recombinant nucleic acid molecule, vector, microbial host cell, recombinant polypeptide with pestici

专利摘要:
AXMI221z, AXMI222z, AXMI223z, AXMI224z AND AXMI225z DELTA-ENDOTOXIN GENES AND METHODS FOR THEIR USE. The present invention relates to compositions and methods for imparting pesticidal activity to bacteria, plants, plant cells, tissues and seeds. Compositions are provided that comprise a sequence encoding a toxin polypeptide. The coding sequences can be used in DNA constructs or expression cassettes for transformation and expression in plants and bacteria. The compositions also comprise transformed bacteria, plants, plant cells, tissues and seeds. In particular, isolated toxin nucleic acid molecules are provided. In addition, amino acid sequences corresponding to polynucleotides and antibodies that specifically bind to those amino acid sequences are covered. In particular, the present invention provides isolated nucleic acid molecules that comprise nucleotide sequences that code for the amino acid sequence shown in any of SEQ ID NO: 21-32, or the nucleotide sequence shown in any of SEQ ID NO: 1-20 , as well as their variants and fragments.
公开号:BR112012020705B1
申请号:R112012020705-9
申请日:2011-02-17
公开日:2021-02-17
发明作者:Kimberly S. Sampson；Daniel J. Tomso
申请人:Athenix Corp.；
IPC主号:

专利说明:

