Novel insect resistance genes and methods of use

Abstract

Compositions and methods for imparting insecticidal activity to bacteria, plants, plant cells, tissues, and seeds are provided. Compositions comprising coding sequences for toxin polypeptides are provided. The coding sequences can be used in DNA constructs or expression cassettes for transformation and expression in plants and bacteria. Compositions also include transformed bacteria, plants, plant cells, tissues, and seeds. Specifically, isolated toxin nucleic acid molecules are provided. Additionally, amino acid sequences corresponding to the polynucleotides are contemplated, and antibodies specifically bind to those amino acid sequences. Specifically, the present invention provides isolated nucleic acid molecules comprising a nucleotide sequence encoding the amino acid sequence set forth in any one of SEQ ID NOs: 16 to 30 or the nucleotide sequence set forth in any one of SEQ ID NOs: 1 to 15, as well as variants and fragments thereof.

Description

Technical Field

[0001] The present invention relates generally to the field of molecular biology. Novel genes encoding insecticidal proteins are provided. These proteins and nucleic acid sequences encoding them can be used to prepare insecticidal formulations and produce transgenic pest-resistant plants. Background Art

[0002] Plant pests are a major cause of crop losses worldwide. In the United States alone, approximately $8 billion is lost annually due to infestations by non-mammalian pests, including insects. Pests are primarily controlled by intensive applications of chemical pesticides, which act by inhibiting insect growth, preventing insect feeding or reproduction, or killing them. While good insect control can be achieved, these chemicals can sometimes also affect other beneficial insects. Another problem associated with the widespread use of chemical pesticides is the emergence of resistant insect variants. Various resistance management practices have partially alleviated this situation, but the demand for alternative pest control agents is increasing. Biological pest control agents, such as strains of Bacillus thuringiensis expressing insecticidal toxins such as delta-endotoxin, have also been applied to crops with satisfactory results, providing an alternative or supplement to chemical pesticides. In particular, expression of insecticidal toxins in transgenic plants, such as Bacillus thuringiensis delta-endotoxins, has provided effective protection against selected pests, and transgenic plants expressing such toxins have been commercialized, allowing farmers to reduce the application of chemical insect control agents.

[0003] Bacillus thuringiensis is a Gram-positive, spore-forming soil bacterium characterized by its ability to produce crystalline inclusions that are particularly toxic to certain insect orders and species, but harmless to plants and other non-target organisms. For this reason, compositions containing Bacillus thuringiensis strains or their insecticidal proteins can be used as environmentally acceptable insecticides to control agricultural pests or insect vectors of various human or animal diseases.

[0004] Crystal (Cry) proteins (delta-endotoxins) from Bacillus thuringiensis have potent insecticidal activity against primarily Lepidopteran, Hemipteran, Dipteran, and Coleopteran larvae. These proteins also show activity against pests of the orders Hymenoptera, Homoptera, Phthiraptera, Mallophaga, and Acari, as well as other invertebrate orders such as Nemathelminthes, Platyhelminthes, and Sarcomastigorphora (Feitelson (1993) The Bacillus Thuringiensis family tree. Advanced Engineered Pesticides, Marcel Dekker, Inc., New York, NY). These proteins were originally classified as CryI to CryV, primarily based on their insecticidal activity. The major classes are Lepidoptera-specific (I), Lepidoptera- and Diptera-specific (II), Coleoptera-specific (III), Diptera-specific (IV), and Nematode-specific (V) and (VI). Proteins are further classified into subfamilies; more highly related proteins within each family are assigned distinguishing letters, such as Cry1A, Cry1B, Cry1C, etc. Even more closely related proteins within each class are given names such as Cry1C1, Cry1C2, etc.

[0005] The nomenclature of Cry genes is described based on amino acid sequence homology rather than insect target specificity (Crickmore et al. (1998) Microbiol. Mol. Biol. Rev. 62: 807-813). In this classification, each toxin is assigned a unique name, combining a primary rank (Arabic numerals), a secondary rank (capital letters), a tertiary rank (lowercase letters), and a quaternary rank (another Arabic numeral). The Roman numerals in the primary rank have been replaced with Arabic numerals. Proteins with sequence identity less than 45% have different primary ranks, and the standards for the secondary and tertiary ranks are 78% and 95%, respectively.

[0006] Crystallins do not exhibit insecticidal activity until they have been ingested and dissolved in the insect midgut. Ingested protoxins are hydrolyzed by proteases in the insect digestive tract to form the active toxic molecule. and Whiteley (1989) Microbiol. Rev. 53:242-255. This toxin binds to the apical brush border receptors in the midgut of target larvae and inserts into the apical membrane, forming ion channels or pores, leading to larval death.

[0007] Delta-endotoxins typically have five conserved sequence domains and three conserved structural domains (see, e.g., de Maagd et al. (2001) Trends Genetics 17: 193-199). The first conserved structural domain consists of seven α helices and is involved in membrane insertion and pore formation. Domain II consists of three β sheets arranged in a Greek key configuration, and domain III consists of two antiparallel β sheets in the form of "jelly rolls" (de Maagd et al., 2001, supra). Domains II and III are involved in receptor recognition and binding and are therefore considered to be determinants of toxin specificity.

[0008] In addition to δ-endotoxins, there are also several other known classes of insecticidal protein toxins. VIP1 / VIP2 toxins (see, for example, U.S. Patent No. 5,770,696) are binary insecticidal toxins that exhibit strong activity against insects through a mechanism believed to involve receptor-mediated endocytosis, followed by cytotoxicity, similar to the mode of action of other binary ("A / B") toxins. A / B toxins, such as VIP, C2, CDT, CST, or B. anthracis edema and lethal toxins, initially interact with target cells as monomers through receptor-mediated specific binding to the "B" component. These monomers then form homologous heptamers. The "B" heptamer-receptor complex then serves as a docking platform that subsequently binds to and allows the enzymatic "A" component to translocate into the cytosol through receptor-mediated endocytosis. Once inside the cytosol of the cell, the "A" component inhibits normal cell function by, for example, ADP-ribosylation of G-actin or by increasing the level of intracellular cyclic AMP (cAMP). See Barth et al. (2004) Critical Reviews in Microbiology and Molecular Biology 68:373-402.

[0009] Intensive use of insecticides based on Bacillus thuringiensis has resulted in resistance in field populations of the diamondback moth (Plutella xylostella) (Ferre and Van Rie (2002) Annu. Rev. Entomol. 47:501-533). The most common resistance mechanism is reduced binding of the toxin to its specific midgut receptor. This may also lead to cross-resistance to other toxins that share the same receptor (Ferre and Van Rie (2002)).

[0010] An additional challenge is the fact that, in some cases, modifications to a pesticidal protein domain may allow for improved control of one pest, but at the same time weaken or reduce resistance to another pest previously controlled by the unmodified protein. Therefore, it is crucial not only to consider new pesticidal activities derived from genetic modification, but also to ensure that the protein maintains resistance to other pests against which it was active prior to genetic modification.

[0011] Because of the damage that insects can cause and the productivity improvements achieved by controlling a variety of pests, there is a continuing need to discover new forms of insecticidal toxins. Summary of the Invention

[0012] Provided are compositions and methods for imparting insecticidal activity to bacteria, plants, plant cells, tissues, and seeds. The compositions comprise nucleic acid molecules encoding sequences of insecticidal and insecticidal polypeptides, vectors comprising these nucleic acid molecules, and host cells comprising these vectors. The compositions also comprise insecticidal polypeptide sequences and antibodies to these polypeptides. The nucleotide sequences can be used in DNA constructs or expression cassettes for transformation and expression in organisms (including microorganisms and plants). The nucleotide or amino acid sequences can be synthetic sequences designed for expression in organisms (including, but not limited to, microorganisms or plants). Compositions also include bacteria, plants, plant cells, tissues, and seeds comprising the nucleotide sequences of the present invention.

[0013] In particular, isolated, recombinant, and chimeric nucleic acid molecules encoding insecticidal proteins that are variants of Axmi486 as disclosed in U.S. Patent Application Publication US2016-0311865 (incorporated herein by reference in its entirety) are provided. Surprisingly, these variants increase resistance to Spodoptera species while maintaining resistance to Plutella xylostella, Anticarsia gemmatalis, Diatraea grandiosella, Diatraea saccharalis, Heliothis virescens, Helicoverpa zea, and Pseudoplusia includens. In addition, amino acid sequences corresponding to the insecticidal proteins are encompassed. In particular, the present invention provides isolated, recombinant or chimeric nucleic acid molecules comprising a nucleotide sequence encoding an amino acid sequence as set forth in any one of SEQ ID NOs: 16-30 or a nucleotide sequence as set forth in SEQ ID NOs: 1-15, as well as biologically active variants and fragments thereof. Also encompassed are nucleotide sequences that are complementary to the nucleotide sequences of the present invention or hybridize with the sequences of the present invention or the complement of the sequences of the present invention. Further provided are vectors, host cells, plants and seeds comprising the nucleotide sequences of the present invention or a nucleotide sequence encoding the amino acid sequence of the present invention, as well as biologically active variants and fragments thereof.

[0014] Provided are methods for producing the polypeptides of the invention and for using these polypeptides to control or kill lepidopteran, hemiptera, coleopteran, nematode, or dipteran pests. Also included are methods and kits for detecting the nucleic acids and polypeptides of the invention in a sample.

[0015] The compositions and methods of the present invention can be used to produce organisms with enhanced pest resistance or tolerance. These organisms and compositions comprising the organisms are desirable for agricultural purposes. The compositions of the present invention can also be used to produce altered or improved proteins having pesticidal activity, or to detect the presence of pesticidal proteins or nucleic acids in products or organisms. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figures 1A-1E Improvements in anti-SAW activity of different mutants were shown;

[0017] Figures 2A-2B Whole culture bioassays of SAW-improved mutants against Helicoverpa armigera (Hz) are shown;

[0018] Figures 3A-3BWhole culture bioassays of SAW-improved mutants against soybean budworm (VBC) are shown;

[0019] Figures 4A-4B Whole culture bioassays of SAW-improved mutants against Spodoptera frugiperda (FAW) are shown;

[0020] Figure 5 Shown is a whole culture bioassay of SAW-improved mutants against Chrysodeixis includens (SBL). DETAILED DESCRIPTION

[0021] The present invention relates to compositions and methods for regulating pest resistance or tolerance in organisms, particularly plants or plant cells. "Resistance" refers to the killing of pests (e.g., insects) upon ingestion or other contact with a polypeptide of the present invention. "Tolerance" refers to the impairment or reduction of a pest's movement, feeding, reproduction, or other functions. The methods involve transforming an organism with a nucleotide sequence encoding a pesticidal protein of the present invention. Specifically, the nucleotide sequences of the present invention can be used to prepare plants and microorganisms with pesticidal activity. Thus, transformed bacteria, plants, plant cells, plant tissues, and seeds are provided. Compositions are pesticidal nucleic acids and proteins of the genus Bacillus or other species. The sequences herein can be used to construct expression vectors for subsequent transformation into an organism of interest, as probes for isolating other homologous (or partially homologous) genes, and to produce altered pesticidal proteins by methods known in the art, such as domain swapping or DNA shuffling. The proteins can be used to control or kill populations of lepidopteran, hemiptera, coleoptera, dipteran, and nematode pests, and to produce compositions with pesticidal activity.

[0022] "Insecticidal toxin" or "insecticide protein" refers to a toxin that has toxic activity against one or more pests, or a protein that has homology to such a protein, said one or more pests including, but not limited to, members of the orders Lepidoptera, Diptera, Hemiptera, and Coleoptera, or the phylum Nematoda. Insecticidal proteins comprise amino acid sequences deduced from the full-length nucleotide sequences disclosed herein, as well as amino acid sequences that are shorter than the full-length sequence due to the use of alternative downstream start sites or due to processing that produces shorter proteins with insecticidal activity. Processing may occur in the organism expressing the protein, or in the pest after ingestion of the protein.

[0023] Thus, provided herein are novel isolated, recombinant, or chimeric nucleotide sequences that confer pesticidal activity. Also provided are amino acid sequences of pesticidal proteins. Translation of such genes produces proteins that allow cells to control or kill pests that ingest the proteins.

[0024] Isolated nucleic acid molecules and variants and fragments thereof

[0025] One aspect of the present invention relates to isolated, recombinant or chimeric nucleic acid molecules comprising nucleotide sequences encoding pesticidal proteins and polypeptides or their biologically active portions, as well as nucleic acid molecules sufficient to be used as hybridization probes to identify nucleic acid molecules encoding proteins with regions of sequence homology. Nucleotide sequences that can hybridize with the nucleotide sequences of the present invention under stringent conditions defined elsewhere herein are also encompassed herein. As used herein, the term "nucleic acid molecule" is intended to include DNA molecules (e.g., recombinant DNA, cDNA, or genomic DNA) and RNA molecules (e.g., mRNA) and analogs of the DNA or RNA produced using nucleotide analogs. Nucleic acid molecules can be single-stranded or double-stranded, but are preferably double-stranded DNA. The term "recombinant" encompasses polynucleotides or polypeptides that have been manipulated relative to natural polynucleotides or polypeptides, such that the polynucleotides or polypeptides (e.g., in chemical composition or structure) are different from the polynucleotides or polypeptides that occur in nature. In another embodiment, "recombinant" polynucleotides do not contain internal sequences (i.e., introns) naturally present in the genomic DNA of the organism from which the polynucleotides originate. A typical example of such polynucleotides is so-called complementary DNA (cDNA).

[0026] An isolated, recombinant or chimeric nucleic acid (or DNA) is used herein to refer to a nucleic acid (or DNA) that is no longer in its natural environment, e.g., in vitro or in a recombinant bacterial or plant host cell. In some embodiments, an isolated, recombinant or chimeric nucleic acid does not contain a sequence (preferably a protein coding sequence) of a nucleic acid in the genomic DNA of the organism from which the nucleic acid is derived (i.e., a sequence located at the 5' and 3' ends of the nucleic acid). For purposes of the present invention, "isolated" does not include an isolated chromosome when used to refer to a nucleic acid molecule. For example, in various embodiments, an isolated Axmi486 variant nucleic acid molecule can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb, or 0.1 kb of a nucleotide sequence of a nucleic acid molecule in the genomic DNA of the cell from which the nucleic acid is derived. Herein, an "Axmi486 variant" refers to a nucleic acid variant as shown in SEQ ID No: 1-15 or a protein encoded by such a nucleic acid variant (e.g., SEQ ID No: 16-30). In various embodiments, the Axmi486 variant protein that is substantially free of cellular material comprises a protein preparation having less than about 30%, 20%, 10%, or 5% (by dry weight) of an Axmi486 variant protein (also referred to herein as a "contaminating protein"). In some embodiments, the recombinant nucleic acid of the invention comprises one or more nucleotide substitutions relative to any one of SEQ ID NOs: 1 to 15, or a variant or fragment thereof, wherein the Axmi486 variant exhibits improved resistance to Spodoptera species compared to an Axmi486 gene (e.g., nucleic acid SEQ ID No: 31) and / or variant protein (such as SEQ ID Nos 32-36) (herein, "Axmi486") that does not comprise the indicated variant.

[0027] The nucleotide sequences encoding the proteins of the present invention comprise the sequences set forth in any one of SEQ ID NOs: 1 to 15, and variants, fragments, and complements thereof, which exhibit increased resistance to Spodoptera litura species compared to the Axmi486 gene without the indicated variants. "Complement" means a nucleotide sequence that is sufficiently complementary to a given nucleotide sequence such that it can hybridize with the given nucleotide sequence to form a stable duplex. The corresponding amino acid sequences of the pesticidal proteins encoded by these nucleotide sequences are set forth in any one of SEQ ID NOs: 16 to 30.

[0028] Nucleic acid molecules that are fragments of these nucleotide sequences encoding pesticidal proteins are also encompassed by the present invention. A "fragment" refers to a portion of a nucleotide sequence encoding a pesticidal protein. A fragment of a nucleotide sequence can encode a biologically active portion of a pesticidal protein, or the fragment can be a fragment that can be used as a hybridization probe or PCR primer using the methods disclosed below. Nucleic acid molecules that are fragments of a nucleotide sequence encoding a pesticidal protein include at least about 50, 100, 200, 300, 400, 500, 600, 700, 800, 900 contiguous nucleotides, or up to the number of nucleotides present in the full-length nucleotide sequence encoding the pesticidal protein disclosed herein, depending on the intended use. "Contiguous" nucleotides refer to nucleotide residues that are immediately adjacent to each other. Fragments of the nucleotide sequences of the present invention will encode protein fragments that retain the biological activity of the pesticidal protein and, therefore, retain pesticidal activity. Thus, biologically active fragments of the polypeptides disclosed herein are also encompassed. By "retaining activity" is meant that the fragment will have at least about 30%, at least about 50%, at least about 70%, 80%, 90%, 95% or more of the pesticidal activity of the pesticidal protein. In one embodiment, the pesticidal activity is coleopteran activity. In another embodiment, the pesticidal activity is lepidopteran activity. In another embodiment, the pesticidal activity is nematicidal activity. In another embodiment, the pesticidal activity is dipteran activity. In another embodiment, the pesticidal activity is hemipteran activity. Methods for measuring pesticidal activity are well known in the art. See, e.g., Czapla and Lang (1990) J. Econ. Entomol. 83:2480-2485; Andrews et al. (1988) Biochem. J. 252:199-206; Marrone et al. (1985) J. Econ. Entomol. 78:290-293; and U.S. Pat. No. 5,743,477, all of which are incorporated herein by reference in their entirety.

[0029] Fragments of the nucleotide sequence encoding the pesticidal protein encoding the biologically active portion of the protein of the present invention will encode at least about 15, 25, 30, 50, 75, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450 contiguous amino acids, or up to the total number of amino acids present in the full-length pesticidal protein of the present invention. In some embodiments, the fragment is a proteolytic cleavage fragment. For example, the proteolytic cleavage fragment can have an N-terminal or C-terminal truncation of at least about 100 amino acids, about 120, about 130, about 140, about 150, or about 160 amino acids relative to any one of SEQ ID: 1 to 15. In some embodiments, the fragments contemplated herein are produced by removal of the C-terminal crystallization domain, for example, by proteolysis or by insertion of a stop codon in the coding sequence.

[0030] In various embodiments, the nucleic acids of the invention include the degenerate nucleic acid of any one of SEQ ID NOs: 1 to 15, wherein the degenerate nucleotide sequence encodes an amino acid sequence identical to any one of SEQ ID NOs: 16 to 30.

[0031] Preferred pesticidal proteins of the present invention are encoded by a nucleotide sequence that is substantially identical to the nucleotide sequence of any one of SEQ ID NOs: 1 to 15, or the pesticidal protein is substantially identical to the amino acid sequence set forth in any one of SEQ ID NOs: 16 to 30. "Substantially identical" refers to an amino acid or nucleotide sequence having at least about 60% or 65% sequence identity, about 70% or 75% sequence identity, about 80% or 85% sequence identity, about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity compared to a reference sequence using one of the alignment programs described herein using standard parameters. Those 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.

