, SEQ ID NO: 246, SEQ ID NO: 248, SEQ ID NO: 250, SEQ ID NO: 252, SEQ ID NO: 254, SEQ ID NO: 256, SEQ ID NO: 258, SEQ ID NO: 260, SEQ ID NO: 262, SEQ ID NO: 264, SEQ ID NO: 266, SEQ ID NO: 268 or SEQ ID NO: 270.

[0083] Variant nucleotide sequences also include synthetically obtained nucleotide sequences, such as those produced by using site-directed mutagenesis, but these sequences still encode the pesticidal proteins of the embodiments, such as mutant toxins. Typically, the variant of the specific nucleotide sequence of the embodiment will have at least about 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with the specific nucleotide sequence, as determined by the sequence alignment program described elsewhere herein using default parameters. The variant of the nucleotide sequence of the embodiment can differ from the sequence by as little as 1-15 nucleotides, as little as 1-10, for example 6-10, as little as 5, as little as 4, 3, 2 or even 1 nucleotide.

[0084] Variants of a specific nucleotide sequence of the embodiments (ie, exemplary nucleotide sequence) can also be evaluated by comparing the percent sequence identity between a polypeptide encoded by the variant nucleotide sequence and a polypeptide encoded by a reference nucleotide sequence. Thus, for example, isolated nucleic acids encoding a polypeptide having a given percent sequence identity to the polypeptide of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:65 NO:66, SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:74, SEQ ID NO:76, SEQ ID NO:78, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:84, SEQ IDNO:86, SEQ ID NO:88, SEQ ID NO:90, SEQ ID NO:92, SEQ ID NO:94, SEQ ID NO:96, SEQ ID NO:98, SEQ ID NO:100, SEQ ID NO:102, SEQ ID NO:104, SEQ ID NO:106, SEQ ID NO:108, SEQ ID NO:110, SEQ ID NO:112, SEQ ID NO:114, SEQ ID NO:116, SEQ ID NO:118, SEQ ID NO:120, SEQ ID NO:122, SEQ ID NO:124, SEQ ID NO:126, SEQ ID NO:128, SEQ ID NO:130, SEQ ID NO:132, SEQ ID NO:134, SEQ IDNO: 136, SEQ ID NO: 138, SEQ ID NO: 140, SEQ ID NO: 142, SEQ ID NO: 144, SEQ ID NO: 146, SEQ ID NO: 148, SEQ ID NO: 150, SEQ ID NO: 152, SEQ ID NO: 154, SEQ ID NO: 156, SEQ ID NO: 158, SEQ ID NO: 160, SEQ ID NO: 162, SEQ ID NO: 164, SEQ ID NO: 166, SEQ ID NO: 168, SEQ ID NO: 170, SEQ ID NO: 172, SEQ ID NO: 174, SEQ ID NO: 176, SEQ ID NO: 178, SEQ ID NO: 180, SEQ ID NO: 182, SEQ ID NO: 184, SEQ ID NO: 186, SEQ ID NO: 188, SEQ ID NO: 190, SEQ ID NO: 192, SEQ ID NO: 194, SEQ ID NO: 196, SEQ ID NO: 198, SEQ ID NO: 200, SEQ ID NO: 202, SEQ ID NO: 204, SEQ ID NO: 206, SEQ ID NO: 208, SEQ ID NO: 210, SEQ ID NO: 212, SEQ ID NO: 214, SEQ ID NO: 216, SEQ ID NO: 218, SEQ ID NO: 220, SEQ ID NO: 222, SEQ ID NO: 224, SEQ ID NO: 226, SEQ ID NO: 228, SEQ ID NO: 230, SEQ ID NO: 232, SEQ ID NO: 234, SEQ ID NO: 236, SEQ ID NO: 238, SEQ ID NO: 240, SEQ ID NO: 242, SEQ ID NO: 244, SEQ ID NO: 246, SEQ ID NO: 248, SEQ ID NO: 250, SEQ ID NO: 252, SEQ ID NO: 254, SEQ ID NO: 256, SEQ ID NO: 258, SEQ ID NO: 260, SEQ ID NO: 262, SEQ ID NO: 264, SEQ ID NO: 266, SEQ ID NO: 268 or SEQ IDNO:270.Can use default parameters to use the sequence alignment program described elsewhere herein to calculate the sequence identity percentage between any two polypeptides.In the case of any given polynucleotide of embodiment, to assessing by comparing the percentage sequence identity shared by two polypeptides of its coding, the percentage sequence identity between two polypeptides of coding is at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, usually at least about 75%, 80%, 85%, at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% or at least about 98%, 99% or higher sequence identity.

[0085] As used herein, the term "variant protein" includes polypeptides derived from natural proteins by: deleting (so-called truncations) one or more amino acids or adding one or more amino acids at the N-terminus and / or C-terminus of natural proteins; deleting or adding one or more amino acids at one or more sites in natural proteins; or replacing one or more amino acids at one or more sites in natural proteins. Therefore, the term "variant protein" encompasses biologically active fragments of natural proteins, which contain a sufficient number of consecutive amino acid residues to retain the biological activity of the natural protein, i.e., have pesticidal activity. Such pesticidal activity may be different or improved relative to the natural protein, or may remain unchanged, as long as the pesticidal activity is retained.

[0086] The variant proteins covered by the embodiments have biological activity, that is, they still have the biological activity of the hope of native protein, that is, the pesticidal activity as described herein.Such variants can be produced by, for example, genetic polymorphism or artificial manipulation.The biological activity variants of the natural pesticidal protein of the embodiment will have at least about 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with the amino acid sequence of native protein, as determined by the default parameters of the sequence alignment program described elsewhere herein.The biological activity variants of the protein of the embodiment can differ from the protein by as little as 1-15 amino acid residue, as little as 1-10, for example 6-10, as little as 5, as little as 4, 3, 2 or even 1 amino acid residue.

[0087] In some embodiments, the insecticidal polypeptide has at least 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity to the following amino acid sequences: SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:74, SEQ ID NO:76, SEQ ID NO:78, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:84, SEQ ID NO:86, SEQ ID NO:88, SEQ ID NO:90, SEQ ID NO:92, SEQ ID NO:94, SEQ ID NO:96, SEQ ID NO:98, SEQ ID NO:100, SEQ ID NO:102, SEQ ID NO:104, SEQ ID NO:106, SEQ ID NO:108, SEQ ID NO:110, SEQ ID NO:112, SEQ ID NO:114, SEQ ID NO:116, SEQ ID NO:118, SEQ ID NO:120, SEQ ID NO:122, SEQ ID NO:124, SEQ ID NO:126, SEQ ID NO:128, SEQ ID NO:130, SEQ ID NO:132, SEQ ID NO:134, SEQ IDNO: 136, SEQ ID NO: 138, SEQ ID NO: 140, SEQ ID NO: 142, SEQ ID NO: 144, SEQ ID NO: 146, SEQ ID NO: 148, SEQ ID NO: 150, SEQ ID NO: 152, SEQ ID NO: 154, SEQ ID NO: 156, SEQ ID NO: 158, SEQ ID NO: 160, SEQ ID NO: 162, SEQ ID NO: 164, SEQ ID NO: 166, SEQ ID NO: 168, SEQ ID NO: 170, SEQ ID NO: 172, SEQ ID NO: 174, SEQ ID NO: 176, SEQ ID NO: 178, SEQ ID NO: 180, SEQ ID NO: 182, SEQ ID NO: 184, SEQ ID NO: 186, SEQ ID NO: 188, SEQ ID NO: 190, SEQ ID NO: 192, SEQ ID NO: 194, SEQ ID NO: 196, SEQ ID NO: 198, SEQ ID NO: 200, SEQ ID NO: 202, SEQ ID NO: 204, SEQ ID NO: 206, SEQ ID NO: 208, SEQ ID NO: 210, SEQ ID NO: 212, SEQ ID NO: 214, SEQ ID NO: 216, SEQ ID NO: 218, SEQ ID NO: 220, SEQ ID NO: 222, SEQ ID NO: 224, SEQ ID NO: 226, SEQ ID NO: 228, SEQ ID NO: 230, SEQ ID NO: 232, SEQ ID NO: 234, SEQ ID NO: 236, SEQ ID NO: 238, SEQ ID NO: 240, SEQ ID NO: 242, SEQ ID NO: 244, SEQ ID NO: 246, SEQ ID NO: 248, SEQ ID NO: 250, SEQ ID NO: 252, SEQ ID NO: 254, SEQ ID NO: 256, SEQ ID NO: 258, SEQ ID NO: 260, SEQ ID NO: 262, SEQ ID NO: 264, SEQ ID NO: 266, SEQ ID NO: 268 or SEQ ID NO: 270.

[0088] In some embodiments, the polypeptide has a modified physical property. As used herein, the term "physical property" refers to any parameter suitable for describing the physicochemical characteristics of a protein. As used herein, "target physical property" and "target property" are used interchangeably to refer to the physical property of a protein being studied and / or modified. Examples of physical properties include, but are not limited to, net surface charge and charge distribution on the protein surface, net hydrophobicity and hydrophobic residue distribution on the protein surface, surface charge density, surface hydrophobic density, total count of surface ionized groups, surface tension, protein size and its distribution in solution, melting temperature, heat capacity, and second virial coefficient. Examples of physical properties also include, but are not limited to, solubility, folding, stability, and digestibility. In some embodiments, the polypeptide has an increased digestibility of proteolytic fragments in the insect gut. In some embodiments, the polypeptide has an increased stability in the insect gut. Models simulating gastric digestion are known to those skilled in the art (Fuchs, RL and JD Astwood. Food Technology 50:83-88, 1996; Astwood, JD et al., Nature Biotechnology 14:1269-1273, 1996; Fu TJ et al., J. Agric Food Chem. 50:7154-7160, 2002).

[0089] Embodiments further encompass microorganisms transformed with at least one nucleic acid of the embodiments, with an expression cassette comprising the nucleic acid, or with a vector comprising the expression cassette. In some embodiments, the microorganism is a microorganism propagated on a plant. Embodiments of the present disclosure relate to encapsulated pesticidal proteins comprising a transformed microorganism capable of expressing at least one pesticidal protein of the embodiments.

[0090] Embodiments provide pesticidal compositions comprising the transformed microorganisms of the embodiments. In such embodiments, the transformed microorganisms are typically present in the pesticidal compositions in pesticidal effective amounts together with a suitable carrier. Embodiments also encompass pesticidal compositions comprising pesticidal effective amounts of the isolated proteins of the embodiments (alone or in combination with the transformed organisms of the embodiments, and / or the encapsulated pesticidal proteins of the embodiments), together with a suitable carrier.

[0091] The embodiments further provide methods for increasing the insect target range by using the pesticidal proteins of the embodiments in combination with at least one other or "second" pesticidal protein. Any pesticidal protein known in the art can be used in the methods of the embodiments. Such pesticidal proteins include, but are not limited to, Bt toxins, protease inhibitors, α-amylases, and peroxidases.

[0092] Embodiments also encompass transformed or transgenic plants comprising at least one nucleotide sequence of the embodiments. In certain embodiments, plants are stably transformed using a nucleotide construct comprising at least one nucleotide sequence of the embodiments, the nucleotide sequence being operably linked to a promoter that drives expression in a plant cell. As used herein, the terms "transformed plant" and "transgenic plant" refer to plants comprising heterologous polynucleotides in their genome. Typically, heterologous polynucleotides are stably integrated into the transgenic or transformed plant genome so that the polynucleotides are passed on to successive generations. Heterologous polynucleotides can be integrated into the genome alone or as part of a recombinant expression cassette.

[0093] It should be understood that the term "transgenic," as used herein, includes any cell, cell line, callus, tissue, plant part, or plant whose genotype has been altered by the presence of heterologous nucleic acid, including those transgenics initially so altered as well as those generated from the initial transgene by sexual crosses or asexual propagation. As used herein, the term "transgenic" does not encompass alterations of the genome (chromosomal or extrachromosomal) caused by conventional plant breeding methods or by naturally occurring events such as random cross-fertilization, non-recombinant viral infection, non-recombinant bacterial transformation, non-recombinant transposition, or spontaneous mutation.

[0094] As used herein, the term "plant" includes whole plants, plant organs (e.g., leaves, stems, roots, etc.), seeds, plant cells and progeny thereof. The parts of transgenic plants are within the scope of the embodiments, and include, for example, plant cells, plant protoplasts, plant cell tissue cultures that can regenerate plants, plant calli, plant masses, and complete plant cells in plants or plant parts (e.g., embryos, pollen, ovules, seeds, leaves, flowers, branches, fruits, cores, ears, corn cobs, shells, stems, roots, root tips, anthers, etc.), which are derived from transgenic plants with the DNA molecules of the embodiments or their previously transformed offspring and are therefore composed of at least a portion of transgenic cells. The plant species that can be used in the methods of the embodiments are generally as broad as the higher plant categories that are suitable for transformation technology, including monocots and dicots.

[0095] Although the embodiment does not rely on the specific biological mechanism for increasing the resistance of plants to plant pests, the expression of the nucleotide sequence of the embodiment in plants can cause the generation of the pest killing protein of the embodiment and the resistance of plants to plant pests to increase. The plant of the embodiment can be applied to the method affecting insect pests in agriculture. Some embodiments provide transformed crop plants, such as maize plants, which can be applied to the method affecting insect pests (such as lepidopteran pests) of plants.

[0096] A "subject plant or plant cell" is a plant or plant cell in which a genetic alteration (e.g., transformation) has been effected with respect to a gene of interest, or a plant or plant cell that is inherited from a plant or cell thus altered and contains the alteration. A "control" or "control plant" or "control plant cell" provides a reference point for measuring changes in the phenotype of a subject plant or plant cell.

[0097] Control plants or plant cells can include, for example: (a) wild-type plants or cells, i.e., plants or plant cells of the same genotype as the starting material used to cause the genetic change in the subject plant or cell; (b) plants or plant cells of the same genotype as the starting material, but which have been transformed with a null construct (i.e., a construct that has no known effect on the trait of interest, such as a construct comprising a marker gene); (c) plants or plant cells that are untransformed segregants in progeny of the subject plant or plant cell; (d) plants or plant cells that are genetically identical to the subject plant or plant cell, but that have not been exposed to conditions or stimuli that would induce expression of the gene of interest; or (e) the subject plant or plant cell itself under conditions where the gene of interest is not expressed.

[0098] Those skilled in the art will readily recognize that advances in the field of molecular biology, such as site-specific and random mutagenesis, polymerase chain reaction methods and protein engineering techniques provide a broad set of tools and protocols suitable for altering or engineering both the amino acid sequence and the underlying genetic sequence of agriculturally beneficial proteins.

[0099] Therefore, the protein of the embodiment can be changed in a variety of ways (including amino acid substitution, deletion, truncation and insertion). The method for such operation is generally known in the art. For example, the amino acid sequence variant of the pesticidal protein can be prepared by introducing mutations into synthetic nucleic acids (e.g., DNA molecules). The methods of mutagenesis and nucleic acid changes are well known in the art. For example, the designed changes can be introduced using oligonucleotide-mediated site-directed mutagenesis techniques. See, for example, Kunkel, (1985) Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences of the United States] 82: 488-492; Kunkel, et al. (1987) Methods in Enzymol. [Enzymology Methods] 154: 367-382; U.S. Patent No. 4,873,192; Walker and Gaastra, ed. (1983) Techniques in Molecular Biology [Molecular Biology Techniques] (MacMillan Publishing Company [MacMillan Publishing Company], New York) and the references cited therein.

[0100] The nucleotide sequence of the mutagenesis of the embodiment can be modified to change about 1, 2, 3, 4, 5, 6, 8, 10, 12 or more amino acids present in the primary sequence of the coded polypeptide. Alternatively, more changes from the native sequence can even be introduced so that the encoded protein can be compared with the corresponding wild-type protein to make at least about 1% or 2%, or about 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12% or even about 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20%, 21%, 22%, 23%, 24%, or 25%, 30%, 35%, or 40% or more codons be changed or otherwise modified. In the same way, the encoded protein can have at least about 1% or 2% or about 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12% or even about 13%, 14%, 15%, 16%, 17%, 18%, 19% or 20%, 21%, 22%, 23%, 24% or 25%, 30%, 35% or 40% or more other codons compared to the corresponding wild-type protein. It should be understood that the mutagenic nucleotide sequence of the embodiment is intended to encompass biological functionality, equivalent peptides with pesticidal activity (for example improved pesticidal activity, as determined by the antifeedant properties for European corn borer larvae). Such sequences may be produced due to known codon redundancy and functionally equivalent results naturally occurring in nucleotide sequences and the proteins encoded thereby.

[0101] Those skilled in the art will recognize that amino acid additions and / or substitutions are generally based on the relative similarity of the amino acid side chain substituents, such as their hydrophobicity, charge, size, etc. Exemplary amino acid substitution groups that take into account various of the foregoing characteristics are well known to those skilled in the art and include: arginine and lysine; glutamic acid and aspartic acid; serine and threonine; glutamine and asparagine; and valine, leucine, and isoleucine.

[0102] Guidance on appropriate amino acid substitutions that do not affect the biological activity of the protein of interest can be found in the model of Dayhoff et al., (1978) Atlas of Protein Sequence and Structure (Natl. Biomed. Res. Found., Washington, DC), incorporated herein by reference. Conservative substitutions can be made, such as exchanging one amino acid for another with similar properties.

[0103] Therefore, the gene and nucleotide sequence of the embodiment include both naturally occurring sequences and mutant forms. Similarly, the protein of the embodiment encompasses both naturally occurring proteins and variants (e.g., truncated polypeptides) and modified forms thereof (e.g., mutants). Such variants will continue to have the desired pesticidal activity. Obviously, the mutations to be carried out in the nucleotide sequence encoding the variant must not place the sequence outside the reading frame, and will not create complementary regions that can produce secondary mRNA structures. See, European Patent Application Publication No. 75,444.