[0001] [0001] This patent application claims the benefit of U.S. Provisional Application Serial No. 61 / 305,802, filed on February 18, 2010, the contents of which are hereby incorporated in full by reference. REFERENCE TO THE LISTING OF SEQUENCES ELECTRONICALLY SUBMITTED
[0002] [0002] The official copy of the sequence list is submitted electronically via EFS-Web as a sequence list in ASCII format with the file name "APA069US01SEQLIST.txt", created on January 10, 2011, with file size of 132 kilobytes and deposited at the same time as the descriptive memory. The list of strings contained in this document in ASCII format is part of the specification and is hereby incorporated in its entirety by reference. FIELD OF THE INVENTION
[0003] [0003] This invention refers to the field of molecular biology. New genes are provided that code for pesticidal proteins. These proteins and the nucleic acid sequences they encode for them are useful in the preparation of pesticidal formulations and in the production of transgenic pest resistant plants. BACKGROUND OF THE INVENTION
[0004] [0004] Bacillus thuringiensis is a gram-positive soil bacteria, spore-forming, characterized by its ability to form crystalline inclusions that are specifically toxic to certain orders and species of insects, but that are harmless to plants and other organisms that are not target. For this reason, compositions including strains of Bacillus thuringiensis or their insecticidal proteins can be used as environmentally acceptable insecticides to control insects that are agricultural pests or insect vectors for a variety of human or animal diseases.
[0005] [0005] The crystal proteins (Cry) (delta-endotoxins) of Bacillus thuringiensis have a potent insecticidal activity against predominantly larvae of Lepidoptera, Hemiptera, Diptera and Coleoptera. These proteins also showed 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. In Advanced Engineered Pestic, Marcel Dekker, Inc., New York, NI). These proteins were originally classified as CryI to CryV based primarily on their insecticidal activity. The main classes were Lepidoptera-specific (I), Lepidoptera- and Diptera-specific (II), Coleoptera-specific (III), Diptera-specific (IV), and nematode-specific (V) and (VI). These proteins were further classified into subfamilies; the most related proteins within each family were assigned division letters such as Cry1A, Cry1B, Cry1C, etc. The even closer proteins within each division were assigned names such as Cry1C1, Cry1C2, etc.
[0006] [0006] A new nomenclature has recently been described for Cry genes based on homology in the amino acid sequence instead of target insect specificity (Crickmore et al. (1998) Microbiol. Mol. Biol. Rev. 62: 807-813) . In the new classification, each toxin is assigned a unique name that incorporates a first classification (an Arabic number), a second classification (an uppercase letter), a third classification (a lowercase letter) and a fourth classification (another Arabic number). In the new classification, Roman numbers were replaced by Arabic numbers in the first classification. Proteins with less than 45% sequence identity have different first classifications, and the criteria for the second and third classification are 78% and 95%, respectively.
[0007] [0007] The crystal protein has no insecticidal activity until it has been ingested or solubilized in the insect's mesentery. The ingested pro-toxin is hydrolyzed by proteases in the insect's digestive tract forming a toxic molecule (Höfte and Whiteley (1989) Microbiol. Rev. 53: 242-255). This toxin binds to apical brush border receptors in the mesentery of the target larvae and introduces itself into the apical membrane creating ion channels or pores, resulting in the death of the larva.
[0008] [0008] Delta-endotoxins generally have five conserved sequence domains, and three conserved structural domains (see, for example, by Maagd et al. (2001) Trends Genetics 17: 193-199). The first conserved structural domain consists of seven alpha helices and is involved in membrane insertion and pore formation. Domain II consists of three beta sheets arranged in a Greek key configuration, and domain III consists of two antiparallel beta sheets in a supercoiled formation (de Maagd et al., 2001, supra). Domains II and III are involved in recognition and binding to the receptor, and are therefore considered determinants for the specificity of the toxin.
[0009] [0009] Due to the devastation that insects can confer and the improvement in yield through the control of insect pests, there is an ongoing need to discover new forms of pesticide toxins. SUMMARY OF THE INVENTION
[0010] [00010] Compositions and methods are provided for imparting pesticidal activity to bacteria, plants, plant cells, tissues and seeds. The compositions include sequences of nucleic acid molecules that code for pesticide and insecticide polypeptides, vectors comprising those nucleic acid molecules, and host cells comprising the vectors. The compositions also include the polypeptide sequences and antibodies against those polypeptides. Nucleotide sequences can be used in DNA constructs or expression cassettes for transformation and expression in organisms, including microorganisms and plants. Nucleotide or amino acid sequences can be synthetic sequences that have been designed for expression in an organism including, but not limited to, a microorganism or a plant. The compositions also comprise transformed bacteria, plants, plant cells, tissues and seeds.
[0011] [00011] In particular, nucleic acid molecules encoding a pesticidal protein are provided. In addition, amino acid sequences corresponding to the pesticidal protein are covered. In particular, the present invention provides an isolated nucleic acid molecule that comprises a nucleotide sequence that encodes the amino acid sequence shown in SEQ ID NO: 21-32, or a nucleotide sequence shown in SEQ ID NO: 1-5, as well as its variants and fragments. Also included are nucleotide sequences that are complementary to a nucleotide sequence of the invention, or that hybridize to a sequence of the invention. Synthetic nucleotide sequences encoding the polypeptides disclosed here are also shown in SEQ ID NO: 6-20.
[0012] [00012] Methods are provided for producing the polypeptides of the invention, and for using those polypeptides for the control or killing of pests of lepidopterans, hemiptera, coleopterans, nematodes or diptera. Also included are methods and kits for detecting nucleic acids and polypeptides of the invention in a sample.
[0013] [00013] The compositions and methods of the invention are useful for the production of organisms with greater resistance or tolerance to pests. These organisms and compositions comprising the organisms are desirable for agricultural purposes. The compositions of the invention are also useful for generating altered or improved proteins that have pesticidal activity, or for detecting the presence of pesticidal proteins or nucleic acids in products or organisms. DETAILED DESCRIPTION
[0014] [00014] The present invention relates to compositions and methods for the regulation of resistance or tolerance to pests in organisms, particularly plants or plant cells. By "resistance" is meant that the pest (for example, an insect) is killed by ingestion or other contact with the polypeptides of the invention. By "tolerance" is meant a limitation or a reduction in movement, feeding, reproduction, or other functions of the pest. The methods involve transforming organisms with a nucleotide sequence that codes for a pesticidal protein of the invention. In particular, the nucleotide sequences of the invention are useful for the preparation of plants and microorganisms that have pesticidal activity. Thus, transformed bacteria, plants, plant cells, plant tissues and seeds are provided. The compositions consist of nucleic acids and pesticidal proteins derived from Bacillus or other species. The sequences are used in the construction of expression vectors for the subsequent transformation into organisms of interest, as probes for the isolation of other homologous (or partially homologous) genes, and for generating altered pesticidal proteins by methods known in the art, such as domains or rearrangement of DNA ("DNA shuffling"), for example, with members of the Cry1, Cry2, and Cry9 endotoxin families. Proteins are used to control or kill populations of pests of lepidoptera, hemiptera, coleoptera, diptera and nematodes and for the production of compositions with pesticidal activity.
[0015] [00015] By "pesticidal toxin" or "pesticidal protein" is meant a toxin that has toxic activity against one or more pests, including, but not limited to, members of the orders Lepidoptera, Diptera and Coleoptera, or the phylum Nematoda, or a protein that has homology to that protein. Pesticidal proteins have been isolated from organisms including, for example, Bacillus sp., Clostridium bifermentans and Paenibacillus popilliae. Pesticidal proteins include amino acid sequences deduced from the complete nucleotide sequences disclosed herein, and amino acid sequences that are shorter than the complete sequences, either due to the use of an alternative downstream initiation site, or due to processing that produces a protein shorter with pesticidal activity. Processing can take place in the organism in which the protein is expressed, or in the pest after ingesting the protein.
[0016] [00016] Pesticidal proteins include delta-endotoxins. Delta-endotoxins include proteins identified as cry1 through cry43, cyt1 and cyt2, and Cyt type toxin. There are currently more than 250 known species of delta-endotoxins with a wide range of specificities and toxicities. For an exhaustive list see Crickmore et al. (1998) Microbiol. Mol. Biol. Rev. 62: 807-813, and for regular updates see Crickmore et al. (2003) "Bacillus thuringiensis toxin nomenclature," at www.biols.susx.ac.uk/Home/Neil_Crickmore/Bt/index.
[0017] [00017] Thus, new isolated nucleotide sequences are provided here that confer pesticidal activity. These isolated nucleotide sequences code for polypeptides with homology to known delta-endotoxins or binary toxins. Also provided are the amino acid sequences of the pesticidal proteins. The protein resulting from the translation of this gene allows cells to control or kill the pests that ingest it. Isolated Nucleic Acid Molecules, and Their Variants and Fragments
[0018] [00018] One aspect of the invention relates to isolated or recombinant nucleic acid molecules comprising nucleotide sequences that code for pesticidal proteins and polypeptides or their biologically active moieties, as well as enough nucleic acid molecules to use as hybridization probes to identify acids nucleic codes encoding proteins with regions of sequence homology. As used herein, the term "nucleic acid molecule" is understood to include DNA molecules (for example, recombinant DNA, cDNA or genomic DNA) and RNA molecules (for example, mRNA) and analogs of the generated DNA or RNA using nucleotide analogs. The nucleic acid molecule can be single-stranded or double-stranded, but is preferably double-stranded DNA.
[0019] [00019] An "isolated" or "recombinant" nucleic acid (or DNA) sequence is used here to refer to a nucleic acid (or DNA) sequence that is no longer in its natural environment, for example, in vitro or in a recombinant bacterial or plant host cell. In some embodiments, an isolated or recombinant nucleic acid is free of sequences (preferably protein-coding sequences) that naturally flank the nucleic acid (that is, sequences located at the 5 'and 3' ends of the nucleic acid) in the organism's genomic DNA from which the nucleic acid is derived. For the purposes of the invention, "isolated" when used to refer to nucleic acid molecules excludes isolated chromosomes. For example, in several embodiments, the nucleic acid molecule encoding the isolated delta-endotoxin may contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb, or 0.1 kb of nucleotide sequences that naturally flank the nucleic acid molecule in the genomic DNA of the cell from which the nucleic acid is derived. In various embodiments, the delta-endotoxin protein which is substantially free of cellular material includes protein preparations with less than 30%, 20%, 10%, or 5% (dry weight) of non-delta-endotoxin protein (also referred to herein) as "recombinant protein").
[0020] [00020] The nucleotide sequences encoding the proteins of the present invention include the sequence shown in SEQ ID NO: 1-20, and its variants, fragments and complements. By "complement" is meant a nucleotide sequence that is sufficiently complementary to a given nucleotide sequence in such a way that it can hybridize with the given nucleotide sequence to thereby form a stable duplex. The corresponding amino acid sequences for the pesticidal protein for which this nucleotide sequence encodes are shown in SEQ ID NO: 21-32.
[0021] [00021] Nucleic acid molecules that are fragments of these nucleotide sequences that code for pesticidal proteins are also encompassed by the present invention. By "fragment" is meant a portion of the nucleotide sequence that codes for a pesticidal protein. A fragment of a nucleotide sequence can code for a biologically active portion of a pesticidal protein, or it can be a fragment that can be used as a hybridization probe or a PCR primer using methods disclosed below. Nucleic acid molecules that are fragments of a nucleotide sequence encoding a pesticidal protein comprise at least about 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1350, 1400 contiguous nucleotides, or even the number of nucleotides present in a complete nucleotide sequence encoding a pesticidal protein disclosed herein, depending on the intended use. By "contiguous" nucleotides is meant nucleotide residues that are immediately adjacent to each other. Fragments of nucleotide sequences of the present invention will encode protein fragments that retain the biological activity of the pesticidal protein and thus retain pesticidal activity. By "retaining activity" is meant that the fragment will have at least about 30%, at least 50%, at least about 70%, 80%, 90%, 95% or more of the pesticidal activity of the pesticidal protein. In one embodiment, pesticidal activity is coleoptericidal activity. In another modality, pesticidal activity is lepidoptericidal activity. In another modality, pesticidal activity is nematocidal activity. In another modality, pesticidal activity is diptericidal activity. In another modality, pesticidal activity is hemiptericidal activity. Methods for measuring pesticidal activity are well known in the art. See, for example, Czapla and Lang (1990) J. Econ. Entomol. 83: 2480-2485; Andrews et al. (1988) Biochem. J. 252: 199-206; Marrone et al. (1985) J.of Economic Entomology 78: 290-293; and U.S. Patent No. 5,743,477, all of which are incorporated herein by reference in their entirety.
[0022] [00022] A fragment of a nucleotide sequence that codes for a pesticidal protein that codes for a biologically active portion of a protein of the invention will code for at least 15, 25, 30, 50, 75, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450 contiguous amino acids, or even the total number of amino acids present in a complete pesticidal protein of the invention. In some embodiments, the fragment is a fragment of proteolytic cleavage. For example, the proteolytic cleavage fragment may 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 SEQ ID NO: 21-32. In some embodiments, the fragments covered here result from the removal of the C-terminal crystallization domain, for example, by means of proteolysis or by inserting a terminating codon into the coding sequence. See, for example, the truncated amino acid sequences shown in SEQ ID NO: 22, 23, 25, 26, and 32. It will be understood that the truncation site can vary by 1, 2, 3, 4, 5, 6, 7 , 8, 9, 10, 11, 12, 13, 14, 15, or more amino acids on either side of the truncation site represented by the terminal SEQ ID NO: 22, 23, 25, 26, and 32 (compared to corresponding complete sequence).
[0023] [00023] It is preferred that the pesticidal proteins of the present invention are encoded by a nucleotide sequence sufficiently identical to the nucleotide sequence of SEQ ID NO: 1-20. By "sufficiently identical" is meant an amino acid or nucleotide sequence that has 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 of sequence identity compared to a reference sequence using a of the alignment programs described here using conventional parameters. One skilled in the art will recognize that these values can be appropriately adjusted to determine the corresponding identity of proteins encoded by two nucleotide sequences taking into account codon degeneration, amino acid similarity, positioning of the reading frame, and the like.
[0024] [00024] To determine the percent identity of two amino acid or nucleic acid sequences, the sequences are aligned for the purpose of optimal comparison. The percentage of identity between two strings is a function of the number of identical positions shared by the strings (that is, percentage of identity = number of identical positions / number of total positions (for example, overlapping positions) x 100). In one embodiment, the two strings are the same length. In another embodiment, the percent identity is calculated over the entire reference sequence (i.e., the sequence shown here as any of SEQ ID NO: 1-20). The percent identity between two sequences can be determined using techniques similar to those described below, whether or not they allow gaps. In calculating the identity percentage, exact matches are typically counted.