[0032] In order to determine the percent identity of two amino acid sequences or two nucleic acids, sequences are compared for the purpose of optimal comparison. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., the total number of the number of positions / positions (e.g., overlapping positions) of percent identity=identical positions × 100). In one embodiment, the two sequences are of the same length. In another embodiment, percent identity is calculated throughout the reference sequence (i.e., sequences disclosed herein are any one of SEQ ID NO: 1 to 30). In the case of allowing or not allowing spaces, techniques similar to those described below can be used to determine the percent identity between the two sequences. When calculating percent identity, exact matching is typically counted. A gap, i.e., a position in the alignment where a residue exists in one sequence but does not exist in another sequence, is considered to be a position with different residues.

[0033] The determination of percent identity between two sequences can be accomplished using a mathematical algorithm. A non-limiting example of a mathematical algorithm for comparing two sequences is the algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 87:2264, as modified in Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877. Such an algorithm is incorporated into the BLASTN and BLASTX programs of Altschul et al. (1990) J. Mol. Biol. 215:403. BLAST nucleotide searches can be performed with the BLASTN program (score = 100, wordlength = 12) to obtain nucleotide sequences homologous to the pesticidal nucleic acid molecules of the present invention. BLAST protein searches can be performed with the BLASTX program (score = 50, wordlength = 3) to obtain amino acid sequences homologous to the pesticidal protein molecules of the present invention. In order 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 utilized. Alternatively, PSI-Blast can be used to perform an iterative search that detects the distance relationship between two molecules. Referring to Altschul et al. (1997) supra. When utilizing BLAST, gapped BLAST, and PSI-Blast programs, the default parameters of each program (e.g., BLASTX and BLASTN) can be used. Alignments can also be performed manually by inspection.

[0034] Another non-limiting example of a mathematical algorithm for sequence comparison is the ClustalW algorithm (Higgins et al. (1994) Nucleic Acids Research 22:4673-4680). ClustalW compares multiple sequences and aligns all amino acid or DNA sequences, and can therefore provide data on the sequence conservation of all amino acid sequences. The ClustalW algorithm is used in several commercially available DNA / amino acid analysis software packages, for example, the ALIGNX module of the Vector NTI program suite (Invitrogen Corporation, Carlsbad, CA). After amino acid sequence alignment with ClustalW, percent amino acid identity can be estimated. A non-limiting example of a software program useful for analysis of ClustalW alignments is GENEDOC TM .GENEDOC TM (Karl Nicholas) allows to assess the amino acid (or DNA) similarity and identity between multiple proteins. Another non-limiting example of a mathematical algorithm for sequence comparison is the algorithm of Myers and Miller (1988) Computer Applications in Biological Sciences (CABIOS) 4: 11-17. Such an algorithm is incorporated into the ALIGN program (Version 2.0), which is a GCG Wisconsin Genetics Software Package, Version 10 (obtained from Accelrys, Inc., 9685 Scranton Rd., San Diego, CA, USA). When comparing amino acid sequences using the ALIGN program, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used.

[0035] Unless otherwise indicated, GAP version 10 using the algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48(3):443-453 will be used to determine sequence identity or similarity using the following parameters: percent identity and percent similarity for nucleotide sequences using GAP weight 50 and length weight 3 and the nwsgapdna.cmp scoring matrix; percent identity or percent similarity for amino acid sequences using GAP weight 8 and length weight 2 and the BLOSUM62 scoring program. Equivalent programs may also be used. "Equivalent program" refers to any sequence comparison program that produces an alignment having identical nucleotide residue matches and the same percent sequence identity for any two sequences in question when compared to the corresponding alignment generated by GAP version 10.

[0036] The present invention also encompasses variant nucleic acid molecules. "Variants" of nucleotide sequences encoding pesticidal proteins include those sequences that encode pesticidal proteins disclosed herein but that are conservatively different due to the degeneracy of the genetic code, as well as those sequences that are fully identical as described above. Molecular biological techniques well known to the public, such as polymerase chain reaction (PCR) and hybridization techniques outlined below, can be used to identify naturally occurring allelic variants. Variant nucleotide sequences also include synthetically derived nucleotide sequences that have been generated, for example, using site-directed mutagenesis but still encode pesticidal proteins disclosed in the present invention as discussed below. The variant proteins encompassed by the present invention have biological activity, i.e., they continue to have the desired biological activity of the native protein, i.e., pesticidal activity. "Retaining activity" means that the variant will have at least about 30%, at least about 50%, at least about 70%, or at least about 80% of the pesticidal activity of the native protein. A preferred embodiment of the present invention is an Axmi486 variant that shows increased activity against pests of the genus Spodoptera litura (e.g., against subtropical armyworms (Spodoptera eriadae)). Methods for measuring pesticidal activity are well known in the art. See, for example, Czapla and Lang (1990) Journal of Economic Entomology 83:2480-2485; Andrews et al. (1988) Journal of Biological Chemistry 252:199-206; Marrone et al. (1985) Journal of Economic Entomology 78:290-293; and U.S. Pat. No. 5,743,477, all of which are incorporated herein by reference in their entirety.

[0037] Those skilled in the art will further understand that changes can be introduced through the mutation of the nucleotide sequence of the present invention, resulting in changes in the amino acid sequence of the encoded pesticidal protein, without changing the biological activity of the protein. Therefore, the nucleic acid molecules separated through the variant can be produced by introducing one or more nucleotide substitutions, additions or deletions into the corresponding nucleotide sequence disclosed herein, so that one or more amino acid substitutions, additions or deletions are introduced into the encoded protein. Mutations can be introduced by standard techniques such as site-directed mutagenesis and PCR-mediated mutagenesis. Such variant nucleotide sequences are also encompassed by the present invention.

[0038] For example, conservative amino acid replacement can be carried out at the non-essential amino acid residues of one or more predictions." non-essential" amino acid residue is a residue that can change and not change biological activity from the wild-type sequence of pesticidal protein, while "essential" amino acid residue is necessary for biological activity." conservative amino acid replacement" is that amino acid residue is replaced by the amino acid residue with similar side chain. In the art, families of amino acid residues with similar side chains have been defined. These families comprise amino acid with basic side chain (for example, lysine, arginine, histidine), amino acid with acidic side chain (for example, aspartic acid, glutamic acid), amino acid with uncharged polar side chain (for example, glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), amino acid with non-polar side chain (for example, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), amino acid with β-branched side chain (for example, threonine, valine, isoleucine) and amino acid with aromatic side chain (for example, tyrosine, phenylalanine, tryptophan, histidine).

[0039] Amino acid substitutions can be made in non-conserved regions that retain function. Typically, such substitutions will not be made to conserved amino acid residues or to amino acid residues residing in conserved motifs (wherein such residues are essential for protein activity). Examples of conservative and potentially essential residues for protein activity include, for example, identical residues between all proteins contained in the comparison of toxins similar to or related to the sequence of the present invention (e.g., identical residues in the comparison of homologous proteins). Examples of conservative but potentially allowable conservative amino acid substitutions and still retain active residues include, for example, only conservatively substituted residues between all proteins contained in the comparison of toxins similar to or related to the sequence of the present invention (e.g., only conservatively substituted residues between all proteins contained in the comparison of homologous proteins). However, it will be understood by those skilled in the art that functional variants may have less conservative or non-conservative changes in conserved residues.

[0040] Alternatively, variant nucleotide sequences can be prepared by randomly introducing mutations along all or part of the coding sequence, such as by saturation mutagenesis, and the resulting mutants can be screened for their ability to confer pesticidal activity to identify mutants that retain activity. Following mutagenesis, the encoded protein can be recombinantly expressed and the activity of the protein can be determined using standard assay techniques.

[0041] Corresponding pesticidal sequences can be identified using methods such as PCR, hybridization, and the like, such sequences being substantially identical to the sequences of the invention (e.g., at least about 70%, at least about 75%, 80%, 85%, 90%, 95% or more sequence identity throughout the reference sequence) and having or conferring pesticidal activity. See, for example, Sambrook and Russell (2001) Molecular Cloning: A Laboratory Manual. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY) and Innis et al. (1990) PCR Protocols: A Guide to Methods and Applications (Academic Press, NY).

[0042] In hybridization methods, all or part of the pesticidal nucleotide sequence can be used to screen cDNA or genomic libraries. Methods for constructing such cDNA and genomic libraries are generally known in the art and are disclosed in Sambrook and Russell, 2001, supra. So-called hybridization probes can be genomic DNA fragments, cDNA fragments, RNA fragments, or other oligonucleotides, and can be used, for example, 32 The probes for hybridization can be labeled with detectable groups such as P or any other detectable markers such as other radioisotopes, fluorescent compounds, enzymes or enzyme cofactors. Probes for hybridization can be prepared by labeling synthetic oligonucleotides based on the known pesticidal protein encoding nucleotide sequences disclosed herein. Degenerate primers designed based on conserved nucleotides or amino acid residues in the nucleotide sequence or the encoded amino acid sequence can also be used. The probes typically include a nucleotide sequence region that hybridizes under stringent conditions to at least about 12, at least about 25, at least about 50, 75, 100, 125, 150, 175 or 200 consecutive nucleotides of the nucleotide sequence encoding the pesticidal protein of the present invention or its fragment or variant. Methods for preparing probes for hybridization are generally known in the art and are disclosed in Sambrook and Russell, 2001, supra, which is incorporated herein by reference.

[0043] For example, the entire insecticidal sequence disclosed herein or one or more portions thereof can be used as probes capable of specific hybridization with corresponding insecticidal protein sequences and messenger RNAs. In order to achieve specific hybridization under a variety of conditions, such probes comprise unique sequences and are preferably at least about 10 nucleotides in length, or at least about 20 nucleotides in length. Such probes can be used to amplify the corresponding insecticidal sequence from a selected organism or sample by PCR. This technology can be used to isolate additional coding sequences from a desired organism or to be used as a diagnostic assay to determine the presence of coding sequences in an organism. Hybridization techniques include hybridization screening of plating DNA libraries (plaques or colonies; see, for example, Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2nd edition, Cold Spring Harbor, New York, Cold Spring Harbor Press)).

[0044] Thus, the present invention encompasses probes for hybridization, as well as nucleotide sequences that are capable of hybridizing to all or part of a nucleotide sequence of the present invention (e.g., at least about 10, 25, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, or up to the full length of a nucleotide sequence disclosed herein). Hybridization of such sequences can be performed under stringent conditions. "Stringent conditions" or "stringent hybridization conditions" refer to conditions under which a probe will hybridize to its target sequence to a detectably greater extent than to other sequences (e.g., at least 2 times the background). Stringent conditions depend on the sequence and will vary in different circumstances. By controlling the stringency of the hybridization and / or washing conditions, a target sequence that is 100% complementary to the probe can be identified (homologous probing). Alternatively, stringency conditions can be adjusted to allow for some mismatches in the sequence so that a lower degree of similarity is detected (heterologous probing). Typically, the probe is less than about 1000 nucleotides in length, preferably less than 500 nucleotides in length.

[0045] Typically, stringent conditions will be those in which the salt concentration is less than about 1.5 M Na ions at pH 7.0 to 8.3, typically about 0.01 to 1.0 M Na ion concentration (or other salts) 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., greater 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. using a buffer solution of 30% to 35% formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulfate), and washing in 1× to 2× SSC (20×SSC=3.0 M NaCl / 0.3 M trisodium citrate) at 50° C. to 55° C. Exemplary moderate stringency conditions include hybridization in 40% to 45% formamide, 1.0 M NaCl, 1% SDS at 37° C., and a wash in 0.5× to 1× SSC at 55° C. to 60° C. Exemplary high stringency conditions include hybridization in 50% formamide, 1 M NaCl, 1% SDS at 37° C., and a wash in 0.1× SSC at 60° C. to 65° C. Optionally, the wash buffer can include about 0.1% to about 1% SDS. The duration of hybridization is generally less than about 24 hours, typically about 4 hours to about 12 hours.

[0046] Specificity is usually a function of post-hybridization washes, with the key factors being the ionic strength and temperature of the final wash solution. For DNA-DNA hybrids, T m This can be approximated from the equation of Meinkoth and Wahl (1984) Anal. Biochem. 138:267-284: m = 81.5°C + 16.6 (log M) + 0.41 (% GC) - 0.61 (% formamide) - 500 / L; where M is the molar concentration of monovalent cations, GC% is the percentage of guanosine and cytosine nucleotides in the DNA, % formamide is the percentage of formamide in the hybridization solution, and L is the length of the hybrid in base pairs. m The temperature (under defined ionic strength and pH) at which 50% of the complementary target sequence hybridizes to a perfectly matched probe. m decrease by about 1°C; therefore, T m , hybridization and / or washing conditions to hybridize to the desired identity sequence. For example, if a sequence with >90% identity is sought, then T m The temperature can be lowered by 10°C. Generally, stringent conditions are selected to be higher than the thermal melting point (T) of the specific sequence and its complement at a defined ionic strength and pH. m However, severe stringency can be achieved at temperatures below the thermal melting point (T m) 1 ° C, 2 ° C, 3 ° C or 4 ° C using hybridization and / or washing; moderate stringency conditions can be below the thermal melting point (T m ) 6 ℃, 7 ℃, 8 ℃, 9 ℃ or 10 ℃, and hybridization and / or washing can be performed at low stringency conditions below the thermal melting point (T m ) 11 ° C, 12 ° C, 13 ° C, 14 ° C, 15 ° C or 20 ° C. Hybridization and / or washing are used. Using the equation, hybridization and wash composition and the desired T m , the skilled artisan will understand that variations in the stringency of hybridization and / or wash solutions are inherently described. m Below 45 ℃ (aqueous solution) or 32 ℃ (formamide solution), then preferably increase SSC concentration, so that higher temperature can be used. Extensive guidance to nucleic acid hybridization is found in Tijssen (1993) " Laboratory Techniques in Biochemistry and Molecular Biology—Hybridization with Nucleic Acid Probes ", Part I, Chapter 2 (Elsevier, New York); and Ausubel et al. (1995) " Current Protocols in Molecular Biology ", Chapter 2 (Greene Publishing and Wiley-Interscience, New York). See Sambrook et al. (1989) " Molecular Cloning: A Laboratory Manual " (2nd ed., Cold Spring Harbor, New York, Cold Spring Harbor, NY, Cold Spring Harbor Laboratory Press).

[0047] Isolated proteins and variants and fragments thereof

[0048] Pesticidal proteins are also encompassed by the present invention. "Pesticidal protein" refers to a protein having an amino acid sequence as set forth in any one of SEQ ID NOs: 16 to 30. Fragments, biologically active portions, and variants thereof are also provided and can be used to practice the methods of the present invention. "Isolated protein" or "recombinant protein" is used to refer to a protein that is no longer in its natural environment, for example, in vitro or in a recombinant bacterial or plant host cell. In some embodiments, the recombinant protein is a variant of any one of SEQ ID NOs: 16 to 30, wherein the variant comprises at least one amino acid substitution, deletion, or insertion relative to any one of SEQ ID NOs: 16 to 30.

[0049] "Fragments" or "biologically active portions" include polypeptide fragments comprising an amino acid sequence that is substantially identical to the amino acid sequence set forth in any one of SEQ ID NOs: 16 to 30 and exhibits pesticidal activity. Biologically active portions of pesticidal proteins can be polypeptides that are, for example, 10, 25, 50, 100, 150, 200, 250, or more amino acids in length. Such biologically active portions can be prepared by recombinant techniques and evaluated for pesticidal activity. Methods for measuring pesticidal activity are well known in the art. See, for example, Czapla and Lang (1990) J. Econ. Entom. 83:2480-2485; Andrews et al. (1988) J. Biol. Chem. 252:199-206; Marrone et al. (1985) J. Econ. Entom. 78:290-293; and U.S. Pat. No. 5,743,477, all of which are incorporated herein by reference in their entirety. As used herein, a fragment includes at least 8 contiguous amino acids of any one of SEQ ID NOs: 16 to 30. However, the invention encompasses other fragments, such as any fragment of a protein that is greater than about 10, 20, 30, 50, 100, 150, 200, 250, or more amino acids in length.

[0050] "Variant" means a protein or polypeptide having an amino acid sequence that is at least about 60%, 65%, about 70%, 75%, about 80%, 85%, about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of any one of SEQ ID NOs: 16 to 30. Variants also include polypeptides encoded by nucleic acid molecules that hybridize under stringent conditions to the nucleic acid molecules of any one of SEQ ID NOs: 16 to 30 or their complement. Variants include polypeptides that differ in amino acid sequence due to mutagenesis. Variant proteins encompassed by the present invention are biologically active, i.e., they continue to possess the desired biological activity of the native protein, i.e., retain pesticidal activity. In some embodiments, the variants have improved activity relative to the native protein. Methods for measuring pesticidal activity are well known in the art. See, e.g., Czapla and Lang (1990) Journal of Economic Entomology 83:2480-2485; Andrews et al. (1988) Journal of Biological Chemistry 252:199-206; Marrone et al. (1985) Journal of Economic Entomology 78:290-293; and U.S. Pat. No. 5,743,477, all of which are incorporated herein by reference in their entirety.

[0051] Bacterial genes, such as genes of the present invention, often have multiple methionine start codons at the beginning of an open reading frame. Typically, translation initiation at one or more of these start codons can result in the production of functional proteins. These start codons can include ATG codons. However, bacteria such as bacillus (Bacillus sp.) also recognize the codon GTG as a start codon, and proteins that begin translation at the GTG codon contain methionine at the first amino acid. In rare cases, translation in bacterial systems can start with the TTG codon, although in this case TTG encodes methionine. In addition, it is generally not possible to determine a priori which of these codons are naturally used in bacteria. Therefore, it should be understood that using a methionine codon alternatively may also result in the production of insecticidal proteins. These insecticidal proteins are encompassed in the present invention and can be used in the method of the present invention. It should be understood that when expressed in plants, for correct translation, it is necessary to change the alternative start codon to ATG.

[0052] In various embodiments of the present invention, the pesticidal protein comprises an amino acid sequence deduced from the full-length nucleotide sequence disclosed herein, as well as an amino acid sequence that is shorter than the full-length sequence due to the use of an alternative downstream start site. Thus, the nucleotide sequence of the present invention and / or the vectors, host cells, and plants comprising the nucleotide sequence of the present invention (as well as methods for preparing and using the nucleotide sequence of the present invention) may comprise a nucleotide sequence encoding an amino acid sequence corresponding to any one of SEQ ID NOs: 16 to 30.

[0053] Also encompassed are antibodies to the polypeptides of the invention or variants or fragments thereof. Methods for generating antibodies are well known in the art (see, e.g., Harlow and Lane (1988) Antibodies: A Laboratory Manual, Cold Spring Harbor, NY; U.S. Patent No. 4,196,265).