[0104] Deletions, insertions, and substitutions of protein sequences encompassed herein are not expected to produce radical changes in protein characteristics. However, when it is difficult to predict in advance the exact effect of a substitution, deletion, or insertion, it will be understood by those skilled in the art that the effect will be evaluated by conventional screening tests (e.g., insect feeding assays). See, e.g., Marrone et al., (1985) J. Econ. Entomol. 78:290-293, and Czapla and Lang (1990) J. Econ. Entomol. 83:2480-2485, incorporated herein by reference.

[0105] Variant nucleotide sequence and protein also comprise by mutagenesis and recombination method, for example sequence and protein that DNA reorganization produces.Use such program, can operate one or more different coding sequences to create the new pest-killing protein with desired property.In this way, produce recombinant polynucleotide library from the colony of related sequence polynucleotide, these related sequence polynucleotide comprise following sequence region, these sequence regions have substantial sequence identity and can carry out homologous recombination in vitro or in vivo.For example, use this method, can reorganize any fragment of the sequence motif of full-length coding sequence, coding purpose domain or the nucleotide sequence of embodiment between the nucleotide sequence of embodiment and the corresponding part of other known Cry nucleotide sequence, to obtain the new gene of protein with improved purpose property of coding.

[0106] The target property includes but is not limited to the pesticidal activity of each unit of pesticidal protein, protein stability and toxicity to non-target species (particularly people, livestock and plants and microorganisms expressing the pesticidal polypeptides of the embodiments). The embodiments are not bound by specific reorganization strategies, and only at least one nucleotide sequence of the embodiments or a part thereof participates in such reorganization strategies. Reorganization may only relate to nucleotide sequences disclosed herein, or may additionally relate to the reorganization of other nucleotide sequences known in the art. The strategy of DNA reorganization is known in the art. See, e.g., Stemmer, (1994) Proc. Natl. Acad. Sci. USA 91:10747-10751; Stemmer (1994) Nature 370:389-391; Crameri et al., (1997) Nature Biotech. 15:436-438; Moore et al., (1997) J. Mol. Biol. 272:336-347; Zhang et al., (1997) Proc. Natl. Acad. Sci. USA 94:4504-4509; Crameri et al., (1998), Nature, 391:288-291; and U.S. Pat. Nos. 5,605,793 and 5,837,458.

[0107] The nucleotide sequence of embodiment can also be used to separate corresponding sequence from other organisms (particularly other bacteria and more particularly other bacillus strains).In this way, such sequence (based on its sequence homology with sequence shown in this article) can be identified using methods such as PCR, hybridization.Sequences selected based on the sequence identity of whole sequence shown in this article or its fragment are contained in embodiment.Such sequence includes the sequence of the straight homologue as disclosed sequence.Term " straight homologue " refers to the gene that is derived from common ancestor gene and is found in different species due to species formation.When its nucleotide sequence and / or its encoded protein sequence have basic identity as defined elsewhere herein, the gene found in different species is considered to be straight homologue.The function of straight homologue is usually highly conservative between species.

[0108] In the PCR method, oligonucleotide primers can be designed for use in the PCR reaction to amplify the corresponding DNA sequence from cDNA or genomic DNA extracted from any organism of interest. Methods for designing PCR primers and PCR cloning are generally known in the art and are disclosed in Sambrook et al., (1989) Molecular Cloning: A Laboratory Manual (2nd ed., Cold Spring Harbor Laboratory Press, Plainview, New York), hereinafter "Sambrook". See also, Innis et al., eds. (1990) PCR Protocols: A Guide to Methods and Applications (Academic Press, New York); Innis and Gelfand, eds. (1995) PCR Strategies (Academic Press, New York); and Innis and Gelfand, eds. (1999) PCR Methods Manual (Academic Press, New York). Known PCR methods include, but are not limited to, methods using paired primers, nested primers, single-specific primers, degenerate primers, gene-specific primers, vector-specific primers, partially mismatched primers, and the like.

[0109] In hybridization techniques, all or part of a known nucleotide sequence is used as a probe that selectively hybridizes to other corresponding nucleotide sequences present in a population of cloned genomic DNA fragments or cDNA fragments (i.e., a genomic or cDNA library) from a selected organism. These hybridization probes can be genomic DNA fragments, cDNA fragments, RNA fragments, or other oligonucleotides and can be labeled with a detectable group such as 32 P or any other detectable marker. Thus, for example, probes for hybridization can be made by oligonucleotides synthesized based on the sequence labels of the embodiments. Methods for preparing hybridization probes and for constructing cDNA and genomic libraries are generally known in the art and are disclosed in Sambrook.

[0110] For example, the complete sequence disclosed herein, or one or more parts thereof can be used as probes, which can be specifically hybridized with corresponding sequences and messenger RNA. In order to achieve specific hybridization under various conditions, such probes include sequences unique to the sequences of the embodiments, and lengths are generally at least about 10 or 20 nucleotides. Such probes can be used to increase the corresponding Cry sequences from selected organisms by PCR. This technology can be used to separate other coding sequences from desired organisms, or as a diagnostic assay for determining the presence of coding sequences in organisms. Hybridization techniques include DNA libraries (plaques or bacterium colonies) of hybridization screening plating; see, for example, Sambrook.

[0111] Hybridization of such sequences can be carried out under stringent conditions. As used herein, the term "stringent conditions" or "stringent hybridization conditions" refers to conditions in which the degree of hybridization of a probe to its target sequence will be detectably higher (e.g., at least 2 times, 5 times, or 10 times over background) than the degree of hybridization of the probe to other sequences. Stringent conditions are sequence dependent and will be different in different situations. By controlling the stringency of hybridization and / or washing conditions, a target sequence that is 100% complementary to the probe can be identified (homologous detection). Alternatively, stringent conditions can be adjusted to allow some mispairing in the sequence so that a lower degree of similarity (heterologous detection) is detected. Typically the probe is less than about 1000 or 500 nucleotides in length.

[0112] Typically, stringent conditions are those under which the salt concentration is less than about 1.5 M sodium ions, typically about 0.01 to 1.0 M sodium ion concentration (or other salts) at pH 7.0 to 8.3, and the temperature is at least about 30° C. for short probes (e.g., 10 to 50 nucleotides), and at least about 60° C. for long probes (e.g., more than 50 nucleotides). Stringent conditions can also be achieved by adding destabilizing agents such as formamide. Exemplary low stringency conditions include hybridization at 37° C. with a buffer solution of 30% to 35% formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulfate), and washing in 1X to 2X SSC (20X SSC=3.0 M NaCl / 0.3 M trisodium citrate) at 50° C. to 55° C. Exemplary moderate stringency conditions include hybridization at 40% to 45% formamide, 1.0 M NaCl, 1% SDS at 37° C., and washing in 0.5X to 1X SSC at 55° C. to 60° C. Exemplary high stringency conditions include hybridization at 50% formamide, 1 M NaCl, 1% SDS at 37° C., and a final wash in 0.1X SSC at 60° C. to 65° C. for at least about 20 minutes. Optionally, the wash buffer may contain about 0.1% to about 1% SDS. Duration of hybridization is typically less than about 24 hours, typically about 4 to about 12 hours.

[0113] The following terms are used to describe sequence relationships between two or more nucleic acids or polynucleotides: (a) "reference sequence," (b) "comparison window," (c) "sequence identity," (d) "percentage of sequence identity," and (e) "substantial identity."

[0114] (a) As used herein, a "reference sequence" is a defined sequence used as a basis for sequence comparison. A reference sequence can be a subset or the entirety of a specified sequence; for example, as a segment of a full-length cDNA or gene sequence, or a complete cDNA or gene sequence.

[0115] (b) As used herein, a "comparison window" refers to a continuous and specified segment of a polynucleotide sequence, wherein the polynucleotide sequence in the comparison window may contain additions or deletions (i.e., gaps) compared to a reference sequence (which does not include additions or deletions) for optimal alignment of the two sequences. Typically, the length of the comparison window is at least 20 consecutive nucleotides, and optionally can be 30, 40, 50, 100 or longer. It will be appreciated by those skilled in the art that, due to the presence of gaps in the polynucleotide sequence, in order to avoid high similarity with the reference sequence, a gap penalty is typically introduced and subtracted from the number of matches.

[0116] Methods of alignment of sequences for comparison are well known in the art. Thus, determination of percent sequence identity between any two sequences can be performed using a mathematical algorithm. A non-limiting example of such a mathematical algorithm is Myers and Miller (1988) CABIOS 4:11-17; the local alignment algorithm in Smith et al., (1981) Adv. Appl. Math. [Advances in Applied Mathematics] 2:482; the global alignment algorithm in Needleman and Wunsch (1970) J. Mol. Biol. [Journal of Molecular Biology] 48:443-453; the search for local alignment method in Pearson and Lipman (1988) Proc. Natl. Acad. Sci. [Proceedings of the National Academy of Sciences of the United States of America] 85:2444-2448; the algorithm in Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences of the United States of America] 872264; as modified in Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences of the United States of America] 90:5873-5877.

[0117] Computer implementations of these mathematical algorithms can be used for sequence comparison to determine sequence identity. These implementations include, but are not limited to: CLUSTAL in the PC / Gene program (available from Intelligenetics, Mountain View, California); GAP, BESTFIT, BLAST, FASTA, and TFASTA in the ALIGN program (version 2.0) and GCG Wisconsin Genetics Software Package (GCG Wisconsin Genetics Software Package) version 10 (available from Accelrys, 9685 Scranton Road, San Diego, California, USA). Alignment using these programs can be performed using default parameters. The CLUSTAL program is fully described in Higgins et al., (1988) Gene 73:237-244 (1988); Higgins et al., (1989) CABIOS 5:151-153; Corpet et al., (1988) Nucleic Acids Res. 16:10881-90; Huang et al., (1992) CABIOS 8:155-65; and Pearson et al., (1994) Meth. Mol. Biol. 24:307-331. The ALIGN program is based on the algorithm of Myers and Miller (1988) (supra). When comparing amino acid sequences, the ALIGN program can use the PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. The BLAST program in Altschul et al., (1990) J. Mol. Biol. 215:403 is based on the algorithm in Karlin and Altschul (1990), supra. BLAST nucleotide searches can be performed using the BLASTN program, score = 100, word length = 12 to obtain nucleotide sequences homologous to the nucleotide sequence encoding the protein of the embodiments. BLAST protein searches can be performed using the BLASTX program, score = 50, word length = 3 to obtain amino acid sequences homologous to the protein or polypeptide of the embodiments. To obtain gapped alignments for comparison purposes, Gapped BLAST (in BLAST 2.0) as described in Altschul et al., (1997) Nucleic Acids Res. 25:3389 can be used.Alternatively, PSI-BLAST (in BLAST 2.0) can be used to perform an iterative search to detect distant relationships between molecules. See Altschul et al., (1997), supra. When using BLAST, gap BLAST, PSI-BLAST, the default parameters of each program (e.g., BLASTN for nucleotide sequences, BLASTX for proteins) can be used. See the website of the National Center for Biotechnology Information, ncbi.hlm.nih.gov on the World Wide Web. Alignment can also be performed manually by inspection.

[0118] (c) As used herein, "sequence identity" or "identity" in the context of two nucleic acid or polypeptide sequences refers to the same residues in the two sequences when aligned for maximum correspondence over a specified comparison window. When using percentages of sequence identity with respect to proteins, it is recognized that residue positions that are not identical generally differ by conservative amino acid substitutions, in which amino acid residues are substituted by other amino acid residues having similar chemical properties (e.g., charge or hydrophobicity), and thus do not alter the functional properties of the molecule. When sequences differ in conservative substitutions, the percentage of sequence identity may be adjusted upward to correct for the conservative nature of the substitution. Sequences that differ by these conservative substitutions are referred to as having "sequence similarity" or "similarity." Methods for making this adjustment are well known to those skilled in the art. Typically, this involves scoring conservative substitutions as partial rather than complete mismatches, thereby increasing the percentage sequence identity. Thus, for example, when identical amino acids are scored as 1, and non-conservative substitutions are scored as zero, conservative substitutions are scored between zero and 1. Scoring of conservative substitutions is calculated, for example, as implemented in the program PC / GENE (Intelligenetics, Mountain View, CA).

[0119] (d) As used herein, "sequence identity percentage" means a value determined by comparing two optimally aligned sequences over a comparison window, wherein the polynucleotide sequence portion in the comparison window may include additions or deletions (i.e., gaps) compared to a reference sequence (which does not include additions or deletions) to perform optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the same nucleic acid base or amino acid residue appears in the two sequences to generate the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window, and then multiplying the result by 100 to generate the sequence identity percentage.

[0120] (e)(i) The term "substantial identity" of a polynucleotide sequence means that the polynucleotides include sequences having at least 70%, 80%, 90%, or 95% or more sequence identity when compared to a reference sequence using one of the described alignment programs using standard parameters. One skilled in the art will recognize that these values ​​can be appropriately adjusted to determine the corresponding identity of proteins encoded by two nucleotide sequences by taking into account codon degeneracy, amino acid similarity, reading frame positioning, etc. For these purposes, substantial identity of amino acid sequences generally means sequence identity of at least 60%, 70%, 80%, 90%, or 95% or more sequence identity.

[0121] Another indication that nucleotide sequences are substantially identical is if two molecules hybridize to each other under stringent conditions. Generally, stringent conditions are selected to be greater than the T value for a specific sequence at a defined ionic strength and pH. m However, stringent conditions include those that are lower than T m The term "strand" refers to a temperature in the range of about 1°C to about 20°C lower, depending on the desired stringency as limited elsewhere herein. If the polypeptides encoded by nucleic acids that do not hybridize to each other under stringent conditions are substantially identical, then the nucleic acids are still substantially identical. For example, this may occur when a copy of a nucleic acid is produced using the maximum codon degeneracy allowed by the genetic code. An indication that two nucleic acid sequences are substantially identical is that the polypeptide encoded by the first nucleic acid is immunologically cross-reactive with the polypeptide encoded by the second nucleic acid.

[0122] (e)(ii) In the context of peptides, the term "substantial identity" means that the peptide comprises a sequence that has at least 70%, 80%, 85%, 90%, 95% or more sequence identity with a reference sequence over a specified comparison window. Optimal alignment for these purposes can be performed using the global alignment algorithm of Needleman and Wunsch (1970), supra. One indication that two peptide sequences are substantially identical is that one peptide is immunoreactive with antibodies directed against the second peptide. Thus, for example, when one peptide differs from a second peptide only by conservative substitutions, the two peptides are substantially identical. "Substantially similar" peptides share the sequences described above, except that residue positions that are not identical may differ by conservative amino acid changes.

[0123] The term "nucleotide construct" is used herein and is not intended to limit the embodiment to the nucleotide construct comprising DNA. It will be appreciated by those of ordinary skill in the art that nucleotide constructs, particularly polynucleotides and oligonucleotides consisting of ribonucleotides and combinations of ribonucleotides and deoxyribonucleotides, can also be used in the method disclosed herein. The nucleotide constructs, nucleic acids and nucleotide sequences of the embodiments additionally encompass all complementary forms of such constructs, molecules and sequences. In addition, the nucleotide constructs, nucleotide molecules and nucleotide sequences of the embodiments encompass all nucleotide constructs, molecules and sequences that can be used for the plant transformation method of the embodiments, including but not limited to those consisting of deoxyribonucleotides, ribonucleotides and combinations thereof. Such deoxyribonucleotides and ribonucleotides include both naturally occurring molecules and synthetic analogs. The nucleotide constructs, nucleic acids and nucleotide sequences of the embodiments also encompass all forms of nucleotide constructs, including but not limited to single-stranded forms, double-stranded forms, hairpins, stem-loop structures, etc.

[0124] Further embodiments relate to transformed organisms, such as organisms selected from the group consisting of plant and insect cells, bacteria, yeast, baculovirus, protozoa, nematodes and algae. The transformed organisms include: a DNA molecule of an embodiment, an expression cassette comprising the DNA molecule, or a vector comprising the expression cassette, which can be stably incorporated into the genome of the transformed organism.

[0125] The sequence of the embodiment is provided in a DNA construct for expression in a target organism. The construct will include 5' and 3' regulatory sequences operably connected to the sequence of the embodiment. As used herein, the term "operably connected" refers to a functional connection between a promoter and a second sequence, wherein the promoter sequence initiates and mediates the transcription of a DNA sequence corresponding to the second sequence. Typically, operably connected means that the nucleic acid sequence connected is continuous, and in the case of being necessary to connect two protein coding regions, is continuous and in the same reading frame. The construct may additionally contain at least one other gene to be co-transformed into an organism. Alternatively, one or more other genes may be provided on a plurality of DNA constructs.

[0126] Such DNA constructs are equipped with a plurality of restriction sites for inserting the Cry toxin sequence so that it is under the transcriptional regulation of these regulatory regions. The DNA construct may additionally comprise a selectable marker gene.

[0127] The DNA construct will include in the 5' to 3' transcription direction: a transcription and translation initiation region (i.e., a promoter), the DNA sequence of the embodiment, and a transcription and translation termination region (i.e., a termination region) that functions in an organism as a host. For the host organism and / or sequence of the embodiment, the transcription initiation region (i.e., a promoter) can be natural, similar, exogenous, or heterologous. In addition, the promoter can be a native sequence, or alternatively, a synthetic sequence. As used herein, the term "exogenous" means that the promoter is not found in the natural organism into which the promoter is introduced. In the case where the promoter is "exogenous" or "heterologous" for the sequence of the embodiment, it refers to a promoter that is not a natural or naturally occurring promoter for the operably connected sequence of the embodiment. As used herein, a chimeric gene comprises a coding sequence operably connected to a transcription initiation region, and the transcription initiation region is heterologous to the coding sequence. When the promoter is a native (native or natural) sequence, the expression of the operably connected sequence changes from wild-type expression, which results in a change in phenotype.