[0025] [00025] The determination of the percentage of identity between two sequences can be achieved using a mathematical algorithm. A non-limiting example of a mathematical algorithm used for the comparison of two sequences is the algorithm by Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 87: 2264, modified according to Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA. 90: 5873-5877. Such an algorithm is incorporated into the BLASTN and BLASTX programs by Altschul et al. (1990) J. Mol. Biol. 215: 403. BLAST nucleotide searches can be performed with the BLASTN program, punctuation = 100, word length = 12, to obtain nucleotide sequences homologous to the pesticide-type nucleic acid molecules of the invention. BLAST protein searches can be performed with the BLASTX program, punctuation = 50, word length = 3, to obtain amino acid sequences homologous to the pesticide protein molecules of the invention. To obtain gap alignments for comparison purposes, Gapped BLAST (in BLAST 2.0) can be used as described in Altschul et al. (1997) Nucleic Acids Res. 25: 3389. Alternatively, PSIBlast can be used to perform an iterated search that detects distant relationships between molecules. See Altschul et al. (1997) supra. When using the BLAST, Gapped BLAST, and PSI-Blast programs, the standard parameters of the respective programs (for example, BLASTX and BLASTN) can be used. Alignment can also be carried out manually by inspection.
[0026] [00026] Another non-limiting example of a mathematical algorithm used for sequence comparison is the ClustalW algorithm (Higgins et al. (1994) Nucleic Acids Res. 22: 4673-4680). ClustalW compares sequences and aligns the entire amino acid or DNA sequence, and can thus provide sequence conservation data for the entire amino acid sequence. The ClustalW algorithm is used in several commercially available DNA / amino acid analysis software packages, such as the ALIGNX module of the Vector NTI Program Suite (Invitrogen Corporation, Carlsbad, CA). After aligning the amino acid sequences with ClustalW, the percentage of amino acid identity can be determined. A non-limiting example of a computer program useful for ClustalW alignment analysis is GENEDOC®. GENEDOC® (Karl Nicholas) allows the evaluation of the similarity and identity of amino acids (or DNA) between multiple proteins. Another non-limiting example of a mathematical algorithm used for sequence comparison is the algorithm by Myers and Miller (1988) CABIOS 4: 11-17. Such an algorithm is incorporated into the ALIGN program (version 2.0), which is part of the GCG Wisconsin Genetics software package, version 10 (made available by Accelrys, Inc., 9685 Scranton Rd., San Diego, CA, USA). When using the ALIGN program to compare amino acid sequences, a PAM120 residue weight table, a gap length penalty of 12 and a gap penalty of 4 can be used.
[0027] [00027] Unless otherwise specified, GAP version 10 will be used, which incorporates the algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48 (3): 443-453 to determine sequence identity or similarity using the following parameters:% identity and% similarity for a nucleotide sequence using a GAP weight of 50 and a Length weight of 3 and the scoring matrix nwsgapdna.cmp; % identity or% similarity for an amino acid sequence using a GAP weight of 8 and a length weight of 2, and the BLOSUM62 scoring program. Equivalent programs can also be used. By "equivalent program" is meant any sequence comparison program that, for any two sequences in question, generates an alignment with matches of identical nucleotide residues and a percentage of identical sequence identity when compared to the corresponding alignment generated by the GAP version 10.
[0028] [00028] The invention also encompasses variant nucleic acid molecules. "Variants" of the pesticide protein coding nucleotide sequences include those sequences which code for the pesticidal proteins presented herein but which differ conservatively due to the degeneracy of the genetic code as well as those which are sufficiently identical as discussed above. Naturally occurring allelic variants can be identified with the use of well-known molecular biology techniques, such as polymerase chain reaction (PCR) and hybridization techniques as shown below. Variant nucleotide sequences also include synthetically derived nucleotide sequences that have been generated, for example, using directed mutagenesis, but which still code for the pesticidal proteins disclosed in the present invention as discussed below. Variant proteins covered by the present invention are biologically active, that is, they continue to possess the desired biological activity of the native protein, that is, retaining pesticidal activity. By "retaining activity" is meant 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. Methods for measuring pesticidal activity are well known in the art. See, for example, Czapla and Lang (1990) J. Econ. Entomol. 83: 2480-2485; Andrews et al. (1988) Biochem. J. 252: 199206; Marrone et al. (1985) J.of Economic Entomology 78: 290-293; and U.S. Patent No. 5,743,477, all of which are incorporated herein by reference in their entirety.
[0029] [00029] Anyone skilled in the art will appreciate that changes can be made by mutating the nucleotide sequences of the invention thereby leading to changes in the amino acid sequence of the encoded pesticidal proteins, without altering the biological activity of the proteins. Thus, isolated variant nucleic acid molecules can be created by introducing one or more nucleotide substitutions, additions, or deletions into the corresponding nucleotide sequence disclosed herein, such that one or more amino acid substitutions, additions or deletions are introduced into the encoded protein. Mutations can be introduced using conventional techniques, such as directed mutagenesis and PCR-mediated mutagenesis. Such variant nucleotide sequences are also encompassed by the present invention.
[0030] [00030] For example, conservative amino acid substitutions can be performed on one or more predicted non-essential amino acid residues. An "non-essential" amino acid is a residue that can be changed from the native sequence of a pesticidal protein without altering biological activity, while an "essential" amino acid residue is necessary for biological activity. A "conservative amino acid substitution" is one in which the amino acid residue is replaced by an amino acid residue with a similar side chain. Families of amino acid residues with similar side chains were defined in the technique. These families include amino acids with basic side chains (for example, lysine, arginine, histidine), acidic side chains (for example, aspartic acid, glutamic acid), uncharged polar side chains (for example, glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and side chains aromatic (eg tyrosine, phenylalanine, tryptophan, histidine).
[0031] [00031] Delta-endotoxins generally have five conserved sequence domains, and three conserved structural domains (see, for example, by Maagd et al. (2001) Trends Genetics 17: 193-199). The first conserved structural domain consists of seven alpha helices and is involved in membrane insertion and pore formation. Domain II consists of three beta sheets arranged in a Greek key configuration, and domain III consists of two antiparallel beta sheets in a supercoiled formation (de Maagd et al., 2001, supra). Domains II and III are involved in recognition and binding to the receptor, and are therefore considered determinants for the specificity of the toxin.
[0032] [00032] Amino acid substitutions can be made in non-conserved regions that retain function. In general, such substitutions would not be made for conserved amino acid residues, or for amino acid residues residing within a conserved motif, where such residues are essential for protein activity. Examples of residues that are conserved and that may be essential for protein activity include, for example, residues that are identical among all proteins contained in an alignment of similar or related toxins in relation to the sequences of the invention (for example, residues that are identical in a homologous protein alignment). Examples of residues that are conserved but that allow conservative amino acid substitutions and still retain activity include, for example, residues that have only conservative substitutions among all proteins contained in an alignment of similar or related toxins of the invention (for example, residues that they only contain conservative substitutions among all proteins contained in the homologous protein alignment). However, one skilled in the art will understand that functional variants may have minor conserved or non-conservative changes in conserved residues.
[0033] [00033] Alternatively, variant nucleotide sequences can be produced by randomly introducing mutations over all or part of the coding sequence, such as by saturation mutagenesis, and the resulting mutants can be screened for their ability to impart pesticidal activity to identify mutants that retain activity. After mutagenesis, the encoded protein can be expressed recombinantly, and the activity of the protein can be determined using conventional testing techniques.
[0034] [00034] Using methods such as PCR, hybridization, and the like, corresponding pesticidal sequences, such as sequences with substantial identity to the sequences of the invention, can be identified. See, for example, Sambrook and Russell (2001) Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NI) and Innis, et al. (1990) PCR Protocols: A Guide to Methods and Applications (Academic Press, NI).
[0035] [00035] In a hybridization method, all or part of the pesticide nucleotide sequence can be used to screen cDNA or genomic libraries. Methods for constructing such cDNA or genomic libraries are generally known in the art and are presented in Sambrook and Russel, 2001, supra. The so-called hybridization probes can be fragments of genomic DNA, fragments of cDNA, fragments of RNA, or other oligonucleotides, and can be labeled with a detectable group such as 32P, or any other detectable marker, such as other radioisotopes, a fluorescent compound , an enzyme or an enzyme cofactor. Hybridization probes can be prepared by labeling synthetic oligonucleotides based on the known nucleotide sequence that codes for the pesticidal protein disclosed herein. Degenerate primers designed on the basis of conserved nucleotides or amino acid residues in the nucleotide sequence or encoded amino acid sequence can additionally be used. The probe typically comprises 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 from the sequence. nucleotides encoding the pesticidal protein of the invention or a fragment or variant thereof. Methods for preparing probes for hybridization are generally known in the art and are presented in Sambrook and Russell, 2001, supra incorporated herein by reference.
[0036] [00036] For example, a complete sequence of pesticidal protein disclosed herein, or one or more portions thereof, can be used as a probe capable of specifically hybridizing to corresponding pesticidal protein-like sequences and messenger RNAs. To achieve specific hybridization under various conditions, such probes include sequences that are unique and are preferably at least 10 nucleotide length, or at least 20 nucleotide length. Such probes can be used to PCR amplify corresponding pesticide sequences from a chosen organism. This technique can be used to isolate additional coding sequences from a desired organism or as a diagnostic test to determine the presence of coding sequences in an organism. Hybridization techniques include screening by hybridization of plated DNA libraries (whether plaques or colonies; see, for example, Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York).
[0037] [00037] The hybridization of such sequences can be performed under stringent conditions. By "stringent conditions" or "stringent hybridization conditions" are meant conditions under which a probe will hybridize to its target sequence to a degree that can be detected to be higher than with other sequences (for example, at least 2 times in background signal). The stringent conditions are sequence dependent and will differ depending on the circumstances. By controlling the stringency of hybridization and / or washing conditions, target sequences can be identified that are 100% complementary to the probe (homologous probe). Alternatively, stringency conditions can be adjusted to allow for some divergence in the sequences so that low degrees of similarity are detected (heterologous probing). Generally, a probe is less than about 1000 nucleotides in length, preferably less than 500 nucleotides in length.
[0038] [00038] Typically, stringent conditions will be chosen in which the salt concentration is less than about 1.5 M of the Na ion, typically about 0.01 to 1.0 M of the Na ion (or other salts) at pH 7.0 to 8.3 and the temperature is at least 30 ° C for short probes (for example 10 to 50 nucleotides) and at least 60 ° C for longer probes (for example, greater than 50 nucleotides). Strict conditions can also be achieved with the addition of destabilizing agents such as formamide. Exemplary low stringency conditions include hybridization with a buffer solution of 30 to 35% formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulfate) at 37 ° C, and a wash in 1X to 2X SSC (20X SSC = 3.0 M NaCl / 0.3 M trisodium citrate) at 50 to 55 ° C. Exemplary conditions of moderate stringency include hybridization to 40 to 45% formamide, 1.0 M NaCl, 1% SDS at 37 ° C and a wash in 0.5X to 1X SSC at 55 to 60 ° C. Exemplary high stringency conditions include hybridization to 50% formamide, 1 M NaCl, 1% SDS at 37 ° C, and a wash in 0.1X SSC at 60 to 65 ° C. Optionally, the wash buffers can comprise about 0.1% to about 1% SDS. The duration of hybridization is generally less than about 24 hours, generally about 4 to about 12 hours.
[0039] [00039] Specificity is typically a function of post-hybridization washes, with ionic strength and temperature of the final wash solution being critical factors. For DNA-DNA hybrids, the Tm can be approximated to the Meinkoth and Wahl (1984) Anal equation. Biochem. 138: 267-284: Tm = 81.5 ° C + 16.6 (log M) + 0.41 (% GC) - 0.61 (% form) - 500 / L; where M is the molarity of monovalent cations,% GC is the percentage of guanosine and cytosine nucleotides in DNA,% form is the percentage of formamide in the hybridization solution, and L is the length of the hybrid in base pairs. Tm is the temperature (under defined ionic strength and pH) at which 50% of a complementary target sequence hybridizes to a perfectly matched probe. The Tm is reduced by about 1 ° C for each 1% discrepancy; thus, Tm, hybridization, and / or washing conditions can be adjusted to hybridize desired identity sequences. For example, if sequences with an identity ≥ 90% are sought, the Tm can be decreased by 10 ° C. Generally stringent conditions are selected at about 5 ° C below the thermal melting point (Tm) for the specific sequence and its complement to defined ionic strength and pH values. However, extremely stringent conditions may use hybridization and / or washing at 1, 2, 3, or 4 ° C lower than the thermal melting point (Tm); moderately stringent conditions you can use hybridization and / or washing at 6, 7, 8, 9, or 10 ° C below the thermal melting point (Tm); low stringency conditions may use hybridization and / or washing at 11, 12, 13, 14, 15, or 20 ° C below the thermal melting point (Tm). Using the equation, the hybridization and washing compositions, and the desired Tm, those skilled in the art will understand that variations in the stringency of the hybridization and / or washing solutions are inherently described. If the desired degree of divergence results in a Tm of less than 45 ° C (aqueous solution) or 32 ° C (formamide solution), it is preferred to increase the concentration of SSC so that a higher temperature can be used. An exhaustive guide to nucleic acid hybridization is found in Tijssen (1993) Laboratory Techniques in Biochemistry and Molecular Biology — Hybridization with Nucleic Acid Probes, Part I, Chapter 2 (Elsevier, New York); and Ausubel et al., eds. (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 Laboratory Press, Cold Spring Harbor, New York). Isolated Proteins and Their Variants and Fragments
[0040] [00040] Pesticidal proteins are also within the scope of the present invention. By "pesticidal protein" is meant a protein containing the amino acid sequence shown in SEQ ID NO: 21-32. Fragments, biologically active portions and variants thereof are also provided and can be used to carry out the methods of the present invention. An "isolated 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. An "isolated protein" or a "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.
[0041] [00041] "Fragments" or "biologically active moieties" includes polypeptide fragments comprising amino acid sequences sufficiently identical to the amino acid sequence shown in SEQ ID NO: 21-32 and which exhibit pesticidal activity. A biologically active portion of a pesticidal protein can be a polypeptide that is, for example, 10, 25, 50, 100, 150, 200, 250 or more amino acids in length. Such biologically active portions can be prepared using recombinant techniques and evaluated for their pesticidal activity. Methods for measuring pesticidal activity are well known in the art. See, for example, Czapla and Lang (1990) J. Econ. Entomol. 83: 2480-2485; Andrews et al. (1988) Biochem. J. 252: 199-206; Marrone et al. (1985) J.of Economic Entomology 78: 290-293; and U.S. Patent No. 5,743,477, all of which are incorporated herein by reference in their entirety. As used herein, a fragment comprises at least 8 contiguous amino acids of SEQ ID NO: 21-32. However, the invention encompasses other fragments, such as any fragment of the protein greater than about 10, 20, 30, 50, 100, 150, 200, 250, or 300 amino acids.