[0054] Therefore, one aspect of the present invention relates to antibodies, single-chain antigen-binding molecules, or other proteins that specifically bind to one or more of the protein or peptide molecules of the present invention and their homologues, fusions, or fragments. In a particularly preferred embodiment, the antibody specifically binds to a protein having an amino acid sequence as shown in any one of SEQ ID NOs: 16 to 30, or a fragment thereof. In another embodiment, the antibody specifically binds to a fusion protein comprising an amino acid sequence selected from the group consisting of an amino acid sequence as shown in any one of SEQ ID NOs: 16 to 30, or a fragment thereof. In various embodiments, the antibody that specifically binds to a protein of the present invention or a fusion protein comprising a protein of the present invention is a non-naturally occurring antibody.

[0055] The antibodies of the present invention can be used to quantitatively or qualitatively detect the protein or peptide molecules of the present invention, or to detect post-translational modifications of proteins. As used herein, an antibody or peptide is said to "specifically bind" to a protein or peptide molecule of the present invention if the presence of non-related molecules does not competitively inhibit such binding.

[0056] The antibodies of the present invention may be included in a kit for detecting a protein or peptide molecule of the present invention. The present invention further includes a method for detecting a protein or peptide molecule of the present invention (particularly a protein encoded by an amino acid sequence shown in any one of SEQ ID NOs: 16 to 30, including variants or fragments of said protein that are capable of specifically binding to an antibody of the present invention), the method comprising contacting a sample with an antibody of the present invention and determining whether the sample contains the protein or peptide molecule of the present invention. Methods for detecting proteins or peptides of interest using antibodies are known in the art.

[0057] Altered or improved variants

[0058] It is recognized that the DNA sequence of a pesticidal protein can be altered in a variety of ways, and that these alterations can result in a DNA sequence encoding a protein having an amino acid sequence that differs from that encoded by the pesticidal protein of the invention. This protein can be altered in various ways, including amino acid substitutions, deletions, truncations, and insertions of one or more amino acids of any one of SEQ ID NOs: 16 to 30, including up to about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 100, about 105, about 110, about 115, about 120, about 125, about 130, about 135, about 140, about 145, about 150, about 155 or more amino acid substitutions, deletions, or insertions. Methods for such manipulations are well known in the art. For example, amino acid sequence variants of insecticidal proteins can be prepared by mutation of DNA. This can also be achieved by one of several forms of mutagenesis and / or directed evolution. In some respects, the variation of encoding in the amino acid sequence will not significantly affect the function of the protein. Such variants will have the desired insecticidal activity. However, it should be understood that the ability of insecticidal proteins to impart insecticidal activity can be improved by using such technology to the composition of the present invention. For example, insecticidal proteins can be expressed in host cells such as XL-1Red (Stratagene, La Jolla, CA), which show a high base error incorporation rate during DNA replication. After breeding in such bacterial strains, DNA can be isolated (for example, by preparing plasmid DNA, or by PCR amplification and cloning the gained PCR fragment into a vector), the insecticidal protein mutation can be cultivated in non-mutagenized strains, and the insecticidal activity of the mutant gene can be identified, for example, by performing assays to test insecticidal activity. Typically, proteins are mixed and used for feeding assays or toxins are directly exposed to insects. See, for example, Marrone et al. (1985) Journal of Economic Entomology 78:290-293 and Cira et al. (2017) J Pest Sci 90:1257–1268. Such assays can include contacting a plant with one or more pests and determining the plant's ability to survive and / or cause the death of the pests. Examples of mutations that increase toxicity are found in Schnepf et al. (1998) Critiques des Microbiol et Molecular Biol 62:775-806.

[0059] Alternatively, changes can be made to the protein sequence at the amino or carboxyl termini of many proteins without significantly affecting activity. This can include insertions, deletions, or changes introduced by modern molecular methods, such as PCR, which involves PCR amplification of protein coding sequences by altering or extending them by including amino acid coding sequences in oligonucleotides used in PCR amplification. Alternatively, the added protein sequence can comprise a complete protein coding sequence, such as protein coding sequences commonly used in the art to generate protein fusions. Such fusion proteins are commonly used to: (1) increase expression of a protein of interest; (2) introduce binding domains, enzymatic activities, or epitopes to facilitate protein purification, protein detection, or other experimental uses known in the art; (3) target protein secretion or translation to subcellular organelles, such as the periplasmic space of Gram-negative bacteria or the endoplasmic reticulum of eukaryotic cells, the latter of which typically results in glycosylation of the protein.

[0060] Variant nucleotide and amino acid sequence of the present invention also encompass sequences derived from mutagenesis and recombination processes (such as DNA reorganization). By this type of program, one or more different pesticidal protein coding regions can be used to produce new pesticidal proteins with desired characteristics. In this way, recombinant polynucleotide libraries are produced by related sequence polynucleotide colonies, and the sequence polynucleotides comprise sequences with substantially sequence identity and capable of homologous recombination in vitro or in vivo. For example, using this method, the sequence motifs encoding paid close attention to domains can be reorganized between pesticidal genes of the present invention and other known pesticidal genes to obtain new genes encoding proteins with improved paid close attention to properties (such as increased insecticidal activity). The strategy for this type 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.

[0061] Domain exchange or reorganization is another mechanism for producing modified insecticidal proteins. Domains can be exchanged between insecticidal proteins to produce hybrid or chimeric toxins with improved insecticidal activity or target spectrum. Methods for producing recombinant proteins and testing their insecticidal activity are well known in the art (see, for example, Naimov et al. (2001) Appl. Environ. Microbiol. 67:5328-5330; de Maagd et al. (1996) Appl. Environ. Microbiol. 62:1537-1543; Ge et al. (1991) J. Biol. Chem. 266:17954-17958; Schnepf et al. (1990) J. Biol. Chem. 265:20923-20930; Rang et al. (1999) Appl. Environ. Microbiol. 65:2918-2925).

[0062] In yet another embodiment, variant nucleotide and / or amino acid sequences can be obtained using one or more of the following: error-prone PCR, oligonucleotide-directed mutagenesis, assembly PCR, sexual PCR mutagenesis, in vivo mutagenesis, cassette mutagenesis, recursive ensemble mutagenesis, exponential ensemble mutagenesis, site-specific mutagenesis, gene reassembly, gene site-saturation mutagenesis, substitution mutagenesis, synthetic ligation reassembly (SLR), recombination, recursive sequence recombination, phosphothioate-modified DNA mutagenesis, uracil-containing template mutagenesis, gapped duplex mutagenesis, point mismatch repair mutagenesis, repair-deficient host strain mutagenesis, chemical mutagenesis, radiation-induced mutagenesis, deletion mutagenesis, restriction selection mutagenesis, restriction purification mutagenesis, artificial gene synthesis, ensemble mutagenesis, chimeric nucleic acid multimer generation, and the like.

[0063] carrier

[0064] The pesticidal sequences of the present invention can be provided in expression cassettes for expression in a host cell of interest, for example, a plant cell or a microorganism. A "plant expression cassette" refers to a DNA construct capable of causing the expression of a protein from an open reading frame in a plant cell. Typically, these contain a promoter and a coding sequence. Typically, such constructs will also contain a 3' untranslated region. Such constructs may contain a "signal sequence" or "leader sequence" to facilitate co-translational or post-translational transport of the peptide to certain intracellular structures, such as chloroplasts (or other plastids), the endoplasmic reticulum, or the Golgi apparatus.

[0065] "Signal sequence" refers to a sequence known or suspected to cause co-translational or post-translational peptide transport across the cell membrane. In eukaryotes, this generally involves secretion into the Golgi apparatus and produces some glycosylation. Bacterial insecticidal toxins are generally synthesized as protoxins that are proteolytically activated in the intestinal tract of target pests (Chang (1987) "Methods Enzymol." 153: 507-516). In some embodiments of the present invention, the signal sequence is located in the native sequence, or can be derived from the sequence of the present invention. "Leader sequence" refers to any sequence that, when translated, produces an amino acid sequence sufficient to trigger the co-translational transport of a peptide chain to a subcellular organelle. Therefore, this includes leader sequences for targeted transport and / or glycosylation by entering the endoplasmic reticulum, entering the vacuole, or plastids comprising chloroplasts, mitochondria, etc. Therefore, further provided herein are polypeptides comprising an amino acid sequence of the present invention, the amino acid sequence being operably linked to a heterologous leader sequence or signal sequence.

[0066] "Plant transformation vector" refers to the DNA molecules necessary for the effective transformation of plant cells. Such molecules can be composed of one or more plant expression cassettes and can be organized into more than one "vector" DNA molecules. For example, a binary vector is a plant transformation vector that utilizes two non-continuous DNA vectors to encode all cis- and trans-acting functions necessary for plant cell transformation (Hellens and Mullineaux (2000) Trends in Plant Science 5:446-451). A "vector" refers to a nucleic acid construct designed to be transferred between different host cells. An "expression vector" refers to a vector that can be incorporated into, integrated into, and expressed in exogenous cells. The box will comprise a 5' and / or 3' regulatory sequence that is operably connected to the sequence of the present invention. "Operably connected" refers to the functional connection between a promoter and a second sequence, wherein the promoter sequence initiates and mediates the transcription of the DNA sequence corresponding to the second sequence. Generally, being operably connected means that the connected nucleic acid sequence is continuous, and when two protein coding regions need to be connected, it is continuous and in the same reading frame. In some embodiments, the nucleotide sequence is operably linked to a heterologous promoter capable of directing expression of the nucleotide sequence in a host cell (e.g., a microbial host cell or a plant host cell). The cassette may additionally contain at least one additional gene to be co-transformed into the organism. Alternatively, the additional genes may be provided on multiple expression cassettes.

[0067] In various embodiments, the nucleotide sequence of the present invention is operably linked to a heterologous promoter capable of directing expression of the nucleotide sequence in a cell, for example, a plant cell or a microorganism. A "promoter" refers to a nucleic acid sequence that acts to direct transcription of a downstream coding sequence. A promoter, along with other transcriptional and translational regulatory nucleic acid sequences (also referred to as "control sequences"), is essential for expression of the DNA sequence of interest.

[0068] Such expression cassettes are provided with a plurality of restriction sites for enabling insertion of the pesticidal sequence under the transcriptional control of the regulatory region.

[0069] The expression cassette will contain, in the 5'-3' direction of transcription, a transcriptional and translational initiation region (i.e., a promoter) that functions in plants, a DNA sequence of the present invention, and a translational and transcriptional termination region (i.e., a terminator). The promoter may be native or analogous, or foreign or heterologous to the plant host and / or the DNA sequence of the present invention. In addition, the promoter may be a native sequence or, alternatively, a synthetic sequence. When a promoter is "native" or "homologous" to the plant host, it means that the promoter is present in the native plant into which the promoter is introduced. When a promoter is "foreign" or "heterologous" to the DNA sequence of the present invention, it means that the promoter is not native or naturally occurring to the operably linked DNA sequence of the present invention. Promoters may be inducible or constitutive. Promoters may be naturally occurring, may be composed of portions of various naturally occurring promoters, or may be partially or completely synthetic. Studies of promoter structure, such as those by Harley and Reynolds (1987) Nucleic Acids Res., 15:2343-2361, provide guidance for promoter design. In addition, the position of the promoter relative to the start of transcription can be optimized. See, for example, Roberts et al. (1979) Proceedings of the National Academy of Sciences of the United States of America, 76:760-764. Many suitable promoters for use in plants are well known in the art.

[0070] For example, suitable constitutive promoters for use in plants include: promoters from plant viruses, such as the peanut chlorotic streak cauliflower mosaic virus (PC1SV) promoter (U.S. Pat. No. 5,850,019); the 35S promoter from cauliflower mosaic virus (CaMV) (Odell et al. (1985) Nature 313:810-812); the 35S promoter described in Kay et al. (1987) Science 236:1299-1302; the chlorella virus promoter described in U.S. Pat. 5,563,328) and the full-length transcriptional promoter from the figwort mosaic virus (FMV) (U.S. Patent No. 5,378,619); promoters from genes such as rice actin (McElroy et al. (1990) Plant Cell 2:163-171 and U.S. Patent No. 5,641,876); ubiquitin (Christensen et al. (1989) Plant Molecular Biology 2:163-171 and U.S. Patent No. 5,641,876); Mol. Biol., 12:619-632 and Christensen et al. (1992) Plant Mol. Biol., 18:675-689 and Grefen et al. (2010) Plant J, 64:355-365; pEMU (Last et al. (1991) Theor. Appl. Genet., 81:581-588); MAS (Velten et al. (1984) EMBO J. 5,510,474); maize H3 histone (Lepetit et al. (1992) Mol. Gen. Genet. 231:276-285 and Atanassova et al. (1992) J. Botanica 2(3):291-300); Brassica napus ALS3 (PCT application WO 97 / 41228); plant ribulose-biscarboxylase / oxygenase (RuBisCO) small subunit gene; orbivirus (AU 689 311) or cassava vein mosaic virus (CsVMV, US 7,053,205); promoters from soybean (Pbdc6 or Pbdc7 described in WO / 2014 / 150449 or the ubiquitin 3 promoter described in U.S. Pat. No. 7393948 and U.S. Pat. No. 8395021); and promoters of various Agrobacterium genes (see U.S. Pat. Nos. 4,771,002; 5,102,796; 5,182,200; and 5,428,147).

[0071] Suitable inducible promoters for use in plants include: a copper-responsive promoter from the ACE1 system (Mett et al. (1993) Proc. Natl. Acad. Sci. USA 90:4567-4571); the promoter of the maize In2 gene that responds to benzenesulfonamide herbicide safeners (Hershey et al. (1991) Mol. Gen. Genetics 227:229-237 and Gatz et al. (1994) Mol. Gen. Genetics 243:32-38); and the promoter from the Tet repressor of Tn10 (Gatz et al. (1991) Mol. Gen. Genetics 243:32-38). Plant Genetics and Genomics 227:229-237). Another type of inducible promoter used in plants is a promoter that responds to an inducing agent to which plants do not normally respond. Exemplary inducible promoters of this type are inducible promoters from steroid hormone genes whose transcriptional activity is induced by glucocorticoids (Schena et al. (1991) Proc. Natl. Acad. Sci. USA 88:10421) or the more recently used chimeric transcriptional activator XVE for use in an inducible plant expression system based on the estrogen receptor activated by estradiol (Zuo et al. (2000) J. Botany, 24:265-273). Other inducible promoters used in plants are described in EP. The promoters described herein are described in PCT WO 93 / 21334 and PCT WO 97 / 06269, which are incorporated herein by reference in their entirety. Promoters composed of portions of other promoters and partially or completely synthetic promoters may also be used. See, for example, Ni et al. (1995) J. Botanica 7:661-676 and PCT WO 95 / 14098 for descriptions of such promoters for use in plants.

[0072] In an embodiment of the present invention, a promoter sequence specific to a particular region or tissue of a plant can be used to express the insecticidal protein of the present invention, such as a seed-specific promoter (Datla, R. et al., 1997, Biotechnology Ann. Rev. 3, 269-296), in particular a rapeseed protein promoter (EP 255 378A1), a phaseolin promoter, a glutenin promoter, a sunflower glycoside promoter (WO92 / 17580), an albumin promoter (WO98 / 45460), an oleosin promoter (WO98 / 45461), a SAT1 promoter, or a SAT3 promoter (PCT / US98 / 06978).

[0073] Inducible promoters advantageously selected from the group consisting of phenylalanine ammonia lyase (PAL), HMG-CoA reductase (HMG), chitinase, glucanase, proteinase inhibitor (PI), PR1 family genes, nopaline synthase (nos) and vspB promoters ( US 5 670 349 , Table 3), HMG2 promoter ( US 5 670 349 ), apple β-galactosidase (ABG1) promoter, and apple aminocyclopropanecarboxylate synthase (ACC synthase) promoter ( WO 98 / 45445 ) can also be used in the constructs of the present invention. Multiple promoters can be used in the constructs of the present invention, including serial use.

[0074] The promoter may comprise or be modified to comprise one or more enhancer elements. In some embodiments, the promoter may comprise multiple enhancer elements. Promoters containing enhancer elements provide higher levels of transcription compared to promoters that do not comprise enhancer elements. Suitable enhancer elements for use in plants include the PC1SV enhancer element (U.S. Pat. No. 5,850,019), the CaMV 35S enhancer element (U.S. Pat. Nos. 5,106,739 and 5,164,316), and the FMV enhancer element (Maiti et al. (1997) Transgenic Res. 6: 143-156); for example, the translational activator of tobacco mosaic virus (TMV) as described in application WO 87 / 07644 or tobacco etch virus (TEV) as described by Carrington and Freed 1990, J. Virol. 64: 1590-1597, or introns such as the adh1 intron of maize or intron 1 of rice actin. See also PCT WO96 / 23898, WO2012 / 021794, WO2012 / 021797, WO2011 / 084370 and WO2011 / 028914.

[0075] Typically, such constructs may contain 5' and 3' untranslated regions. Such constructs may contain a "signal sequence" or "leader sequence" to promote the co-translational or post-translational transport of the peptide of interest to certain intracellular structures, such as chloroplasts (or other plastids), the endoplasmic reticulum or the Golgi apparatus, or to be secreted. For example, the construct may be engineered to contain a signal peptide to promote transfer of the peptide to the endoplasmic reticulum. A "signal sequence" refers to a sequence known or suspected to cause co-translational or post-translational peptide transport across the cell membrane. In eukaryotes, this typically involves secretion into the Golgi apparatus and produces some glycosylation. A "leader sequence" refers to any sequence that, when translated, produces an amino acid sequence sufficient to trigger the co-translational transport of the peptide chain to a subcellular organelle. Thus, this includes leader sequences targeted for transport and / or glycosylation by entering the endoplasmic reticulum, entering the vacuole, plastids including chloroplasts, mitochondria, etc. It may also be preferred that the plant expression cassette be engineered to contain introns so that expression requires intron-mediated mRNA processing.

[0076] The term "3' untranslated region" refers to the polynucleotides located downstream of the coding sequence. The polyadenylation signal sequence and other sequences encoding regulatory signals that affect the addition of polyadenylic acid tracts to the 3' end of the mRNA precursor are considered 3' untranslated regions. The term "5' untranslated region" refers to the polynucleotides located upstream of the coding sequence.

[0077] Other upstream or downstream non-translated elements include enhancers. Enhancers are polynucleotides used to increase expression of a promoter region. Enhancers are well known in the art and include, but are not limited to, the SV40 enhancer region and the 35S enhancer element.

[0078] 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, the DNA sequence of interest, the plant host, or any combination thereof). Convenient termination regions are available from the Ti plasmid of Agrobacterium tumefaciens, such as the octopine synthase and nopaline synthase termination regions. See also Guerineau et al. (1991) Mol. Genet. Genomics 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 Acids Res. 15:9627-9639.