[0128] The termination region may be native to the transcriptional initiation region, may be native to the operably linked DNA sequence of interest, may be native to the plant host, or may be derived from another source (i.e., foreign or heterologous to the promoter, sequence of interest, plant host, or any combination thereof).

[0129] Convenient termination regions are available from the Ti-plasmid of A. tumefaciens, such as the octopine synthase and nopaline synthase termination regions. See Guerineau et al. (1991) Mol. Gen. Genet. 262:141-144; Proudfoot (1991) Cell 64:671-674; Sanfacon et al. (1991) Genes Dev. 5:141-149; Mogen et al. (1990) Plant Cell 2:1261-1272; Munroe et al. (1990) Gene, 91:151-158; Ballas et al. (1989) Nucleic Acids Res. 17:7891-7903; and Joshi et al. (1987) Nucleic Acid Res. 15:9627-9639.

[0130] When appropriate, nucleic acid can be optimized to increase expression in the host organism. Therefore, when the host organism is a plant, synthetic nucleic acids can be synthesized using plant preference codons to improve expression. For discussion of the use of host preference codons, see, for example, Campbell and Gowri (1990) Plant Physiol. [Plant Physiology] 92: 1-11. For example, although the nucleotide sequence of the embodiment can be expressed in monocotyledonous and dicotyledonous plant species, the sequence can be modified to explain the codon preference and GC content preference specific to monocotyledonous or dicotyledonous plants, because these preferences have shown differences (Murray et al. (1989) Nucleic Acids Res. [Nucleic Acids Research] 17: 477-498). Thus, the maize preference codons of specific amino acids can be derived from the known gene sequences of maize. The maize codons from 28 kinds of genes of maize plants are used in Murray et al., and are listed in Table 4 (ibid). Methods for synthesizing plant-preferred genes are available in the art. See, for example, U.S. Pat. Nos. 5,380,831 and 5,436,391, and Murray et al. (1989) Nucleic Acids Res. 17:477-498, incorporated herein by reference.

[0131] It is known that there are other sequence modifications that can enhance gene expression in a cell host. These include eliminating the following sequences: sequences encoding false polyadenylation signals, sequences encoding exon-intron splice site signals, sequences encoding transposon-like repetitive sequences, and other sequences that are fully characterized and may be detrimental to gene expression. The GC content of the sequence can be adjusted to the average level of a given cell host, as calculated by reference to known genes expressed in the host cell. As used herein, the term "host cell" refers to a cell that contains a vector and supports replication and / or expression of an expression vector. The host cell can be a prokaryotic cell such as Escherichia coli, or a eukaryotic cell such as yeast, insect, amphibian or mammalian cell, or a monocot or dicot plant cell. The example of a monocot host cell is a maize host cell. When possible, the modified sequence is to avoid the occurrence of predictable hairpin secondary mRNA structures.

[0132] The expression cassette may additionally comprise a 5' leader sequence. Such leader sequences may serve to enhance translation. Translation leader sequences are known in the art and include: picornavirus leader sequences, e.g., EMCV leader sequence (encephalomyocarditis 5' non-coding region) (Elroy-Stein et al. (1989) Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences of the United States] 86: 6126-6130); Potyvirus leader sequences, e.g., TEV leader sequence (tobacco etch virus) (Gallie et al. (1995) Gene [Gene] 165 (2): 233-238); MDMV leader sequence (maize dwarf mosaic virus), human immunoglobulin heavy chain binding protein (BiP) (Macejak et al. (1991) Nature [Nature] 353: 90-94); non-translated leader sequence from coat protein mRNA of alfalfa mosaic virus (AMV RNA 4) (Jobling et al. (1987) Nature 325:622-625); tobacco mosaic virus leader sequence (TMV) (Gallie et al. (1989) in Molecular Biology of RNA, ed. Cech (Liss, New York), pp. 237-256); and maize chlorotic mottle virus leader sequence (MCMV) (Lommel et al. (1991) Virology 81:382-385). See also, Della-Cioppa et al., (1987) Plant Physiol. 84:965-968.

[0133] In preparing the expression cassette, the various DNA fragments may be manipulated to provide a DNA sequence in the proper orientation and, where appropriate, in the proper reading frame. To this end, adapters or linkers may be employed to connect the DNA fragments, or other manipulations may be involved to provide convenient restriction sites, remove excess DNA, remove restriction sites, etc. For this purpose, in vitro mutagenesis, primer repair, restriction, annealing, resubstitution (e.g., conversion and transversion) may be involved.

[0134] Many promoters can be used to implement the embodiments. The promoter can be selected based on the desired results. The nucleic acid can be combined with a constitutive promoter, a tissue-preferred promoter, an inducible promoter, or other promoters for expression in a host organism. Suitable constitutive promoters for use in plant host cells include, for example, the core promoter of the Rsyn7 promoter and other constitutive promoters disclosed in WO 99 / 43838 and U.S. Patent No. 6,072,050; the core CaMV 35S promoter (Odell et al., (1985) Nature 313:810-812); rice actin (McElroy et al., (1990) Plant Cell 2:163-171); ubiquitin (Christensen et al., (1989) Plant Mol. Biol. 12:619-632 and Christensen et al., (1992) Plant Mol. Biol. 13:619-632). Mol. Biol. [Plant Molecular Biology] 18: 675-689); pEMU (Last et al., (1991) Theor. Appl. Genet. [Theoretical and Applied Genetics] 81: 581-588); MAS (Velten et al., (1984) EMBO J. [Journal of the European Molecular Biology Association] 3: 2723-2730); ALS promoter (U.S. Pat. No. 5,659,026), etc. Other constitutive promoters include, for example, those discussed in U.S. Pat. Nos. 5,608,149; 5,608,144; 5,604,121; 5,569,597; 5,466,785; 5,399,680; 5,268,463; 5,608,142; and 6,177,611.

[0135] Depending on the desired outcome, it may be beneficial to express the gene from an inducible promoter. Of particular interest for use in regulating expression of the nucleotide sequences of the embodiments in plants is a wound-inducible promoter. Such wound-inducible promoters may respond to damage caused by insect feeding and include potato protease inhibitors (PPIs). II) genes (Ryan (1990) Ann. Rev. Phytopath. [Annual Review of Plant Pathology] 28:425-449; Duan et al. (1996) Nature Biotechnology [Nature Biotechnology] 14:494-498); wun1 and wun2 (U.S. Patent No. 5,428,148); win1 and win2 (Stanford et al. (1989) Mol. Gen. Genet. [Molecular Genetics and General Genetics] 215:200-208); systemin (McGurl et al. (1992) Science [Science] 225:1570-1573); WIP1 (Rohmeier et al. (1993) Plant Mol. Biol. [Plant Molecular Biology] 22:783-792; Eckelkamp et al. (1993) FEBS Letters [European Biochemical Society Newsletter] 323: 73-76); MPI gene (Corderok et al. (1994) Plant J. [Plant Journal] 6 (2): 141-150), etc., which are incorporated herein by reference.

[0136] In addition, pathogen-inducible promoters can be used in the methods and nucleotide constructs of the embodiments. Such pathogen-inducible promoters include those from pathogenesis-related proteins (PR proteins) (e.g., PR proteins, SAR proteins, β-1,3-glucanase, chitinase, etc.). See, for example, Redolfi et al. (1983) Neth. J. Plant Pathol. [Dutch Journal of Plant Pathology] 89: 245-254; Uknes et al. (1992) Plant Cell [Plant Cell] 4: 645-656; and Van Loon (1985) Plant Mol. Virol. [Plant Molecular Virology] 4: 111-116. See also, WO 99 / 43819, which is incorporated herein by reference.

[0137] Of interest are promoters that are expressed locally at or near the site of pathogen infection. See, for example, Marineau et al., (1987) Plant Mol. Biol. 9:335-342; Matton et al., (1989) Molecular Plant-Microbe Interactions 2:325-331; Somsisch et al., (1986) Proc. Natl. Acad. Sci. USA 83:2427-2430; Somsisch et al., (1988) Mol. Gen. Genet. 2:93-98; and Yang (1996) Proc. Natl. Acad. Sci. USA 93:14972-14977. See also Chen et al., (1996) Plant J. 10:955-966; Zhang et al., (1994) Proc. Natl. Acad. Sci. USA 91:2507-2511; Warner et al., (1993) Plant J. 3:191-201; Siebertz et al., (1989) Plant Cell 1:961-968; U.S. Pat. No. 5,750,386 (inducible nematodes); and references cited therein. Of particular interest is the inducible promoter of the maize PRms gene, expression of which is induced by the pathogen Fusarium moniliforme (see, eg, Cordero et al., (1992) Physiol. Mol. Plant Path. 41: 189-200).

[0138] Chemically regulated promoters can be used to regulate gene expression in plants by using exogenous chemical regulators. Depending on the target, the promoter can be a chemically induced promoter when the application chemical induces gene expression, or the promoter can be a chemically repressed promoter when the application chemical suppresses gene expression. Chemically induced promoters are known in the art, and include but are not limited to the maize In2-2 promoter activated by benzenesulfonamide herbicide safeners, the maize GST promoter activated by the hydrophobic electrophilic compound used as pre-emergence herbicide, and the tobacco PR-1a promoter activated by salicylic acid. Other chemical-regulated promoters of interest include steroid-responsive promoters (see, e.g., Schena et al., (1991) Proc. Natl. Acad. Sci. USA 88:10421-10425 and McNellis et al., (1998) Plant J. 14(2):247-257 for glucocorticoid-inducible promoters) and tetracycline-inducible and tetracycline-repressible promoters (see, e.g., Gatz et al., (1991) Mol. Gen. Genet. 227:229-237, and U.S. Pat. Nos. 5,814,618 and 5,789,156), which are incorporated herein by reference.

[0139] Tissue-preferred promoters can be used to target enhanced pesticidal protein expression within specific plant tissues. Tissue-preferred promoters include those discussed in: Yamamoto et al. (1997) Plant J. 12(2):255-265; Kawamata et al. (1997) Plant Cell Physiol. 38(7):792-803; Hansen et al. (1997) Mol. Gen Genet. 254(3):337-343; Russell et al. (1997) Transgenic Res. 6(2):157-168; Rinehart et al. (1996) Plant Physiol. 112(3):1331-1341; Van Camp et al. (1996) Plant Physiol. 112(2):525-535; Canevascini et al. (1996) Plant Physiol. Physiol. 112(2):513-524; Yamamoto et al. (1994) Plant Cell Physiol. 35(5):773-778; Lam (1994) Results Probl. Cell Differ. 20:181-196; Orozco et al. (1993) Plant Mol Biol. 23(6):1129-1138; Matsuoka et al. (1993) Proc Natl. Acad. Sci. USA 90(20):9586-9590; and Guevara-Garcia et al. (1993) Plant J. 4(3):495-505. If necessary, such promoters can be modified for weak expression.

[0140] Leaf-preferred promoters are known in the art. See, for example, Yamamoto et al. (1997) Plant J. 12(2):255-265; Kwon et al. (1994) Plant Physiol. 105:357-67; Yamamoto et al. (1994) Plant Cell Physiol. 35(5):773-778; Gotor et al. (1993) Plant J. 3:509-18; Orozco et al. (1993) Plant Mol. Biol. 23(6):1129-1138; and Matsuoka et al. (1993) Proc. Natl. Acad. Sci. USA 90(20):9586-9590.

[0141] Root-preferred or root-specific promoters are known and can be selected from the many available promoters from the literature or re-isolated from different compatible species. See, e.g., Hire et al. (1992) Plant Mol. Biol. 20(2):207-218 (soybean root-specific glutamine synthetase gene); Keller and Baumgartner (1991) Plant Cell 3(10):1051-1061 (root-specific control elements in the GRP 1.8 gene of French bean); Sanger et al. (1990) Plant Mol. Biol. 14(3):433-443 (root-specific promoter of mannopine synthase (MAS) from Agrobacterium tumefaciens); and Miao et al. (1991) Plant Cell 3(1):11-22 (full-length cDNA clone encoding cytosolic glutamine synthetase (GS) expressed in soybean roots and root nodules). See also, Bogusz et al., (1990) Plant Cell 2(7):633-641, which describes two root-specific promoters isolated from the hemoglobin gene from the nitrogen-fixing non-legume Parasponia andersonii and the related non-nitrogen-fixing non-legume Trema tomentosa. The promoters of these genes were linked to a β-glucuronidase reporter gene and introduced into both the non-legume Nicotiana tabacum and the legume Lotus corniculatus, and in both cases retained root-specific promoter activity. Leach and Aoyagi (1991) describe their analysis of the promoters of the highly expressed rolC and rolD root-inducible genes of Agrobacterium rhizogenes (see Plant Science (Limerick) 79(1):69-76). They concluded that the enhancer and tissue-preferential DNA determinants were dissociated in the promoter. Teeri et al. (1989) used gene fusions with lacZ to show that the Agrobacterium T-DNA gene encoding octopine synthase was active especially in the epidermis of the root tip, and that the TR2' gene was root-specific in intact plants and stimulated by wounding in leaf tissue, a combination of characteristics particularly desirable for use with insecticidal or larvicidal genes (see, EMBO J. [Journal of the European Molecular Biology Association] 8(2): 343-350). The TR1' gene fused with nptII (neomycin phosphotransferase II) showed similar characteristics.Additional root-preferred promoters include the VfENOD-GRP3 gene promoter (Kuster et al., (1995) Plant Mol. Biol. 29(4):759-772); and the rolB promoter (Capana et al., (1994) Plant Mol. Biol. 25(4):681-691. See also U.S. Pat. Nos. 5,837,876; 5,750,386; 5,633,363; 5,459,252; 5,401,836; 5,110,732; and 5,023,179.

[0142] "Seed-preferred" promoters include "seed-specific" promoters (those active during seed development, such as promoters of seed storage proteins) and "seed germination" promoters (those active during seed germination). See, Thompson et al., (1989) BioEssays [Biological Assays] 10: 108, which is incorporated herein by reference. Such seed-preferred promoters include, but are not limited to, Cim1 (cytokinin-induced signal); cZ19B1 (maize 19 kDa zein); and milps (inositol-1-phosphate synthase) (see U.S. Patent No. 6,225,529, incorporated herein by reference). γ-zein and Glob-1 are endosperm-specific promoters. For dicotyledons, seed-specific promoters include, but are not limited to, bean β-phaseolin, napin, β-conglycinin, soybean lectin, cruciferin, etc. For monocots, seed-specific promoters include, but are not limited to, maize 15 kDa zein, 22 kDa zein, 27 kDa zein, g-zein, wax, contractin 1, contractin 2, globulin 1, and the like. See also WO 00 / 12733, which discloses seed-preferred promoters from end1 and end2 genes; incorporated herein by reference. Promoters with "preferential" expression in a particular tissue are expressed to a greater extent in the tissue than in at least one other plant tissue. Some tissue-preferred promoters are expressed almost exclusively in a particular tissue.

[0143] When low-level expression is needed, a weak promoter can be used. Generally, as used herein, the term "weak promoter" refers to a promoter that drives the expression of a coding sequence at a low level. Low-level expression refers to a level of about 1 / 1000 transcripts to about 1 / 100,000 transcripts to about 1 / 500,000 transcripts. Alternatively, it should be appreciated that the term "weak promoter" also encompasses promoters that drive expression only in a few cells but not in other cells, thereby having a low-level total expression. When a promoter drives expression at an unacceptably high level, a portion of the promoter sequence can be deleted or modified to reduce the level of expression.

[0144] Such weak constitutive promoters include, for example: the core promoter of the Rsyn7 promoter (WO 99 / 43838 and U.S. Pat. No. 6,072,050), the core 35S CaMV promoter, etc. Other constitutive promoters include, for example, those disclosed in the following U.S. Pat. Nos.: 5,608,149; 5,608,144; 5,604,121; 5,569,597; 5,466,785; 5,399,680; 5,268,463; 5,608,142; and 6,177,611; incorporated herein by reference.