[0042] [00042] By "variants" are meant proteins or polypeptides containing an amino acid sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92% , 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of any of SEQ ID NO: 21-32. Variants also include polypeptides encoded by a nucleic acid molecule that hybridizes to the nucleic acid molecules of SEQ ID NO: 1-20, or its complement, under stringent conditions. Variants include polypeptides that differ in the amino acid sequence due to mutagenesis. Variant proteins covered by the present invention are biologically active, that is, they continue to possess the desired biological activity of the native protein, that is, retaining pesticidal activity. In some embodiments, the variants have improved activity over the native protein. Methods for measuring pesticidal activity are well known in the art. See, for example, Czapla and Lang (1990) J. Econ. Entomol. 83: 2480-2485; Andrews et al. (1988) Biochem. J. 252: 199-206; Marrone et al. (1985) J.of Economic Entomology 78: 290-293; and U.S. Patent No. 5,743,477, all of which are incorporated herein by reference in their entirety.
[0043] [00043] Bacterial genes, such as the axmi genes of this invention, very often have multiple methionine initiation codons near the beginning of the open reading frame. Often, initiation of translation into one or more of these initiation codons will lead to the generation of a functional protein. These initiation codons can include ATG codons. However, bacteria such as Bacillus sp. they also recognize the codon GTG as the initiation codon, and proteins that initiate translation in the GTG codons contain a methionine in the first amino acid. On rare occasions, translation into bacterial systems can start at a TTG codon, through which TTG codes for a methionine. In addition, it is often not determined a priori which of these codons are used naturally in the bacterium. Thus, it is understood that the use of one of the alternative codons of methionine can also lead to the generation of pesticidal proteins. See, for example, the alternative initiation site for the AXMI223z protein shown in SEQ ID NO: 28 and the alternative initiation site for the AXMI224z protein shown in SEQ ID NO: 30. These pesticidal proteins are encompassed in the present invention and can be used in the methods of the present invention. It should be understood that, when expressed in plants, it will be necessary to change the alternative initiation codon to ATG for an appropriate translation.
[0044] [00044] Antibodies against the polypeptides of the present invention, or against variants or fragments thereof, are also encompassed. Methods for producing antibodies are well known in the art (see, for example, Harlow and Lane (1988) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NI; U.S. Patent No. 4,196,265). Changed or Improved Variants
[0045] [00045] It is recognized that the DNA sequences of a pesticidal protein can be altered by several methods, and that these alterations can result in DNA sequences that code for different amino acid sequences than those encoded by a pesticidal protein of the present invention. This protein can be altered in several ways including amino acid substitutions, deletions, truncations, and insertions of one or more amino acids of SEQ ID NO: 21-32 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 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 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 generally known in the art. For example, sequences of variant amino acids from a pesticidal protein can be prepared by mutating DNA. This can also be achieved by one of several forms of mutagenesis and / or directed evolution. In some respects, the changes encoded in the amino acid sequence will not substantially affect the function of the protein. Such variants will possess insecticidal activity. However, it is understood that the ability of a pesticidal protein to impart pesticidal activity can be improved by using such techniques in the compositions of this invention. For example, one can express the pesticidal protein in host cells that show high rates of incorrect incorporation of bases during DNA replication, such as XL-1 Red (Stratagene, La Jolla, CA). After propagation in such strains, DNA can be isolated from (for example, by preparing plasmid DNA, or by PCR amplification and cloning of the resulting PCR fragment into a vector), culture of the pesticide protein mutations in a strain non-mutagenic, and identification of genes mutated with pesticidal activity, for example through the modality of a test for pesticidal activity. Usually, the protein is mixed and used in ingestion tests. See, for example, Marrone et al. (1985) J.of Economic Entomology 78: 290-293. Such tests may include putting the plants in contact 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 result in increased toxicity are found in Schnepf et al. (1998) Microbiol. Mol. Biol. Rev. 62: 775-806.
[0046] [00046] Alternatively, changes can be made to the protein sequence of various proteins at the amino- or carboxy-terminal end without substantially affecting activity. These may include insertions, deletions, or changes introduced by modern molecular methods, such as PCR, including PCR amplifications that alter or extend the protein coding sequence by virtue of the inclusion of amino acid coding sequences in the oligonucleotides used in the amplification by PCR. Alternatively, the protein sequences added may include complete protein-encoding sequences, such as those commonly used in the art to generate protein fusions. Such fusion proteins are often used to (1) increase the expression of a protein of interest, (2) introduce a binding domain, enzyme activity, or epitope to facilitate either the purification of the protein, or the detection of the protein, or other experimental uses known in the art, (3) direct the secretion or translation of a protein to a subcellular organelle, such as the periplasmic space of Gram-negative bacteria, or the endoplasmic reticulum of eukaryotic cells, which in the latter case often results in glycosylation of the protein.
[0047] [00047] Nucleotide and variant amino acid sequences of the present invention also encompass sequences derived from mutagenesis and recombination procedures such as "DNA array". With such a procedure, one or more regions that code for the pesticidal protein can be used to create a new pesticidal protein having the desired properties. In this way, libraries of recombinant polynucleotides are generated from a population of related sequence polynucleotides comprising sequence regions that have substantial sequence identity and can be homologously recombined in vitro or in vivo. For example, using this approach, sequence motifs encoding a domain of interest can be randomly distributed between a pesticidal gene of the invention and other known pesticidal genes to obtain a new gene encoding a protein with an improved property of interest, such as as an increased insecticidal activity. Strategies for this DNA rearrangement are known in the prior art. See, for example, Stemmer (1994) Proc. Natl. Acad. Sci. USA 91: 10747-10751; Stemmer (1994) Nature 370: 389-391; Crameri et al. (1997) Nature Biotech. 15: 436-438; Moore et al. (1997) J. Mol. Biol. 272: 336347; Zhanget al. (1997) Proc. Natl. Acad. Sci. USA94: 4504-4509; Crameri et al. (1998) Nature 391: 288-291; and U.S. Patent Nos. 5,605,793 and 5,837,458.
[0048] [00048] The exchange or random distribution of domains is another mechanism for the generation of altered delta-endotoxin proteins. Domains can be exchanged between pesticidal proteins, resulting in hybrid or chimeric toxins with an improved pesticidal activity or target spectrum. Methods for generating recombinant proteins and testing them for 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: 2092320930; Rang et al. (91999) Appl. Environ. Microbiol. 65: 2918-2925). Vector
[0049] [00049] A pesticidal sequence of the invention can be provided in an expression cassette for expression in a plant of interest. By "plant expression cassette" is meant a DNA construct that is able to result in the expression of a protein from an open reading frame in a plant cell. These typically contain a promoter and a coding sequence. Often2, such constructions will also contain an untranslated 3 'region. Such constructs may contain a "signal sequence" or a "leader sequence" to facilitate targeting during translation or post-translation of the peptide to certain intracellular structures such as the chloroplast (or other plastids), endoplasmic reticulum, or Golgi apparatus.
[0050] [00050] By "signal sequence" is meant a sequence that is known or suspected to result in transport during translation or post-translation of the peptide across the cell membrane. In eukaryotes, this typically involves secretion into the Golgi apparatus, with some resulting in glycosylation. Insecticidal bacterial toxins are often synthesized as pro-toxins, which are proteolytically activated in the intestine of the target pest (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 a sequence of the invention. By "leader sequence" is meant any sequence that, when translated, results in a sequence of amino acids sufficient to trigger the co-translation of the peptide chain to a subcellular organelle. Thus, this includes leader sequences directed to transport and / or glycosylation by passage to the endoplasmic reticulum, passage to vacuoles, plastids including chloroplasts, mitochondria, and the like.
[0051] [00051] By "plant transformation vector" is meant a DNA molecule that is necessary for the efficient transformation of a plant cell. Such a molecule can consist of one or more plant expression cassettes, and can be organized into more than one "vector" DNA molecule. For example, binary vectors are plant transformation vectors that use two non-contiguous DNA vectors that encode all cis-acting and trans-acting functions necessary for the transformation of plant cells (Hellens and Mullineaux (2000) Trends in Plant Science 5: 446-451). "Vector" refers to a nucleic acid construct designed for transfer between different host cells. "Expression vector" refers to a vector that has the ability to incorporate, integrate and express heterologous DNA sequences or fragments in a foreign cell. The cassette will include regulatory sequences 5 'and 3' operably linked to a sequence of the invention. By "operably linked" is meant a functional link 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, operably linked means that the nucleic acid sequences to be linked are contiguous and, when necessary to join two regions that code for the protein, contiguous and in the same open reading frame. The cassette can additionally contain at least one additional gene to be co-transformed in the organism. Alternatively, the additional gene (s) can be supplied on multiple expression cassettes.
[0052] [00052] "Promoter" refers to a nucleic acid sequence that functions to direct the transcription of a downstream coding sequence. The promoter in conjunction with other transcriptional and translation regulatory nucleic acid sequences (also called "control sequences") are necessary for the expression of a DNA sequence of interest.
[0053] [00053] Such an expression cassette is provided with a plurality of restriction sites for the insertion of the pesticide sequence to be under the transcriptional regulation of the regulatory regions.
[0054] [00054] The expression cassette will include in the 5'-3 'transcription direction, a transcription initiation and translation region (i.e., a promoter), a DNA sequence of the invention and a transcription and translation termination region. functional translation (ie termination region) in plants. The promoter can be native, or analogous, or foreign or heterologous, with respect to the host plant and / or the DNA sequence of the invention. In addition, the promoter can be the natural sequence or alternatively a synthetic sequence. When the promoter is "native" or "homologous" to the host plant, the promoter is intended to be obtained from the native plant into which the promoter is introduced. When the promoter is "foreign" or "heterologous" to the DNA sequence of the invention, it is intended that the promoter is not the native or naturally occurring promoter for the operably linked DNA sequence of the invention.
[0055] [00055] The termination region may be native to the transcription initiation region, may be native with the operably linked DNA sequence of interest, may be native with the host plant, or may be derived from another source (ie foreign or heterologous to the promoter, the DNA sequence of interest, the host plant, or any combination thereof). Convenient termination regions are available from the A. plasmidaciens Ti plasmid, such as the octopine synthase and nopaline synthase termination regions. See also Guerineau et al. (1991) Mol. Gen. Genet. 262: 141-144; Proudfoot (1991) Cell 64: 671-674; Sanfacon et al. (1991) Genes Dev. 5: 141-149; Mogen et al. (1990) Plant Cell 2: 12611272; Munroe et al. (1990) Gene 91: 151-158; Ballas et al. (1989) Nucleic Acids Res. 17: 7891-7903; and Joshi et al. (1987) Nucleic Acid Res. 15: 9627-9639.
[0056] [00056] When appropriate, the gene (s) can be optimized for increased expression in the transformed host cell. That is, the genes can be synthesized using codons preferred by the host cell for improved expression, or they can be synthesized using codons at a preferred codon utilization frequency of the host cell. Generally, the GC content of the gene will be increased. See, for example, Campbell and Gowri (1990) Plant Physiol. 92: 1-11 for a discussion of host use of preferred codons. Methods are available in the art for the synthesis of genes preferred by plants. See, for example, U.S. Patent Nos. 5,380,831 and 5,436,391 and Murray et al. (1989) Nucleic Acids Res. 17: 477-498, incorporated herein by reference.
[0057] [00057] In one embodiment, the pesticidal protein is directed to the chloroplast for expression. Thus, in cases where the pesticidal protein is not inserted directly into the chloroplast, the expression cassette will additionally contain a nucleic acid that codes for a transport peptide to direct the pesticidal protein to the chloroplasts. Such transporter peptides are known in the art. See, for example, 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. Commun. 196: 1414-1421; and Shah et al. (1986) Science 233: 478-481.
[0058] [00058] The pesticidal gene to be targeted to the chloroplast can be optimized for expression in the chloroplast to take into account differences in the use of codons between the nucleus of the plant and this organelle. In this way, the nucleic acids of interest can be synthesized using codons preferred by chloroplasts. See, for example, U.S. Patent No. 5,380,831, incorporated herein by reference. Plant Transformation
[0059] [00059] The methods of the invention involve introducing a nucleotide construct into a plant. By "introduction" is meant to present the nucleotide construct to the plant in such a way that the construct gains access to the interior of a plant cell. The methods of the invention do not require that a particular method for introducing a nucleotide construct be used, only that the nucleotide construct gains access to the interior of at least one plant cell. Methods for introducing nucleotide constructs into plants are known in the art including, but not limited to, stable transformation methods, transient transformation methods, and virus-mediated methods.
[0060] [00060] "Plant" means whole plants, plant organs (for example, leaves, stems, roots, etc.), seeds, plant cells, propagules, embryos and their progeny. Plant cells can be differentiated or undifferentiated (for example, calluses, cells in suspension culture, protoplasts, leaf cells, root cells, phloem cells, pollen).
[0061] [00061] "Transgenic plants" or "transformed plants" or "stably transformed" plants or cells or tissues refer to plants that have incorporated or integrated nucleic acid sequences or exogenous DNA fragments into the plant cell. These nucleic acid sequences include those that are exogenous, or not present in the untransformed plant cell, as well as those that may be endogenous, or present in the untransformed plant cell. "Heterologist" generally refers to nucleic acid sequences that are not endogenous to the cell or the part of the native genome in which they are present, and have been added to the cell by infection, transfection, microinjection, electroporation, microprojection, or the like.
[0062] [00062] The transgenic plants of the invention express one or more of the new toxin sequences disclosed herein. In several embodiments, the transgenic plant also comprises one or more additional genes for insect resistance (for example, Cry1, as members of the Cry1A, Cry1B, Cry1C, Cry1D, Cry1E, and Cry1F; Cry2, as members of the Cry2A; Cry9 family , as members of the Cry9A, Cry9B, Cry9C, Cry9D, Cry9E, and Cry9F families, etc.). One skilled in the art will understand that the transgenic plant can contain any gene that confers an agronomic trait of interest.