[0079] In appropriate cases, the gene can be optimized to improve the expression (synthetic DNA sequence) in the transformed host cell. That is, the preferred codons of the host cell can be used to synthesize the gene to improve expression, or the codons can be used to synthesize the gene with the preferred codon usage frequency of the host. The expression of the open reading frame of the synthetic DNA sequence in the cell results in the production of a polypeptide of the present invention. The synthetic DNA sequence can be used to simply remove unwanted restriction endonuclease sites, promote DNA cloning strategies, change or remove any potential codon bias, change or improve GC content, remove or change alternative reading frames and / or change or remove intron / exon splicing recognition sites, polyadenylation sites, Shine-Delgarno sequences, unwanted promoter elements, etc. that may be present in the native DNA sequence. Generally, the GC content of the gene will increase. Referring to, for example, Campbell and Gowri (1990) Plant Physiology 92:1-11 discuss the preferred codon usage of the host. Methods for synthesizing preferred genes of plants can be obtained in this area. See, e.g., U.S. Patent Nos. 5,380,831 and 5,436,391, U.S. Patent Publication No. 20090137409, and Murray et al. (1989) Nucleic Acids Res. 17:477-498, herein incorporated by reference.

[0080] In one embodiment, the present invention provides the invention claims a kind of synthetic DNA sequence that can be used for the present invention.Also might use synthetic DNA sequence to other improvement to be introduced into DNA sequence, as introducing intron sequence, generation is expressed as the DNA sequence of the protein fusion to organelle targeting sequence, as chloroplast transit peptide, apoplast / vacuole targeting peptide or make gained peptide remain in the peptide sequence in the endoplasmic reticulum.Therefore, in one embodiment, pesticidal protein targeting chloroplast is expressed.In this way, when pesticidal protein is not directly inserted into the chloroplast, expression cassette will contain the nucleic acid of coding transit peptide additionally so that pesticidal protein is guided to chloroplast.This type of transit peptide is known in the art. See, e.g., Von Heijne et al. (1991) Plant Mol. Biol. Rep. 9: 104-126; Clark et al. (1989) J. Biol. Chem. 264: 17544-17550; Della-Cioppa et al. (1987) Plant Physiol. 84: 965-968; Romer et al. (1993) Biochem. Biophys. Res. Commun. 196: 1414-1421; and Shah et al. (1986) Science 233: 478-481.

[0081] Insecticidal genes to be targeted to the chloroplast can be optimized for expression in the chloroplast to account for the differences in codon usage between the plant nucleus and this organelle. In this way, the nucleic acid of interest can be synthesized using chloroplast-preferred codons. See, for example, U.S. Patent No. 5,380,831, which is incorporated herein by reference.

[0082] Plant transformation

[0083] The methods of the present invention relate to introducing a nucleotide construct into a plant. "Introducing" refers to presenting a nucleotide construct to a plant in such a manner that the construct enters the interior of a plant cell. The methods of the present invention do not require the use of a specific method for introducing a nucleotide construct into a plant; they only require that the nucleotide construct enter the interior of at least one cell of the plant. Methods for introducing a nucleotide construct into a plant are known in the art and include, but are not limited to, stable transformation methods, transient transformation methods, and viral-mediated methods.

[0084] "Plant" refers to whole plants, plant organs (e.g., leaves, stems, roots, etc.), seeds, plant cells, propagules, embryos, and progeny thereof. Plant cells can be differentiated or undifferentiated (e.g., callus, suspension culture cells, protoplasts, leaf cells, root cells, phloem cells, pollen).

[0085] "Transgenic plants" or "transformed plants" or "stably transformed" plants or cells or tissues are plants that have had exogenous nucleic acid sequences or DNA fragments incorporated or integrated into the plant cells. These nucleic acid sequences include nucleic acid sequences that are exogenous or not present in the untransformed plant cells, as well as nucleic acid sequences that are endogenous or present in the untransformed plant cells. "Heterologous" generally refers to a nucleic acid sequence that is not endogenous to the cell or part of its native genome and that has been added to the cell by infection, transfection, microinjection, electroporation, microprojection, or the like.

[0086] The transgenic plants of the present invention express one or more novel toxin sequences disclosed herein. In some embodiments, the protein or nucleotide sequence of the present invention is advantageously combined in plants with other genes encoding proteins or RNA that confer useful agronomic properties on such plants. Among the genes encoding proteins or RNA that confer useful agronomic properties on transformed plants, DNA sequences encoding proteins that confer tolerance to one or more herbicides, proteins that confer tolerance to certain insects, proteins that confer tolerance to certain diseases, DNA encoding RNA that provides nematode or insect control, etc. Such genes are specifically described in disclosed PCT patent applications WO91 / 02071 and WO95 / 06128, as well as U.S. Patent No. 7,923,602 and U.S. Patent Application Publication No. 20100166723, each of which is incorporated herein by reference in its entirety.

[0087] Among the DNA sequences encoding proteins that confer tolerance to certain herbicides on transformed plant cells and plants, mention may be made of: the bar or PAT gene or the Streptomyces coelicolor gene that confers tolerance to the glufosinate herbicide described in WO 2009 / 152359; genes encoding suitable EPSPS that confer tolerance to herbicides that target EPSPS, such as glyphosate and its salts ( US 4,535,060 , US 4,769,061 , US 5,094,945 , US 4,940,835 , US 5,188,642 , US 4,971,908 , US 5,145,783 , US 5,310,667 , US 5,312,910 , US 5,627,061, US 5,633,435); genes encoding glyphosate-n-acetyltransferase (e.g., US 8,222,489, US 8,088,972, US 8,044,261, US 8,021,857, US 8,008,547, US 7,999,152, US 7,998,703, US 7,863,503, US 7,714,188, US 7,709,702, US 7,666,644, US 7,666,643, US 7,531,339, US 7,527,955, and US 7,405,074); genes encoding glyphosate oxidoreductase (e.g., US 5,463,175); or a gene encoding an HPPD inhibitor-resistant protein (e.g., the HPPD inhibitor-resistant genes described in WO 2004 / 055191, WO 199638567, US 6791014, WO2011 / 068567, WO2011 / 076345, WO2011 / 085221, WO2011 / 094205, WO2011 / 068567, WO2011 / 094199, WO2011 / 094205, WO2011 / 145015, WO2012 / 056401, and WO2014 / 043435).

[0088] Among the DNA sequences encoding suitable EPSPSs conferring tolerance to herbicides targeting EPSPS, mention will be made more particularly of the gene encoding a plant EPSPS, in particular a maize EPSPS, in particular a maize EPSPS comprising two mutations, in particular a mutation at amino acid position 102 and a mutation at amino acid position 106 (WO 2004 / 074443), and described in patent application US 6566587, hereinafter referred to as double mutant maize EPSPS or 2mEPSPS, or the gene encoding the EPSPS isolated from Agrobacterium, described by sequence ID No. 2 and sequence ID No. 3 of U.S. Pat. No. 5,633,435, also referred to as CP4.

[0089] Among the DNA sequences encoding suitable EPSPSs conferring tolerance to herbicides targeting EPSPS, mention will be made more particularly of the genes encoding the EPSPS GRG23 from Arthrobacter globiformis and mutants GRG23 ACE1, GRG23 ACE2 or GRG23 ACE3, in particular mutants or variants of GRG23 as described in WO 2008 / 100353, such as GRG23 (ace3) R173K of SEQ ID No. 29 in WO 2008 / 100353.

[0090] In the case of DNA sequences encoding EPSPS, and more particularly encoding the above-mentioned genes, the sequences encoding these enzymes are advantageously preceded by sequences encoding a transit peptide, in particular an "optimized transit peptide" as described in US Pat. Nos. 5,510,471 or 5,633,448.

[0091] Exemplary herbicide tolerance traits that can be combined with the nucleic acid sequences of the present invention further include at least one ALS (acetolactate synthase) inhibitor (WO2007 / 024782); a mutated Arabidopsis ALS / AHAS gene (U.S. Patent 6,855,533); a gene encoding 2,4-D-monooxygenase that confers tolerance to 2,4-D (2,4-dichlorophenoxyacetic acid) by metabolizing (U.S. Patent 6,153,401); and a gene encoding dicamba monooxygenase that confers tolerance to dicamba (3,6-dichloro-2-methoxybenzoic acid) by metabolizing (US2008 / 0119361 and US2008 / 0120739).

[0092] In various embodiments, nucleic acids of the invention are stacked with one or more herbicide tolerance genes, comprising one or more HPPD inhibitor herbicide tolerance genes, and / or one or more glyphosate and / or glufosinate tolerance genes.

[0093] Among the DNA sequences encoding proteins associated with insect resistance properties, mention should be made more particularly of the Bt proteins that are widely described in the literature and are well known to those skilled in the art. Mention should also be made of proteins extracted from bacteria such as Photorhabdus (WO 97 / 17432 and WO 98 / 08932).

[0094] Among the DNA sequences encoding proteins of interest conferring novel insect tolerance properties, more particular mention will be made of the Bt Cry or VIP proteins which are widely described in the literature and are well known to those skilled in the art. These include Cry1F proteins or hybrids derived from Cry1F proteins (e.g., hybrid Cry1A-Cry1F proteins or toxic fragments thereof described in U.S. Pat. No. 6,326,169; U.S. Pat. No. 6,281,016; U.S. Pat. No. 6,218,188); Cry1A-type proteins or toxic fragments thereof, preferably Cry1Ac proteins or hybrids derived from Cry1Ac proteins (e.g., hybrid Cry1Ab-Cry1Ac proteins described in U.S. Pat. No. 5,880,275) or Cry1Ab or Bt2 proteins or insecticidal fragments thereof as described in EP451878; Cry2Ae, Cry2Af or Cry2Ag proteins or toxic fragments thereof as described in WO2002 / 057664; WO 2007 / 140256 (SEQ ID NO: 140256). No.7); VIP3Aa19 protein of NCBI accession number ABG20428; VIP3Aa20 protein of NCBI accession number ABG20429 (SEQ ID No. 2 in WO 2007 / 142840); VIP3A protein produced in COT202 or COT203 cotton events (WO2005 / 054479 and WO2005 / 054480, respectively); Cry proteins as described in WO2001 / 47952; as described in Estruch et al. (1996), Proceedings of the National Academy of Sciences of the United States of America, 28; 93(11):5389-94 and US Pat. 6,291,156; an insecticidal protein from a strain of Xenorhabdus (as described in WO 98 / 50427), Serratia (particularly from S. entomophila), or Photorhabdus species, such as the Tc protein from Photorhabdus as described in WO 98 / 08932 (e.g., Waterfield et al., 2001, Appl Environ Microbiol. 67(11):5017-24; Ffrench-Constant and Bowen, 2000, Cell Mol Life Sci. 57(5):828-33). Also encompassed herein are any variants or mutants of any of these proteins that differ from any of the above sequences, especially the sequences of toxic fragments thereof, in a few (1-10, preferably 1-5) amino acids, or are fused to a transit peptide, such as a plastid transit peptide or another protein or peptide.

[0095] In another embodiment, the sequence encompassed herein is an MTX-like sequence. The term "MTX" is used in the art to describe a group of insecticidal proteins produced by Bacillus sphaericus. The first of these insecticidal proteins, commonly referred to as MTX1 in the art, is synthesized as parasporal crystals that are toxic to mosquitoes. The main components of the crystals are two proteins of 51kDa and 42kDa. Since the presence of both proteins is necessary for toxicity, MTX1 is considered to be a "binary" toxin (Baumann et al. (1991) Microbiology Reviews 55:425-436).

[0096] By analyzing different strains of Bacillus sphaericus with different toxicities, two new classes of MTX toxins have been identified. MTX2 and MTX3 represent different, related classes of insecticidal toxins that exhibit insecticidal activity. See, for example, Baumann et al. (1991) Microbiol. Rev. 55:425-436, which is incorporated herein by reference in its entirety. MTX2 is a 100-kDa toxin. Recently, MTX3 has been identified as a separate toxin, although the amino acid sequence of MTX3 from Bacillus sphaericus is 38% identical to the MTX2 toxin of Bacillus sphaericus SSII-1 (Liu et al. (1996) Appl. Environ. Microbiol. 62:2174-2176). MTX toxins may be used to both increase the insecticidal activity of Bacillus sphaericus strains and manage the evolution of resistance to Bin toxins in mosquito populations (Wirth et al. (2007) Appl. Environ. Microbiol. 73(19):6066-6071).

[0097] In various embodiments, the MTX-like sequences include the nucleotide sequences shown in SEQ ID Nos: 1-15, the amino acid sequences shown in SEQ ID Nos: 16-30, and biologically active variants and fragments thereof.

[0098] In various embodiments, the nucleic acids of the invention can be combined in plants with one or more genes that confer desirable traits, such as herbicide tolerance, insect tolerance, drought tolerance, nematode control, water use efficiency, nitrogen use efficiency, improved nutritional value, disease resistance, improved photosynthesis, improved fiber quality, stress tolerance, improved reproduction, and the like.