[0145] Typically, the expression cassette will contain a selective marker gene for selecting transformed cells. Selectable marker genes are used to select transformed cells or tissues. Marker genes include genes encoding antibiotic resistance, such as genes encoding neomycin phosphotransferase II (NEO) and hygromycin phosphotransferase (HPT), and genes that confer resistance to herbicidal compounds such as glufosinate, bromoxynil, imidazolinone, and 2,4-dichlorophenoxyacetic acid (2,4-D). Other examples of suitable selectable marker genes include, but are not limited to, genes encoding resistance to chloramphenicol (Herrera Estrella et al., (1983) EMBO J. [Journal of the European Molecular Biology Association] 2:987-992); methotrexate (Herrera Estrella et al. (1983) Nature 303:209-213 and Meijer et al. (1991) Plant Mol. Biol. [Plant Molecular Biology] 16:807-820); streptomycin (Jones et al. (1987) Mol. Gen. Genet. [Molecular Genetics and General Genetics] 210:86-91); spectinomycin (Bretagne-Sagnard et al. (1996) Transgenic Res. [Transgenic Research] 5:131-137); bleomycin (Hille et al. (1990) Plant Mol. Biol. [Plant Molecular Biology] 16:807-820); Mol. Biol. 7:171-176); sulfonamides (Guerineau et al. (1990) Plant Mol. Biol. 15:127-136); bromoxynil (Stalker et al. (1988) Science 242:419-423); glyphosate (Shaw et al. (1986) Science 233:478-481; and U.S. Pat. Nos. 7,709,702 and 7,462,481); glufosinate (DeBlock et al. (1987) EMBO J. 6:2513-2518). See generally Yarranton (1992) Curr. Opin. Biotech. 3:506-511; Christopherson et al. (1992) Proc. Natl. Acad. Sci. USA 89:6314-6318; Yao et al. (1992) Cell 71:63-72; Reznikoff (1992) Mol. Microbiol.[Molecular Microbiology] 6:2419-2422; Barkley et al., (1980) The Operon, pp. 177-220; Hu et al., (1987) Cell 48:555-566; Brown et al., (1987) Cell 49:603-612; Figge et al., (1988) Cell 52:713-722; Deuschle et al., (1989) Proc. Natl. Acad. Sci. USA 86:5400-5404; Fuerst et al., (1989) Proc. Natl. Acad. Sci. USA 86:2549-2553; Deuschle et al., (1990) Science 248:480-483; Gossen (1993) Ph.D. Thesis, University of Heidelberg [University of Heidelberg, Germany]; Reines et al., (1993) Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences of the United States of America] 90:1917-1921; Labow et al., (1990) Mol. Cell. Biol. [Molecular and Cellular Biology] 10:3343-3356; Zambretti et al., (1992) Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences of the United States of America] 89:3952-3956; Baim et al., (1991) Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences of the United States of America] 88:5072-5076; Wyborski et al., (1991) Nucleic Acids Res. [Nucleic Acids Research] 19:4647-4653; Hillenand-Wissman (1989) Topics Mol. Struc. Biol. [Hotspot Mol. Structural Biology] 10:143-162; Degenkolb et al., (1991) Antimicrob. Agents Chemother. [Antimicrob Agents and Chemotherapy] 35:1591-1595; Kleinschnidt et al., (1988) Biochemistry [Biochemistry] 27:1094-1104; Bonin (1993) Ph.D. Thesis [Doctoral Dissertation] University of Heidelberg [Germany Heidelberg University]; Gossen et al., (1992) Proc. Natl. Acad. Sci.USA [Proceedings of the National Academy of Sciences of the United States of America] 89:5547-5551; Oliva et al., (1992) Antimicrob. Agents Chemother. [Antimicrob agents and chemotherapy] 36:913-919; Hlavka et al., (1985) Handbook of Experimental Pharmacology [Handbook of Experimental Pharmacology], Vol. 78 (Springer-Verlag, Berlin); and Gill et al., (1988) Nature [Nature] 334:721-724. Such disclosures are incorporated herein by reference. .

[0146] The above list of selectable marker genes is not intended to be limiting. Any selectable marker gene can be used in the embodiments.

[0147] The methods of these embodiments involve introducing a polypeptide or polynucleotide into a plant. "Introducing" is intended to mean providing a polynucleotide or polypeptide into a plant in such a way that the sequence is able to enter the interior of a cell of the plant. The methods of these embodiments do not depend on a specific method for introducing a polynucleotide or polypeptide into a plant, as long as the polynucleotide or polypeptide enters the interior of at least one cell of the plant. Methods for introducing a polynucleotide or polypeptide into a plant are known in the art, including but not limited to stable transformation methods, transient transformation methods, and virus-mediated methods.

[0148] "Stable transformation" is intended to mean that a nucleotide construct introduced into a plant is integrated into the genome of the plant and can be inherited by its progeny. "Transient transformation" is intended to mean that a polynucleotide is introduced into a plant and does not integrate into the genome of the plant, or that a polypeptide is introduced into a plant.

[0149] Transformation protocols and protocols for introducing nucleotide sequences into plants can vary depending on the type of plant or plant cell (ie, monocot or dicot) being targeted for transformation. Suitable methods for introducing nucleotide sequences into plant cells and subsequent insertion into the plant genome include microinjection (Crossway et al., (1986) Biotechniques 4:320-334), electroporation (Riggs et al., (1986) Proc. Natl. Acad. Sci. USA 83:5602-5606), Agrobacterium-mediated transformation (U.S. Pat. Nos. 5,563,055 and 5,981,840), direct gene transfer (Paszkowski et al., (1984) EMBO J 3:2717-2722), and ballistic particle acceleration (see, e.g., U.S. Pat. Nos. 4,945,050, 5,879,918, 5,886,244, and 5,932,782; Tomes et al., (1995) Plant Cell, Tissue, and Organ Culture: Fundamental Biology. Methods [Plant Cell, Tissue and Organ Culture: Basic Methods], ed. Gamborg and Phillips (Springer-Verlag, Berlin); and McCabe et al., (1988) Biotechnology 6:923-926); and Lec1 transformation (WO 00 / 28058). For potato transformation methods, see Tu et al., (1998) Plant Molecular Biology 37:829-838 and Chong et al., (2000) Transgenic Research 9:71-78. Additional transformation methods can be found in Weissinger et al. (1988) Ann. Rev. Genet. 22:421-477; Sanford et al. (1987) Particulate Science and Technology 5:27-37 (onion); Christou et al. (1988) Plant Physiol. 87:671-674 (soybean); McCabe et al. (1988) Bio / Technology 6:923-926 (soybean); Finer and McMullen (1991) In Vitro Cell Dev. Biol.In Vitro Cell Biology and Developmental Biology 27P:175-182 (soybean); Singh et al. (1998) Theor. Appl. Genet. 96:319-324 (soybean); Datta et al. (1990) Biotechnology 8:736-740 (rice); Klein et al. (1988) Proc. Natl. Acad. Sci. USA 85:4305-4309 (maize); Klein et al. (1988) Biotechnology 6:559-563 (maize); U.S. Patents 5,240,855, 5,322,783 and 5,324,646; Klein et al. (1988) Plant Genet. Physiol. 91:440-444 (maize); Fromm et al. (1990) Biotechnology 8:833-839 (maize); Hooykaas-Van Slogteren et al. (1984) Nature (London) 311:763-764; U.S. Pat. No. 5,736,369 (cereals); Bytebier et al. (1987) Proc. Natl. Acad. Sci. USA 84:5345-5349 (Liliaceae); De Wet et al. (1985) The Experimental Manipulation of Ovule Tissues [Experimental Manipulation of Ovule Tissues], Chapman et al., eds. (Longman, New York), pp. 197-209 (pollen); Kaeppler et al., (1990) Plant Cell Reports [Plant Cell Reports] 9:415-418 and Kaeppler et al., (1992) Theor. Appl. Genet.Theoretical and Applied Genetics 84:560-566 (whisker-mediated transformation); D'Halluin et al., (1992) Plant Cell 4:1495-1505 (electroporation); Li et al., (1993) Plant Cell Reports 12:250-255 and Christou and Ford, (1995) Annals of Botany 75:407-413 (rice); Osjoda et al., (1996) Nature Biotechnology 14:745-750 (maize via Agrobacterium tumefaciens); all of which are incorporated herein by reference.

[0150] In certain embodiments, various transient transformation methods can be used to provide the sequences of these embodiments to plants. Such transient transformation methods include, but are not limited to, introducing Cry toxin proteins or variants and fragments thereof directly into plants or introducing Cry toxin transcripts into plants. Such methods include, for example, microinjection or particle bombardment. See, for example, Crossway et al., (1986) Mol Gen. Genet. [Molecular Genetics and General Genetics] 202: 179-185; Nomura et al., (1986) Plant Sci. [Plant Science] 44: 53-58; Hepler et al., (1994) Proc. Natl. Acad. Sci. [Proceedings of the National Academy of Sciences of the United States] 91: 2176-2180 and Hush et al., (1994) The Journal of Cell Science [Cell Science Magazine] 107: 775-784, all of which are incorporated herein by reference. Alternatively, Cry toxin polynucleotides can be transiently transformed into plants using techniques known in the art. Such techniques include viral vector systems, and precipitating polynucleotides in a manner that prevents subsequent release of the DNA. Thus, transcription can occur from particle-bound DNA, but the frequency of its release for integration into the genome is greatly reduced. Such methods include the use of particles coated with polyethyleneimine (PEI; Sigma #P3143).

[0151] The method for inserting polynucleotides in the specific position targeting of plant genome is known in the art. In one embodiment, the insertion of polynucleotides at the required genome position is realized by utilizing site-specific recombination system. See, for example, WO 99 / 25821, WO 99 / 25854, WO 99 / 25840, WO 99 / 25855 and WO 99 / 25853, which are all incorporated herein by reference. In short, the polynucleotides of the present embodiment can be included in the transfer box of two different recombination sites of side joints. The transfer box is introduced into the plant, and the plant has stably incorporated the target site into its genome, and the target site flanks two different recombination sites corresponding to the site of the transfer box. Suitable recombinase is provided, and the transfer box is integrated into the target site. Thus, the purpose polynucleotide is integrated into the specific chromosome position in the plant genome.

[0152] The transformed cells can be grown into plants in a conventional manner. See, for example, McCormick et al., (1986) Plant Cell Reports 5:81-84. These plants can then be grown and pollinated with the same transformed strain or a different strain, and the resulting hybrids with constitutive or inducible expression of the desired phenotypic characteristic identified. Two or more generations can be grown to ensure that expression of the desired phenotypic characteristic is stably maintained and inherited, and then seeds are harvested to ensure that expression of the desired phenotypic characteristic has been achieved.

[0153] The nucleotide constructs of the embodiments can be provided to plants by contacting the plants with viruses or viral nucleic acids. Typically, such methods involve incorporating the target nucleotide construct into viral DNA or RNA molecules. It has been recognized that the recombinant protein of the embodiments may initially be synthesized as part of a viral polyprotein, which may then be processed by in vivo or in vitro proteolysis to produce the desired pesticidal protein. It is also recognized that such a viral polyprotein comprising at least a portion of the amino acid sequence of the pesticidal protein of the embodiments may have the desired pesticidal activity. Such viral polyproteins and the nucleotide sequences encoding them are encompassed in the embodiments. Methods for providing plants with nucleotide constructs and producing encoded proteins in plants are known in the art and involve viral DNA or RNA molecules. See, for example, U.S. Patent Nos. 5,889,191; 5,889,190; 5,866,785; 5,589,367; and 5,316,931; incorporated herein by reference.

[0154] The embodiments further relate to plant propagation material of the transformed plants of the embodiments, including but not limited to seeds, tubers, corms, bulbs, leaves, and root and shoot cuttings.

[0155] These embodiments can be used to transform any plant species, including but not limited to monocots and dicots. Examples of plants of interest include, but are not limited to, corn (Zea mays), Brassica species (Brassica sp.) (e.g., B. napus, B. rapa, B. juncea) (particularly those Brassica species that can be used as a source of seed oil), alfalfa (Medicago sativa), rice (Oryza sativa), rye (Secale cereale), sorghum (Sorghum bicolor, Sorghum vulgare), millet (e.g., pearl millet (Pennisetum glaucum), millet (Panicum miliaceum), foxtail millet (Setaria italica), millet (Eleusine coracana), sunflower (Helianthus annuus), safflower (Carthamus tinctorius, wheat (Triticum aestivum), soybean (Glycine max), tobacco (Nicotiana tabacum), potato (Solanumtuberosum), peanut (Arachis hypogaea), cotton (Gossypium barbadense, Gossypium hirsutum), sweet potato (Ipomoea batatas), cassava (Manihotesculenta), coffee (Coffea spp.), coconut (Cocos nucifera), pineapple (Ananas comosus), citrus (Citrus spp.), cocoa (Theobroma cacao), tea (Camellia sinensis), banana (Musa spp.), avocado (Persea americana), fig (Ficus casica), guava (Psidiumguajava), mango (Mangifera indica), olive (Olea europaea), papaya (Carica papaya), cashew (Anacardium occidentale), macadamia (Macadamia integrifolia), almond (Prunus amygdalus), sugar beets (Betavulgaris), sugarcane (Saccharum spp.), oats, barley, vegetables, ornamentals and conifers.

[0156] Vegetables include tomatoes (Lycopersicon esculentum), lettuce (e.g., Lactucasativa), green beans (Phaseolus vulgaris), lima beans (Phaseolus limensis), peas (Lathyrus spp.), and members of the genus Cucumber such as cucumber (C. sativus), cantaloupe (C. cantalupensis), and musk melon (C. melo). Ornamental plants include azaleas (Rhododendron species), hydrangea (Macrophylla hydrangea), hibiscus (Hibiscus rosasanensis), roses (Rosa species), tulips (Tulipa species), daffodils (Narcissus species), petunias (Petunia hybrida), carnations (Dianthus caryophyllus), poinsettias (Euphorbia pulcherrima), and chrysanthemums. Conifers that can be used to practice embodiments include, for example, pines such as loblolly pine (Pinus taeda), slash pine (Pinus elliotii), ponderosa 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); true firs such as silver fir (Abies amabilis) and balsam fir (Abies balsamea); and cedars such as western red cedar (Thuja plicata) and Alaska yellow cedar (Chamaecyparis nootkatensis).Plants of the embodiments include crop plants, including, but not limited to, corn, alfalfa, sunflower, Brassica species, soybean, cotton, safflower, peanut, sorghum, wheat, millet, tobacco, sugarcane, and the like.

[0157] Turfgrasses include, but are not limited to: annual bluegrass (Poa annua); annual ryegrass (Lolium multiflorum); Canada bluegrass (Poa compressa); Chewings fescue (Festuca rubra); colonial bentgrass (Agrostis tenuis); creeping bentgrass (Agrostis palustris); crested wheatgrass (Agropyron desertorum); fairway wheatgrass (Agropyron cristatum); hard fescue (Festuca longifolia); Kentucky bluegrass (Poa pratensis); orchardgrass (Dactylis glomerata; perennial ryegrass (Lolium perenne); red foxgrass (Festuca rubra); redtop (Agrostis alba); rough bluegrass (Poa trivialis); sheep fescue (Festuca ovina); smooth bromegrass (Bromus inermis); tall fescue (Festuca arundinacea); timothy (Phleum pratense); velvet bentgrass (Agrostis canina); weeping alkaligrass (Puccinellia distans); western wheatgrass (Agropyron smithii); Cynodon dactylon (Cynodon species); St. Augustineweed (St.Augustine grass (Stenotaphrum secundatum); Zoysia grass (Zoysia species); Bahia grass (Paspalum notatum); Carpetgrass (Axonopus affinis); Centipede grass (Eremochloa ophiuroides); Kikuyu grass (Pennisetumclandesinum); Seashore paspalum (Paspalumvaginatum); Blue gramma (Bouteloua gracilis); Buffalo grass (Buchloe dactyloids); Sideoats gramma (Bouteloua curtipendula).

[0158] Target plants include cereal plants, oil seed plants and legumes that provide target seeds. Target seeds include cereal seeds, such as corn, wheat, barley, rice, sorghum, rye, millet, etc. Oil seed plants include cotton, soybean, safflower, sunflower, Brassica, maize, alfalfa, palm, coconut, flax, castor, olive, etc. Legumes include beans and peas. Legumes include guar beans, locust beans, fenugreek, soybeans, French beans, cowpeas, mung beans, lima beans, broad beans, lentils, chickpeas, etc.

[0159] In certain embodiments, the nucleic acid sequences of the embodiments can be stacked with any combination of target polynucleotide sequences to produce plants with desired phenotypes. For example, the polynucleotides of the embodiments can be stacked with any other polynucleotides encoding polypeptides with pesticidal and / or insecticidal activity, such as other Bt toxin proteins (described in U.S. Pat. Nos. 5,366,892; 5,747,450; 5,736,514; 5,723,756; 5,593,881; and Geiser et al. (1986) Gene 48:109), pentin (described in U.S. Pat. No. 5,981,722), etc. The resulting combination can also include multiple copies of any one of the target polynucleotides. The polynucleotides of the embodiments can also be stacked with any other gene or combination of genes to produce plants with various desired trait combinations, including but not limited to desirable traits for animal feed such as high oil genes (e.g., U.S. Pat. No. 6,232,529); balanced amino acids (e.g., hordothionin (U.S. Pat. Nos. 5,990,389; 5,885,801; 5,885,802; and 5,703,049); barley high lysine (Williamson et al. (1987) Eur. J. Biochem. [European Journal of Biochemistry] 165:99-106; and WO 98 / 20122) and high methionine proteins (Pedersen et al. (1986) J. Biol. Chem. [Journal of Biological Chemistry] 261:6279; Kirihara et al. (1988) Gene [Gene] 71:359; and Musumura et al. (1989) Plant Mol. Biol. [Plant Molecular Biology] 12:123)); increased digestibility (e.g., improved storage proteins (U.S. Patent 6,858,778)); and thioredoxin (U.S. Patent No. 7,009,087), the disclosures of which are incorporated herein by reference.

[0160] The polynucleotides of the embodiments can also be stacked with the following traits: desirable traits for disease or herbicide resistance (e.g., fumonisin detoxification genes (U.S. Pat. No. 5,792,931); avirulence and disease resistance genes (Jones et al., (1994) Science 266:789; Martin et al., (1993) Science 262:1432; and Mindrinos et al. (1994) Cell 78:1089); traits that cause herbicide resistance acetolactate synthase (ALS) mutants, such as S4 and / or Hra mutations; glutamine synthase inhibitors, such as glufosinate or Basta (e.g., bar gene); and glyphosate resistance (such as EPSPS genes and GAT genes disclosed in U.S. Pat. Nos. 7,709,702 and 7,462,481; and characteristics desired for processing or processed products, such as high oil (e.g., U.S. Pat. No. 6,232,529); modified oils (e.g., fatty acid desaturase genes (U.S. Pat. Nos. 5,952,544; WO 94 / 11516)); modified starch (e.g., ADPG pyrophosphatase (AGPase), starch synthase (SS), starch branching enzyme (SBE) and starch debranching enzyme (SDBE)); and polymers or bioplastics (e.g., U.S. Pat. No. 5,602,321); β-ketothiolase, polyhydroxybutyrate synthase and acetoacetyl-CoA reductase (Schubert et al., (1988) J. Bacteriol. [Journal of Bacteriology] 170: 5837-5847) promote the expression of polyhydroxyalkanoates (PHA)), the disclosure of which is incorporated herein by reference. The polynucleotides of the embodiments can also be used with plants that provide agronomic traits (e.g., male sterility (e.g., see U.S. Pat. No. 5.583,210), stem strength, flowering time or transformation technology traits (e.g., cell cycle regulation or gene targeting (e.g., WO 99 / 61619; WO 00 / 17364; WO 99 / 25821)), the disclosure of which is incorporated herein by reference.