[0063] [00063] The transformation of plant cells can be carried out by one of several techniques known in the art. The pesticidal gene of the invention can be modified to promote or increase expression in plant cells. Typically, a construct that expresses such a protein would contain a promoter to drive transcription of the gene, as well as a 3 'untranslated region to allow termination of transcription and polyadenylation. The organization of such constructions is well known in the art. In some cases, it may be useful to modify the gene in such a way that the resulting peptide is secreted, or otherwise targeted within the plant cell. For example, the gene can be modified to contain a signal peptide to facilitate the transfer of the peptide to the endoplasmic reticulum. It may also be preferable to modify the plant expression cassette to contain an intron, such that processing of the intron mRNA is necessary for expression.
[0064] [00064] Typically, this "plant expression cassette" will be introduced into a "plant transformation vector". This plant transformation vector can be understood by one or more DNA vectors necessary to achieve the transformation of plants. For example, it is common practice in the art to use plant transformation vectors that are comprised of more than one contiguous segment of DNA. These vectors are often referred to in the art as "binary vectors". Binary vectors as well as vectors with helper plasmids are often used for Agrobacterium-mediated transformation, in which the size and complexity of the DNA segments required to achieve efficient transformation are significantly high, and it is advantageous to separate functions in separate DNA molecules. Binary vectors typically contain a plasmid vector that contains the cis-acting sequences necessary for the transfer of T-DNA (such as left and right margins), a selection marker that is modified to be capable of expression in a plant cell, and a "gene of interest" (a gene modified to be capable of expression in a plant cell for which the generation of transgenic plants is desired). Sequences necessary for bacterial replication are also present in this plasmid vector. The cis-acting sequences are distributed in such a way as to allow efficient transfer to and the expression of plant cells. For example, the genetic marker that can be selected and the pesticide gene are located between the left and right margins. Often, a second plasmid vector contains the transacting factors that mediate the transfer of T-DNA from Agrobacterium to plant cells. This plasmid often contains virulence functions (Vir genes) that allow infection of plant cells by Agrobacterium, and the transfer of DNA by cleavage into flanking sequences and the transfer of mediated DNA transfer, as is understood in the art (Hellens and Mullineaux ( 2000) Trends in Plant Science 5: 446-451). Various types of Agrobacterium strains (for example, LBA4404, GV3101, EHA101, EHA105, etc.) can be used for the transformation of plants. The second plasmid vector is not necessary to transform plants by other methods such as microprojection, microinjection, electroporation, polyethylene glycol, etc.
[0065] [00065] In general, plant transformation methods involve transferring heterologous DNA into target plant cells (eg, immature or mature embryos, suspension cultures, undifferentiated calluses, protoplasts, etc.), followed by application of an appropriate upper limit level of selection (depending on the selection marker gene) to recover transformed plant cells from a mass group of untransformed cells. Explants are typically transferred to a fresh supply of the same medium and grown on a routine basis. Subsequently, the transformed cells are differentiated into sprouts after being placed in regeneration medium supplemented with a maximum level of selection agent. The shoots are then transferred to a selective rooting medium for the recovery of rooted shoots or seedlings. The transgenic seedling then grows into a mature plant and produces fertile seeds (for example, Hiei et al. (1994) The Plant Journal 6: 271-282; Ishida et al. (1996) Nature Biotechnology 14: 745-750) . Explants are typically transferred to a fresh supply of the same medium and grown on a routine basis. A general description of the techniques and methods for generating transgenic plants can be found in Ayres and Park (1994) Critical Reviews in Plant Science 13: 219-239 and Bommineni and Jauhar (1997) Maydica 42: 107-120. The transformed material contains many cells; both transformed and untransformed cells are present in any subject callus or tissue piece or group of target cells. The ability to kill untransformed cells and allow proliferation of transformed cells results in transformed plant cultures. Often, the ability to remove untransformed cells is a limitation for the rapid recovery of cells from transformed plants and the generation of successful transgenic plants.
[0066] [00066] Transformation protocols as well as protocols for the introduction of nucleotide sequences in plants may vary depending on the type of plant or plant cell, that is, monocot or dicot, which is the target of the transformation. The generation of transgenic plants can be carried out by one of several methods, including, but not limited to, microinjection, electroporation, direct genetic transfer, the introduction of heterologous DNA by Agrobacterium in plant cells (transformation mediated by Agrobacterium), bombardment of plant cells with heterologous foreign DNA adhered to particles, ballistic particle acceleration, aerosol beam transformation (US Published Application No. 20010026941; US Patent No. 4,945,050; International Publication No. WO 91/00915; US Published Application No. 2002015066), transformation with Lec1, and several other methods of direct mediation without particles to transfer DNA.
[0067] [00067] Methods for the transformation of chloroplasts are well known in the art. See, for example, Svab et al. (1990) Proc. Natl. Acad. Sci. USA 87: 8526-8530; Svab and Maliga (1993) Proc. Natl. Acad. Sci. USA 90: 913-917; Svab and Maliga (1993) EMBO J. 12: 601-606. The method is based on the delivery of DNA containing a selection marker with a particle gun and directing the DNA to the plastidic genome by homologous recombination. In addition, plastid transformation can be performed by transactivating an inactive plastid transgene by preferential expression in a tissue of a plastid-directed RNA polymerase and encoded by nuclear genes. Such a system was presented in McBride et al. (1994) Proc. Natl. Acad. Sci. USA. 91: 7301-7305.
[0068] [00068] After the integration of heterologous foreign DNA into plant cells, it is then necessary to apply an appropriate upper limit level of selection in the medium to kill the untransformed cells and to separate and proliferate the putatively transformed cells that survive this selection treatment by transferring regularly to a cool environment. Through continuous passage and testing with the appropriate selection, cells that are transformed with the plasmid vector are identified and proliferated. Molecular and biochemical methods can then be used to confirm the presence of the heterologous gene of interest integrated in the genome of the transgenic plant.
[0069] [00069] The cells that have been transformed can be grown to produce plants according to conventional methods. See, for example, McCormick et al. (1986) Plant Cell Reports 5: 81-84. These plants can then be grown, and or pollinated with the same transformed strain or different strains, and the resulting hybrid with expression constituting the desired phenotypic characteristic can be identified. Two or more generations can be grown to ensure that the expression of the desired phenotypic characteristic is maintained with stability and inherited and then the seeds collected to ensure that the expression of the desired phenotypic characteristic has been achieved. Thus, the present invention provides transformed seeds (also referred to as "transgenic seeds") with a nucleotide construct of the invention, for example, an expression cassette of the invention, stably incorporated into its genome. Plant Transformation Assessment
[0070] [00070] After the introduction of foreign heterologous DNA into plant cells, the transformation or integration of the heterologous gene into the plant genome is confirmed by several methods such as the analysis of nucleic acids, proteins and metabolites associated with the integrated gene.
[0071] [00071] PCR analysis is a fast method for screening transformed cells, tissues, or shoots for the presence of an incorporated gene at an early stage before transplanting into soil (Sambrook and Russell (2001) Molecular Cloning: A Laboratory Manual Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NI). The PCR reaction is performed using specific oligonucleotide primers for the gene of interest or a region of the Agrobacterium vector, etc.
[0072] [00072] Plant transformation can be confirmed by Southern blot analysis of genomic DNA (Sambrook and Russell, 2001, supra). In general, the total DNA is extracted from the transformant, digested with appropriate restriction enzymes, resolved on an agarose gel and transferred to a nitrocellulose or nylon membrane. The membrane or "blot" is then probed with, for example, a target DNA fragment radiolabelled with 32P to confirm the integration of the gene introduced into the plant's genome according to conventional techniques (Sambrook and Russell, 2001, supra).
[0073] [00073] In Northern blot analysis, RNA is isolated from specific tissues of a transformant, fractionated on an agarose and formaldehyde gel, and transferred to a nylon filter according to conventional procedures that are routinely used in the art ( Sambrook and Russell, 2001, supra). The expression of RNA encoded by the pesticidal gene is then tested by hybridizing the filter to a radioactive probe derived from a pesticidal gene, by methods known in the art (Sambrook and Russell, 2001, supra).
[0074] [00074] Western blotting, biochemical and similar assays of transgenic plants can be performed to confirm the presence of the protein encoded by the pesticide gene by conventional procedures (Sambrook and Russell, 2001, supra) using antibodies that bind to one or more epitopes present in the pesticidal protein. Pesticidal Activity in Plants
[0075] [00075] In another aspect of the invention, transgenic plants can be generated that express a pesticidal protein that has pesticidal activity. The methods described above for generating transgenic plants can be used as an example, but the way in which transgenic plants are generated is not critical to this invention. Methods known or described in the art such as Agrobacterium-mediated transformation, biological transformation, and non-particle-mediated methods can be used at the discretion of the experimenter. Plants that express a pesticidal protein can be isolated by conventional methods described in the art, for example, by transformation of corns, selection of transformed corns, and regeneration of fertile plants from such transgenic corns. In such a process, any gene can be used as a selection marker as long as its expression in plant cells confers the ability to identify or select transformed cells.
[0076] [00076] Several markers have been developed for use with plant cells such as resistance to chloramphenicol, aminoglycoside G418, hygromycin, or the like. Other genes that code for a product involved in the metabolism of chloroplasts can also be used as selection markers. For example, genes that provide resistance to plant herbicides such as glyphosate, bromoxynil, or imidazolinone can be particularly useful. Such genes have been reported (Stalker et al. (1985) J. Biol. Chem. 263: 6310-6314 (bromoxynil resistance nitrilase gene); and Sathasivan et al. (1990) Nucl. Acids Res. 18: 2188 ( AHAS imidazolinone resistance gene.) Additionally, the genes disclosed herein are useful as markers for evaluating the transformation of bacterial or plant cells. Methods for detecting the presence of a transgene in a plant, plant organ (for example, leaves, stems, roots, etc.), seed, plant cell, propagule, embryo or progeny thereof are well known in the art In one embodiment, the presence of the transgene is detected by testing its pesticidal activity.
[0077] [00077] Fertile plants that express a delta-endotoxin can be tested for pesticidal activity, and plants that have an optimal activity selected for further improvement. Methods for testing pest activity are available in the art. Usually, the protein is mixed and used in ingestion tests. See, for example, Marrone et al. (1985) J.of Economic Entomology 78: 290-293.
[0078] [00078] The present invention can be used for the transformation of any species of plant, including, but not limited to, monocots and dicots. Examples of plants of interest include, but are not limited to, corn, sorghum, wheat, sunflower, tomatoes, crucifers, peppers, potatoes, cotton, rice, soybeans, beets, sugar cane, tobacco, barley and rapeseed, Brassica sp. , alfalfa, rye, millet, saffron, peanut, sweet potato, cassava, coffee, coconut, pineapple, citrus, cocoa, tea, banana, avocado, fig, guava, mango, olive, papaya, cashew, macadamia, almond, oat , legumes and vegetables, ornamental plants, and conifers.
[0079] [00079] Vegetables and greens include, but are not limited to, tomatoes, lettuce, green beans, lima beans, peas and members of the genus Curcumistais like cucumber, cantaloupe melon and melon bark. Ornamental plants include, but are not limited to, azaleas, hydrangeas, hibiscus, roses, tulips, daffodils, petunias, carnations, poinsettias, and chrysanthemums. Preferably, plants of the present invention are cultivated plants (e.g., corn, sorghum, wheat, sunflower, tomato, cruciferous, peppers, potato, cotton, rice, soy, beet, sugar cane, tobacco, barley, rapeseed, etc. .). Use in Pesticide Control
[0080] [00080] General methods for using strains comprising a nucleotide sequence of the present invention, or a variant thereof, in the pesticide control or in the modification of other organisms as pesticide agents are known in the art. See, for example, U.S. Patent No. 5,039,523 and EP 0480762A2.
[0081] [00081] Bacillus strains containing a nucleotide sequence of the present invention, or a variant thereof, or microorganisms that have been genetically altered to contain a pesticidal gene and protein can be used to protect crops and agricultural products from pests. In one aspect of the invention, whole cells, that is to say non-lysed, of a toxin-producing organism (pesticide) are treated with reagents to prolong the activity of the toxin produced in the cell when the cell is applied to the insect's environment (s) ( saved.
[0082] [00082] Alternatively, the pesticide is produced by introducing a pesticidal gene into a cell host. The expression of the pesticide gene results, directly or indirectly, in an intracellular production and maintenance of the pesticide. In one aspect of this invention, these cells are then treated under conditions that prolong the activity of the toxin produced in the cell when the cell is applied to the environment of the target insect (s). The resulting product retains the toxicity of the toxin. These naturally encapsulated pesticides can then be formulated according to conventional techniques for application to the environment that houses the target pest, for example, soil, water, and plant foliage. See, for example, EPA 0192319, and the references cited there. Alternatively, cells expressing a gene of this invention can be formulated to, for example, allow application of the resulting material as a pesticide.
[0083] [00083] The active ingredients of the present invention are normally applied in the form of compositions and can be applied to the area of cultivation or to the plant to be treated, simultaneously or in sequence, with other compounds. These compounds can be fertilizers, herbicides, cryoprotectants, surfactants, detergents, pesticidal soaps, dormant oils, polymers and / or biodegradable controlled release vehicle formulations that allow the prolonged dosing of the target area after a single application of the formulation. They can also be herbicides, chemical insecticides, virucides, microbicides, amoebicides, pesticides, fungicides, bacteriocides, nematocides, selective molluscicides or mixtures of several of these preparations, if desired, together with more acceptable vehicles in agriculture, surfactants or adjuvants that promote application normally employed in the formulation technique. Suitable vehicles and adjuvants can be solid or liquid and correspond to substances normally used in formulation technology, for example, natural or regenerated mineral substances, solvents, dispersants, wetting agents, adhesion-promoting agents, binders or fertilizers. Likewise, formulations can be prepared in edible "baits" or processed in pest "traps" to allow feeding or ingestion by a target pest of the pesticidal formulation.
[0084] [00084] Methods for applying an active ingredient of the present invention or an agrochemical composition of the present invention containing at least one of the pesticidal proteins produced by the bacterial strains of the present invention include application to leaves, seed coating and application to soil . The number of applications and the rate of application depend on the intensity of infestation by the corresponding pest.