[0099] Particularly useful transgenic events that can be combined with the genes of the present invention in plants of the same species (e.g., by crossing or retransforming a plant containing another transgenic event with a chimeric gene of the present invention) include: event 531 / PV-GHBK04 (cotton, insect control, described in WO2002 / 040677); event 1143-14A (cotton, insect control, not deposited, described in WO2006 / 128569); event 114 3-51B (cotton, insect control, not deposited, described in WO2006 / 128570); Event 1445 (cotton, herbicide tolerance, not deposited, described in US-A2002-120964 or WO2002 / 034946); Event 17053 (rice, herbicide tolerance, deposited as PTA-9843, described in WO2010 / 117737); Event 17314 (rice, herbicide tolerance, deposited as PTA-9843, described in WO2010 / 117737); 9844, described in WO2010 / 117735); Event 281-24-236 (cotton, insect control - herbicide tolerance, deposited as PTA-6233, described in WO2005 / 103266 or US-A2005-216969); Event 3006-210-23 (cotton, insect control - herbicide tolerance, deposited as PTA-6233, described in US-A2007-14387); 6 or described in WO2005 / 103266); Event 3272 (cotton, quality traits, deposited as PTA-9972, described in WO2006 / 098952 or US-A2006-230473); Event 33391 (wheat, herbicide tolerance, deposited as PTA-2347, described in WO2002 / 027004); Event 40416 (corn, insect control-herbicide tolerance, deposited as ATCC PTA-11508, described in WO 11 / 075593); Event 43A47 (corn, insect control-herbicide tolerance, deposited as ATCC PTA-11509, described in WO 2011 / 075595); Event 5307 (corn, insect control, deposited as ATCC PTA-9561, described in WO 2010 / 077816); Event ASR-368 (evergreen grass, herbicide tolerance, deposited as ATCC PTA-4816, described in US-A 2006-162007 or WO 2004 / 053062); Event B16 (corn, herbicide tolerance, not deposited, described in US-A 2003-126634); event BPS-CV127-9 (soybean, herbicide tolerant, deposited as NCIMB No. 41603, described in WO 2010 / 080829);Event BLR1 (rape, restoration of male sterility, deposited as NCIMB 41193, described in WO2005 / 074671); Event CE43-67B (cotton, insect control, deposited as DSMACC2724, described in US-A2009-217423 or WO2006 / 128573); Event CE44-69D (cotton, insect control, not deposited, described in US-A2010-0024077); Event CE44-69D (cotton, insect control, not deposited, described in WO2006 / 128571); Event CE46-02A (cotton, insect control, not deposited, described in WO2006 / 128572); Event COT102 (cotton, insect control, not deposited, described in US-A 2006-130175 or WO2004 / 039986); Event COT202 (cotton, insect control, not deposited, described in US-A 2007-067868 or WO2005 / 054479); Event COT203 (cotton, insect control, not deposited, described in WO2005 / 054480); Event DAS21606-3 / 1606 (soybean, herbicide tolerance, deposited as PTA-11028, described in WO2012 / 033794); Event DAS40278 (corn, herbicide tolerance, deposited as ATCC PTA-10244, described in WO2011 / 022469); event DAS-44406-6 / pDAB8264.44.06.1 (soybean, herbicide tolerant, deposited as PTA-11336, described in WO2012 / 075426); event DAS-14536-7 / pDAB8291.45.36.2 (soybean, herbicide tolerant, deposited as PTA-11335, described in WO2012 / 075429); event DAS-59122-7 (corn, insect control - herbicide tolerant, deposited as ATCC PTA 11384, described in US-A2006-070139); event DAS-59132 (corn, insect control - herbicide tolerance, not deposited, described in WO2009 / 100188); event DAS68416 (soybean, herbicide tolerance, deposited as ATCC PTA-10442, described in WO2011 / 066384 or WO2011 / 066360); event DP-098140-6 (corn, herbicide tolerance, deposited as ATCC PTA-8296, described in US-A2009-137395 or WO 08 / 112019); Event DP-305423-1 (soybean, quality traits, undeposited, described in US-A2008-312082 or WO2008 / 054747);Event DP-32138-1 (corn, hybrid system, deposited as ATCC PTA-9158, described in US-A2009-0210970 or WO2009 / 103049); Event DP-356043-5 (soybean, herbicide tolerant, deposited as ATCC PTA-8287, described in US-A2010-0184079 or WO2008 / 002872); Event EE-1 (eggplant, insect control, not deposited, described in WO 07 / 091277); Event FI117 (corn, herbicide tolerant, deposited as ATCC 209031, described in US-A2006-059581 or WO 98 / 044140); event FG72 (soybean, herbicide tolerant, deposited as PTA-11041, described in WO 2011 / 063413); event GA21 (corn, herbicide tolerant, deposited as ATCC 209033, described in US-A 2005-086719 or WO 98 / 044140); event GG25 (corn, herbicide tolerant, deposited as ATCC 209032, described in US-A 2005-188434 or WO 98 / 044140); event GHB119 (cotton, insect control-herbicide tolerant, deposited as ATCC PTA-8398, described in WO 2008 / 151780); event GHB614 (cotton, herbicide tolerant, deposited as ATCC PTA-6878, described in US-A 2010-050282 or WO 2007 / 017186); event GJ11 (maize, herbicide tolerant, deposited as ATCC 209030, described in US-A 2005-188434 or WO 98 / 044140); event GM RZ13 (sugar beet, virus resistant, deposited as NCIMB-41601, described in WO 2010 / 076212); event H7-1 (sugar beet, herbicide tolerant, deposited as NCIMB 41158 or NCIMB 41159, described in US-A 2004-172669 or WO 2004 / 074492); event JOPLIN1 (wheat, disease tolerance, not deposited, described in US-A 2008-064032); event LL27 (soybean, herbicide tolerance, deposited as NCIMB 41658, described in WO 2006 / 108674 or US-A 2008-320616); event LL55 (soybean, herbicide tolerance, deposited as NCIMB 41660, described in WO 2006 / 108675 or US-A 2008-196127);Event LLcotton25 (cotton, herbicide tolerant, deposited as ATCC PTA-3343, described in WO 2003 / 013224 or US-A 2003-097687); Event LLRICE06 (rice, herbicide tolerant, deposited as ATCC 203353, described in US 6,468,747 or WO 2000 / 026345); Event LLRice62 (rice, herbicide tolerant, deposited as ATCC 203352, described in WO 2000 / 026345); Event LLRICE601 (rice, herbicide tolerant, deposited as ATCC PTA-2600, described in US-A 2008-2289060 or WO2000 / 026356); event LY038 (corn, quality traits, deposited as ATCC PTA-5623, described in US-A 2007-028322 or WO2005 / 061720); event MIR162 (corn, insect control, deposited as PTA-8166, described in US-A2009-300784 or WO2007 / 142840); event MIR604 (corn, insect control, not deposited, described in US-A 2008-167456 or WO2005 / 103301); event MON15985 (cotton, insect control, deposited as ATCC PTA-2516, described in US-A 2004-250317 or WO 2002 / 100163); event MON810 (maize, insect control, not deposited, described in US-A 2002-102582); event MON863 (maize, insect control, deposited as ATCC PTA-2605, described in WO 2004 / 011601 or US-A 2006-095986); event MON87427 (maize, pollination control, deposited as ATCC PTA-7899, described in WO 2011 / 062904); event MON87460 (maize, stress tolerance, deposited as ATCC PTA-8910, described in WO2009 / 111263 or US-A2011-0138504); event MON87701 (soybean, insect control, deposited as ATCC PTA-8194, described in US-A2009-130071 or WO2009 / 064652); event MON87705 (soybean, quality trait - herbicide tolerance, deposited as ATCC PTA-9241, described in US-A 2010-0080887 or WO2010 / 037016); event MON87708 (soybean, herbicide tolerance, deposited as ATCC PTA-9670, described in WO2011 / 034704);Event MON87712 (soybean, yield, deposited as PTA-10296, described in WO2012 / 051199); Event MON87754 (soybean, quality traits, deposited as ATCC PTA-9385, described in WO2010 / 024976); Event MON87769 (soybean, quality traits, deposited as ATCC PTA-8911, described in US-A 2011-0067141 or WO2009 / 102873); Event MON88017 (corn, insect control-herbicide tolerance, deposited as ATCC PTA-5582, described in US-A 2008-028482 or WO2005 / 059103); Event MON88913 (cotton, herbicide tolerance, deposited as ATCC PTA-4854, described in WO 2004 / 072235 or US-A 2006-059590); event MON88302 (canola, herbicide tolerant, deposited as PTA-10955, described in WO 2011 / 153186); event MON88701 (cotton, herbicide tolerant, deposited as PTA-11754, described in WO 2012 / 134808); event MON89034 (corn, insect control, deposited as ATCC PTA-7455, described in WO 07 / 140256 or US-A 2008-260932); event MON89788 (soybean, herbicide tolerant, deposited as ATCC PTA-6708, described in US-A 2006-282915 or WO2006 / 130436); event MS11 (oilseed rape, pollination control-herbicide tolerance, deposited as ATCC PTA-850 or PTA-2485, described in WO2001 / 031042); event MS8 (oilseed rape, pollination control-herbicide tolerance, deposited as ATCC PTA-730, described in WO2001 / 041558 or US-A2003-188347); event NK603 (maize, herbicide tolerance, deposited as ATCC PTA-2478, described in US-A 2007-292854); event PE-7 (rice, insect control, not deposited, described in WO2008 / 114282); event RF3 (oilseed rape, pollination control-herbicide tolerance, deposited as ATCC PTA-730, described in WO 2001 / 041558 or US-A 2003-188347); Event RT73 (oilseed rape, herbicide tolerant, undeposited, described in WO 2002 / 036831 or US-A 2008-070260);Event SYHT0H2 / SYN-000H2-5 (soybean, herbicide tolerant, deposited as PTA-11226, described in WO 2012 / 082548); Event T227-1 (sugar beet, herbicide tolerant, not deposited, described in WO 2002 / 44407 or US-A 2009-265817); Event T25 (corn, herbicide tolerant, not deposited, described in US-A 2001-029014 or WO 2001 / 051654); Event T304-40 (cotton, insect control-herbicide tolerant, deposited as ATCC PTA-8171, described in US-A 2010-077501 or WO2008 / 122406); event T342-142 (cotton, insect control, not deposited, described in WO2006 / 128568); event TC1507 (corn, insect control-herbicide tolerance, not deposited, described in US-A 2005-039226 or WO2004 / 099447); event VIP1034 (corn, insect control-herbicide tolerance, deposited as ATCC PTA-3925, described in WO2003 / 052073); Event 32316 (corn, insect control - herbicide tolerance, deposited as PTA-11507, described in WO2011 / 084632); Event 4114 (corn, insect control - herbicide tolerance, deposited as PTA-11506, described in WO2011 / 084621); Event EE-GM3 / FG72 (soybean, herbicide tolerance, ATCC Accession No. PTA-11041), optionally stacked with Event EE-GM1 / LL27 or Event EE-GM2 / LL55 (WO2011 / 063413A2); Event DAS-68416-4 (soybean, herbicide tolerance, ATCC Accession No. PTA-10442, described in WO2011 / 066360A 1); event DAS-68416-4 (soybean, herbicide tolerance, ATCC Accession No. PTA-10442, WO2011 / 066384A1); event DP-040416-8 (corn, insect control, ATCC Accession No. PTA-11508, WO2011 / 075593A1); event DP-043A47-3 (corn, insect control, ATCC Accession No. PTA-11509, WO2011 / 075595A1); event DP-004114-3 (corn, insect control, ATCC Accession No. PTA-11506, WO2011 / 084621A1); event DP-032316-8 (corn, insect control, ATCC Accession No. PTA-11507, WO2011 / 084632A1);Event MON-88302-9 (canola, herbicide tolerance, ATCC Accession No. PTA-10955, WO2011 / 153186A1); Event DAS-21606-3 (soybean, herbicide tolerance, ATCC Accession No. PTA-11028, WO2012 / 033794A2); Event MON-87712-4 (soybean, quality traits, ATCC Accession No. PTA-10296, WO2012 / 051199A2); Event DAS-44406-6 (soybean, stacked herbicide tolerance, ATCC Accession No. PTA-11336, WO2012 / 075426A1); Event DAS-14536-7 (soybean, stacked herbicide tolerance, ATCC Accession No. PTA-11335, WO2012 / 075429A1); Event SYN-000H2-5 (soybean, herbicide tolerance, ATCC Accession No. PTA-11226, WO2012 / 082548A2); Event DP-061061-7 (canola, herbicide tolerance, no available deposit number, WO2012071039A1); Event DP-07 3496-4 (canola, herbicide tolerance, no available deposit number, US2012131692); event 8264.44.06.1 (soybean, stacked herbicide tolerance, accession number PTA-11336, WO2012075426A2); event 8291.45.36.2 (soybean, dexamethasone tolerance, accession number PTA-11335, WO2012075429A2); event SYHT0H2 (soybean, ATCC accession number PTA-11226, WO2012 / 082548A2); event MON 88701 (cotton, ATCC Accession No. PTA-11754, WO2012 / 134808A1); event KK179-2 (alfalfa, ATCC Accession No. PTA-11833, WO2013 / 003558A1); event pDAB8264.42.32.1 (soybean, stacked herbicide tolerance, ATCC Accession No. PTA-11993, WO2013 / 010094A1); event MZDT09Y (corn, ATCC Accession No. PTA-13025, WO2013 / 012775A1).

[0100] The transformation of plant cells can be accomplished by one of several techniques known in the art. The pesticidal gene of the present invention can be modified to obtain or enhance expression in plant cells. Typically, the construct expressing this type of protein will contain a promoter for driving the transcription of the gene and a 3' non-translational region for allowing transcriptional termination and polyadenylation. The organization of this type of construct is well known in the art. In some cases, it may be useful to engineer the gene so that the resulting peptide is secreted or otherwise targeted in the plant cell. For example, the gene can be engineered to contain a signal peptide to promote the transfer of the peptide to the endoplasmic reticulum. It may also be preferred that the plant expression cassette be engineered to contain introns so that expression requires intronic mRNA processing.

[0101] Typically, this "plant expression cassette" will be inserted into a "plant transformation vector." This plant transformation vector can be composed of one or more DNA vectors required to achieve plant transformation. For example, it is common practice in the art to utilize plant transformation vectors composed of more than one contiguous DNA segment. These vectors are commonly referred to in the art as "binary vectors." Binary vectors and vectors with helper plasmids are most commonly used for Agrobacterium-mediated transformation, where the size and complexity of the DNA segments required for efficient transformation are very large, and it is advantageous to separate the functions onto separate DNA molecules. Binary vectors typically contain a plasmid vector containing the cis-acting sequences required for T-DNA transfer (such as the left and right borders), a selectable marker engineered to be expressed in plant cells, and a "gene of interest" (a gene engineered to be expressed in the plant cells in which it is desired to produce transgenic plants). This plasmid vector also contains sequences required for bacterial replication. The cis-acting sequences are arranged in a manner that allows for efficient transfer and expression in plant cells. For example, a selectable marker gene and an insecticidal gene are located between the left and right borders. Typically, a second plasmid vector contains the trans-acting factors that mediate T-DNA transfer from Agrobacterium to plant cells. This plasmid typically contains virulence functions (Vir genes) that allow Agrobacterium to infect plant cells and transfer DNA by cleavage at border sequences and vir-mediated DNA transfer, as understood in the art (Hellens and Mullineaux (2000) Trends Plant Sci 5:446-451). Several types of Agrobacterium strains (e.g., LBA4404, GV3101, EHA101, EHA105, etc.) can be used for plant transformation. A second plasmid vector is not necessary for transformation of plants by other methods such as microprojection, microinjection, electroporation, polyethylene glycol, etc.

[0102] Typically, plant transformation methods involve transferring heterologous DNA to target plant cells (e.g., immature or mature embryos, suspension cultures, undifferentiated callus, protoplasts, etc.), and then applying the maximum threshold level (depending on the selectable marker gene) of appropriate selection to recover the transformed plant cells from a group of untransformed cell masses. Explants are typically transferred to the same medium of a fresh supply and routinely cultured. Subsequently, the transformed cells are differentiated into buds after being placed on a regeneration medium supplemented with a selection agent of the maximum threshold level. The buds are then transferred to a selective rooting medium to recover rooted buds or plantlets. The transgenic plantlets then grow into mature plants and produce fertile seeds (e.g., Hiei et al. (1994) Journal of Botany 6:271-282; Ishida et al. (1996) Nature Biotechnology 14:745-750). Explants are typically transferred to the same medium of a fresh supply and routinely cultured. A general description of techniques and methods for producing transgenic plants is found in Ayres and Park (1994) Critical Reviews in Plant Science 13:219-239 and Bommineni and Jauhar (1997) Maydica 42:107-120. Since the transformed material contains many cells; both transformed and untransformed cells are present in any one target callus or tissue or cell mass. The ability to kill untransformed cells and allow transformed cells to proliferate results in a transformed plant culture. Often, the ability to remove untransformed cells is a limitation to the rapid recovery of transformed plant cells and the successful generation of transgenic plants.

[0103] Transformation protocols and protocols for introducing nucleotide sequences into plants may vary depending on the type of plant or plant cell being targeted for transformation (i.e., monocot or dicot). Generation of transgenic plants can be performed by one of several methods, including but not limited to microinjection, electroporation, direct gene transfer, introduction of heterologous DNA into plant cells by Agrobacterium (Agrobacterium-mediated transformation), bombardment of plant cells with heterologous exogenous DNA attached to particles, ballistic particle acceleration, aerosol beam transformation (U.S. Published Application No. 20010026941; U.S. Patent No. 4,945,050; International Publication No. WO 91 / 00915; U.S. Published Application No. 2002015066), Lec1 transformation, and various other non-particle-mediated DNA transfer methods.

[0104] Methods for the transformation of chloroplasts are known in the art. See, for example, Svab et al. (1990) Proceedings of the National Academy of Sciences of the United States of America 87:8526-8530; Svab and Maliga (1993) Proceedings of the National Academy of Sciences of the United States of America 90:913-917; Svab and Maliga (1993) Journal of the European Molecular Biology Association 12:601-606. The method relies on the delivery of DNA containing a selectable marker by a particle gun and targets the DNA to the plastid genome by homologous recombination. In addition, plastid transformation can be accomplished by transactivating silent plastid-borne transgenes through tissue-preferred expression of nuclear-encoded and plastid-directed RNA polymerases. Such systems have been reported in McBride et al. (1994) Proceedings of the National Academy of Sciences of the United States of America 91:7301-7305.

[0105] After the heterologous exogenous DNA is integrated into the plant cells, an appropriately selected maximum threshold level is then applied to the culture medium to kill the non-transformed cells, and the putatively transformed cells that survive this selection treatment are propagated by periodic transfer to fresh culture medium. Through serial passage and appropriately selected challenges, cells transformed with the plasmid vector can be identified and propagated. Molecular and biochemical methods can then be used to confirm that the integrated heterologous gene of interest is present in the genome of the transgenic plant.

[0106] The cell that has been transformed can be cultivated into plant in a conventional manner.See, for example, McCormick et al. (1986) " Plant Cell Reports (Plant Cell Reports) " 5:81-84. These plants can then be grown, and with identical transformed strains or different strains pollination, and identification is carried out with the resulting hybrid of the constitutive expression of desired phenotypic characteristics. Two or more generations can be cultivated to ensure stable maintenance and the expression of hereditary desired phenotypic characteristics, and then seeds are gathered in the crops to ensure the expression of desired phenotypic characteristics. In this way, the invention provides the seed (also referred to as " transgenic seed ") of transformation, the seed of described transformation has nucleotide construct of the present invention, for example, and expression cassette of the present invention is stably incorporated into its genome.

[0107] Plant transformation evaluation

[0108] After the heterologous foreign DNA is introduced into plant cells, the transformation or integration of the heterologous gene into the plant genome is confirmed by various methods, such as analysis of nucleic acids, proteins, and metabolites associated with the integrated gene.

[0109] PCR analysis is a rapid method for screening transformed cells, tissues, or shoots for the presence of the incorporated gene at an early stage prior to transplantation into soil (Sambrook and Russell (2001) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, NY). PCR is performed using oligonucleotide primers specific for the gene of interest or the Agrobacterium vector background.

[0110] Plant transformation can be confirmed by Southern blot analysis of genomic DNA (Sambrook and Russell, 2001, supra). Typically, total DNA is extracted from transformants, digested with appropriate restriction enzymes, fractionated on an agarose gel, and transferred to a nitrocellulose or nylon membrane. The transformed cells are then stained with, for example, a radiolabeled 32 The membrane or "blot" is probed with the P target DNA fragment to confirm integration of the introduced gene into the plant genome (Sambrook and Russell, 2001, supra).

[0111] In Northern blot analysis, RNA is isolated from specific tissues of the transformants, fractionated in formaldehyde agarose gels, and blotted onto nylon filters according to standard procedures routinely used in the art (Sambrook and Russell, 2001, supra). The filter is then hybridized with a radioactive probe derived from the pesticidal gene by methods known in the art to test for expression of RNA encoded by the pesticidal gene (Sambrook and Russell, 2001, supra).

[0112] Transgenic plants can be subjected to Western blots, biochemical assays, and the like to confirm the presence of the protein encoded by the pesticidal gene by standard procedures (Sambrook and Russell, 2001, supra) using antibodies that bind to one or more epitopes present on the pesticidal protein.

[0113] Insecticidal activity in plants

[0114] In another aspect of the present invention, transgenic plants expressing pesticidal proteins with pesticidal activity can be produced. The method described above by way of example can be used to produce transgenic plants, but the mode of producing transgenic plant cells is not critical for the present invention. The experimenter can use methods known or described in the art as appropriate, such as transformation, bioballistic transformation and non-particle-mediated methods of Agrobacterium-mediated transformation. The plant expressing pesticidal proteins can be separated by the common method described in this area, for example, by the selection of callus, transformed callus and regeneration of fertile plants from this type of transgenic callus. In this type of process, any gene can be used as selectable marker, as long as the expression of the gene in the plant cell gives the ability to identify or select transformed cells.

[0115] Many markers have been developed for use with plant cells, such as resistance to chloramphenicol, aminoglycoside G418, hygromycin, and the like. Other genes encoding products involved in chloroplast metabolism may also be used as selectable markers. For example, genes that provide resistance to plant herbicides such as glyphosate, bromoxynil, or imidazolinones may be particularly useful. Such genes have been reported (Stalker et al. (1985) Journal of Biological Chemistry 263: 6310-6314 (bromoxynil-resistant nitrilase gene); and Sathasivan et al. (1990) Nucleic Acids Research 18: 2188 (AHAS imidazolinone resistance gene). In addition, the genes disclosed herein can be used as markers for evaluating bacterial or plant cell transformation. Methods for detecting the presence of transgenes in plants, plant organs (e.g., leaves, stems, roots, etc.), seeds, plant cells, propagules, embryos, or their progeny are well known in the art. In one embodiment, the presence of transgenes is detected by testing for insecticidal activity.

[0116] Fertile plants expressing the insecticidal protein can be tested for insecticidal activity, and plants showing the best activity can be selected for further breeding. The method is available in the art to measure pest activity. Typically, the protein is mixed and used in feeding assays. See, for example, Marrone et al. (1985) Journal of Economic Entomology 78:290-293.

[0117] The present invention 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 (maize), sorghum, wheat, sunflower, tomato, crucifers, peppers, potatoes, cotton, rice, soybeans, sugar beets, sugarcane, tobacco, barley, as well as rapeseed, Brassica plants, alfalfa, rye, millet, safflower, peanuts, sweet potatoes, cassava, coffee, coconut, pineapple, citrus trees, cocoa, tea, banana, avocado, fig, guava, mango, olive, papaya, cashew, macadamia, almonds, oats, vegetables, ornamentals, and conifers.

[0118] Vegetables include, but are not limited to, tomatoes, lettuce, green beans, lima beans, peas, and members of the genus Curcumis, such as cucumbers, cantaloupes, and cantaloupes. Ornamental plants include, but are not limited to, azaleas, hydrangeas, hibiscus, roses, tulips, daffodils, petunias, carnations, poinsettias, and chrysanthemums. Preferably, the plants of the present invention are crops (e.g., maize, sorghum, wheat, sunflowers, tomatoes, crucifers, peppers, potatoes, cotton, rice, soybeans, sugar beets, sugarcane, tobacco, barley, rapeseed, etc.).

[0119] Use in pest control

[0120] General methods for employing strains comprising the nucleotide sequences of the present invention or variants thereof as pesticides in pest control or in engineering other organisms are known in the art. See, for example, US Pat. No. 5,039,523 and EP 0 480 762 A2.

[0121] Bacillus strains containing nucleotide sequences of the present invention or variants thereof, or microorganisms that have been genetically altered to contain the insecticidal genes and proteins of the present invention can be used to protect crops and products from insect pests. In one aspect of the invention, intact cells, i.e., unlyzed cells, of the organism producing the toxin (pesticide) are treated with an agent that prolongs the activity of the toxin produced in the cell when the cell is applied to the environment of the target pest.