[0161] In one embodiment, the stacked traits can be traits or events that have received regulatory approval, which are well known to those skilled in the art and can be found at the Center for Environmental Risk Assessment (cera-gmc.org / ?action=gm_crop_database, which can be accessed using the www prefix) and at the International Service for the Acquisition of Agri-biotech Applications (isaaa.org / gmapprovaldatabase / default.asp, which can be accessed using the www prefix).

[0162] These stacked combinations can be produced by any method, including but not limited to: by any conventional or Plant cross breeding or genetic transformation carried out by the method.If these proterties are stacked by genetic transformation to plants, the purpose polynucleotide sequence can be combined at any time and in any order.For example, a transgenic plant comprising one or more desired proterties can be used as a target to introduce other proterties by subsequent conversion.The purpose polynucleotide provided by any combination of the proterties and the transformation box can be introduced with a co-transformation scheme.For example, if two sequences are introduced, the two sequences can be included in a separate transformation box (trans) or included in the same transformation box (cis).The expression of the sequence can be driven by the same promoter or by different promoters.In some cases, it may be necessary to introduce a transformation box that will suppress the expression of the purpose polynucleotide.This can be combined with any combination of other suppression boxes or overexpression boxes to produce a desired proterties combination in the plant.It should be further recognized that a site-specific recombination system can be used to stack polynucleotide sequences at a desired genome position. See, e.g., WO 99 / 25821, WO 99 / 25854, WO 99 / 25840, WO 99 / 25855, and WO 99 / 25853, all of which are incorporated herein by reference.

[0163] In some embodiments, the disclosed polynucleotide composition can be introduced into the genome of the plant using genome editing technology, or the polynucleotides previously introduced into the plant genome can be edited using genome editing technology. For example, the disclosed polynucleotides can be introduced into the desired position in the plant genome by using double-strand break technology (such as TALEN, large-range nucleases, zinc finger nucleases, CRISPR-Cas, etc.). For example, for the purpose of site-specific insertion, the disclosed polynucleotides can be introduced into the desired position in the genome using the CRISPR-Cas system. The required position in the plant genome can be any target site required for insertion, such as a genomic region suitable for breeding, or it can be a target site located in a genomic window with an existing target trait. The existing target trait may be an endogenous trait or a previously introduced trait.

[0164] In some embodiments, in the case where the disclosed polynucleotides have been previously introduced into the genome, genome editing techniques can be used to change or modify the introduced polynucleotide sequence. Site-specific modifications can be introduced into the disclosed polynucleotide composition, and the site-specific modifications include modifications produced using any method for introducing site-specific modifications, including but not limited to using gene repair oligonucleotides (e.g., U.S. Publication 2013 / 0019349), or by using double-strand break techniques, such as TALEN, meganucleases, zinc finger nucleases, CRISPR-Cas, etc. Such techniques can be used to modify the previously introduced polynucleotides by inserting, deleting or replacing the nucleotides in the introduced polynucleotides. Alternatively, double-strand break techniques can be used to add additional nucleotide sequences to the introduced polynucleotides. Additional sequences that can be added include additional expression elements (such as enhancer sequences and promoter sequences). In another embodiment, genome editing techniques can be used to locate additional insecticidal active proteins adjacent to the polynucleotide compositions disclosed herein in the plant genome to produce molecular stacks of insecticidal active proteins.

[0165] "Altered target site", "altered target sequence", "modified target site" and "modified target sequence" are used interchangeably herein and refer to a target sequence as disclosed herein, which comprises at least one change when compared to an unaltered target sequence. Such "changes" include, for example: (i) substitution of at least one nucleotide, (ii) deletion of at least one nucleotide, (iii) insertion of at least one nucleotide, or (iv) any combination of (i)-(iii).

[0166] The compositions of the embodiments can be applied to protect plants, seeds and plant products in various ways. For example, the compositions can be used in a method involving placing an effective amount of the pesticidal composition in a pest environment by a procedure selected from the group consisting of spraying, dusting, broadcasting or seed coating.

[0167] In plant propagation material (fruit, tuber, bulb, corm, particle, seed), particularly before seed is sold as commercial product, usually with comprising herbicide, insecticide, fungicide, bactericide, nematicide, molluscicide or several mixtures in these preparations to handle, if desired, also can be handled with other carrier, surfactant or application promotion auxiliary agent commonly used in formulation field to provide protection thereby not being damaged by bacteria, fungi or animal pest.In order to handle seed, protective agent coating can be applied to seed by dipping tuber or grain with liquid preparation or by coating it with the wet or dry preparation of combination.In addition, in special case, other methods that are applied to plant are possible, for example for the processing of bud or fruit.

[0168] Plant seeds of embodiments comprising nucleotide sequences encoding pesticidal proteins of embodiments can be treated with seed protectant coatings comprising seed treatment compounds, including, for example, captan, carboxin, thiram, methalaxyl, methyl pirimiphos, and other agents commonly used for seed treatment. In one embodiment, the seed protectant coating comprising the pesticidal composition of embodiments is used alone or in combination with one of the seed protectant coatings commonly used for seed treatment.

[0169] It is known that genes encoding pesticidal proteins can be used to transform insect pathogenic protozoa, including baculoviruses, fungi, protozoa, bacteria, and nematodes.

[0170] The gene encoding the pesticidal protein of the embodiment can be introduced into a microbial host via a suitable vector, and the host is applied to an environment or a plant or an animal. In the context of inserting nucleic acid into a cell, the term "introducing" means "transfection" or "conversion" or "transduction" and includes reference to incorporating nucleic acid into a eukaryotic or prokaryotic cell, wherein the nucleic acid can be incorporated into the genome (e.g., chromosome, plasmid, plastid or mitochondrial DNA) of the cell, converted into an autonomous replicon, or transiently expressed (e.g., transfected mRNA).

[0171] Microbial hosts known to occupy the "phyllosphere" (foliage, phyllosphere, rhizosphere and / or rhizosphere) of one or more target crops can be selected. These microorganisms are selected so as to be able to successfully compete with wild-type microorganisms in a particular environment, provide stable maintenance and expression of genes expressing pesticidal proteins, and, desirably, provide improved protection of pesticides from environmental degradation and inactivation.

[0172] Such microorganisms include bacteria, algae and fungi. Of particular interest are microorganisms, for example bacteria, such as Pseudomonas, Erwinia, Serratia, Klebsiella, Xanthomonas, Streptomyces, Rhizobium, Rhodopseudomonas, Methylius, Agrobacterium, Acetobacter, Lactobacillus, Arthrobacter, Azotobacter, Leuconostoc and Alcaligenes; fungi, in particular yeasts, such as Saccharomyces, Cryptococcus, Kluyveromyces, Sporobolomyces, Rhodotorula and Aureobasidium.Of particular interest are phyllospheric bacterial species such as Pseudomonas syringae, Pseudomonas fluorescens, Serratia marcescens, Acetobacter xylinum, Agrobacteria, Rhodopseudomonas spheroides, Xanthomonas campestris, Rhizobium melioti, Alcaligenes entrophus, Clavibacter xyli and Azotobacter vinelandii. vinelandii), and phyllosphere yeast species such as Rhodotorularubra, R. glutinis, R. marina, R. aurantiaca, Cryptococcus albidus, C. diffluens, C. laurentii, Saccharomyces rosei, S. pretoriensis, S. cerevisiae, Sporobolomyces roseus, S. odorus, Kluyveromyces veronae, and Aureobasidium pollulans. Of particular interest are colored microorganisms.

[0173] Under conditions that allow stable maintenance and expression of genes, many methods can be obtained to introduce genes expressing pesticidal proteins into microbial hosts. For example, expression cassettes can be constructed that include a target nucleotide construct operably connected to transcription and translation regulatory signals for expressing the nucleotide construct, and a nucleotide sequence homologous to a sequence in the host organism (where integration will occur), and / or a functional replication system in the host (where integration or stable maintenance will occur).

[0174] Transcriptional and translational regulatory signals include, but are not limited to, promoters, transcription start sites, operators, activators, enhancers, other regulatory elements, ribosome binding sites, start codons, termination signals, etc. See, e.g., U.S. Pat. Nos. 5,039,523 and 4,853,331; EPO 0480762A2; Sambrook; Maniatis et al. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York); Davis et al., eds. (1980) Advanced Bacterial Genetics (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York), and references cited therein.

[0175] Suitable host cells (wherein when the treated cells are applied to the environment of one or more target pests, the cells containing the pesticidal protein will be treated to prolong the activity of the pesticidal protein in the cell) can include prokaryotes or eukaryotes, usually limited to those cells that do not produce toxic substances to higher organisms (such as mammals). However, organisms that produce toxic substances to higher organisms can be used, wherein the toxin is unstable or its application level is low enough to avoid any possibility of toxicity to the mammalian host. As hosts, prokaryotes and lower eukaryotes, such as fungi, are particularly noteworthy. Exemplary prokaryotes (Gram-negative and Gram-positive prokaryotes) include Enterobacteriaceae, such as Escherichia, Erwinia, Shigella, Salmonella, and Proteus; Bacillaceae; Rhizobiaceae, such as Rhizobium; Spirillaceae, such as Photobacterium, Fermentobacterium; Zymomonas, Serratia, Aeromonas, Vibrio, Desulfovibrio, Spirillum; Lactobacillaceae; Pseudomonadaceae, such as Pseudomonas and Acetobacter; Azotobacteraceae and Nitrobacteraceae. Fungi in eukaryotic organisms, such as Phycomycetes and Ascomycetes (including yeasts, such as Saccharomyces and Schizosaccharomyces; and Basidiomycetes yeasts, such as Rhodotorula, Aureobasidium, Sporobolomyces, etc.

[0176] Features of particular interest selected in host cells for the purpose of pesticidal protein production include ease of introduction of pesticidal protein genes into the host, availability of expression systems, efficiency of expression, stability of the protein in the host, and the presence of auxiliary gene functions. Features of interest for use as microcapsules for pesticides include protective properties for the pesticide, such as cell wall thickness, pigmentation, and formation of intracellular packaging or inclusion bodies; leaf affinity; no mammalian toxicity; attracting pest uptake; easy killing and repair without damaging the toxin; etc. Other considerations include ease of formulation and handling, economy, storage stability, etc.

[0177] Host organisms of particular interest include yeasts, such as Rhodotorula species, Atractylodes species, Saccharomyces species (e.g., Saccharomyces cerevisiae), Sporobloid Saccharomyces; foliar organisms, such as Pseudomonas species (e.g., Pseudomonas aeruginosa, Pseudomonas fluorescens), Erwinia species, and Flavobacterium spp., as well as other such organisms, including Bt, Escherichia coli, Bacillus subtilis, etc.

[0178] The gene encoding the pesticidal protein of the embodiment can be introduced into microorganisms (epiphytes) that reproduce on plants to deliver the pesticidal protein to potential target pests. The epiphytes can be, for example, Gram-positive or Gram-negative bacteria.

[0179] For example, root-colonizing bacteria can be isolated from target plants by methods known in the art. Specifically, Bacillus cereus strains that colonize roots can be isolated from the roots of plants (see, for example, Handelsman et al. (1991) Appl. Environ. Microbiol. [Application and Environmental Microbiology] 56: 713-718). Genes encoding the pest-killing proteins of the embodiments can be introduced into Bacillus cereus that colonize roots by standard methods known in the art.

[0180] Genes encoding pesticidal proteins can be introduced into the Bacillus planted in the roots, for example, by electroporation. Specifically, genes encoding pesticidal proteins can be cloned into shuttle vectors, such as pHT3101 (Lerecius et al., (1989) FEMS Microbiol. Letts. [FEMS Microbiology Communications] 60: 211-218). The shuttle vector pHT3101 containing the coding sequence of a specific pesticidal protein gene can be transformed into the Bacillus planted in the roots, for example, by electroporation (Lerecius et al., (1989) FEMS Microbiol. Letts. [FEMS Microbiology Communications] 60: 211-218).

[0181] The expression system can be designed so that the pesticidal protein is secreted outside the cytoplasm of Gram-negative bacteria such as E. coli. The advantages of having a secreted pesticidal protein are: (1) avoiding the potential cytotoxic effects of the expressed pesticidal protein; and (2) improving the purification efficiency of the pesticidal protein, including but not limited to improving the efficiency of protein recovery and purification per volume of cell culture fluid, and reducing the time and / or cost of recovery and purification per unit of protein.

[0182] The pest-killing protein can be made to be secreted in Escherichia coli, for example, by fusing a suitable Escherichia coli signal peptide to the amino terminus of the pest-killing protein. The signal peptide identified by Escherichia coli can be found in the known protein (e.g., OmpA protein) secreted in Escherichia coli (Ghrayeb et al., (1984) EMBO J [European Molecular Biology Association Journal] 3: 2437-2442). OmpA is the main protein of the outer membrane of Escherichia coli, and therefore its signal peptide is considered to be effective in the translocation process. In addition, the OmpA signal peptide does not need to be modified before processing, as for other signal peptides, for example, lipoprotein signal peptide (Duffaud et al., (1987) Meth. Enzymol. [enzymology method] 153: 492).

[0183] The pesticidal proteins of the embodiments can be fermented in bacterial hosts, and the resulting bacteria are treated and used as microbial sprays in the same manner as Bt strains have been used as insecticide sprays. In the case of one or more pesticidal proteins secreted from Bacillus, the secretion signal is removed or mutated using procedures known in the art. Such mutations and / or deletions prevent one or more pesticidal proteins from being secreted into the growth medium during the fermentation process. The pesticidal proteins are retained in the cells, and the cells are then treated to produce encapsulated pesticidal proteins. Any suitable microorganism can be used for this purpose. Pseudomonas has been used to express Bt toxins as encapsulated proteins, and the resulting cells are treated and sprayed as insecticides (Gaertner et al., (1993), in: Advanced Engineered Pesticides [Advanced Engineered Pesticides], edited by Kim).

[0184] Alternatively, the pesticidal protein is produced by introducing a heterologous gene into a cell host. The expression of the heterologous gene directly or indirectly causes the pesticide to be produced and maintained in the cell. Then, when the cell is applied to the environment of one or more target pests, the cells are processed under the conditions of extending the activity of the toxin produced in the cell. The resulting product retains the toxicity of the toxin. Then, the pesticidal protein of these natural encapsulations can be prepared according to conventional techniques for application to the environment (for example, soil, water and leaves of plants) hosting the target pest. See, for example, EP 0192319, and the document cited therein.

[0185] In an embodiment, the transformed microorganism (which includes the whole organism, cell, one or more spores, one or more pesticidal proteins, one or more pesticidal components, one or more components affecting harmful organisms, one or more mutants, living cells or dead cells and cell components, including mixtures of living cells and dead cells and cell components, and including broken cells and cell components) or separated pesticidal proteins can be formulated into one or more pesticidal compositions (i.e., such as suspensions, solutions, emulsions, dusting powders, dispersible granules or pills, wettable powders and emulsifiable concentrates, aerosols or sprays, impregnated granules, adjuvants, coating pastes, colloids) with an acceptable carrier, and are also encapsulated in, for example, polymeric substances. Such formulated compositions can be prepared by conventional methods such as drying, freeze drying, homogenization, extraction, filtration, centrifugation, precipitation or concentration of cell cultures comprising polypeptides.

[0186] The composition disclosed above can be obtained by adding surfactant, inert carrier, preservative, wetting agent, feeding stimulant, attractant, encapsulating agent, adhesive, emulsifier, dye, UV protective agent, buffer, flow agent or fertilizer, micronutrient donor or other preparations that affect plant growth. Including but not limited to one or more agricultural chemicals of herbicides, insecticides, fungicides, bactericides, nematicides, molluscicides, acaricides, plant growth regulators, harvesting aids and fertilizers can be combined with carriers, surfactants or auxiliary agents commonly used in preparations or other component fields to promote the application of product processing and specific target harmful organisms. Suitable carriers and auxiliary agents can be solid or liquid, and corresponding to the materials often used in formulation technology, such as natural or regenerated minerals, solvents, dispersants, wetting agents, tackifiers, adhesives or fertilizers. The active ingredient of the embodiment is usually applied in the form of composition, and can be applied to crop areas to be treated, plants or seeds. For example, the composition of the embodiment can be applied to grain in the preparation of barns or silos etc. or during storage. The composition of the embodiment can be applied simultaneously or successively with other compounds. The method of applying the active ingredient of the embodiment or the agricultural chemical composition of the embodiment (the composition comprises at least one of the pesticidal proteins produced by the bacterial strain of the embodiment) includes but is not limited to foliar application, seed coating and soil application. The number of applications and the speed of application depend on the intensity of the corresponding pest infection.