[0085] [00085] The composition can be formulated as a powder, dust, grains, granules, spray, emulsion, colloid, solution or the like, and can be prepared by conventional methods such as dissection, lyophilization, homogenization, extraction, filtration, centrifugation , sedimentation, or concentration of a cell culture comprising the polypeptide. In all of these compositions that contain at least one of these pesticidal polypeptides, the polypeptide can be present in a concentration of about 1% to about 99% by weight.
[0086] [00086] Pests of lepidopterans, hemiptera, dipterans, or coleopterans can be killed or their numbers reduced in a given area by the methods of the invention, or they can be applied prophylactically to an environmental area to prevent infestation by a susceptible pest. Preferably, the pest ingests, or comes in contact with, an effective pesticidal amount of the polypeptide. By "effective pesticide amount" is meant an amount of pesticide that is capable of causing death to at least one pest, or significantly reducing the growth of the pest, its diet, or its normal physiological development. This amount will vary depending on factors such as, for example, the specific target pests to be controlled, the specific environment, the location, the plant, the crop, or the agricultural site to be treated, the environmental conditions, and the method, rate, concentration, stability and amount of application of the polypeptide composition with effective pesticidal activity. The formulations can also vary in relation to climatic conditions, environmental considerations and / or frequency of application and / or severity of infestation by the pest.
[0087] [00087] The described pesticidal compositions can be prepared through the formulation or bacterial cells, suspension of crystals and / or spores, or the protein component isolated with the desired agricultural acceptable vehicle. The compositions can be formulated prior to administration by an appropriate medium such as lyophilized, dried, or in a suitable aqueous vehicle, medium or diluent, such as saline or other buffer. The formulated compositions can be in the form of a powder or granular material, or a suspension in an oil (vegetable or mineral), or water or oil / water emulsions, or in the form of a wettable powder, or in combination with any other vehicle material suitable for agricultural application. Suitable vehicles for agricultural application can be solid or liquid and are well known in the art. The term "acceptable vehicle for agricultural application" covers all adjuvants, inert components, dispersants, surfactants, adhesion-promoting agents, binders, etc. which are normally used in pesticide formulation technology; these are well known to those skilled in pesticide formulation. The formulations can be mixed with one or more solid or liquid adjuvants and prepared by various means, for example, by mixing, combining and / or grinding the pesticidal composition with suitable adjuvants using conventional formulation techniques. Suitable formulations and methods of application are described in U.S. Patent No. 6,468,523, incorporated herein by reference.
[0088] [00088] "Plague" includes, but is not limited to, insects, fungi, bacteria, nematodes, mites, ticks, and the like. Insect pests include insects selected from the orders Coleoptera, Diptera, Hymenoptera, Lepidoptera, Mallophaga, Homoptera, Hemiptera, Orthroptera, Thysanoptera, Dermaptera, Isoptera, Anoplura, Siphonaptera, Trichoptera, etc., particularly Coleoptera, Lepidoptera and Diptera.
[0089] [00089] The order Coleoptera includes the suborders Adephaga and Polyphaga. The suborder Adephaga includes the superfamilies Caraboidea and Gyrinoidea, while the suborder Polyphaga includes the superfamilies Hydrophiloidea, Staphylinoidea, Cantharoidea, Cleroidea, Elateroidea, Dascilloidea, Dryopoidea, Byrrhoidea, Cucujoidea, Meloidea, Meandeaeaeideaeaideaeaideaeaideaeaideaeaideaeaideaeaideaeaeeaeaeeaeaeeaeaeeaeaeeaeaceaeeaeaceae; . The Caraboidea superfamily includes the families Cicindelidae, Carabidae and Dytiscidae. The Gyrinoidea superfamily includes the family Gyrinidae. The superfamily Hydrophiloidea includes the family Hydrophilidae. The Staphylinoidea superfamily includes the families Silphidae and Staphylinidae. The Cantharoidea superfamily includes the families Cantharidae and Lampyridae. The superfamily Cleroidea includes the families Cleridae and Dermestidae. The Elateroidea superfamily includes the families Elateridae and Buprestidae. The superfamily Cucujoidea includes the family Coccinellidae. The superfamily Meloidea includes the family Meloidae. The superfamily Tenebrionoidea includes the family Tenebrionidae. The Scarabaeoidea superfamily includes the families Passalidae and Scarabaeidae. The superfamily Cerambycoidea includes the family Cerambycidae. The Chrysomeloidea superfamily includes the Chrysomelidae family. The superfamily Curculionoidea includes the families Curculionidae and Scolytidae.
[0090] [00090] The order Diptera includes the suborders Nematocera, Brachycera, and Cyclorrhapha. The suborder Nematocera includes the families Tipulidae, Psychodidae, Culicidae, Ceratopogonidae, Chironomidae, Simuliidae, Bibionidae and Cecidomyiidae. The suborder Brachycera includes the families Stratiomyidae, Tabanidae, Therevidae, Asilidae, Mydidae, Bombyliidae and Dolichopodidae. The subclause Cyclorrhapha includes the Aschiza and Aschiza divisions. The Aschiza division includes the families Phoridae, Syrphidae and Conopidae. The Aschiza division includes the sections Acalyptratae and Calyptratae. The Acalyptratae section includes the families Otitidae, Tephritidae, Agromyzidae and Drosophilidae. The Calyptratae section includes the families Hippoboscidae, Oestridae, Tachinidae, Anthomyiidae, Muscidae, Calliphoridae and Sarcophagidae.
[0091] [00091] The order Lepidoptera includes the families Papilionidae, Pieridae, Lycaenidae, Nymphalidae, Danaidae, Satyridae, Hesperiidae, Sphingidae, Saturniidae, Geometridae, Arctiidae, Noctuidae, Lymantriidae, Sesiidae and Tineidae.
[0092] [00092] Insect pests of the invention for the main crops include: Maize: Ostrinia nubilalis, European corn borer; Agrotis ipsilon, black agrotis; Helicoverpa zea, corn lipid; Spodoptera frugiperda, cartridge caterpillar; Diatraea grandiosella, southwestern corn borer; Elasmopalpus lignosellus, corn cane borer; Diatraea saccharalis, sugar cane borer; Diabrotica virgifera, western pinworm larva; Diabrotica longicornis barberi, northern pinworm larva; Diabrotica undecimpunctata howardi, southern pinworm larva; Melanotus spp., Elaterid larva; Cyclocephala borealis, northern beetle (white larva); Cyclocephala immaculata, southern beetle (white larva); Popillia japonica, Japanese beetle; Chaetocnema pulicaria, corn beetle; Sphenophorus maidis, corn weevil; Rhopalosiphum maidis, corn leaf aphid; Anuraphis maidiradicis, corn root aphid; Blissus leucopterus leucopterus, stink bug; Melanoplus femurrubrum, red-legged locust; Melanoplus sanguinipes, migratory locust; Hylemya platura, corn seed fly larvae; Agromyza parvicornis, a corn fly; Anaphothrips obscrurus, thrips of herbs; Solenopsis milesta, ant thief; Tetranychus urticae, striped mite; Sorghum: Chilo partellus, sorghum borer; Spodoptera frugiperda, cartridge caterpillar; Helicoverpa zea, corn lipid; Elasmopalpus lignosellus, corn cane borer; Feltia subterranea, thread; Phyllophaga crinita, chorus; Eleodes, Conoderus and Aeolus spp., Elaterid larvae; Oulema melanopus, cereal leaf beetle; Chaetocnema pulicaria, corn beetle; Sphenophorus maidis, corn weevil; Rhopalosiphum maidis; corn leaf aphid; Sipha flava, yellow aphid; Blissus leucopterus leucopterus, stink bug; Contarinia sorghicola, sorghum fly; Tetranychus cinnabarinus, red mite; Tetranychus urticae, striped mite; Wheat: Pseudaletia unipunctata, military caterpillar; Spodoptera frugiperda, cartridge caterpillar; Elasmopalpus lignosellus, lesser cornstalk borer; Agrotis orthogonia, western cutworm; Elasmopalpus lignosellus, corn cane borer; Oulema melanopus, cereal leaf beetle; Hypera punctata, stalk leaf weevil; Diabrotica undecimpunctata howardi, southern pinworm larva; Russian wheat aphid; Schizaphis graminum, green aphid; Macrosiphum avenae, ear aphid; Melanoplus femurrubrum, red-legged locust; Melanoplus differentialis, differential grasshopper; Melanoplus sanguinipes, migratory locust; Mayetiola destructor, Hessian fly; Sitodiplosis mosellana, wheat mosquito; American Meromyza, larva of the wheat stem; Hylemya coarctata, wheat bulb fly; Frankliniella fusca, tobacco thrips; Cephus cinctus, hornet hornet hornet; Aceria tulipae, Mite-do-rattle; Sunflower: Suleima helianthana, sunflower shoot moth; Homoeosoma electellum, sunflower moth; Zygogramma exclamationis, sunflower beetle; Bothyrus gibbosus, carrot beetle; Neolasioptera murtfeldtiana, sunflower seed mosquito; Cotton: Heliothis virescens, apple caterpillar; Helicoverpa zea, earworm; Spodoptera exigua, cartridge caterpillar; Pectinophora gossypiella, pink cotton caterpillar; Anthonomus grandis, cotton weevil; Aphis gossypii, cotton aphid; Pseudatomoscelis seriatus, cotton flea; Trialeurodes abutilonea, whitefly; Lygus lineolaris, stink bug; Melanoplus femurrubrum, red-legged locust; Melanoplus differentialis, differential grasshopper; Thrips tabaci, onion thrips; Franklinkiella fusca, tobacco thrips; Tetranychus cinnabarinus, red mite; Tetranychus urticae, striped mite; Rice: Diatraea saccharalis, sugar cane borer; Spodoptera frugiperda, military caterpillar; Helicoverpa zea, corn lipid; Colaspis brunnea, grape beetle; Lissorhoptrus oryzophilus, North American root bug; Sitophilus oryzae, rice weevil; Nephotettix nigropictus, rice leafhopper; Blissus leucopterus leucopterus, stink bug; Acrosternum hilare, green stink bug; Soy: Pseudoplusia includens, false medium caterpillar; Anticarsia gemmatalis, soybean caterpillar; Plathypena scabra, green clover larva; Ostrinia nubilalis, European corn borer; Agrotis ipsilon, threaded caterpillar; Spodoptera exigua, cartridge caterpillar; Heliothis virescens, apple caterpillar; Helicoverpa zea, earworm; Epilachna varivestis, Mexican bean beetle; Myzus persicae, light green aphid; Empoasca fabae, potato-sucking insect; Acrosternum hilare, green stink bug; Melanoplus femurrubrum, red-legged locust; Melanoplus differentialis, differential grasshopper; Hylemya platura, corn seed fly larvae; Sericothrips variabilis, soybean thrips; Thrips tabaci, onion thrips; Tetranychus turkestani, strawberry mite; Tetranychus urticae, striped mite; Barley: Ostrinia nubilalis, European corn borer; Agrotis ipsilon, black agrotis; Schizaphis graminum, green aphid; Blissus leucopterus leucopterus, stink bug; Acrosternum hilare, green stink bug; Euschistus servus, nearctic stink bug; Delia platura, corn seed fly larvae; Mayetiola destructor, Hessian fly; Petrobia latens, brown wheat moth; Rapeseed: Brevicoryne brassicae, cabbage aphid; Phyllotreta cruciferae, beetle; Mamestra configurata, Bertha's caterpillar; Plutella xylostella, cruciferous moth; Delia ssp., Larvae of the roots.
[0093] [00093] Nematodes include parasitic nematodes such as root nodule, cyst and lesion nematodes, including Heterodera spp., Meloidogyne spp. and Globodera spp .; particularly members of cyst nematodes, including, but not limited to, Heterodera glycines (soy cyst nematode); Heterodera schachtii (beet cyst nematode); Heterodera avenae (cereal cyst nematode); and Globodera rostochiensis and Globodera pailida (potato cyst nematode). Lesion nematodes include Pratylenchus spp. Methods to increase plant yield
[0094] [00094] Methods are provided to increase the yield of the plant. The methods comprise providing a plant or plant cell that expresses a polynucleotide that codes for the pesticide polypeptide sequence disclosed herein, and growing the plant or its seed in a field infested by a pest against which said polypeptide has pesticidal activity. In some modalities, the polypeptide has pesticidal activity against a pest of lepidopterans, coleopterans, diptera, hemiptera or nematodes, and the said field is infested by a pest of lepidopterans, coleopterans, diptera, hemiptera or nematodes. As defined herein, the "yield" of the plant refers to the quality and / or quantity of biomass produced by the plant. "Biomass" means any product of the plant measured. An increase in biomass production is any improvement in the measured product yield of the plant. The increase in plant yield has several commercial applications. For example, increasing the plant's leaf biomass can increase the yield of leafy vegetables for human or animal consumption. In addition, the increase in leaf biomass can be used to increase the production of pharmaceutical or industrial products derived from the plant. An increase in income may comprise any statistically significant increase including, but not limited to, at least a 1% increase, at least a 3% increase, at least a 5% increase, at least a 10% increase, at least an increase of 20%, at least a 30% increase, at least 50%, at least 70%, at least 100% or higher in yield compared to a plant that does not express the pesticide sequence.
[0095] [00095] Plants can also be treated with one or more chemical compositions, including one or more herbicides, insecticides, or fungicides. Examples of chemical compositions include: Herbicides for Fruits / Vegetables and Vegetables: Atrazine, Bromacil, Diuron, Glyphosate, Linuron, Metribuzin, Simazine, Trifluralin, Fluazifope, Glufosinate, Halosulfuron Gowan, Paraquato, Propizamida, Setoxidame, Halifuramide, Halifuram; Insecticides for Fruits / Vegetables and Vegetables: Aldicarb, Bacillus thuringiensis, Carbaryl, Carbofuran, Chlorpyrifos, Cypermethrin, Deltamethrin, Diazinon, Malathion, Abamectin, Cyfluthrin / beta-cyfluthrin, Esfenvalerato, Lambda-ciflazine; , Thiacloprid, Dinotefuron, Fluacripirim, Tolfenpirad, Clothianidin, Spirocyclofen, Gamma-cyhalothrin, Spiromesifen, Spinosad, Rinaxipir, Ciazipir, Spinoteram, Triflumuron, Spirotetrama, Imidaclidide, Citronidone, Flodendloride, Flubendiamide, Flubendiamide , Spinach, Tiodicarb, Flonicamide, Metiocarb, Emamectin Benzoate, Indoxacarb, Foztiazate, Fenamiphos, Cadusafos, Pyriproxifene, Fenbutatin Oxide, Hextiazox, Metomil, 4 - [[(6-Chloropyridin-3-yl) -3-yl) difluorethyl) amino] furan-2 (5H) -one; Fungicides for Fruits / Vegetables and Vegetables:
[0096] [00096] Carbendazim, Chlorothalonil, EBDCs, Sulfur, Thiophanate-methyl, Azoxystrobin, Cymoxanil, Fluazinam, Fosetil, Iprodione, Crosoxime-methyl, Metalaxyl / mefenoxam, Trifloxystrobin, Etaboxamide, Iprovalamide, Phenoxyamide, Phyoxyamide, Trifoxamamide, Phenoxyamide, Phyoxyamide, Phyoxyamide, Phyoxyamide , Zoxamide, Picoxystrobin, Pyraclostrobin, Cyflufenamide, Boscalide; Herbicides for Cereals: Isoproturon, Bromoxynil, Ioxinil, Fenoxis, Chlorosulfuron, Clodinafope, Diclofope, Diflufenicano, Fenoxaprope, Florasulame, Fluroxipir, Metsulfuron, Triasulfuron, Flucarbazone, Iodosulfuron, Phosphururon, Phosphorphine, Phosphorphine, Phosphorphine, Phosphorphine, Phosphorphine Flupirsulfuron, Sulfosulfuron, Pirasulfotole, Piroxsulam, Flufenacet, Tralcoxidime, Piroxasulfone; Cereal Fungicides: Carbendazim, Chlorothalonil, Azoxystrobin, Ciproconazole, Ciprodinil, Fenpropimorph, Epoxiconazole, Cresoxime-methyl, Quinoxyfen, Tebuconazole, Trifloxystrobin, Simeconazole, Picoxystrobin, Pyroxoxobobin, Pyroxoxobobin, Pyroxoxobobin, Pyroxoxobobin, Pyroxoxobobin, Pyroxoxobobin, Pyroxoxobobin, Pyroxoxobobin, Pyroxoxobobin, Pyroxoxobobin, Pyroxoxobobin; Insecticides for Cereals: Dimetoate, Lambda-cyhaltrin, Deltamethrin, alpha-Cypermethrin, β-cyfluthrin, Bifenthrin, Imidacloprid, Clothianidin, Thiamethoxam, Tiaclopride, Acetamipride, Dinetofuron, Chlorphyrifides, Metamidimide, Metamidimide, Pyridine; Corn Herbicides: Atrazine, Alachlor, Bromoxynil, Acetochlor, Dicamba, Clopyralide, (S-) Dimetenamide, Glufosinate, Gliphosate, Isoxaflutole, (S-) Metolachlor, Mesotrione, Nicosulfuron, Primisulfuron, Tromsone, Saflufenacil, Tiencarbazone, Flufenacete, Piroxasulfone; Corn Insecticides: Carbofuran, Chlorpirifos, Bifentrina, Fipronil, Imidaclopride, Lambda-Ci-halotrina, Teflutrina, Terbufos, Thiametoxame, Clotianidina, Spiromasifene, Flubendiamide, Triflumurão, Rinaxipina, Citrine, Tetra, Triflumorão, Teflutrina, Tebupirimfos, Etiprole, Ciazipir, Tiaclopride, Acetamipride, Dinetofurano, Avermectina, Metiocarb, Espirodiclofeno, Espirotetramate; Corn Fungicides: Fenitropane, Tirame, Protioconazole, Tebuconazole, Trifloxystrobin; Herbicides for Rice: Butachloro, Propanil, Azimsulfurão, Bensulfurão, Cialofope, Daimurão, Fentrazamida, Imazosulfurão, Mefenacete, Oxaziclomefona, Pirazosulfurão, Piributicarb, Quincloraque, Tiobencarben, Indanofane, Piranamide, Oxadiargyl, Etoxisulfuron, Pretilachlor, Mesotrione, Tefuriltrione, Oxadiazone, Fenoxaprope, Pyrimisulfan; Insecticides for Rice: Diazinão, Fenitrotiona, Fenobucarb, Monocrotofos, Benfuracarbe, Buprofezina, Dinotefurano, Fipronil, Imidaclopride, Isoprocarb, Tiaclopride, Chromafenozide, Tiaclopride, Dinotefuran, Clotiamidina, Clotiamidina, Tothylidine, Chloramydine, Chloramydine, Chloramydine, Chlamydine, Chloride Spinach, Emamectin-Benzoate, Cypermethrin, Chlorpyrifos, Cartape, Metamidofos, Etofenprox, Triazofos, 4 - [[((6-Chloropyridin-3-yl) methyl] (2,2-difluoroethyl) amino] furan-2 (5H) -one , Carbofurão, Benfuracarbe; Rice Fungicides: Thiophanate-methyl, Azoxystrobin, Carpropamide, Edifenfos, Ferimzona, Iprobenfos, Isoprothiolana, Pencicurão, Probenazole, Piroquilona, Triciclazole, Trifloxistrobina, Diclocimete, Fenoxanil, Simeconole; Herbicides for Cotton: Diuron, Fluometuron, MSMA, Oxifluorfen, Promethrin, Trifluralin, Carfentrazone, Cletodime, Fluazifop-butyl, Glyphosate, Norflurazone, Pendimethalin, Piritiobac-sodium, Trifloxysulfuron, Tepraloxidine, Tufaloxidine, Flufos; Cotton Insecticides: Aphosphate, Aldicarb, Chlorpyrifos, Cypermethrin, Deltamethrin, Malathione, Monocrotophos, Abamectin, Acetamipride, Emamectin Benzoate, Imidacloprid, Indoxacarb, Lambda-Cialothrin, Spinosad, Flaminamide, Flamylamide, Flamylamide, Piramide, Flamylamide, Flaminamide Triflumuron, Rinaxipir, beta-cyfluthrin, Spirotetramate,