[0122] Alternatively, the insecticide is produced by introducing an insecticide gene into a cell host. The expression of the insecticide gene directly or indirectly causes the intracellular production and maintenance of the insecticide. In one aspect of the present invention, these cells are then processed under the conditions of the activity of the toxin produced in the extended cell when the cell is applied to the environment of the target pest. Products obtained therefrom has retained the toxicity of the toxin. The insecticide of these natural encapsulations can then be deployed according to routine techniques to be applied to the environment of the target pest, for example, soil, water and the leaf of a plant. Referring to for example EPA0192319 and the references cited therein. Alternatively, the cell expressing the gene of the present invention can be deployed to allow the resulting material to be used as an insecticide.

[0123] Active component of the present invention is used with the form of composition usually, and can be applied to crop area or the plant to be processed simultaneously or in succession with other compounds.These compounds can be fertilizers, weed killer herbicides, cryoprotectants, surfactants, washing compositions, insecticide soaps, dormancy oils, polymers and / or slow-release or biodegradable carrier formulations, and described formulations allow that after its single application, target area is carried out long-term administration.Described compound can also be several mixtures in selective herbicides, chemical insecticides, virucides, microbicides, amoebicides, insecticides, fungicides, bactericides, nematocides, molluscicides or these preparations, if desired, together with other agriculturally acceptable carriers, surfactants or the application promotion adjuvant that is generally used in formulation field.Suitable carrier and adjuvant can be solid or liquid and corresponding to commonly used material in the formulation technology, for example natural or regenerated mineral matter, solvent, dispersant, wetting agent, tackifier, adhesive or fertilizer. Likewise, the formulations may be prepared as edible "baits" or formulated into pest "traps" to permit feeding or ingestion by the target pest of the pesticide formulation.

[0124] Methods for applying the active ingredient of the present invention or the agricultural chemical composition of the present invention containing at least one insecticidal protein produced by the strain of the present invention include foliar application, seed coating, and soil application. The number of applications and the application rate depend on the intensity of the corresponding pest infestation.

[0125] The compositions can be formulated into powders, dusts, pellets, granules, sprays, emulsions, colloids, solutions, and the like, and can be prepared by conventional methods such as drying, lyophilizing, homogenizing, extracting, filtering, centrifuging, settling, or concentrating cell cultures containing the polypeptide. In all such compositions containing at least one such pesticidal polypeptide, the polypeptide can be present at a concentration of about 1% to about 99% by weight.

[0126] Lepidoptera, Hemiptera, Diptera or Coleoptera pests can be killed or reduced in number in a given area by the method of the present invention, or the method of the present invention can be preventively applied to environmental areas to prevent the infestation of susceptible pests. Preferably, the pest ingests the polypeptide of an insecticidal effective dose or contacts the pest with the polypeptide of an insecticidal effective dose. "Insecticidal effective dose" refers to the amount of the insecticide that can cause at least one insect to die or significantly reduce insect growth, feeding or normal physiological development. For example, the insecticide can cause the egg hatching of the insect to reduce, the death of any developmental stage, molting to reduce and / or the insect to reduce the feeding of the target organism (for example, reducing the number of feeding sites of plants or plant cells and / or reducing the damage to plants or plant cells). This quantity will vary depending on the following factors, for example, the specific target pest to be controlled, the specific environment to be processed, position, plant, crop or agricultural site, environmental conditions and the application method, rate, concentration, stability and quantity of the insecticidal effective polypeptide composition. The formulation can also change with respect to the severity of climatic conditions, environmental considerations and / or application frequency and / or pest infestation.

[0127] The described insecticide compositions can be prepared by blending a suspension of bacterial cells, crystals, and / or spores or an isolated protein component with a desired agriculturally acceptable carrier. The composition can be formulated in an appropriate manner prior to application, such as by freeze-drying, lyophilization, drying, or in an aqueous carrier, medium, or suitable diluent (e.g., saline or other buffer). The formulated composition can be in the form of a powder or granular material, or a suspension in an oil (vegetative or mineral), or in water or an oil / water emulsion, or as a wettable powder, or in combination with any other carrier material suitable for agricultural applications. Suitable agricultural carriers can be solid or liquid and are well known in the art. The term "agriculturally acceptable carrier" covers all adjuvants, inert components, dispersants, surfactants, tackifiers, adhesives, etc. commonly used in insecticide formulation technology; these are well known to those skilled in the art of insecticide formulation. The formulation can be mixed with one or more solid or liquid adjuvants and prepared in various ways, for example, by uniformly mixing, blending, and / or grinding the insecticide composition with a suitable adjuvant using conventional formulation techniques. Suitable formulations and methods of administration are described in US Patent No. 6,468,523, which is incorporated herein by reference.

[0128] "Pests" include, but are not limited to, insects, fungi, bacteria, nematodes, mites, ticks, etc. Pests include insects selected from the following orders: Coleoptera, Diptera, Hymenoptera, Lepidoptera, Trichophaga, Homoptera, Hemiptera, Orthroptera, Thysanoptera, Dermaptera, Isoptera, Anoplura, Siphonaptera, Trichoptera, etc., especially Coleoptera, Lepidoptera and Diptera.

[0129] The order Coleoptera includes the suborders Adephaga and Polyphaga. The suborder Carnivora includes the superfamily Caraboidea and Gyrinoidea, while the suborder Polyphaga includes the superfamily Hydrophiloidea, Staphylinoidea, Cantharoidea, Cleroidea, Elateroidea, Dascilloidea, Dryopoidea, Byrrhoidea, Cucujoidea, Meloidea, Mordelloidea, Tenebrionoidea, Bostrichoidea, Scarabaeoidea, Cerambycoidea, Chrysomeloidea, and Curculionoidea. The Caraboidea includes the Cicindelidae, Carabidae, and Dytiscidae. The Gyrinoidea includes the Gyrinidae. The Hydrophiloidea includes the Hydrophilidae. The Pederoidea includes the Silphidae and Staphylinidae. The Asteroidea includes the Cantharidae and Lampyridae. The Cleroidea includes the Cleridae and Dermestidae. The Elateridae includes the Buprestidae. The Coccinellidae includes the Meloidoidea. The Tenebrionoidea includes the Tenebrionidae. The superfamily Scaraboidea includes the families Passalidae and Scarabaeidae. The superfamily Cerambycidae includes the family Cerambycidae. The superfamily Chrysomelidae includes the family Chrysomelidae. The superfamily Curculionidae includes the family Curculionidae and the family Scolytidae.

[0130] The order Diptera includes the suborders Nematocera, Brachycera, and Cyclorrhapha. The Nematocera includes the families Tipulidae, Psychodidae, Culicidae, Ceratopogonidae, Chironomidae, Simuliidae, Bibionidae, and Cecidomyiidae. The Brachycera includes the families Stratiomyidae, Tabanidae, Therevidae, Asilidae, Mydidae, Bombyliidae, and Dolichopodidae. The Cyclorrhapha includes the Aschiza and Aschiza groups. The seamless group includes the Phoridae, Syrphidae, and Conopidae. The seamless group includes the Acalyptratae and Calyptratae. The apterous groups include the Otitidae, Tephritidae, Agromyzidae, and Drosophilidae. The Calyptratae include the Hippoboscidae, Oestridae, Tachinidae, Anthomyiidae, Muscidae, Calliphoridae, and Sarcophagidae.

[0131] The order Lepidoptera includes the families Papilionidae, Pieridae, Lycaenidae, Nymphalidae, Danaidae, Satyridae, Hesperiidae, Sphingidae, Saturniidae, Geometridae, Arctiidae, Noctuidae, Lymantriidae, Sesiidae, and Tineidae.

[0132] Nematodes include parasitic nematodes, such as root-knot nematodes, cyst nematodes, and decay nematodes, including cyst nematodes (Heterodera spp.), root-knot nematodes (Meloidogyne spp.), and Globodera spp.; in particular, members of the cyst nematode family, including but not limited to soybean cyst nematode (Heterodera glycines / soybean cyst nematode); beet cyst nematode (Heterodera schachtii / beet cyst nematode); cereal cyst nematode (Heterodera avenae / cereal cyst nematode); and potato golden wireworm (Globodera rostochiensis) and potato cyst nematode (Globodera pailida / potato cyst nematodes). Decay nematodes include Pratylenchus spp.

[0133] Hemipteran pests (including species designated as Hemiptera, Homoptera, or Heteroptera) include, but are not limited to, Lygus spp., such as the Western tarnished plant bug (Lygus hesperus), the tarnished plant bug (Lygus lineolaris), and the green plant bug (Lygus elisus); aphids, such as the green peach aphid (Myzus persicae), the cotton aphid (Aphis gossypii), the cherry aphid or black cherry aphid (Myzus cerasi), the soybean aphid (Aphis glycines Matsumura); the brown plant hopper (Brown planthopper); and stink bugs, such as the green stink bug (Acrosternum hilare), the brown marmorated stink bug (Halyomorpha halys), the southern green stink bug (Nezara viridula), the rice stinkbug (Oebalus pugnax), the forest bug (Pentatomarufipes), the European stink bug (Rhaphigaster nebulosa), and the shield bug Troilus luridus.

[0134] The main crop pests of the present invention include: corn: European corn borer (Ostrinia nubilalis); black cutworm (Agrotis ipsilon); corn earworm (Helicoverpa zea); fall armyworm (Spodoptera frugiperda); southwestern corn borer (Diatraea grandiosella); lesser cornstalk borer (Elasmopalpus lignosellus); surgarcane borer (Diatraea saccharalis); western corn rootworm (Diabrotica virgifera); northern corn rootworm (Diabrotica longicornis barberi); southern corn rootworm (Diabrotica undecimpunctata howardi); and comb-clawed clickworms (Melanotus spp.), wireworms; northern masked chafer (Cyclocephala borealis) (white grub); southern masked chafer (Cyclocephala immaculata) (white grub); Japanese beetle (Popillia japonica); cornflea beetle (Chaetocnema pulicaria); maize billbug (Sphenophorus maidis); corn leaf aphid (Rhopalosiphum maidis); corn root aphid (Anuraphis maidiradicis); chinch bug (Blissus leucopterus leucopterus); redlegged grasshopper (Melanoplus femurrubrum); black locust (Melanoplus sanguinipes / migratory grasshopper; Hylemya platura / seedcorn maggot; Agromyza parvicornis / corn blot leafminer; Anaphothrips obscrurus / grass thrips; Solenopsis milesta / thief ant; Tetranychus urticae / twospotted spider mite; sorghum: Chilo partellus (sorghum borer); fall armyworm; Spodoptera cosmioides; subtropical armyworm; corn earworm; lesser corn stalk borer; Feltia subterranea (granulate cutworm); Phyllophaga crinita (white grub); Eleodes, Conoderus, and Aeolus spp. (wireworms); Oulemamelanopus (cereal leaf beetle); corn flea beetle; maize weevil; corn aphid; Siphaflava (yellow sugarcane aphid); wheat stink bug; sorghum gall midge (Contarinia spp.). sorghicola / sorghummidge); Tetranychus cinnabarinus / carmine spider mite; Two-spotted spider mite; wheat:Army worm (Pseudaletia unipunctata); Fall armyworm; Lesser corn stalk borer; Western cutworm (Agrotis orthogonia); Lesser corn stalk borer; Black-horned mudworm; Clover leaf weevil (Hypera punctata); Southern corn rootworm; Russian wheat aphid; Schizaphis graminum (greenbug); English grain aphid (Macrosiphumavenae); Red-legged grasshopper; Differential grasshopper (Melanoplus differentialis); Black locust; Hessian fly (Mayetiola destructor); Wheat midge (Sitodiplosis mosellana); Wheat stem maggot (Meromyza americana); Wheat ball fly (Hylemya coarctata / wheat bulb fly); Frankliniella fusca / tobacco thrips; Cephus cinctus / wheat stemsawfly; Aceria tulipae / wheat curl mite; sunflower Sunflower bud moth (Suleimahelianthana); sunflower borer (Homoeosoma electellum); sunflower beetle (Zygogramma exclamationis); carrot beetle (Bothyrus gibbosus); sunflower seed midge (Neolasioptera murtfeldtiana); cotton:Cotton budworm (Heliothis virescens); cotton bollworm (Helicoverpa zea); beet armyworm (Spodoptera exigua); pink bollworm (Pectinophora gossypiella); boll weevil (Anthonomus grandis); cotton aphid (Aphis gossypii); cotton fleahopper (Pseudatomoscelis seriatus); banded-winged whitefly (Trialeurodesabutilonea); American meadow locust (Pseudatomoscelis seriatus); red-legged grasshopper (Pseudatomoscelis seriatus); onion thrips (Thripstabaci); tobacco thrips (Franklinkiella fusca); two-spotted spider mite; rice :Sucrose borer; Fall armyworm; Related armyworm; Subtropical armyworm; Corn earworm; Grape colaspis brunnea; Rice water weevil (Lissorhoptrus oryzophilus); Rice weevil (Sitophilus oryzae); Rice leafhopper (Nephotettix nigropictus); Wheat stink bug; Green stink bug; Asiatic rice borer (Chilu suppressalis); soybeans: soybean armyworm; soybean armyworm (Anticarsia gemmatalis), velvet bean caterpillar; alfalfa armyworm (Plathypena scabra / green cloverworm); European corn borer; black cutworm; beet armyworm; related armyworm; subtropical armyworm; green bollworm; cotton bollworm; Mexican bean beetle (Epilachna varivestis); peach aphid; potato leafhopper (Empoasca fabae); green stink bug; red-legged grasshopper; special grasshopper; corn seed fly; soybean thrips (Sericothrips variabilis / soybean thrips); onion thrips; strawberry spider mite (Tetranychus turkestani / strawberry spider mite); two-spotted spider mite; barley : European corn borer; black cutworm; wheat aphid; wheat stink bug; green stink bug; brown stink bug (Euschistus servus); neotropical brown stink bug (Euschistus heros); seedcorn maggot (Delia platura); black midge; brown wheat mite (Petrobia latens); rape : Cabbage aphid (Brevicorynebrassicae / cabbage aphid); Turnip flea beetle (Phyllotreta cruciferae / Flea beetle); Bertha armyworm (Mamestra configurata / Bertha armyworm); Diamondback moth; Ground fly (Delia ssp.), Root maggot (Root maggot).

[0135] Method for increasing plant yield

[0136] A method for increasing plant yield is provided. The method comprises providing a plant or plant cell expressing a polynucleotide encoding a pesticidal polypeptide sequence disclosed herein, and growing the plant or its seeds in a field infested with (or susceptible to infestation by) pests to which the polypeptide has pesticidal activity. In some embodiments, the polypeptide has pesticidal activity against Lepidoptera, Coleoptera, Diptera, Hemiptera, or nematode pests, and the field is infested with Lepidoptera, Hemiptera, Coleoptera, Diptera, or nematode pests. As defined herein, the "yield" of a plant refers to the quality and / or quantity of biomass produced by the plant. "Biomass" refers to any measured plant product. An increase in biomass yield is any improvement in the yield of a measured plant product. Increasing plant yield has several commercial applications. For example, increasing plant leaf biomass can increase the yield of leafy vegetables consumed by humans or animals. In addition, increasing leaf biomass can be used to increase the yield of plant-derived pharmaceuticals or industrial products. Increased yield can include any statistically significant increase, including but not limited to, at least 1% increase, at least 3% increase, at least 5% increase, at least 10% increase, at least 20% increase, at least 30% increase, at least 50% increase, at least 70% increase, at least 100% increase or more, compared to plants that do not express the pesticidal sequence. In specific methods, plant yield is increased due to improved pest resistance in plants expressing the pesticidal proteins disclosed herein. Expression of the pesticidal protein results in a reduction in the ability of the pest to infest or feed.