[0187] Suitable surfactants include, but are not limited to, anionic compounds such as carboxylates, such as metal carboxylates; carboxylates of long-chain fatty acids; N-acyl sarcosinates; monoesters or diesters of phosphoric acid with fatty alcohol ethoxylates or salts of these esters; fatty alcohol sulfates, such as sodium lauryl sulfate, sodium octadecyl sulfate or sodium hexadecyl sulfate; ethoxylated fatty alcohol sulfates; ethoxylated alkylphenol sulfates; lignin sulfonates; petroleum sulfonates; alkylaryl sulfonates, such as alkylbenzene sulfonates or lower alkylnaphthalene sulfonates, such as butylnaphthalene sulfonate; salts of sulfonated naphthalene-formaldehyde condensates; salts of sulfonated phenol-formaldehyde condensates; more complex sulfonates, such as amide sulfonates, such as the sulfonated condensation products of oleic acid and N-methyltaurine; or dialkyl sulfosuccinates, for example, sodium sulfonate of dioctyl succinate. Nonionic agents include condensation products of fatty acid esters, fatty alcohols, fatty acid amides or fatty-alkyl- or alkenyl-substituted phenols with ethylene oxide; fatty esters of polyol ethers, such as sorbitan fatty acid esters; condensation products of such esters with ethylene oxide, such as polyoxyethylene sorbitan fatty acid esters; block copolymers of ethylene oxide and propylene oxide; acetylenic diols such as 2,4,7,9-tetraethyl-5-decyne-4,7-diol or ethoxylated acetylenediol. Examples of cationic surfactants include, for example, aliphatic monoamines, diamines or polyamines, such as acetates, cyclopentaneates or oleates; or oxygen-containing amines, such as amine oxides of polyoxyethylene alkylamines; amide-linked amines prepared by condensation of carboxylic acids with diamines or polyamines; or quaternary ammonium salts.

[0188] Examples of inert materials include, but are not limited to, inorganic minerals such as kaolin, layered silicates, carbonates, sulfates, phosphates; or plant materials such as cork, powdered corn cobs, peanut shells, rice hulls, and walnut shells.

[0189] The composition of the embodiment can be in a suitable form for direct application or as a concentrate of the main composition, which requires dilution with an appropriate amount of water or other diluent before application. The pesticidal concentration will vary depending on the nature of the specific formulation (specifically, whether it is a concentrate or direct application). The composition comprises 1% to 98% of a solid or liquid inert carrier, and 0 to 50% or 0.1% to 50% of a surfactant. These compositions will be given at the label rate of commercial products, for example, about 0.01lb-5.0lb / acre when in dry form, and about 0.01pts.-10pts. / acre when in liquid form.

[0190] In a further embodiment, the compositions and transformed microorganisms and pesticidal proteins of the embodiments may be treated prior to formulation to prolong pesticidal activity when applied to the environment of the target pest, provided that the pretreatment is not detrimental to the pesticidal activity. Such treatment may be performed by chemical and / or physical methods, provided that the treatment does not adversely affect the properties of one or more compositions. Examples of chemical agents include, but are not limited to, halogenating agents; aldehydes such as formaldehyde and glutaraldehyde; anti-infective agents such as zephiran chloride; alcohols such as isopropanol and ethanol; and histological fixatives such as Bouin's fixative and Helly's fixative (see, e.g., Humason (1967) Animal Tissue Techniques (WH Freeman and Co.)).

[0191] In other embodiments, before the pesticidal protein composition of the embodiment is applied to the environment of the target pest, it may be beneficial to treat the Cry toxin polypeptide with a protease (e.g., trypsin) to activate the protein. Methods for activating protoxins by serine proteases are well known in the art. See, for example, Cooksey (1968) Biochem. J. [Journal of Biological Chemistry] 6: 445-454, and Carroll and Ellar (1989) Biochem. J. [Journal of Biological Chemistry] 261: 99-105, which are incorporated herein by reference. For example, suitable activation protocols include, but are not limited to, combining a polypeptide to be activated (e.g., a purified novel Cry polypeptide (e.g., having an amino acid sequence shown in SEQ ID NO: 4 or SEQ ID NO: 8)) and trypsin at a weight ratio of 1 / 100 of protein / trypsin in 20 nM NaHCO3 (pH 8), and digesting the sample at 36°C for 3 hours.

[0192] Composition (comprising the microorganism and the pest killing protein of the conversion of embodiment) can be applied to the environment of insect pests by, for example, spraying, atomizing, dusting, scattering, coating or pouring, at the time when pests have begun to appear or before pests appear as a protective measure introduced into soil or on soil, introduced into irrigation water, by seed treatment or general application or dusting.For example, the pest killing protein of embodiment and / or the microorganism of conversion can be mixed with grain to protect grain during storage.It is generally important to obtain good prevention and control of pests in the early stage of plant growth, because this is when plants may be most seriously damaged.If it is considered necessary, the composition of embodiment can conveniently include another insecticide.In one embodiment, the composition is directly applied to soil in the granular form of the composition of the dead cells of the microorganism of carrier and the bacillus strain of embodiment or conversion when planting.Another embodiment is the granular form of the composition of the dead cells of the microorganism of the bacillus strain of embodiment or conversion.Another embodiment is the granular form of the composition of the dead cells of the microorganism of the bacillus strain of embodiment or conversion comprising agricultural chemicals (for example as herbicides, insecticides, fertilizers, inert carriers) and the bacillus strain of embodiment or conversion.

[0193] Those skilled in the art will appreciate that not all compounds are equally effective for all harmful organisms. The compounds of the embodiments show activity against insect pests, which may include economically important agronomy, forests, greenhouses, nurseries, ornamental plants, food and fiber, public and animal health, home and commercial buildings, home and stored product pests. Insect pests include insects selected from the following orders: Coleoptera, Diptera, Hymenoptera, Lepidoptera, Mallophaga, Homoptera, Hemiptera, Orthroptera, Thysanoptera, Dermaptera, Isoptera, Anoplura, Siphonaptera, Trichoptera, etc., especially Coleoptera and Lepidoptera.

[0194] Insects of the order Lepidoptera include, but are not limited to, armyworms, noctuids, loopers, and bollworms of the family Noctuidae: Agrotis ipsilon Hufnagel (black cutworm); A. orthogonia Morrison (western cutworm); A. segetum Denis & Schiffermüller (radish moth); A. subterranea Fabricius (granulate cutworm); Alabama argillacea Hübner (cottonleaf worm); Anticarsia gemmatalis Hübner (velvet bean caterpillar); Athetis mindara Barnes and McDunnough (rough skinned cutworm); Earias insulana Boisduval (spiny bollworm); E. vittella Fabricius (spotted bollworm); Egira (Xylomyges) curialis Grote (citrus cutworm); Euxoa messoria Harris (darksided cutworm); Helicoverpaarmigera Hübner (American bollworm); Corn earworm (corn earworm or cotton bollworm); Heliothis virescens Fabricius (tobacco budworm); Hypena scabra Fabricius) (greencloverworm); Mamestra configurata Walker (berthaarmyworm); cabbage armyworm (M.brassicae Linnaeus (cabbage moth); Melanchra picta Harris (zebra caterpillar); Pseudaletia unipuncta Haworth (snodus); Pseudoplusia includens Walker (soybean looper); Richia albicosta Smith (Western bean cutworm); Spodoptera frugiperda JE Smith (fall armyworm); S. exigua Hübner (beet armyworm); S. litura Fabricius (tobacco cutworm, cluster caterpillar); Trichoplusia ni Hübner (cabbage cutworm, cabb ... looper); borers, sheath moths, web-spinners, coneworms and leaf-feeding insects from the family Pyralidae; and the family Pyralidae such as Achroia grisella Fabricius (lesser wax moth); Amyelois transitella Walker (naval orangeworm); Anagasta kuehniella Zeller (Mediterranean flour moth); Cadra cautella Walker (almond moth); Chilo partellus Swinhoe (stem borer); C. suppressalis Walker (striped stem / rice borer); C. terrenellus Walker (striped stem / rice borer); Pagenstecher (sugarcane stemp borer); Corcyra cephalonica Stainton (rice moth); Crambus caliginosellus Clemens (corn root webworm); Poa annua (C.teterrellus Zincken (bluegrass webworm); Cnaphalocrocis medinalis Guenée (rice leaf roller); Desmia funeralis Hübner (grape leaffolder); Diaphaniahyalinata Linnaeus (melon worm); D. nitidalis Stoll (pickleworm); Diatraea grandiosella Dyar (southwestern corn borer); D.saccharalis Fabricius (surgarcane borer); Elasmopalpus lignosellus Zeller (lesser cornstalk borer); Eoreuma loftini Dyar (Mexican rice borer); Ephestia elutella Hübner (tobacco (cacao) moth); Galleria mellonella Linnaeus (greater wax moth); Hedylepta accepta Butler (sugarcane leafroller); Herpetogrammalicarsisalis Walker (sod webworm); Homoeosoma electellum Hulst (sunflower moth); Loxostege sticticalis Linnaeus (beet webworm); Maruca testulalis Geyer (bean podborer); Orthaga thyrisalis Walker (tea tree web moth); Ostrinia nubilalis Hübner (European corn borer); Plodia interpunctella Hübner (Indian meal moth); Scirpophaga incertulas Walker (yellow stem borer); Udea rubigalis Guenée (celery leaf roller); leaftier); and leaf rollers, aphids, seed and fruit insects in the family Tortricidae, Acleris gloverana Walsingham (Western blackheaded budworm); Eastern blackheaded budworm (A.variana Fernald)(Eastern blackheaded budworm); Adoxophyes orana Fischer von. ) (summer fruit tortrix moth); Archips spp., including A. argyrospila Walker (fruit tree leaf roller) and A. rosana Linnaeus (European leaf roller); Argyrotaenia spp.; Bonagota salubricola Meyrick (Brazilian apple leaf roller); Choristoneura spp.; Cochylis hospes Walsingham (banded sunflower moth); Cydia latiferreana Walsingham (filbertworm); C. pomonella Linnaeus (apple codling moth); Endopiza viteana Clemens (grapeberry moth); Eupoecilia ambiguella Hübner (grape vine moth); Grapholita molesta Busck (oriental fruit moth); Lobesia botrana Denis & Schiffermüller (European grape vinemoth); Platynota flavedana Clemens (variegated leafroller); P. stultana Walsingham (omnivorous leafroller); Spilonota ocellana Denis & Schiffermüller (eyespotted bud moth); and Suleima helianthana Riley) (sunflower bud moth).

[0195] Other selected agronomic pests from the order Lepidoptera include, but are not limited to, Alsophilapometaria Harris (fall cankerworm); Anarsia lineatella Zeller (peach twig borer); Anisota senatoria JESmith (orange striped oakworm); Antheraea pernyi Guérin-Méneville (Chinese oak moth); Bombyx mori Linnaeus (Silkworm); Bucculatrix thurberiella Busck (cotton leaf perforator); Colias eurytheme Boisduval (alfalfa caterpillar); Datanaintegerrima Grote & Robinson (walnut caterpillar); Dendrolimus sibiricus Tschetwerikov (Siberian silk moth), Ennomos subsignaria Hübner (elm looper) spanworm); Erannistiliaria Harris (linden looper); Erechthias flavistriata Walsingham (sugarcane bud moth); Euproctis chrysorrhoea Linnaeus (browntail moth); Harrisina americana Guérin-Méneville (grapeleaf skeletonizer); Heliothis subflexa Guenée; Hemileucaoliviae Cockrell (range caterpillar); Hyphantria cunea Drury (fall webworm); Keiferia lycopersicella Walsingham (tomato wireworm); pinworm); Lambdina fiscellariafiscellaria Hulst (Eastern hemlock looper); Western hemlock looper (L.fiscellaria lugubrosa Hulst (Western hemlock looper); Leucoma salicis Linnaeus (satin moth); Lymantria disparLinnaeus (gypsy moth); Malacosoma spp.; Manduca quinquemaculata Haworth (five spotted hawk moth, tomato hornworm); M. sexta Haworth (tomato hornworm, tobacco hornworm); Operophtera brumata Linnaeus (winter moth); Orgyia spp.; Paleacrita vernata Peck (spring cankerworm); Papilio cresphontes Cramer (giant swallowtail, orange dog); Phryganidia californica Packard (California oakworm); Phyllocnistiscitrella Stainton (citrus leafminer); Phyllonorycterblancardella Fabricius (spotted tentiform leafminer); Pieris brassicae Linnaeus (large white butterfly); P. rapae Linnaeus (small white butterfly); P.napi Linnaeus (green veined white butterfly); Platyptilia carduidactyla Riley (artichoke plume moth); Plutella xylostella Linnaeus (diamondback moth); Pectinophora gossypiella Saunders (pink bollworm); Pontia protodice Boisduval & Leconte (Southern cabbageworm); Sabulodes aegrotata Guenée (omnivorous looper); Schizura concinna JESmith (redhumped caterpillar); Sitotroga cerealella Olivier (Angoumois grainmoth); Thaumetopoea pityocampa Schiffermuller (pineprocessionary caterpillar); Tineola bisselliella Hummel (webbing clothesmoth); Tuta absoluta Meyrick (tomatoleafminer); and Yponomeuta padella Linnaeus (ermine moth).

[0196] Of concern are larvae and adults of the order Coleoptera, including weevils from the families Anthribidae, Bruchidae, and Curculionidae, including, but not limited to: Anthonomus grandis Boheman (boll weevil); Cylindrocopturus adspersus LeConte (sunflower stem weevil); Diaprepes abbreviatus Linnaeus (root weevil); Hyperapunctata Fabricius (clover leaf weevil); Lissorhoptrusoryzophilus Kuschel (rice water weevil); Metamasius hemipterus hemipterus Linnaeus (West Indian cane weevil); weevil); silky cane weevil (M. hemipterus sericeus Olivier); granary weevil (Sitophilus granarius Linnaeus); rice weevil (S. oryzae Linnaeus); red sunflower seed weevil (Smicronyx fulvus LeConte); gray sunflower seed weevil (S. sordidus LeConte); maize billbug (Sphenophorus maidis Chittenden); Guinea sugarcane weevil (Rhabdoscelus obscurus Boisduval); New Guinea sugarcane weevil (Rhabdoscelus obscurus Boisduval); sugarcaneweevil); flea beetles, cucumber beetles, rootworms, leaf beetles, potato leaf beetles, and leaf miners of the family Chrysomelidae, including but not limited to: Chaetocnema ectypa Horn (desert corn flea beetle); corn copper flea beetle (C.pulicaria Melsheimer (corn flea beetle); Colaspis brunnea Fabricius (grape colaspis); Diabrotica barberi Smith & Lawrence (northern corn rootworm); D. undecimpunctata howardi Barber (southern corn rootworm); D. virgifera virgifera LeConte (western corn rootworm); Leptinotarsa ​​decemlineata Say (Colorado potato beetle); Oulema melanopus Linnaeus (cereal leafbeetle); Phyllotreta cruciferae Goeze (corn flea beetle); Zygogramma exclamationis Fabricius (sunflower beetle); beetles from the family Coccinellidae, including but not limited to: Epilachna varivestis Mulsant (Mexican bean beetle); chafers and other beetles from the family Scarabaeidae, including but not limited to: Antitrogus parvulus Britton (Childers sugarcane grub); Cyclocephala borealis Arrow (northernmasked chafer, white grub); southern rhinoceros beetle (C.immaculata Olivier (southern masked chafer, white grub); Dermolepida albohirtum Waterhouse (grey-backed sugarcane beetle); Euetheola humilis rugiceps LeConte (sugarcane beetle); Lepidiota frenchi Blackburn (French sugarcane grub); Tomarus gibbosus De Geer (carrot beetle); T. subtropicus Blatchley (sugarcane grub); Phyllophaga crinita Burmeister (white grub); P. latifrans LeConte (June beetle); Popillia japonica Newman (Japanese beetle); Rhizotrogus majalis Razoumowsky (European chafer); carpet beetles from the family Dermestidae; wireworms from the family Elateridae, Eleodes spp., Melanotus spp. (including M. communis Gyllenhal); Conoderus spp.; Limonius spp.; Agriotes spp.; Ctenicera spp.; Aeolus spp. spp.); bark beetles from the family Scolytidae; beetles from the family Tenebrionidae; beetles from the family Cerambycidae, such as but not limited to Migdolus fryanus Westwood (longhorn beetle); and beetles from the family Buprestidae, including but not limited to Aphanisticus cochinchinae seminulum Obenberger (leaf-mining buprestid beetle).

[0197] Of interest are adults and immatures of the order Diptera, including the leafminer Agromyza parvicornis Loew (corn blotch leafminer); chironomids, including but not limited to: Contarinia sorghicola Coquillett (sorghum midge); Mayetiola destructor Say (Hessian fly); Neolasiopteramurtfeldtiana Felt, (sunflower seed midge); Sitodiplosis mosellana Géhin (wheat midge); fruit flies (Tephritidae), Oscinella frit Linnaeus (frit flies); maggots, including but not limited to: Delia species (Delia spp.), including Delia platura Meigen (seedcorn maggot); D. coarctata Fallen (wheat bulb fly); Fannia canicularis Linnaeus, F. femoralis Stein (lesser houseflies); Meromyza americana Fitch (American wheat stem maggot); Musca domestica Linnaeus (house flies); Stomoxys calcitrans Linnaeus (stable flies); Fall fly, horn fly, blowfly, Chrysomya spp.; Phormia spp.; and other muscoid fly pests, horse flies Tabanus spp.); bot flies Gastrophilus spp.; Oestrus spp.; Hypoderma spp.; deer flies Chrysops spp.); Melophagus ovinus Linnaeus (sheep ticks) and other Brachycera, mosquitoes Aedes species; Anopheles spp.; Culex spp.; black flies Prosimulium spp.; Simulium spp.; blood-sucking midges, sand flies, sciarids and other Nematocera. .