[0097] [00097] Clothianidin, Thiamethoxam, Thiacloprid, Dinetofuran, Flubendiamide, Ciazipir, Spinosadam, Spininosam, Gamma Cyhalothrin, 4 - [[(6-Chloropyridin-3-yl) methyl] (2,2-difluoroethyl) amino] furan-2 ( 5H) -one, Tiocarb, Avermectin, Flonicamide, Pyridalil, Spiromesifene, Sulfoxaflor, Profenofos, Triazofos, Endosulfan; Fungicides for cotton: Etridiazole, Metalaxil, Quintozeno; Soy Herbicides: Alachlor, Bentazone, Trifluralin, Chlorimuron-Ethyl, Chloransulam-Methyl, Fenoxaprope, Fomesafen, Fluazifope, Glyphosate, Imazamox, Imazaquin, Imazetapyr, (S-) Metolachlor, Metribuzin, Tehran, Methylamine, Pendin; Insecticides for soybeans: Lambda-cyhalothrin, Methomyl, Parathion, Tiocarb, Imidaclopride, Clothianidin, Thiamethoxam, Tiaclopride, Acetamipride, Dinetofuran, Flubendiamide, Rinaxipir, Ciazipir, Espinosade, Spininame, Ethylamine, Eylamine, Eylamine; gamma and lambda Cyhalothrin, 4 - [[((6-Chloropyridin-3-yl) methyl] (2,2-difluorethyl) amino] furan-2 (5H) -one, Spirotetramate, Spinodiclofen, Triflumuron, Flonicamide, Thiodicarb, beta- Cyfluthrin; Soy Fungicides: Azoxystrobin, Ciproconazole, Epoxiconazole, Flutriafole, Pyraclostrobin, Tebuconazole, Trifloxystrobin, Protioconazole, Tetraconazole; Beet Herbicides: Chloridazone, Desmedipham, Etofumesate, Fenmedipham, Trialate, Clopyralide, Fluazifope, Lenacil, Metamitron, Quinmera, Cycloxidime, Triflusulfuron, Tepraloxidime, Quizalofope; Beet Insecticides: Imidacloprid, Clothianidin, Thiamethoxam, Thiacloprid, Acetamipride, Dinetofuran, Deltamethrin, ß-Cyfluthrin, gamma / lambda Cyhalothrin, 4 - [[(6-Chloropyridin-3-yl) methyl] (2,2-difluor ] furan-2 (5H) -one, Teflutrin, Rinaxipir, Ciaxipir, Fipronil, Carbofuron; Canola Herbicides: Clopiralid, Diclofope, Fluazifope, Glufosinate, Glyphosate, Metazachlor, Trifluralin Etametsulfuron, Quinmeraque, Quizalofope, Cletodime, Tepraloxidime; Canola Fungicides: Azoxystrobin, Carbendazime, Fludioxonil, Iprodione, Procloraz, Vinclozolina; Canola Insecticides:
[0098] [00098] Carbofuran, Organophosphates, Pyrethroids, Thiacloprid, Deltamethrin, Imidacloprid, Clothianidin, Thiamethoxam, Acetamipride, Dinetofuran, ß-Cyphlutrin, Gamma and lambda Cyalothrin, Tau-Fluvaleriato, Flame-eminate, Spade, Rhein, Spine, [[((6-Chloropyridin-3-yl) methyl] (2,2-difluorethyl) amino] furan-2 (5H) -one.
[0099] [00099] The following examples are presented for purposes of illustration and not as a limitation. EXPERIMENTAL EXAMPLES Example 1. Discovery of new pesticidal genes from Bacillus thuringiensis
[0100] • Preparação de DNA extracromossômico da estirpe. DNA extracromossômico contém uma mistura de alguns ou todos os seguintes: plasmídeos de vários tamanhos; cromossomos fágicos; fragmentos de DNA genômico não separados pelo protocolo de purificação; outras moléculas extracromossômicas não caraterizadas. • Corte mecânico ou enzimático do DNA extracromossômico para gerar fragmentos com uma distribuição de comprimentos. • Sequenciação do DNA fragmentado por métodos de pirossequenciação de elevado rendimento. • Identificação de genes de toxina putativos através de homologia e/ou outras análises computacionais. • Quando necessário, acabamento das sequências do gene de interesse por uma de várias estratégias de PCR ou de clonagem (por exemplo, TAIL-PCR).
[0101] [000101] The toxin gene disclosed herein is amplified by PCR from pAX980 and the PCR reaction product is cloned into the Bacillus expression vector pAX916, or another appropriate vector, by methods well known in the art. The resulting Bacillus strain, which contains the vector with the axmi gene, is grown in a conventional growth medium, such as CYS medium (10 g / l of Bacto-casitone; 3 g / l of yeast extract; 6 g / l KH2PO4; 14 g / l K2HPO4; 0.5 mM MgSO4; 0.05 mM MnCl2; 0.05 mM FeSO4) until spore formation is evident by microscope analysis. Samples are prepared and tested for activity in bioassays. Example 2. Tests for pesticidal activity
[0102] [000102] The nucleotide sequences of the invention can be tested for their ability to produce pesticidal proteins. The ability of a pesticidal protein to act as a pesticide in a pest is often assessed in several ways. One way well known in the art is to perform an intake test. In one of these intake tests, the pest is exposed to a sample containing either compounds to be tested, or control samples. This is often accomplished by placing the material to be tested, or an appropriate dilution of such material in a material that the pest will ingest, such as an artificial diet. The material to be tested can be in the form of a liquid, a solid or a suspension. The material to be tested can be placed on the surface and then allowed to dry. Alternatively, the material to be tested can be mixed with a melted artificial diet, and then served in the test chamber. The test chamber can be, for example, a bowl, a plate, or a well from a microplate.
[0103] [000103] Tests for sucking pests (for example, aphids) may involve separating the material to be tested from the insect through a partition, ideally a portion that can be punctured by the sucking parts of the sucking insect's mouth, to allow ingestion of the insect. material to be tested. The material to be tested is often mixed with an intake stimulator, such as sucrose, to promote the intake of the test compound.
[0104] [000104] Other types of tests may include the microinjection of the material to be tested in the mouth, or in the intestine of the pest, as well as the development of transgenic plants, followed by a test for the pest's ability to feed on the transgenic plant. Plant testing may involve isolating parts of plants normally consumed, for example, small cages attached to a leaf, or isolating whole plants in cages containing insects.
[0105] [000105] Other methods and approaches for testing pests are known in the art, and can be found, for example, in Robertson, J. L. & H. K. Preisler, eds. (1992) Pesticide bioassays with arthropods.CRC, Boca Raton, FL. Alternatively, tests are commonly described in the journals Arthropod Management Tests and Journal of Economic Entomology or in discussion with members of the Entomological Society of America (ESA).
[0106] [000106] In some embodiments, the regions of DNA encoding the delta-endotoxin toxin domains disclosed here are cloned into the E. coli expression vector pMAL-C4x behind the malE gene encoding the maltose-binding protein ( MBP). These fusions, which are in the same reading phase, result in the expression of MBP-Axmi fusion proteins in E. coli.
[0107] [000107] For expression in E.coli, BL21 * DE3 is transformed with individual plasmids. Isolated colonies are inoculated on LB supplemented with carbenicillin and glucose, and grown overnight at 37 ° C. On the following day, fresh medium is inoculated with 1% of the culture grown overnight and grown at 37 ° C until reaching the logarithmic stage. Subsequently, cultures are induced with 0.3 mM IPTG overnight at 20 ° C. Each cell pellet is suspended in 20 mM Tris-Cl buffer, pH 7.4 + 200 mM NaCl + 1 mM DTT + protease inhibitors, and sonicated. Analysis using SDS-PAGE can be used to confirm the expression of the fusion proteins.
[0108] [000108] Total cell-free extracts are passed through an amylose column linked to fast protein liquid chromatography (FPLC) for affinity purification of MBP-axmi fusion proteins. Fusion proteins bound to the resin are eluted with 10 mM maltose solution. The purified fusion proteins are then cleaved with factor Xa or trypsin to remove the MBP amino terminal marker from the Axmi protein. Protein cleavage and solubility can be determined by SDS-PAGE. Example 3. Expression and purification of axmiz genes
[0109] [000109] Truncated versions of axmi221z and axmi222z were cloned into the maltose binding protein (MBP) expression vector, resulting in pAX5092 and pAX5093, respectively. The expression of the resulting fusion protein was induced by means of IPTG. The protein was then purified on a maltose column and was cleaved with Protease Factor Xa to generate the purified, unlabeled protein. The truncated 6-his proteins axmi221z and axmi222z were also purified on a cobalt column and subjected to bioassays.
[0110] [000110] Complete and truncated versions of some genes were cloned into the vector pRSF-1b as shown in Table 2. Due to cloning in that vector, the resulting expressed protein contains six additional N-terminal histidine residues.
[0111] [000111] The DNA regions encoding the toxin domains of some genes have been cloned separately into an E. coli pMAL-C4x expression vector behind the malE gene encoding the maltose-binding protein (MBP) as shown in Table 2. These fusions, which are in the same reading phase, resulted in the expression of MBP-AXMI fusion proteins in E. coli. Each of the proteins produced from the constructions above was tested in bioassays in the form of a 10x concentrated pellet.
[0112] [000112] For protein expression in E.coli, BL21 * DE3 was transformed with individual plasmids. An isolated colony was inoculated in LB medium supplemented with carbenicillin and glucose, and grown overnight at 37 ° C. The following day, fresh medium was inoculated with 1% of the culture grown overnight and grown at 37 ° C until reaching the logarithmic phase. Subsequently, cultures were induced with 0.3 mM IPTG overnight at 20 ° C. Each cell pellet was suspended in 20 mM Tris-Cl buffer, pH 7.4 + 200 mM NaCl + 1 mM DTT + protease inhibitors, and sonicated. An analysis by SDS-PAGE confirmed the expression of fusion proteins.
[0113] [000113] Total cell-free extracts were loaded onto an FPLC equipped with an amylose column, and the MBP-AXMI fusion proteins were purified by affinity chromatography. The resin-bound fusion protein was eluted with 10 mM maltose solution. The purified fusion proteins were then cleaved with factor Xa or trypsin to remove the MBP amino terminal marker from the AXMIz protein. Protein cleavage and solubility were determined by SDS-PAGE. Example 4. Activity of proteins expressed from axmiz genes in Bioassays
[0114] [000114] The bioassay of the expressed Axmiz genes resulted in observation of the following activities in insect pests:
[0115] [000115] The coding regions of the invention are linked with promoter and terminator sequences suitable for expression in plants. Such sequences are well known in the art and can include the rice actin promoter or the corn ubiquitin promoter for monocot expression, the UBQ3 promoter or the Arabidopsis CaMV 35S promoter for dicot expression, and the nos or PinII terminators. Techniques for the production and confirmation of promoter - gene - terminator constructs are also well known in the art.
[0116] [000116] In one aspect of the invention, synthetic DNA sequences are designed and generated. These synthetic sequences have an altered nucleotide sequence relative to the parent sequence, but they encode proteins that are essentially identical to the parent sequence. See, for example, Table 4.
[0117] [000117] In another aspect of the invention, modified versions of the synthetic genes are designed in such a way that the resulting peptide is directed to a plant organelle, such as the endoplasmic reticulum or the apoplast. Peptide sequences that are known to result in targeting fusion proteins to plant organelles are known in the art. For example, the N-terminal region of the Lupine White Lupinus albus acid phosphatase gene (Genebank® ID GI: 14276838, Miller et al. (2001) Plant Physiology 127: 594-606) is known in the art to result in targeting heterologous proteins to the endoplasmic reticulum. If the resulting fusion protein also contains a retention sequence in the endoplasmic reticulum comprising the N-terminal peptide lysine-aspartic acid-glutamic acid-leucine (i.e., the "KDEL" motif, SEQ ID NO: 33) at the C-terminus, the fusion protein will be directed to the endoplasmic reticulum. If the fusion protein does not have a sequence for targeting the endoplasmic reticulum at the C-terminus, the protein will be directed to the endoplasmic reticulum, but will eventually be sequestered in the apoplast.
[0118] [000118] Thus, this gene codes for a fusion protein that contains the thirty-one N-terminus amino acids of the Lupine White Lupinus albus acid phosphatase gene (GENBANK® ID GI: 14276838, Miller et al., 2001, supra) fused to the N-terminus amino acid sequence of the invention, as well as the C-terminus KDEL sequence. Thus, the resulting protein is predicted to target the endoplasmic reticulum of the plant upon expression in a plant cell.
[0119] [000119] The plant expression cassettes described above are combined with an appropriate selection marker to assist in the selection of transformed cells and tissues, and linked into plant transformation vectors. These can include agrobacterium-mediated binary transformation vectors or simple plasmid vectors for aerosol or biolistic transformation. Example 6. Transformation of Corn Cells with the pesticide protein genes described here
[0120] [000120] Ears are collected 8-12 days after pollination. Embryos from the ears are isolated, and these embryos with a size of 0.8-1.5 mm are preferred for use for transformation. Embryos are plated with the scutellum facing upwards in a suitable incubation medium, such as DN62A5S medium (3.98 g / L N6 salts; 1 mL / L (1000x stock) Vitamins N6; 800 mg / L L- Asparagine; 100 mg / L Myo-inositol; 1.4 g / L L-Proline; 100 mg / L Casamino acids; 50 g / L sucrose; 1 mL / L (from 1 mg / mL Stock) 2,4-D ). However, media and salts other than DN62A5S are suitable and are known in the art. Embryos are incubated overnight at 25 ° C in the dark. However, it is not necessary per se to incubate embryos overnight.
[0121] [000121] The resulting explants are transferred to grid squares (30-40 per plate), transferred to osmotic medium for 30-45 minutes, then transferred to a bombardment plate (see, for example, PCT Publication No. WO / 0138514 and US Patent No. 5,240,842).
[0122] [000122] DNA constructs designed for the genes of the invention in plant cells are accelerated in plant tissue using an accelerator with aerosol bombardment, using conditions essentially as described in PCT Publication No. WO / 0138514. After bombardment, the embryos are incubated for about 30 minutes in an osmotic medium, and are placed on incubation medium overnight at 25 ° C in the dark. In order to avoid bombs exploding improperly damaged, they are incubated for at least 24 hours before transfer to recovery medium. The embryos are then spread over a recovery period medium, for 5 days, 25 ° C in the dark, then transferred to a selection medium. The explants are incubated in a selection medium for up to eight weeks, depending on the nature and characteristics of the particular selection used. After the selection period, the resulting callus is transferred to the embryo's maturation medium, until the formation of mature somatic embryos is observed. The resulting mature somatic embryos are then placed in low intensity light, and the regeneration process is initiated as known in the art. The resulting shoots are allowed to take root in the rooting medium, and the resulting plants are transferred to nursery pots and propagated as transgenic plants. Materials DN62A5S medium
[0123] [000123] Adjust the pH of the solution to pH 5.8 with 1N KOH / 1N KCl, add Gelrite (Sigma) to 3 g / L, and autoclave. After cooling to 50 ° C, add 2 ml / L of a 5 mg / ml stock solution of silver nitrate (Phytotechnology Labs). Example 7. Transformation of genes of the invention into Plant Cells by Agrobacterium-Mediated Transformation
[0124] [000124] Ears are collected 8-12 days after pollination. Embryos from the ears are isolated, and these embryos with a size of 0.8-1.5 mm are preferred for use for transformation. The embryos are plated with the scutellum facing upwards in a suitable incubation medium, and are incubated overnight at 25 ° C in the dark. However, it is not necessary per se to incubate embryos overnight. The embryos are contacted with an Agrobacterium strain containing the appropriate vectors for transfer mediated by the plasmid Ti for 5-10 min, and then plated in co-culture medium for 3 days (25 ° C in the dark). After co-cultivation, the explants are transferred to a medium recovery period for 5 days (25 ° C in the dark). The explants are incubated in a selection medium for up to eight weeks, depending on the nature and characteristics of the particular selection used. After the selection period, the resulting callus is transferred to the embryo's maturation medium, until the formation of mature somatic embryos is observed. The resulting mature somatic embryos are then placed in low intensity light, and the regeneration process is initiated as known in the art.
[0125] [000125] All publications and patent applications mentioned in the specification are indicative of the level of technique of those skilled in the art to which this invention belongs. All publications and patent applications are hereby incorporated by reference in the same scope as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference.
[0126] [000126] Although the previous invention has been described in some detail by way of illustration and example for the sake of clarity of understanding, it will be obvious that certain changes and modifications can be practiced within the scope of the appended claims.