[0137] Plants can also be treated with one or more chemical compositions comprising one or more herbicides, insecticides, or fungicides. Exemplary chemical compositions include: Fruit / vegetable herbicides: Atrazine, Bromacil, Diuron, Glyphosate, Linuron, Metribuzin, Simazine, Trifluralin, Fluazifop, Glufosinate, Halosulfuron Gowan, Propyzamide, Sethoxydim, Butafenacil, Halosulfuron, Indaziflam; Fruits / vegetables kill insects Dosage:Bacillus thuringiensis, Carbaryl, Carbofuran, Chlorpyrifos, Cypermethrin, Deltamethrin, Abamectin, Cyfluthrin / beta-cyfluthrin, Esfenvalerate, Lambda-cyhalothrin, Acequinocyl, Bifenazate, Methoxyfenozide, Novaluron, Chromafenozide, Thiacloprid, Dinotefuran, Fluacrypyrim, Spirodiclofen, Gamma-cyhalothrin, Spiromesifen, Spinosad, Rynaxypyr, Cyazypyr, Triflumuron, Spirotetramat, Imidacloprid, Flubendiamide, Thiodicarb, Metaflumizone, Sulfoxaflor, Cyflumetofen, Cyanopyrafen, Clothianidin, Thiamethoxam, Spinotoram, Thiodicarb, Flonicamid, Methiocarb, Emamectin-benzoate, Indoxacarb, Pyriproxifen, Fenbutatin-oxid; Fruit / Vegetable Fungicides:Ametoctradin, Azoxystrobin, Benthiavalicarb, Boscalid, Captan, Carbendazim, Chlorothalonil, Copper, Cyazofamid, Cyflufenamid, Cymoxanil, Cyproconazole, Cyprodinil, Difenoconazole azole), Dimetomorph, Dithianon, Fenamidone, Fenhexamid, Fluazinam, Fludioxonil, Fluopicolide, Fluopyram, Fluoxastrobin, Fluxapyroxad, Folpet, Fosetyl, Iprodione, Propineb provalicarb), Isopyrazam, Kresoxim-methyl, Mancozeb, Mandipropamid, Metalaxyl / mefenoxam, Metiram, Metrafenone, Myclobutanil, Penconazole, Penthiopyrad, Picoxystrobin, Propamocarb Propamocarb), Propiconazole, Propineb, Proquinazid, Prothioconazole, Pyraclostrobin, Pyrimethanil, Quinoxyfen, Spiroxamine, Sulfur, Tebuconazole, Thiophanate-methyl, Trifloxystrobin; Cereal herbicides:2.4-D, Amidosulfuron, Bromoxanil, Carfentrazone-E, Chlorotoluron, Chlorsulfuron, Clodinafop-P, Clopyralid, Dicamba, Diclofop-M, Diflufenican, Fenoxaprop, Florasulam, Flucarbazone-NA, Flufenacet, Flupyrosulfuron-M, Fluroxypyr, Fluroxypyr Flurtamone, Glyphosate, Iodosulfuron, Ioxynil, Isoproturon, MCPA, Mesosulfuron, Metsulfuron, Pendimethalin, Pinoxaden, Propoxycarbazone, Prosulfocarb, Pyroxsulam, Sulfosulfuron, Thifensulfuron, Tralkoxydim, Triasulfuron, Tribenuron, Trifluralin, Tritosulfuron; Cereal fungicides: Azoxystrobin, Bixafen, Boscalid, Carbendazim, Chlorothalonil, Cyflumilast, Cyproconazole, Cyprodinil, Dimoxystrobin, Epoxiconazole, Fenpropidin, Fenpropimorph, Fluopyram, Fluoxastrobin, Fluquinconazole, Fluopyram, Pyraclostrobin, Kresoxim-methyl, Metconazole, Metrafenone, Penthiopyrad, Picoxystrobin, Prochloraz, Propiconazole, Proquinoxal, Prothioconazole, Pyraclostrobin, Quinoxyfen, Spirocyclanil, Tebuconazole, Thiophanate-methyl, Trifloxystrobin; valley Insecticides:Dimethoate, Lambda-cyhalthrin, Deltamethrin, alpha-Cypermethrin, beta-Cyhalthrin, Bifenthrin, Imidacloprid, Clothianidin, Thiamethoxam, Thiacloprid, Acetamiprid, Dinetofuran, Chlorpyriphos, Pirimicarb, Methiocarb, and Sulfenpyroxil; Maize Herbicides: Atrazine, Alachlor, Bromoxynil, Acetochlor, Dicamba, Clopyralid, (S-)Dimethenamid, Glufosinate, Glyphosate, Isoxaflutole, (S-)Metolachlor, Mesotrione, Nicosulfuron, Primisulfuron, Rimsulfuron, Sulcotrione, Foramsulfuron, Topramezone, Tembotrione, Saflufenacil, Thiencarbazone, Flufenacet, Pyroxasulfon; Corn Insecticide: Carbofuran, chlorpyrifos, bifenthrin, fipronil, imidacloprid, lambda-cyfluthrin, tefluthrin, thiamethoxam, clothianidin, spiromesifen, flubendiamide, triflumuron, chlorantraniliprole, deltamethrin, thiodicarb, beta-cyfluthrin, cypermethrin, bifenthrin, lufenuron, tebupirimphos, ethiprole, cyanamide, thiacloprid, acetamiprid, dinotefuran, avermectin; Corn Killer Fungal agents: Azoxystrobin, Bixafen, Boscalid, Cyproconazole, Diastereoisomerase, Effexor, Fenitropan, Fluopyram, Fluoxastrobin, Fluopyram, Pyraclostrobin, Metconazole, Penthiopyrad, Picoxystrobin, Propiconazole, Prothioconazole, Pyraclostrobin, Tebuconazole, Trifloxystrobin; Rice herbicides:Butachlor, Propanil, Azimsulfuron, Bensulfuron, Cyhalofop, Daimuron, Fentrazamide, Imazosulfuron, Mefenacet, Oxaziclomefone, Pyrazosulfuron, Pyributicarb, Quinclorac, Thiobencarb, Indanofan, Flufenacet, Tetrazodone, Halosulfuron-methyl, Debiril, Benzobicyclon, Pyriftalid, Penoxsulam, Bispyribac, Oxadiargyl, Ethoxysulfuron, Pretilachlor, Mesotrione, Tefuryltrione, Oxadiazone, Fenoxaprop, Pyrimisulfan; Rice insecticide: Diazinon, Fenobucarb, Benfuracarb, Buprofezin, Dinotefuran, Fipronil, Imidacloprid, Isoprocarb, Thiacloprid, Chlorfenapyr, Clothianidin, Ethylthiocyanate, Flubendiamide, Chlorantraniliprole, Deltamethrin, Acetamiprid, Thiamethoxam, Cyantraniliprole, Spinosad, Spinetoram, Emamectin, Cypermethrin, Chlorpyrifos, Etofenprox, Carbofuran, Benfuracarb, Sulfenpyroxil; Rice fungicide:Azoxystrobin, Carbendazim, Carpropamid, Diclocymet, Difenoconazole, Edifenphos, Ferimzone, Gentamycin, Hexaconazole, Hymexazol, Iprobenfos (IBP), Isoprothiolane, Isotianil, Kasugamycin, Mancozeb, Metominostrobin, Orysastrobin, Pencycuron, Probenazole, Propiconazole, Propineb, Pyroquilon, Tebuconazole, Thiophanate-methyl, Tiadinil, Tricyclazole, Trifloxystrobin, Validamycin; Cotton herbicides: Diuron, Fluometuron, MSMA, Oxyfluorfen, Prometryn, Trifluralin, Methaclonide, Clethodim, Fluazifop-butyl, Glyphosate, Norflurazon, Pendimethalin, Pyrithiobac-sodium, Trifloxysulfuron, Tepraloxydim, Glufosinate, Flumioxazin, Thidiazuron; Cotton insecticide: Acephate, chlorpyrifos, cypermethrin, deltamethrin, abamectin, acetamiprid, emamectin, imidacloprid, indoxacarb, lambda-cyfluthrin, spinosad, thiodicarb, gamma-cyfluthrin, spiromesifen, pyridalyl, flonicamid; flubendiamide, triflumuron, chlorantraniliprole, beta-cyfluthrin, spirotetramat, clothianidin, thiamethoxam, thiacloprid, dinotefuran, flubendiamide, cyanamide, spinosad, ethyl spinosad, gamma-cyfluthrin, 4-[[(6-chloropyridin-3-yl)methyl](2,2-difluoroethyl)amino]furan-2(5H)-one), thiodicarb, abamectin, flonicamid, pyridalyl, spiromesifen, sulfoxaflor; Cotton fungicide:Azoxystrobin, bixafen, boscalid, carbendazim, chlorothalonil, copper, cyproconazole, difenoconazole, difenoconazole, oxazolidinone, epoxiconazole, fenamid, fluazinam, fluopyram, fluoxastrobin, flupyrad, iprodione, pyraclostrobin, isothiocarb, mancozeb, maneb, fenoxam, penthiopyrad, picoxystrobin, propineb, prothioconazole, pyraclostrobin, quintozene, tebuconazole, tetraconazole, thiophanate-methyl, trifloxystrobin; Soybean herbicide: Alachlor, Bentazone, Trifluralin, Chlorimuron-Ethyl, Cloransulam-Methyl, Oxaprop-Phthiocarb, Fomesafen, Roundup, Glyphosate, Imazamox, Imazaquin, Imazethapyr, (S-)Metolachlor, Metribuzin, Pendimethalin, Pyriltrione, Glufosinate-ammonium; Soybean Insecticide: λ-Cyfluthrin, Methomyl, Imidacloprid, Clothianidin, Thiamethoxam, Thiamethoxam, Acetamiprid, Dinotefuran, Flubendiamide, Chlorantraniliprole, Cyantraniliprole, Spinosad, Spinetoram, Emamectin, Fipronil, Ethylthiocyanate, Deltamethrin, β-Cyfluthrin, (γ and λ-Cyfluthrin), 4-[[(6-chloropyridin-3-yl)methyl](2,2-difluoroethyl)amino]furan-2(5H)-one, Spirotetramat, Spirodiclofen, Triflumuron, Flonax, Thiodicarb, β-Cyfluthrin; Soybean fungicides: Azoxystrobin, Bixafen, Boscalid, Carbendazim, Chlorothalonil, Copper, Cyproconazole, Difenoconazole, Difenoconazole, Esterstrobin, Epoxiconazole, Fluazinam, Fluopyram, Fluoxastrobin, Flutriafol, Flupyrad, Pyraclostrobin, Iprodione, Isothiazolin, Mancozeb, Maneb, Metconazole, Methiconazole, Myclobutanil, Penthiopyrad, Picoxystrobin, Propiconazole, Propineb, Prothioconazole, Pyraclostrobin, Tebuconazole, Tetraconazole, Thiophanate-methyl, Trifloxystrobin; beet herbicide:Chloridazon, Desmedipham, Ethofumesate, Phenmedipham, Triallate, Clopyralid, Lenacil, Metamitron, Quinmerac, Cycloxydim, Triflusulfuron, Pyrrolidone, Quizalofop; Beet Insecticide: Imidacloprid, clothianidin, thiamethoxam, thiacloprid, acetamiprid, dinotefuran, deltamethrin, β-cyfluthrin, γ / λ-cyfluthrin, 4-[[(6-chloropyridin-3-yl)methyl](2,2-difluoroethyl)amino]furan-2(5H)-one, tefluthrin, chlorantraniliprole, cyanamide (Cyaxypyr), fipronil, carbofuran; Canola herbicide: Clopyralid, diclofenac, cypermethrin, glufosinate, glyphosate, metazachlor, trifluralin, ethametsulfuron, quinclorac, quizalofop-p-ethyl, clethodim, pyraclostrobin; Canola Fungicides: Azoxystrobin, bixafen, boscalid, carbendazim, cyproconazole, difenoconazole, difenoconazole, oxazolidinone, epoxiconazole, fluazinam, fluopyram, fluoxastrobin, flusilazole, flupyraclostrobin, iprodione, pyraclostrobin, mepiquat-chloride, metconazole, pyraclostrobin, paclobutrazole, penthiopyrad, picoxystrobin, prochloraz, prothioconazole, pyraclostrobin, tebuconazole, thiophanate-methyl, trifloxystrobin, and vinclozolin; Canola Insecticide: Carbofuran, Thiacloprid, Deltamethrin, Imidacloprid, Clothianidin, Thiamethoxam, Acetamiprid, Dinotefuran, Beta-Cyfluthrin, (Gamma and Lambda Cyfluthrin), Tau-Fluvaleriate, Ethylthiocyanate, Spinosad, Spinetoram, Flubendiamide, Chlorantraniliprole, Cyantraniliprole, 4-[[(6-Chloropyridin-3-yl)methyl](2,2-difluoroethyl)amino]furan-2(5H)-one.

[0138] Method for introducing the gene of the present invention into another plant

[0139] Also provided herein are methods for introducing a nucleic acid of the invention into another plant. The nucleic acid of the invention or a fragment thereof can be introduced into a second plant by recurrent selection, backcrossing, pedigree breeding, strain selection, mass selection, mutation breeding and / or genetic marker enhancement selection.

[0140] Therefore, in one embodiment, the method of the present invention comprises hybridizing a first plant comprising nucleic acid of the present invention with a second plant to produce an F1 progeny plant and selecting an F1 progeny plant comprising nucleic acid of the present invention. The method may further comprise hybridizing a selected progeny plant with a first plant comprising nucleic acid of the present invention to produce a backcross progeny plant and selecting a backcross progeny plant comprising nucleic acid of the present invention. This paper provides a method for evaluating pesticidal activity elsewhere. The method may further comprise continuously repeating these steps one or more times to produce a selected second or higher backcross progeny plant comprising nucleic acid of the present invention.

[0141] Any breeding method involving selection of plants for a desired phenotype can be used in the methods of the present invention. In some embodiments, F1 plants can be self-pollinated to produce segregating F2 generations. Individual plants expressing the desired phenotype (e.g., pesticidal activity) can then be selected in each generation (F3, F4, F5, etc.) until the trait is homozygous or fixed within the breeding population.

[0142] The second plant can be a plant with a desirable trait, such as herbicide tolerance, insect tolerance, drought tolerance, nematode control, water use efficiency, nitrogen use efficiency, improved nutritional value, disease resistance, improved photosynthesis, improved fiber quality, stress tolerance, improved reproduction, etc. The second plant can be an elite event as described elsewhere herein.

[0143] In various embodiments, plant parts (whole plants, plant organs (e.g., leaves, stems, roots, etc.), seeds, plant cells, propagules, embryos, etc.) can be harvested from the resulting hybrids and propagated or collected for downstream uses (e.g., food, feed, biofuel, oil, flour, meal, etc.).

[0144] Methods of obtaining plant products

[0145] The present invention also relates to a method for obtaining a commodity product comprising harvesting and / or grinding grain from a crop comprising the nucleic acid of the present invention to obtain a commodity product. Agronomically and commercially important products and / or compositions of matter include, but are not limited to, animal feed, commodities and plant products and by-products intended for use as food for human consumption or for compositions and commodities for human consumption, in particular deactivated seed / grain products, including (semi-)processed products produced from such grains / seeds, wherein the product is or comprises whole or processed seeds or grains, animal feed, corn meal or soy meal, corn flour or soy meal, corn, corn starch, soy meal, soy flour, oatmeal, soy protein concentrate, soy protein isolate, textured soy protein concentrate, Products and compositions of matter that are agriculturally and commercially important are intended to be within the scope of the present invention if they contain detectable amounts of the nucleotide and / or amino acid sequences described herein as diagnostic for any plant containing such nucleotide sequences.

[0146] The following examples are offered by way of illustration and not by way of limitation.

[0147] Experimental Examples

[0148] Example 1. Discovery of a new insecticide gene

[0149] Axmi486 was identified from the Bacillus thuringiensis ATX65002 strain using the steps described in U.S. Patent Publication US20180371032 (incorporated herein by reference in its entirety) and selected for further modification. As previously shown, Axmi486 showed activity against Plutella xylostella, Spodoptera frugiperda, Southwestern corn borer, Small sugarcane borer, Tobacco budworm, Corn earworm, and Spodoptera frugiperda (herein, "target insects"; see Table 7 of US20180371032). It is further desired to produce Axmi486 variants that show increased resistance, mortality, and / or tolerance to insects belonging to species of the genus Spodoptera litura (e.g., Spodoptera eridania (herein, "SAW")) while maintaining effectiveness against previously observed pests.

[0150] Example 2. Protein engineering of the toxin gene Axmi486 for improving biological activity against soybean pest SAW

[0151] Based on the protein structure of Axmi486, internal functional information, and experimental data from alanine scanning mutagenesis studies, target positions for mutagenesis were selected. The key goal of this experiment was to produce Axmi variants with increased resistance to SAW compared to Axmi486, while ensuring that the Axmi variants did not reduce their resistance to the target insect compared to Axmi486. Approximately 3,600 mutants were generated for screening against SAW. Alanine mutants were identified and tested for their activity against Hz and their stability in Hz intestinal fluid. Mutants that did not destroy activity and retained wild-type activity were further screened for improved SAW activity. The results of these experiments enabled the prioritization and selection of amino acids for targeted site-saturation mutagenesis. 47 relevant positions were targeted.

[0152] Mutant library creation

[0153] Mutagenesis was used to create a single-position mutation saturation library. A combination of primers containing degenerate codons (NDT / VHG / TGG) was used to reduce redundancy at the codon level. The Axmi486_1Pb plasmid DNA template and the Agilent Technologies Quick-Change Lightning Site-Directed Mutagenesis Kit were used to create the library. The mutant library was transformed in T7 Express competent cells, and random colonies were subjected to DNA sequencing to confirm the mutations.

[0154] Example 3. Bioassay Screening for SAW Activity

[0155] For expression in E. coli, T7 Express competent cells were transformed with individual plasmids from the 47 relevant positions and plated onto LB agar carbenicillin plates. A single colony from each library was inoculated in Instant TB supplemented with carbenicillin and glycerol and grown at 37°C with shaking at 250 rpm for 24 hours until cell saturation was reached.

[0156] Whole cultures expressing variants identified in Example 2, inoculated from a freshly transformed single bacterial colony library and wild-type Axmi486, were screened for SAW resistance. Forty-seven single-site saturation libraries were screened in the SAW bioassay. Variants that outperformed wild-type Axmi486 were selected as primary hits to confirm improved activity across the entire culture. Glycerol stocks of the primary hits were streaked onto LB agar carbenicillin plates. These selected hits were inoculated into fresh Instant TB medium supplemented with carbenicillin and glycerol and grown at 37°C with shaking at 250 rpm for 24 hours. Once saturation was reached, whole culture samples were aliquoted from each variant culture, mixed with the appropriate LDS sample buffer, and boiled at 95°C for 10 minutes. Optical density analysis was performed using 4%-12% bis-tris SDS-PAGE to quantify the 79 kDa Axmi486 protein band of interest. A range of protein concentrations (1 mg / ml, 0.1 mg / ml, 0.03 mg / ml, 0.02 mg / ml) with multiple replicates was used to determine the SAW activity improvement (see Figures 1a-1e). The improvement in SAW activity was confirmed in at least three independent biological replicates for five variants including three single position variants and two stacked / combined variants. The improvement in SAW activity was confirmed for the cotton bollworm (Hz) (see Figures 2a and 2b), soybean armyworm (VBC) (see Figures 3a and 3b), fall armyworm (FAW) (see Figures 4a and 4b), soybean silver moth (Chrysodeixis includens) (SBL) (see Figures 4a and 4b). Figure 5 Whole culture bioassays of SAW-modified mutants were performed in Heliothis virescens (Hv) and Heliothis virescens (Hv) to determine the effect of the mutations on activity.

[0157] Example 3b. Assay Description

[0158] Bioassay plates are imaged with Lemnatec to score insecticidal activity. Lemnatec images the plates, and a trained algorithm generates a pixel score for each treated well infested with larvae. The mean object size is the average object area, in pixels, of the larvae after protein treatment. A larger mean object area indicates that the larvae are growing and developing, while a smaller mean object area means that the larvae are stunted and / or not growing or developing. Therefore, the smaller the mean object area, the greater the insecticidal activity of the protein.

[0159] Table 1a. Identified Axmi486 variants with increased resistance to SAW while maintaining resistance to target insects

[0160]

[0161]

[0162] Table 1b. References between internal names and serial ID numbers

[0163] Internal name DNA SEQ ID NO Amino acid SEQ ID NO WT N / A N / A R60S 1 16 R60T 2 17 E275D 3 18 R60S E275D 4 19 R60T E275D 5 20 R60S H97L T100N D96P 6 21 R60T H97L T100N D96P 7 22 E275D H97L T100N D96P 8 23 R60S E275D H97L T100N D96P 9 24 R60T E275D H97L T100N D96P 10 25 R60S T100N D96P 11 26 R60T T100N D96P 12 27 E275D T100N D96P 13 28 R60S E275D T100N D96P 14 29 R60T E275D T100N D96P 15 30

[0164] Table 2. Bioassay results

[0165] mutation SEQ ID NO % SAW hypoplasia compared with WT WT 0％ R60S 16 75％ R60T 17 78％ E275D 18 69％ R60S E275D 19 87％ R60T E275D 20 83％

[0166] Percentages of treatment based on 1 mg / ml confluent Axmi486 whole culture. Percent stunting equals the percentage of how much smaller the insects are compared to WT size.

[0167] Example 4. Plant expression gene vector

[0168] Hz improved version

[0169] Natural and improved insecticidal variants of Axmi486 were nominated for testing in plants.Plant transformation vectors and transgenic events were generated using techniques consistent with typical practice of those skilled in the art.