[0198] Insects of the order Hemiptera include, but are not limited to, the following families: Adelgidae, Aleyrodidae, Aphididae, Asterolecaniidae, Cercopidae, Cicadellidae, Cicadidae, Cixiidae, Coccidae, Coreidae, Dactylopiidae, Delphacidae, Diaspididae, Eriococcidae, Flatidae, Fulgoridae, Issidae, Lygaeidae, Margarodidae, Membracidae, Miridae, Ortheziidae, Pentatomidae, Phoenicococcidae, Phylloxeridae, Pseudococcidae, Psyllidae, Pyrrhocoridae and Tingidae.

[0199] Agriculturally important members from the order Hemiptera include, but are not limited to: Acrosternum hilareSay (green stink bug); Acyrthisiphon pisum Harris (peaaphid); Adelges spp. (adelgids); Adelphocoris rapidus Say (rapid plant bug); Anasa tristis De Geer (squash bug); Aphis craccivora Koch (cowpea aphid); A. fabae Scopoli (black bean aphid); A. gossypii Glover (cotton aphid, melon aphid); A. maidiridicis (corn root aphid). Forbes, corn root aphid; A. pomi De Geer (apple aphid); A. spiraecola Patch (spirea aphid); Aulacaspis tegalensis Zehntner (sugarcane scale insect); Aulacorthum solani Kaltenbach (foxglove aphid); Bemisia tabaci Gennadius (tobacco whitefly, sweetpotato whitefly); B. argentifolii Bellows & Perring (silverleaf whitefly); Blissus leucopterus leucopterus Say (chinchilla bug); Blostomatidae spp.); Brevicoryne brassicae Linnaeus (cabbage aphid); Cacopsylla pyricola Foerster (pearpsylla); Calocoris norvegicus Gmelin (potato capsid bug); Chaetosiphon fragaefolii Cockerell (strawberry aphid); Cimicidae spp.; Coreidae spp.; Corythuca gossypii Fabricius (cotton lace bug); Cyrtopeltis modesta Distant (tomatobug); C. notatus Distant (suckfly); Deois flavopicta. (spittlebug); Dialeurodes citri Ashmead (citrus whitefly); Diaphnocoris chlorionis Say (honeylocust plant bug); Diuraphis noxia Kurdjumov / Mordvilko (Russian wheat aphid); Duplachionaspis divergens Green (armored scale); Dysaphis plantaginea Paaserini (rosy apple aphid); Dysdercus suturellus Herrich- ) (cotton stainer); Dysmicoccus boninsis Kuwana (gray sugarcane mealybug); Empoasca fabae Harris (potato leafhopper); Eriosomalanigerum Hausmann (woolly apple aphid); Erythroneoura spp. (grape leafhoppers); Eumetopina flavipes Muir (Island sugarcane planthopper); Eurygaster spp.; Euschistus servus Say (brown stink bug); E. variolarius Palisot de Beauvois (one-spotted stink bug); bug); Graptostethus spp.) (complex of seedbugs); and Hyalopterus pruni Geoffroy (mealy plumaphid); Icerya purchasi Maskell (cottony cushion scale); Labopidicola allii Knight (onion plant bug); Laodelphaxstriatellus Fallen (smaller brown planthopper); Leptoglossus corculusSay (leaf-footed pine seed bug); Leptodictya tabida Herrich-Schaeffer (sugarcane lace bug); Lipaphis erysimi Kaltenbach (turnip aphid); Lygocoris pabulinus Linnaeus (common green capsid); Lygus lineolaris Palisot de Beauvois (tarnished plant bug); L. Hesperus Knight (Western tarnished plant bug); L. pratensis Linnaeus (common meadow bug); L. rugulipennis Poppius (European tarnished plant bug); Macrosiphum euphorbiae Thomas (potato aphid); Macrosteles quadrilineatus Forbes (aster leafhopper); Magicicada septendecim Linnaeus (periodical cicada); Mahanarva fimbriolata. (sugarcane aphid); Melanaphissacchari Zehntner (sugarcane aphid); Melanapsis glomerata Green (black scale); Metopolophium dirhodum Walker (rosegrain aphid); Myzus persicae Sulzer (peach-potato aphid, green peach aphid); Nasonovia ribisnigri Mosley (lettuce aphid); Nephotettix cinticeps Uhler (green leafhopper); N. nigropictus )(rice leafhopper); Nezara viridula Linnaeus (southern green stink bug); Nilaparvata lugens brown planthopper; Nysius ericae Schilling (false chinch bug); Nysius raphanus Howard (false chinch bug); Oebalus pugnax Fabricius (rice stink bug); Oncopeltus fasciatus Dallas (large milkweed bug); Orthops campestris Linnaeus; Graptostethus spp. (root aphids and gall aphids); Peregrinus maidis Ashmead (corn planthopper); Perkinsiella saccharicida Kirkaldy (sugarcane delphacid); Phylloxera devastatrix Pergande (pecan phylloxera); Planococcus citri Risso (citrus mealybug); Plesiocorisrugicollis Fallen (apple capsid); Poecilocapsus lineatus Fabricius (four-lined plant bug); Pseudatomoscelis seriatus Reuter (cotton fleahopper); Pseudococcus spp. (other mealybug groups); Pulvinaria elongata Newstead (cottony grass scale); scale); Pyrilla perpusilla Walker (Sugarcane leafhopper); Pyrrhocoridae spp.; Quadraspidiotus perniciosus Comstock (San Jose scale); Reduviidae spp.); Rhopalosiphum maidis Fitch (corn leaf aphid); R. padi Linnaeus (bird cherry-oat aphid); Saccharicoccus sacchariCockerell (sugarcane mealybug); Schizaphis graminum Rondani (greenbug); Sipha flava Forbes (yellow sugarcane aphid); Sitobionavenae Fabricius (English grain aphid); Sogatella furcifera Horvath (white-backed planthopper); Sogatodes oryzicola Muir (rice planthopper); Spanagonicus albofasciatus Reuter (whitemarked flyhopper); Therioaphis maculata Buckton, spotted alfalfa aphid; Tinidae spp.; Toxoptera aurantii Boyer de Fonscolombe (black citrus aphid); and T. citricida Kirkaldy (brown citrus aphid); Trialeurodes abutiloneus (bandedwinged whitefly) and T. vaporariorum Westwood (greenhouse whitefly); Trioza diospyri Ashmead (persimmon psylla); and Typhlocyba pomaria McAtee (white appleleafhopper).

[0200] In addition, adults and larvae of the order Acari (Mites) are included, such as Aceriatosichella Keifer (wheat curl mite); Panonychus ulmiKoch (European red mite); Petrobia latens Müller (brown wheat mite); Steneotarsonemus bancrofti Michael (sugarcane stem mite); spider mites and spider mites in the family Tetranychidae, Oligonychus grypus Baker & Pritchard, O. indicus Hirst (sugarcane spider mite), O. pratensis Banks (Banks grass mite), Oligonychus stickneyi McGregor (sugarcane spider mite); Tetranychus urticae Koch (two spotted spider mite); mite); T. mcdanieli McGregor (McDaniel mite); T. cinnabarinus Boisduval, carmine spider mite; T. turkestani Ugarov & Nikolski (strawberry spider mite), flat mites in the Tenuipalpidae, Brevipalpus lewisi McGregor (citrus flat mite); rust mites and bud gall mites in the Eriophyidae and other leaf feeding mites and mites important to human and animal health, namely dust mites in the Epidermoptidae, hair follicle mites in the Demodicidae, grain mites in the Glycyphagidae, ticks in the Ixodidae.Blacklegged tick (Ixodesscapularis Say) (deer tick); I. holocyclus Neumann (Australian paralysis tick); Dermacentor variabilis Say (American dog tick); Amblyomma americanum Linnaeus (lone star tick); and itch mites and sarcoptes in the families Psoroptidae, Pyemotidae and Sarcoptidae.

[0201] Of concern are insect pests of the order Thysanura, eg, Lepisma saccharina Linnaeus (silverfish); Thermobia domestica Packard (firebrat).

[0202] Other arthropod pests covered include spiders in the order Araneae, such as Loxosceles reclusa Gertsch & Mulaik (brown recluse spider); and Latrodectus mactans Fabricius (black widow spider); and centipedes in the order Scutigeromorpha, such as Scutigera coleoptrata Linnaeus (house centipede). In addition, insect pests of the order Isoptera are of concern, including insect pests of the family termitidae, such as, but not limited to, Cylindrotermes nordenskioeldi Holmgren and Pseudacanthotermes militaris Hagen (sugar cane termite). Insects of the order Thysanoptera are also of concern, including, but not limited to, thrips, such as Stenchaetothrips minutus van Deventer (sugar cane thrips).

[0203] The pesticidal activity of the compositions of the embodiments against insect pests can be tested at an early developmental stage (e.g., as larvae or other immature forms). The insects can be reared in complete darkness at from about 20°C to about 30°C and from about 30% to about 70% relative humidity. Bioassays can be conducted as described in Czapla and Lang (1990) J. Econ. Entomol. [Journal of Economic Entomology] 83(6): 2480-2485. Methods for rearing insect larvae and conducting bioassays are well known to those of ordinary skill in the art.

[0204] Various bioassay techniques are known to those skilled in the art. The general procedure involves adding the test compound or organism to a feed source in a closed container. Pesticide activity can be measured by, but is not limited to, mortality, weight loss, changes in attractiveness, repulsion, and other behavioral and physical changes after feeding and exposure for an appropriate length of time. The bioassays described herein can be used for any feeding insect pest in the larval or adult stages.

[0205] The present invention also relates to the following embodiments:

[0206] 1. A recombinant polypeptide, said recombinant polypeptide comprising the following mature Bt toxin moieties: SEQ ID NO:64, SEQ ID NO:152, SEQ ID NO:170, SEQ ID NO:184, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:74, SEQ ID NO:76, SEQ ID NO:78, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:84, SEQ ID NO:86, SEQ ID NO:88, SEQ ID NO:90, SEQ ID NO:92, SEQ ID NO:94, SEQ ID NO:96, SEQ ID NO:98, SEQ ID NO:100, SEQ ID NO:102, SEQ ID NO:104, SEQ ID NO:106, SEQ ID NO:108, SEQ ID NO:110, SEQ ID NO:112, SEQ ID NO:114, SEQ ID NO:116, SEQ ID NO:118, SEQ ID NO:120, SEQ ID NO:122, SEQ ID NO:124, SEQ ID NO:126, SEQ ID NO:128, SEQ ID NO:130, SEQ ID NO:132, SEQ ID NO:134, SEQ ID NO:136, SEQ ID NO:138, SEQ ID NO:140, SEQ IDNO:142, SEQ ID NO:144, SEQ ID NO:146, SEQ ID NO:148, SEQ ID NO:150, SEQ ID NO:154, SEQ ID NO:156, SEQ ID NO:158, SEQ ID NO:160, SEQ ID NO:162, SEQ ID NO:164, SEQ ID NO:166, SEQ ID NO:168, SEQ ID NO:172, SEQ ID NO:174, SEQ ID NO:176, SEQ ID NO:178, SEQ ID NO:180, SEQ ID NO:182, SEQ ID NO:186, SEQ ID NO:188, SEQ ID NO:190, SEQ ID NO:192, SEQ ID NO:194, SEQ ID NO:196, SEQ ID NO:198, SEQ ID NO:200, SEQ ID NO:202, SEQ ID NO:204, SEQ ID NO:206, SEQ ID NO:208, SEQ ID NO:210, SEQ ID NO:212, SEQ ID NO:214, SEQ ID NO:216, SEQ ID NO:218, SEQ ID NO:220, SEQ ID NO:222, SEQ ID NO:224, SEQ ID NO:226, SEQ ID NO:228, SEQ ID NO:230, SEQ ID NO:232, SEQ ID NO:234, SEQ ID NO:236, SEQ ID NO:238, SEQ ID NO:240, SEQ ID NO:242, SEQ ID NO:244, SEQ ID NO:246, SEQ ID NO:248, SEQ ID NO:250, SEQ ID NO:252, SEQ IDNO:254, SEQ ID NO:256, SEQ ID NO:258, SEQ ID NO:260, SEQ ID NO:262, SEQ ID NO:264, SEQ ID NO:266, SEQ ID NO:268 or SEQ ID NO:270.

[0207] 2. The recombinant polypeptide of embodiment 1, wherein the mature Bt toxin is operably linked to a heterologous protoxin region.

[0208] 3. A recombinant polypeptide, the recombinant polypeptide comprising an amino acid sequence having at least 95% sequence identity with the following: SEQ ID NO:64, SEQ ID NO:152, SEQ ID NO:170, SEQ ID NO:184, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:74, SEQ ID NO:76, SEQ ID NO:78, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:84, SEQ ID NO:86, SEQ ID NO:88, SEQ ID NO:90, SEQ ID NO:92, SEQ ID NO:94, SEQ ID NO:96, SEQ ID NO:98, SEQ ID NO:100, SEQ ID NO:102, SEQ ID NO:104, SEQ ID NO:106, SEQ ID NO:108, SEQ ID NO:110, SEQ ID NO:112, SEQ ID NO:114, SEQ ID NO:116, SEQ ID NO:118, SEQ ID NO:120, SEQ ID NO:122, SEQ ID NO:124, SEQ ID NO:126, SEQ ID NO:128, SEQ ID NO:130, SEQ ID NO:132, SEQ ID NO:134, SEQ ID NO:136, SEQ ID NO:138, SEQ IDNO:140, SEQ ID NO:142, SEQ ID NO:144, SEQ ID NO:146, SEQ ID NO:148, SEQ ID NO:150, SEQ ID NO:154, SEQ ID NO:156, SEQ ID NO:158, SEQ ID NO:160, SEQ ID NO:162, SEQ ID NO:164, SEQ ID NO:166, SEQ ID NO:168, SEQ ID NO:172, SEQ ID NO:174, SEQ ID NO:176, SEQ ID NO:178, SEQ ID NO:180, SEQ ID NO:182, SEQ ID NO:186, SEQ ID NO:188, SEQ ID NO:190, SEQ ID NO:192, SEQ ID NO:194, SEQ ID NO:196, SEQ ID NO:198, SEQ ID NO:200, SEQ ID NO:202, SEQ ID NO:204, SEQ ID NO:206, SEQ ID NO:208, SEQ ID NO:210, SEQ ID NO:212, SEQ ID NO:214, SEQ ID NO:216, SEQ ID NO:218, SEQ ID NO:220, SEQ ID NO:222, SEQ ID NO:224, SEQ ID NO:226, SEQ ID NO:228, SEQ ID NO:230, SEQ ID NO:232, SEQ ID NO:234, SEQ ID NO:236, SEQ ID NO:238, SEQ ID NO:240, SEQ ID NO:242, SEQ ID NO:244, SEQ ID NO:246, SEQ ID NO:248, SEQ ID NO:250, SEQ ID NO:252, SEQ ID NO:254, SEQ ID NO:256, SEQ ID NO:258, SEQ ID NO:260, SEQ ID NO:262, SEQ ID NO:264, SEQ ID NO:266, SEQ ID NO:268 or SEQ ID NO:270.

[0209] 4. The recombinant insecticidal polypeptide of embodiment 1, 2 or 3, wherein the insecticidal polypeptide is operably linked to a heterologous signal sequence or transport sequence.

[0210] 5. An agricultural composition comprising at least one recombinant insecticidal polypeptide according to embodiment 1, 2, 3 or 4.

[0211] 6. A recombinant polynucleotide encoding the insecticidal polypeptide of embodiment 1, 2, 3 or 4.

[0212] 7. The recombinant polynucleotide according to embodiment 6, wherein the polynucleotide is selected from: SEQ ID NO:63, SEQ ID NO:151, SEQ ID NO:169, SEQ ID NO:183, SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:71, SEQ ID NO:73, SEQ ID NO:75, SEQ ID NO:77, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:89, SEQ ID NO:91, SEQ ID NO:93, SEQ ID NO:95, SEQ ID NO:97, SEQ ID NO:99, SEQ ID NO:101, SEQ ID NO:103, SEQ ID NO:105, SEQ ID NO:107, SEQ ID NO:109, SEQ ID NO:111, SEQ ID NO:113, SEQ ID NO:115, SEQ ID NO:117, SEQ ID NO:119, SEQ ID NO:121, SEQ ID NO:123, SEQ ID NO:125, SEQ ID NO:127, SEQ ID NO:129, SEQ ID NO:131, SEQ ID NO:133, SEQ ID NO:135, SEQ ID NO:137, SEQ ID NO:139, SEQ IDNO:141, SEQ ID NO:143, SEQ ID NO:145, SEQ ID NO:147, SEQ ID NO:149, SEQ ID NO:153, SEQ ID NO:155, SEQ ID NO:157, SEQ ID NO:159, SEQ ID NO:161, SEQ ID NO:163, SEQ ID NO:165, SEQ ID NO:167, SEQ ID NO:171, SEQ ID NO:173, SEQ ID NO:175, SEQ ID NO:177, SEQ ID NO:179, SEQ ID NO:181, SEQ ID NO:185, SEQ ID NO:187, SEQ ID NO:189, SEQ ID NO:191, SEQ ID NO:193, SEQ ID NO:195, SEQ ID NO:197, SEQ ID NO:199, SEQ ID NO:201, SEQ ID NO:203, SEQ ID NO:205, SEQ ID NO:207, SEQ ID NO:209, SEQ ID NO:211, SEQ ID NO:213, SEQ ID NO:215, SEQ ID NO:217, SEQ ID NO:219, SEQ ID NO:221, SEQ ID NO:223, SEQ ID NO:225, SEQ ID NO:227, SEQ ID NO:229, SEQ ID NO:231, SEQ ID NO:233, SEQ ID NO:235, SEQ ID NO:237, SEQ ID NO:239, SEQ ID NO:241, SEQ ID NO:243, SEQ ID NO:245, SEQ ID NO:247, SEQ ID NO:249, SEQ ID NO:251, SEQ IDNO:253, SEQ ID NO:255, SEQ ID NO:257, SEQ ID NO:259, SEQ ID NO:261, SEQ ID NO:263, SEQ ID NO:265, SEQ ID NO:267 or SEQ ID NO:269.