权利要求:
Claims (9)
[0001]
Recombinant nucleic acid molecule, characterized by the fact that it consists of a nucleotide sequence that encodes a sequence of amino acids with pesticidal activity, in which said nucleotide sequence is presented in any of SEQ ID NO: 8, 13 or 18, or the sequences degenerate from those encoding the same polypeptide as SEQ ID NO: 27 or 28, where the nucleotide sequence is a synthetic sequence that was designed for expression in a plant, wherein said nucleotide sequence is operably linked to a promoter capable of directing the expression of said nucleotide sequence in a plant cell.
[0002]
Recombinant nucleic acid molecule according to claim 1, characterized by the fact that it is inserted in the genome of a plant, plant cell or seed.
[0003]
Vector, characterized by the fact that it comprises the recombinant nucleic acid molecule as defined in claim 1.
[0004]
Vector according to claim 3, characterized by the fact that it further comprises a nucleic acid molecule that encodes a heterologous polypeptide.
[0005]
Microbial host cell, characterized by the fact that it contains the vector as defined in claim 3.
[0006]
Method for controlling a population of lepidopteran pests, or for killing a lepidopteran pest, characterized by the fact that it comprises contacting said population or plague with a pesticide-effective amount of a polypeptide having pesticidal activity, in which said polypeptide is the amino acid sequence of SEQ ID NO: 27 or 28, in which the aforementioned lepidopteran pest is selected from the caterpillar, potato beetle, cruciferous moth, European corn borer, corn cartridge caterpillar , apple caterpillar, corn cob caterpillar, southern corn borer, southwest corn borer, and soybean caterpillar.
[0007]
Method for producing a polypeptide with pesticidal activity, characterized in that it comprises culturing the host cell, as defined in claim 5, under conditions in which the nucleic acid molecule encoding the polypeptide is expressed.
[0008]
Method for the protection of a plant from a pest, characterized by the fact that it comprises expressing in a plant or its cell a nucleotide sequence that encodes a pesticidal polypeptide, in which the said nucleotide sequence is presented in any of SEQ ID NO: 8, 13 or 18, or the degenerate sequences thereof that encode the same polypeptide as SEQ ID NO: 27 or 28, wherein the nucleotide sequence is a synthetic sequence that has been designed for expression in a plant; in which the said plant produces a polypeptide with pesticidal activity against a lepidopteran pest, in which the said lepidopteran pest is selected from the caterpillar, potato beetle, crucifer moth, European corn borer, caterpillar corn canister, apple caterpillar, corn cob caterpillar, southern corn borer, southwestern corn borer, and soybean caterpillar.
[0009]
Method to increase the yield in a plant, characterized by the fact that it comprises growing in a field a plant or a seed thereof that has a DNA construct stably incorporated in its genome, which comprises a nucleotide sequence that encodes a protein with activity pesticide, wherein said nucleotide sequence is presented in any of SEQ ID NO: 8, 13 or 18, or the degenerate sequences thereof that encode the same polypeptide as SEQ ID NO: 27 or 28, where the nucleotide sequence is a synthetic sequence that was designed for expression in a plant; and in which the said field is infested by a pest against which the said polypeptide has pesticidal activity, in which the said pest is selected from the caterpillar, potato beetle, cruciferous moth, European corn borer, caterpillar corn maize, apple caterpillar, corn cob caterpillar, southern corn borer, southwest corn borer, and soybean caterpillar.

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法律状态:
2018-03-06| B06T| Formal requirements before examination [chapter 6.20 patent gazette]|

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2018-04-10| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|

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2018-12-18| B06A| Notification to applicant to reply to the report for non-patentability or inadequacy of the application [chapter 6.1 patent gazette]|

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2020-06-02| B06A| Notification to applicant to reply to the report for non-patentability or inadequacy of the application [chapter 6.1 patent gazette]|

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2020-12-22| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|

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2021-02-17| B16A| Patent or certificate of addition of invention granted|Free format text: PRAZO DE VALIDADE: 10 (DEZ) ANOS CONTADOS A PARTIR DE 17/02/2021, OBSERVADAS AS CONDICOES LEGAIS. |

优先权:

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申请号 | 申请日 | 专利标题

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US30580210P| true| 2010-02-18|2010-02-18|

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US61/305,802|2010-02-18|

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PCT/US2011/025172|WO2011103248A2|2010-02-18|2011-02-17|AXMI221z, AXMI222z, AXMI223z, AXMI224z, AND AXMI225z DELTA-ENDOTOXIN GENES AND METHODS FOR THEIR USE|

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