[0170] Synthetic sequences encoding the essential region of insecticidal traits were designed and produced as a key component of the described invention. The goal of generating synthetic sequences was to achieve satisfactory protein expression in selected target plant species, as opposed to using native bacterial sequences. Synthetic versions consisted of altered nucleotide sequences that either retained the native protein sequence or introduced targeted mutations, altering specific amino acids associated with improved pest control. The following versions were produced:

[0171] A version encoding the 315 amino acid sequence of the native axmi486 gene from Bacillus thuringiensis

[0172] A version encoding the 315 amino acid sequence of the axmi486 gene from Bacillus thuringiensis containing a single point mutation at position 96 (aspartic acid to proline)

[0173] A version encoding the 315 amino acid sequence of the axmi486 gene from Bacillus thuringiensis containing a single point mutation at position 97 (histidine to aspartic acid)

[0174] A version encoding the 315 amino acid sequence of the axmi486 gene from Bacillus thuringiensis containing a single point mutation at position 97 (histidine to leucine)

[0175] A version encoding a 315 amino acid sequence of the axmi486 gene from Bacillus thuringiensis containing double point mutations at position 96 (aspartic acid to proline) and at position 100 (threonine to asparagine)

[0176] A version encoding a 315 amino acid sequence of the axmi486 gene from Bacillus thuringiensis containing double point mutations at position 96 (aspartic acid to proline) and at position 97 (histidine to aspartic acid)

[0177] A version encoding a 315 amino acid sequence of the axmi486 gene from Bacillus thuringiensis containing double point mutations at position 96 (aspartic acid to proline) and at position 97 (histidine to leucine)

[0178] A version encoding a 315 amino acid sequence of the axmi486 gene from Bacillus thuringiensis containing three point mutations at position 96 (aspartic acid to proline), at position 97 (histidine to leucine), and at position 100 (threonine to asparagine)

[0179] The described coding region is configured with the appropriate promoter and terminator sequences required for plant expression of the desired transgene. Examples of expression in dicots can include the promoter region of ubiquitin 10 (UBQ10) from Arabidopsis thaliana (A.thaliana) (Grefen et al., 2010) and the 3' untranslated region (3'nos) of the nopaline synthase gene from Agrobacterium tumefaciens (Depicker A. et al., 1982) functionally combined with one of the described insecticide synthetic sequences. Successful configuration will not be limited to the described examples expressed in dicots or monocots. Methods and techniques for combining regulatory sequences with coding regions are well known in the art.

[0180] To produce the desired transgenic plants, the described or similar pest control cassettes will need to be combined with a suitable selectable marker cassette in an appropriate plant transformation vector. The resulting complete vector can be used in a variety of transformation techniques, including but not limited to Agrobacterium-mediated or bioballistic techniques performed by those skilled in the art.

[0181] Improved version of SAW

[0182] Natural and improved insecticidal variants of Axmi486 were nominated for testing in plants.Plant transformation vectors and transgenic events were generated using techniques consistent with typical practice of those skilled in the art.

[0183] Synthetic sequences encoding the essential region of insecticidal traits were designed and produced as a key component of the described invention. The goal of generating synthetic sequences was to achieve satisfactory protein expression in selected target plant species, as opposed to using native bacterial sequences. Synthetic versions consisted of altered nucleotide sequences that either retained the native protein sequence or introduced targeted mutations, altering specific amino acids associated with improved pest control. The following versions were produced:

[0184] A version encoding the 315 amino acid sequence of the native axmi486 gene from Bacillus thuringiensis

[0185] A version encoding the 315 amino acid sequence of the axmi486 gene from Bacillus thuringiensis containing a single point mutation at position 60 (arginine to serine)

[0186] A version encoding the 315 amino acid sequence of the axmi486 gene from Bacillus thuringiensis containing a single point mutation at position 60 (arginine to threonine)

[0187] A version encoding the 315 amino acid sequence of the axmi486 gene from Bacillus thuringiensis containing a single point mutation at position 275 (glutamic acid to aspartic acid)

[0188] A version encoding a 315 amino acid sequence of the axmi486 gene from Bacillus thuringiensis containing double point mutations at position 60 (arginine to serine) and at position 275 (glutamate to aspartate)

[0189] A version encoding a 315 amino acid sequence of the axmi486 gene from Bacillus thuringiensis containing double point mutations at position 60 (arginine to threonine) and at position 275 (glutamate to aspartate)

[0190] The described coding region is configured with the appropriate promoter and terminator sequences required for plant expression of the desired transgene. Examples of expression in dicots can include the promoter region of ubiquitin 10 (UBQ10) from Arabidopsis thaliana (A.thaliana) (Grefen et al., 2010) and the 3' untranslated region (3'nos) of the nopaline synthase gene from Agrobacterium tumefaciens (Depicker A. et al., 1982) functionally combined with one of the described insecticide synthetic sequences. Successful configuration will not be limited to the described examples expressed in dicots or monocots. Methods and techniques for combining regulatory sequences with coding regions are well known in the art.

[0191] To produce the desired transgenic plants, the described or similar pest control cassettes will need to be combined with a suitable selectable marker cassette in an appropriate plant transformation vector. The resulting complete vector can be used in a variety of transformation techniques, including but not limited to Agrobacterium-mediated or bioballistic techniques performed by those skilled in the art.

[0192] Hz+SAW improved version

[0193] Natural and improved insecticidal variants of Axmi486 were nominated for testing in plants.Plant transformation vectors and transgenic events were generated using techniques consistent with typical practice of those skilled in the art.

[0194] Synthetic sequences encoding the essential region of insecticidal traits were designed and produced as a key component of the described invention. The goal of generating synthetic sequences was to achieve satisfactory protein expression in selected target plant species, as opposed to using native bacterial sequences. Synthetic versions consisted of altered nucleotide sequences that either retained the native protein sequence or introduced targeted mutations, altering specific amino acids associated with improved pest control. The following versions were produced:

[0195] A version encoding the 315 amino acid sequence of the native axmi486 gene from Bacillus thuringiensis

[0196] A version encoding a 315 amino acid sequence of the axmi486 gene from Bacillus thuringiensis containing four point mutations at position 60 (arginine to serine), at position 96 (aspartic acid to proline), at position 97 (histidine to leucine), and at position 100 (threonine to asparagine)

[0197] A version encoding the 315 amino acid sequence of the axmi486 gene from Bacillus thuringiensis containing four point mutations at position 96 (aspartic acid to proline), at position 97 (histidine to leucine), at position 100 (threonine to asparagine), and at position 275 (glutamate to aspartic acid)

[0198] A version encoding the 315 amino acid sequence of the axmi486 gene from Bacillus thuringiensis containing five point mutations at position 60 (arginine to serine), at position 96 (aspartic acid to proline), at position 97 (histidine to leucine), at position 100 (threonine to asparagine), and at position 275 (glutamic acid to aspartic acid)

[0199] A version encoding the 315 amino acid sequence of the axmi486 gene from Bacillus thuringiensis containing five point mutations at position 60 (arginine to threonine), at position 96 (aspartic acid to proline), at position 97 (histidine to leucine), at position 100 (threonine to asparagine), and at position 275 (glutamic acid to aspartic acid)

[0200] The described coding region is configured with the appropriate promoter and terminator sequences required for plant expression of the desired transgene. Examples of expression in dicots can include the promoter region of ubiquitin 10 (UBQ10) from Arabidopsis thaliana (A.thaliana) (Grefen et al., 2010) and the 3' untranslated region (3'nos) of the nopaline synthase gene from Agrobacterium tumefaciens (Depicker A. et al., 1982) functionally combined with one of the described insecticide synthetic sequences. Successful configuration will not be limited to the described examples expressed in dicots or monocots. Methods and techniques for combining regulatory sequences with coding regions are well known in the art.

[0201] To produce the desired transgenic plants, the described or similar pest control cassettes will need to be combined with a suitable selectable marker cassette in an appropriate plant transformation vector. The resulting complete vector can be used in a variety of transformation techniques, including but not limited to Agrobacterium-mediated or bioballistic techniques performed by those skilled in the art.

[0202] Examples of common components of a pest control box:

[0203] UBQ10:

[0204] The promoter region of Arabidopsis thaliana ubiquitin 10 (UBQ10) (Grefen et al., 2010)

[0205] Axmi486 WT:

[0206] Coding sequence of the axmi486 gene of Bacillus thuringiensis

[0207] A variant of axmi486-1Pb with silent mutations was introduced to remove allergen hits. axmi486 encodes a novel insecticidal protein that is approximately 49% similar to MTX3. axmi486-1Pb is a variant of axmi486 optimized for soybean expression (JCeasar 46%).

[0208] 3'nos:

[0209] 3' untranslated region of the nopaline synthase gene of Agrobacterium tumefaciens (Depicker A. et al., 1982)

[0210] Example 5. Soybean transformation

[0211] Soybean transformation is realized using methods well known in the art, as described substantially using the method of transformation soybean half-seed explants mediated by Agrobacterium tumefaciens as described by Paz et al. (2006), Plant Cell Reports 25:206. Cyclosulfuron is used as a selective marker to identify transformants. The appearance of green shoots is observed and recorded as an index of tolerance to the herbicides isoxazolidinone or cyclosulfuron. The tolerant transgenic shoots will show normal greening comparable to the wild-type soybean shoots not treated with isoxazolidinone or cyclosulfuron, while the wild-type soybean shoots treated with the same amount of isoxazolidinone or cyclosulfuron will be completely decolorized. This indicates that the presence of the HPPD protein enables tolerance to HPPD inhibitor herbicides, such as isoxazolidinone or cyclosulfuron.

[0212] Tolerant green shoots are transferred to a rooting medium or grafted. After an acclimatization period, rooted plantlets are transferred to a greenhouse. Transgenic plants are then sprayed with an HPPD inhibitor herbicide, such as tembotrione at a rate of 100 g AI / ha or mesotrione supplemented with rapeseed oil containing methyl ammonium sulfate at a rate of 300 g AI / ha. Ten days after application, symptoms resulting from the herbicide application are evaluated and compared with those observed in wild-type plants under the same conditions.

[0213] Example 6. Transformation of maize cells with the insecticidal protein genes described herein

[0214] Maize ears are preferably collected 8-12 days after pollination. Embryos are isolated from the ears, and those 0.8-1.5 mm in size are preferred for transformation. The embryos are plated with the scutellum facing up on a suitable incubation medium, such as DN62A5S medium (3.98 g / L N6 salts; 1 mL / L (1000× stock) N6 vitamins; 800 mg / L L-asparagine; 100 mg / L inositol; 1.4 g / L L-proline; 100 mg / L casamino acids; 50 g / L sucrose; 1 mL / L (1 mg / mL stock) 2,4-D). However, media and salts other than DN62A5S are suitable and known in the art. The embryos are incubated overnight in the dark at 25°C. However, it is not necessary for the embryos themselves to be incubated overnight.

[0215] The resulting explants are transferred to grid squares (30-40 per plate), placed on infiltration medium for approximately 30-45 minutes, and then transferred to irradiated plates (see, eg, PCT Publication No. WO / 0138514 and US Patent No. 5,240,842).

[0216] Use aerosol beam accelerator, use basically as PCT discloses the condition described in No. WO / 0138514, the DNA construct designed for the gene of the present invention in the plant cell is accelerated to enter the plant tissue. After the irradiation, the embryo is incubated on the infiltration medium for about 30 minutes, and then placed on the incubation medium in the dark at 25 ℃ and spend the night. To avoid excessive damage through irradiation explant, before transferring to the recovery medium, it is incubated for at least 24 hours. Then in the dark at 25 ℃, the embryo is spread on the recovery phase medium for about 5 days, then transferred to the selection medium. The explant is incubated in the selection medium for up to eight weeks, depending on the nature and characteristic of the specific selection used. After the selection period, the gained callus is transferred to the embryo maturation medium, until the formation of mature somatic embryos is observed. Then the gained mature somatic embryo is placed under low light, and the regeneration process is started by methods known in the art. The gained bud is allowed to take root on the rooting medium, and the gained plant is transferred to the seedling pot and propagated as a transgenic plant.

[0217] Table 3: Materials, DN62A5S culture medium

[0218]

[0219] The pH of the solution was adjusted to 5.8 with 1N KOH / 1N KCl, Gelrite (Sigma) was added at a concentration of up to 3 g / L, and the medium was autoclaved. After cooling to 50°C, 2 ml / L of a 5 mg / ml silver nitrate stock solution (Phytotechnology Labs) was added.

[0220] Example 7. Transformation of the Gene of the Invention into Plant Cells by Agrobacterium-Mediated Transformation

[0221] It is best to collect ears 8-12 days after pollination. From ear, isolate embryo, and preferably those 0.8-1.5mm size embryos are used for transformation. Embryo is plated on a suitable incubation medium with the scutellum upward, and incubated overnight at 25 ℃ in the dark. However, it is not necessary for the embryo itself to be incubated overnight. Embryo is contacted with the Agrobacterium strain containing the appropriate carrier for Ti plasmid-mediated transfer for about 5-10 minutes, then plated on the co-cultivation medium for about 3 days (in the dark at 22 ℃). After co-cultivation, explant is transferred to the recovery phase medium for 5-10 days (in the dark at 25 ℃). Explant is incubated in the selection medium for up to eight weeks, depending on the nature and characteristic of the specific selection used. After the selection period, the gained callus is transferred to the embryo maturation medium, until the formation of mature somatic embryos is observed. Then the gained mature somatic embryo is placed under low light, and the regeneration process is started as known in the art.

[0222] All variants were aligned with the protein of Axmi486 WT.

[0223] All publications and patents mentioned in this specification are indicative of the levels of those skilled in the art to which this invention pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

[0224] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be apparent that certain changes and modifications may be practiced within the scope of the appended claims.

Claims

1. A recombinant nucleic acid molecule consisting of a nucleotide sequence selected from the group consisting of: a) the nucleotide sequences shown in SEQ ID NOs: 1-2, 4-7, 9-12 and 14-15; and b) a nucleotide sequence encoding a polypeptide consisting of the amino acid sequence of SEQ ID NOs: 16-17, 19-22, 24-27 and 29-30.

2. The recombinant nucleic acid molecule according to claim 1, wherein the nucleotide sequence encodes an amino acid sequence having pesticidal activity.

3. An expression cassette comprising the recombinant nucleic acid according to any one of claims 1 to 2. The expression cassette according to claim 3 , which is designed for expression in plants.

5. The expression cassette of claim 3 or 4, wherein the nucleic acid is operably linked to a promoter capable of directing expression of the nucleic acid in a plant cell.

6. A DNA construct comprising the recombinant nucleic acid according to any one of claims 1 to 2.

7. The DNA construct according to claim 6, which is designed for expression in plants.

8. according to the DNA construct described in claim 6 or 7, wherein said nucleic acid is operably linked to the promoter that can direct said nucleic acid to be expressed in plant cells.

9. A vector comprising the recombinant nucleic acid molecule according to any one of claims 1 to 2, the expression cassette according to any one of claims 3 to 5, or the DNA construct according to any one of claims 6 to 8.

10. The vector of claim 9, further comprising a nucleic acid molecule encoding a heterologous polypeptide.

11. A host cell comprising the recombinant nucleic acid molecule according to any one of claims 1 to 2 or the expression cassette according to any one of claims 3 to 5 or the DNA construct according to any one of claims 6 to 8, wherein the host cell is not a plant cell.

12. The host cell according to claim 11, which is a bacterial host cell.

13. A method of producing a plant comprising the recombinant nucleic acid molecule according to any one of claims 1 to 2 or the expression cassette according to any one of claims 3 to 5 or the DNA construct according to any one of claims 6 to 8, the method comprising growing the plant or its seeds in a field.

14. The method of claim 13, wherein the plant is selected from the group consisting of maize, sorghum, wheat, sunflower, tomato, crucifers, pepper, potato, cotton, rice, soybean, sugar beet, sugar cane, tobacco, or barley. The method according to claim 14 , wherein the cruciferous plant is cabbage or rapeseed.

16. A recombinant polypeptide having insecticidal activity, which is selected from the polypeptides consisting of the amino acid sequences of SEQ ID NOs: 16-17, 19-22, 24-27 and 29-30.

17. The recombinant polypeptide according to claim 16, wherein the recombinant polypeptide is more effective against subtropical armyworms ( Spodoptera eridania ) increases the resistance granted by the 18. A composition comprising the polypeptide according to any one of claims 16 to 17.

19. The composition of claim 18, wherein the composition is selected from the group consisting of a powder, a granule, a spray, an emulsion, a colloid, and a solution.

20. The composition according to claim 18, wherein the composition is selected from a powder or a granule.

21. The composition of claim 18, wherein the composition is prepared by drying, lyophilizing, homogenizing, extracting, filtering, centrifuging, sedimenting, and concentrating a bacterial cell culture.

22. The composition of claim 18, comprising 1% to 99% by weight of the polypeptide.

23. A method for controlling a population of lepidopteran pests, the method comprising contacting the population with a pesticidally effective amount of a polypeptide according to any one of claims 16 to 17.

24. The method according to claim 23, wherein the pest is subtropical armyworm ( Spodoptera eridania ).

25. A method for killing a lepidopteran pest, the method comprising contacting the pest with a pesticidally effective amount of the polypeptide according to any one of claims 16 to 17 or feeding the pest with a pesticidally effective amount of the polypeptide.

26. The method according to claim 25, wherein the pest is subtropical armyworm ( Spodoptera eridania ).

27. A method for producing a polypeptide having pesticidal activity, the method comprising culturing the host cell according to claim 11 or 12 under conditions such that a nucleic acid molecule encoding the polypeptide is expressed.

28. A method of producing a plant, wherein the method comprises integrating the recombinant nucleic acid molecule according to any one of claims 1 to 2 or the expression cassette according to any one of claims 3 to 5 or the DNA construct according to any one of claims 6 to 8 into the genome of the plant.

29. The method of claim 28, wherein the plant is selected from the group consisting of maize, sorghum, wheat, sunflower, tomato, crucifers, pepper, potato, cotton, rice, soybean, sugar beet, sugar cane, tobacco, or barley.

30. The method of claim 29, wherein the cruciferous plant is cabbage or rapeseed.

31. A method for protecting a plant from lepidopteran pests, the method comprising expressing the nucleic acid molecule according to any one of claims 1 to 2 in a plant or a cell thereof.

32. The method of claim 31, wherein the plant produces a pesticidal polypeptide having pesticidal activity against lepidopteran pests.

33. The method according to claim 31 or 32, wherein the lepidopteran pest is subtropical armyworm.

34. A method for increasing plant yield, the method comprising growing a plant or its seeds in a field having stably incorporated into its genome a recombinant nucleic acid molecule according to any one of claims 1 to 2, an expression cassette according to any one of claims 3 to 5, or a DNA construct according to any one of claims 6 to 8, wherein the field is infested with a lepidopteran pest and the polypeptide has pesticidal activity against the lepidopteran pest.

35. The method of claim 34, wherein the lepidopteran pest is subtropical armyworm.

36. Use of a nucleic acid molecule according to any one of claims 1 to 2, an expression cassette according to any one of claims 3 to 5, or a DNA construct according to any one of claims 6 to 8 for protecting plants from lepidopteran pests, against which the amino acid encoded by the nucleic acid has insecticidal activity.

37. The use according to claim 36, wherein the lepidopteran pest is subtropical armyworm.

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