[0213] 8. The recombinant polynucleotide of embodiment 6 or 7, wherein the polynucleotide is codon-optimized for expression in a crop plant.

[0214] 9. A DNA construct comprising the recombinant polynucleotide of embodiment 6, 7 or 8 operably linked to a heterologous regulatory element.

[0215] 10. A host cell comprising the recombinant polynucleotide of embodiment 6, 7 or 8.

[0216] 11. A host cell comprising the DNA construct according to embodiment 9.

[0217] 12. A transgenic plant comprising the polynucleotide of embodiment 6, 7 or 8.

[0218] 13. A transgenic plant comprising the DNA construct of embodiment 9.

[0219] 14. A method of inhibiting the growth of or killing an insect pest or pest population, the method comprising contacting the insect pest with an insecticidal polypeptide according to embodiment 1, 2, 3 or 4.

[0220] 15. A method of inhibiting the growth of or killing an insect pest or pest population, the method comprising expressing in a plant the polynucleotide of embodiment 6, 7 or 8.

[0221] 16. A method for controlling an infestation by a pest, the method comprising providing the transgenic plant of embodiment 12 or 13, or a part thereof, in the diet of the pest.

[0222] 17. A method for increasing crop yield, the method comprising growing the transgenic plant of embodiment 12 or 13, wherein the yield of the crop is increased in the presence of an insect pest relative to a crop not comprising the transgenic plant.

[0223] 18. The method of embodiment 14, 15, 16 or 17, wherein the insect pest or pest population is resistant to at least one Cry insecticidal protein.

[0224] The following examples are offered by way of illustration and not by way of limitation.

[0225] experiment

[0226] Example 1 - Gene identification and E. coli expression

[0227] The polynucleotide encoding the insecticide was obtained from a screen of Bacillus thuringiensis isolates from the DuPont internal proprietary collection of Bacillus thuringiensis isolates. Table 1 shows the insecticidal protein identifier, the sequence identifier number of the polynucleotide encoding the insecticidal protein, the sequence identifier number of the insecticidal protein, and the internal strain name.

[0228] Table 1

[0229]

[0230]

[0231]

[0232]

[0233] The polynucleotide encoding the insecticidal polypeptide in Table 1 was cloned into the pET28a vector coli BL21 cells (Invitrogen). Large-scale 1.0L cultures were grown to an OD600nm of approximately 0.8, and then the cultures were induced with isopropyl β-D-1-thiogalactopyranoside (IPTG) 1mM and grown at 16°C for 16 hours. The cell pellet was lysed with 50mL of 500mM NaCl / 20mM Tris / 5mM imidazole / pH 7.9 (supplemented with 0.02% lysozyme (w / v) and 0.1% Tween-20 and 1 tablet of Complete Protease Inhibitor, Roche). After lysis, the solution was sonicated and the lysate was centrifuged at 25,000rpm for 30 minutes. The supernatant containing the soluble protein fraction was filtered through a 0.45u vacuum filter, and then 1ml Talon (Clontech) slurry was added, and then incubated at 100rpm on a rotor for 1 hour for binding. The lysate was then added to the column, and the bound protein was separated and washed with 20ml of 50mmM NaCl / 20mM Tris / 5mM imidazole / pH7.9, and then eluted with 1.5ml of 50mmM NaCl / 20mM Tris / 500mM imidazole / pH 7.9. The purified protein was then dialyzed into a 50mM sodium carbonate buffer at pH 10. In the Lepidoptera and Coleoptera in vitro feeding assay panel, the purified protein was submitted for insecticidal activity.

[0234] Example 2 - Insect Assays with Partially Purified Proteins

[0235] Insecticidal activity bioassay screening was performed to evaluate the effects of insecticidal proteins on various species of the Lepidoptera, European corn borer (Ostrinia nubilalis), corn ear moth (Helicoverpazea), black cutworm (Agrotis ipsilon), fall armyworm (Spodopterafrugiperda), soybean armyworm (Pseudoplusia includens), and bean armyworm (Anticarsia gemmatalis); and Coleopteran species (Western corn rootworm (Diabrotica virgifera).

[0236] In a 96-well plate device, lepidopteran feeding assay is performed on an artificial diet containing purified protein. Then the purified protein (25ul) is added to the artificial diet. Two to five newborn larvae are placed in each hole and allowed to feed arbitrarily for 5 days. The results are expressed as larval reactions (such as stunted development) and / or mortality. If the larvae are similar to the negative control of the feed only using the above-mentioned buffer for feeding, the results are expressed as negative. For European corn borer (Ostrinia nubilalis), corn ear moth (Helicoverpa zea), black cutworm (Agrotis ipsilon), fall armyworm (Spodoptera frugiperda), soybean armyworm (Pseudoplusia includens) and bean armyworm (Anticarsia gemmatalis), each purified protein is screened for the first time in a single concentration. The insect assay was scored as follows: 1 = insecticidal activity at the highest concentration tested; 0 = no activity.

[0237] Table 2 shows the results of the initial insecticidal assay of the insecticidal polypeptides.

[0238] Table 2

[0239]

[0240]

[0241]

[0242]

[0243]

[0244] Example 3 - Transient Expression in Soybean Leaves and Insect Bioassay

[0245] For soybean expression, an optimized coding sequence was designed. To demonstrate the activity of the insecticidal polypeptides of the present disclosure, a transient expression system under the control of the AtUBQ10 promoter can be utilized (Dav, et al., (1999) Plant Mol. Biol. [Plant Molecular Biology] 40: 771-782; Norris SR et al. (1993) Plant Mol Biol. [Plant Molecular Biology] 21 (5): 895-906). The agroinfiltration method, which introduces a suspension of Agrobacterium cells into intact plant cells so that reproducible infection and subsequent plant-derived transgene expression can be measured or studied, is well known in the art (Kapila et al., (1997) Plant Science [Plant Science] 122: 101-108). Briefly, excised leaf discs of soybean (soybean, Glycinemax) are agroinfiltrated with standardized bacterial cell cultures of test and control strains. After 4 days, leaf discs were infested with two newly emerged soybean armyworms (SBL) (Chrysodeixis includens), corn earworms (CEW) (Helicoverpa zea), edamame caterpillars (VBC) (Anticarsia gemmatalis), or fall armyworms (Spodoptera frugiperda). Control leaf discs were inoculated with Agrobacterium containing only the DsRed2 fluorescent marker (Clontech TM , 1290 Terra Bella Ave. Mountain View, CA 94043)) expression vector. Leaf discs of non-infiltrated plants were included as a second control. Green leaf tissue consumption was scored three days after infection and given a score of 0 to 9. Example 4 Transient expression on maize leaf tissue and insect bioassay

[0246] The polynucleotides of the present disclosure are cloned into transient expression vectors under the control of the maize ubiquitin promoter (Christensen and Quail, (1996) Transgenic Research 5:213-218) and a repeat version of the promoter from mirabilis mosaic virus (DMMV PRO; Dey and Maiti, (1999) Plant Mol. Biol., 40:771-82). The agroinfiltration method, in which a suspension of Agrobacterium cells is introduced into intact plant cells so that reproducible infection and subsequent plant-derived transgenic expression can be measured or studied, is well known in the art (Kapila et al., (1997) Plant Science 122:101-108). Briefly, young maize plants are agroinfiltrated with standardized bacterial cell cultures of test and control strains. Leaf disks were produced from each plantlet and infested with WCRW (corn rootworm (Diabrotica virgifera)) along with appropriate controls. The extent of consumption of green leaf tissue was scored 2 days after infestation.

[0247] Example 5 - Agrobacterium-mediated transformation of maize and regeneration of transgenic plants

[0248] For Agrobacterium-mediated transformation of maize with the polynucleotides of the present disclosure, the method of Zhao (U.S. Pat. No. 5,981,840 and PCT Patent Publication WO 98 / 32326; the contents of which are hereby incorporated by reference) can be used. In short, immature embryos are separated from maize, and then these immature embryos are contacted with an Agrobacterium suspension under specific conditions, whereby the bacteria are able to transfer the polynucleotides to at least one cell of at least one immature embryo (step 1: infection step). In this step, the immature embryos can be immersed in the Agrobacterium suspension to begin inoculation. The embryos are co-cultured with Agrobacterium for a period of time (step 2: co-cultivation step). After the infection step, the immature embryos can be cultured on a solid medium. After this co-cultivation period, an optional "resting" step is envisioned. In this resting step, these embryos are incubated in the presence of at least one antibiotic (without adding a selection agent for plant transformants), which is known to inhibit the growth of Agrobacterium (step 3: resting step). The immature embryos are cultured on a solid medium with an antibiotic but without a selective agent to eliminate the resting stage of Agrobacterium and the persistent infected cells. Next, the inoculated embryos are cultured on a medium containing a selective agent, and the growing transformed callus is recovered (step 4: selection step). The immature embryos are cultured on a solid medium containing a selective agent to selectively grow the transformed cells. Then, the callus is regenerated into a plant (step 5: regeneration step), and the callus grown on the selective medium is cultured on a solid medium to be regenerated into a plant.

[0249] Example 6 - Transformation of soybean embryos

[0250] Soybean embryos are bombarded with plasmids containing polynucleotides of the present disclosure, which are operably linked to suitable promoters, as follows. To induce somatic embryos, cotyledons 3-5 mm in length cut from surface-sterilized immature seeds of appropriate soybean cultivars are cultured on appropriate agar medium at 26° C. in light or darkness for six to ten weeks. Somatic embryos that produce secondary embryos are then excised and placed in an appropriate liquid culture medium. After repeated screening of somatic embryo clusters that are amplified into early globular stage embryos, the suspension is maintained as described below.

[0251] Soybean embryogenic suspension cultures were maintained in 35 mL liquid medium on a rotary shaker at 26°C at 150 rpm with anthesis lighting on a 16:8 hour day / night schedule. Cultures were subcultured every two weeks by inoculating approximately 35 mg of tissue into 35 mL liquid medium.

[0252] Soybean embryogenic suspension cultures are then transformed by particle gun bombardment (Klein et al., (1987) Nature (London) 327:70-73; U.S. Pat. No. 4,945,050). These transformations can be performed using a Du Pont Biolistic PDS1000 / HE instrument (helium improved).

[0253] Selectable marker genes that can be used to promote soybean transformation include, but are not limited to, the 35S promoter from cauliflower mosaic virus (Odell et al., (1985) Nature 313:810-812), the hygromycin phosphotransferase gene from plasmid pJR225 (from Escherichia coli; Gritz et al., (1983) Gene 25:179-188), and the 3' region of the nopaline synthase gene from the T DNA of the Ti plasmid of Agrobacterium tumefaciens. An expression cassette comprising a polynucleotide of the present disclosure operably linked to a suitable promoter can be isolated as a restriction fragment. The fragment can then be inserted into a unique restriction site of a vector carrying the marker gene.

[0254] To 50 μL of a 60 mg / mL 1 μm gold particle suspension was added (in order): 5 μL DNA (1 μg / μL), 20 μL spermidine (0.1 M), and 50 μL CaCl2 (2.5 M). The particle preparation was then stirred for three minutes, centrifuged in a microcentrifuge for 10 seconds, and the supernatant removed. The DNA-coated particles were then washed once in 400 μL 70% ethanol and resuspended in 40 μL absolute ethanol. The DNA / particle suspension was sonicated three times for one second each. Five microliters of DNA-coated gold particles were then added to each macrocarrier disk.

[0255] About 300-400mg of two-week-old suspension culture is placed in an empty 60x 15mm culture dish, and residual liquid is removed from the tissue with a pipette. For each transformation experiment, about 5-10 tissue plates are usually bombarded. The membrane disruption pressure is set to 1100psi, and the container is evacuated to a vacuum of 28 inches of mercury. The tissue is placed about 3.5 inches away from the blocking screen and bombarded three times. After the bombardment, the tissue is divided into two halves and placed back in the liquid and cultivated as described above.

[0256] The selective culture medium of the present invention is replaced with fresh culture medium after bombardment for five to seven days, and can be replaced with fresh culture medium containing 50mg / mL hygromycin after bombardment for 11 to 12 days.This selective culture medium is renewed weekly.Seven to eight weeks after bombardment, observe and grow the green tissue through conversion from the embryonic cluster of unconverted necrosis.Get the green tissue of separation and inoculate each shaking bottle, to produce new, asexual propagation, through the embryonic suspension culture of conversion.Each new strain is treated as independent transformation event processing.These suspension passages are cultivated and remain as immature embryonic cluster then, perhaps regenerate as whole plant through the maturation and budding of each somatic embryo.

[0257] Example 7 - Regeneration of soybean somatic embryos into plants

[0258] In order to obtain whole plants from embryogenic suspension cultures, the tissue must be regenerated.

[0259] Embryo maturation

[0260] Under cool white fluorescent (Phillips Cool White Econowatt F40 / CW / RS / EW) and Agro (Phillips F40Agro) bulbs (40 watts), with 90-120 μE / m 2s light intensity of 16: 8hr photoperiod, in SB196, at 26 ℃, embryo culture 4-6 weeks. After this time, the embryo cluster is taken out to solid agar medium SB166 for 1-2 weeks. The cluster is then subcultured to medium SB103 for 3 weeks. During this period, single embryos are taken out from the cluster and screened for ABA accumulation. It should be noted that any detectable phenotype produced by the expression of the target gene can be screened at this stage.

[0261] Embryo drying and germination

[0262] By placing the mature single embryo in an empty small culture dish (35x 10mm) for about 4-7 days, make them dry. With fiber tape, the plate is sealed (producing a small humidity box). The dried embryo is planted in the SB71-4 culture medium, and they are allowed to germinate under the above-mentioned same culture conditions. From the germination medium, take out the sprouted plantlets, and fully rinse with water, and then plant in a 24-hole packaging tray (pack tray), cover with a transparent plastic dome. After 2 weeks, remove the dome and plant is subjected to cold-resistant training (hardened off) for one week again. If the plantlets appear to be cold-resistant, they are transplanted to Redi-Earth TM 10" pots with up to 3 plantlets / pot.

[0263] Analysis of leaf tissue from transgenic soybean events

[0264] Leaf tissue was harvested at about V3 / V4 and fed to the target Lepidoptera species. Leaf discs were infested individually with 2 newborn soybean armyworms (SBL) (Chrysodeixis includens), corn earworm (CEW) (Helicoverpa zea), veggie caterpillars (VBC) (Anticarsia gemmatalis) or fall armyworm (Spodopterafrugiperda). Leaf discs of wild-type plants were included as controls. Green leaf tissue consumption was scored five days after infection and given a score of 0 to 9. Protein expression can be confirmed by western blotting using antibodies against the polypeptides.

[0265] The above description of the various illustrated embodiments of the present disclosure is not intended to be exhaustive or to limit the scope to the precise form disclosed. Although specific embodiments and examples are described herein for illustrative purposes, various equivalent modifications within the scope of the present disclosure are possible as will be appreciated by those skilled in the relevant art. The teachings provided herein may be applied to other purposes in addition to the above examples. Based on the above teachings, many modifications and variations are possible, and are therefore within the scope of the appended claims.

[0266] These and other changes can be made in light of the above detailed description.In general, in the following claims, the terms used should not be construed to limit the scope to the specific embodiments disclosed in the specification and the claims.

[0267] The entire disclosure of each document (including patents, patent applications, journal articles, abstracts, manuals, books, or other publications) cited in the Background, Detailed Description, and Examples is incorporated herein by reference in its entirety.

[0268] With respect to numbers used (e.g., amounts, temperatures, concentrations, etc.), efforts have been made to ensure accuracy, but some experimental error and deviation should be allowed for. Unless otherwise indicated, parts are parts by weight, molecular weight is average molecular weight; temperature is in degrees Celsius; and pressure is at or near atmospheric.

Claims

1. A recombinant polypeptide, which consists of the amino acid sequence of SEQ ID NO:

268.

2. The recombinant polypeptide of claim 1, wherein the recombinant polypeptide is operably linked to a heterologous signal sequence or a transport sequence.

3. An agricultural composition comprising at least one recombinant polypeptide according to claim 1 or 2.

4. A recombinant polynucleotide encoding the recombinant polypeptide according to claim 1 or 2.

5. The recombinant polynucleotide of claim 4, wherein the polynucleotide is SEQ ID NO:

267.

6. The recombinant polynucleotide of claim 4 or 5, wherein the polynucleotide is codon-optimized for expression in a crop plant.

7. A DNA construct comprising the recombinant polynucleotide of claim 4, 5 or 6 operably linked to a heterologous regulatory element.

8. A host cell comprising the recombinant polynucleotide of claim 4, 5 or 6.

9. A host cell comprising the DNA construct according to claim 7.

10. A method for producing a transgenic plant comprising the polynucleotide of claim 4, 5 or 6.

11. A method for producing a transgenic plant comprising the DNA construct of claim 7.

12. A method for inhibiting the growth of or killing an insect pest or a pest population, the method comprising contacting the insect pest with the recombinant polypeptide of claim 1 or 2.

13. A method of inhibiting the growth of or killing an insect pest or pest population, the method comprising expressing in a plant a polynucleotide according to claim 4, 5 or 6.

14. A method for controlling an insect pest infestation, the method comprising providing a transgenic plant according to claim 10 or 11, or a part thereof, in the diet of the insect pest.

15. A method for increasing crop yield, the method comprising growing the transgenic plant of claim 10 or 11, wherein the yield of the crop is increased in the presence of an insect pest relative to a crop not comprising the transgenic plant.

16. The method of any one of claims 12, 13, 14 or 15, wherein the insect pest or pest population is resistant to at least one Cry insecticidal protein.

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