Axmi-192 pesticidal genes, expression cassette, bacterial host cell, polypeptide with pesticidal act

专利摘要:
axmi-192 pesticides, its expression cassette, its use, host cell and its production method, production methods of a transgenic plant and seeds, pesticide polypeptide and its production method, composition, methods for pest control and killing , to increase the production and yield of a plant, as well as to isolate a nucleic acid sequence. The present invention relates to compositions and methods for imparting pesticidal activity to bacteria, plants, plant cells, tissues and seeds. compositions are provided which 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. additionally, amino acid sequences corresponding to the polynucleotides and antibodies that specifically bind to those amino acid sequences are encompassed. in particular, the present invention provides isolated nucleic acid molecules comprising nucleotide sequences encoding the amino acid sequence shown in seq id no:28-62, or the nucleotide sequence shown in seq id no:1-27, as well as their variants and fragments.
公开号:BR112012002275B1
申请号:R112012002275-0
申请日:2010-07-30
公开日:2022-02-01
发明作者:Kimberly S. Sampson；Daniel John Tomso；Rong Guo
申请人:Athenix Corporation；
IPC主号:

专利说明:

Cross-reference to Related Orders
[0001] This patent application claims the benefit of the U.S. 61/230,659, filed on July 31, 2009, the contents of which are hereby fully incorporated by citation. Reference to Electronically Submitted Sequence List
[0002] The official copy of the sequence list is submitted electronically via the EFS-Web as a sequence list in ASCII format with the filename "APA067US01_SEQLIST.txt", created on July 27, 2010, with a file size of 273 kilobytes and deposited at the same time as the descriptive memory. The list of sequences contained in this document in ASCII format forms part of the specification and is hereby incorporated by reference in its entirety. Field of Invention
[0003] The present invention relates to the field of molecular biology. New genes coding for pesticidal proteins are provided. Such proteins and the nucleic acid sequences encoding them are useful in the preparation of pesticide formulations and in the production of pest-resistant transgenic plants. Context of the Invention
[0004] Bacillus thuringiensis is a spore-forming Gram-positive soil bacterium characterized by its ability to form crystalline inclusions that are specifically toxic to certain insect orders and species, but which 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] Bacillus thuringiensis crystal proteins (Cry) have a potent insecticidal activity against predominantly Lepidoptera, Diptera and Coleopteran larvae. These proteins also showed activity against pests of the Hymenoptera, Homoptera, Phthiraptera, Mallophaga, and Acari orders, as well as other invertebrate orders such as Nemathelminthes, Platyhelminthes, and Sarcomastigorphora (Feitelson (1993) The Bacillus Thuringiensis family tree. In Advanced Engineered Pesticides, Marcel Dekker, Inc., New York, NY). 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, Cry 1C, etc. Even closer proteins within each division were given names such as Cry1C1, Cry1C2, etc.
[0006] A new nomenclature was recently described for Cry genes based on amino acid sequence homology rather than target insect specificity (Crickmore et al. (1998) Microbiol. Mol. Biol. Rev. 62:807-813). In the new classification, each toxin is given 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 numerals were replaced by Arabic numerals in the first classification. Proteins with less than 45% sequence identity have different first ranks, and the criteria for second and third rank are 78% and 95%, respectively.
[0007] The crystal protein does not show insecticidal activity until it has been ingested or solubilized in the insect's mesentery. The ingested protoxin is hydrolyzed by proteases in the insect's digestive tract to form a toxic molecule (Hofte and Whiteley (1989) Microbiol. Rev. 53:242-255). This toxin binds to apical brush border receptors in the mesentery of target larvae and enters the apical membrane creating ion channels or pores, resulting in larval death.
[0008] Delta-endotoxins generally have five conserved sequence domains, and three conserved structural domains (see, for example, from 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 receptor recognition and binding, and are therefore considered to be determinants of toxin specificity.
[0009] In addition to delta-endotoxins, there are several other classes of pesticidal protein toxins. VIP1/VIP2 toxins (see, for example, US patent 5,770,696) are binary pesticidal toxins that exhibit high activity in insects by a mechanism believed to - if it involves receptor-mediated endocytosis followed by cell toxicity, similar to the mechanism of action of other binary ("A/B") toxins. A/B toxins such as VIP, C2, CDT, CST, or the lethal and edema-promoting B. anthracis toxins initially interact with target cells through the binding of "B" components in the form of monomers in a specific and mediated way. receiver. These monomers then form homoheptamers. The heptamer "B"-receptor complex then acts as a support base which then binds and allows the translocation of an "A" enzymatic component(s) into the cytosol via receptor-mediated endocytosis. . Once in the cellular cytosol, the "A" components inhibit normal cellular function by, for example, promoting ADP-ribosylation of G-actin or leading to an increase in intracellular levels of cyclic AMP (cAMP). See Barth et al. (2004) Microbiol.Mol. Biol. Rev. 68:373-402.
[00010] The intensive use of insecticides based on B. thuringiensis has already led to resistance in field populations of the diamondback moth, Plutella xylostella (Ferré and Van Rie (2002) Annu. Rev. Entomol. 47:501-533). The most common mechanism of resistance is decreased binding of the toxin to its specific receptor(s) in the mesentery. This may further promote cross-resistance to other toxins that share the same receptor (Ferré and Van Rie (2002)). Summary of the Invention
[00011] Compositions and methods for imparting pest resistance to bacteria, plants, plant cells, tissues and seeds are provided. The compositions include sequences of nucleic acid molecules encoding toxin polypeptides, vectors comprising those nucleic acid molecules, and host cells comprising the vectors. The compositions also include the toxin 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. The 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.
[00012] In particular, isolated or recombinant nucleic acid molecules corresponding to the toxin nucleic acid sequences are provided. In addition, amino acid sequences corresponding to the polynucleotides are encompassed. In particular, the present invention provides an isolated nucleic acid molecule that comprises a nucleotide sequence encoding the amino acid sequence shown in any of SEQ ID NO:28-62, or a nucleotide sequence shown in any of SEQ ID NO:1 -27, as well as its variants and fragments. Also encompassed are nucleotide sequences that are complementary to a nucleotide sequence of the invention, or that hybridize to a sequence of the invention.
[00013] The compositions and methods of the invention are useful for producing organisms, particularly bacteria and plants, with pesticide resistance. These organisms and compositions derived therefrom are desirable for agricultural purposes. The compositions of the invention are also useful for generating altered or improved toxin proteins that have pesticidal activity, or for detecting the presence of toxin proteins or nucleic acids in products or organisms. Detailed Description
[00014] The present invention relates to compositions and methods for regulating resistance or tolerance to pests in organisms, particularly plants or plant cells. By "resistance" it is meant that the pest (e.g. 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 the movement, feeding, reproduction, or other functions of the pest.The methods involve transforming organisms with a nucleotide sequence that encodes for a pesticidal protein of the invention. In particular, the nucleotide sequences of the invention are useful for preparing plants and microorganisms that have pesticidal activity. Thus, transformed bacteria, plants, plant cells, plant tissues and seeds are provided. Compositions are nucleic acids and pesticidal proteins derived from bacterial species. The sequences find use in the construction of expression vectors for subsequent transformation into organisms of interest, as probes for isolating other homologous (or partially homologous) genes, and for generating altered insecticidal proteins by methods known in the art, such as exchange of domains or DNA rearrangement ("DNA shuffling"). The proteins are used to control or kill lepidopteran, coleopteran, dipteran and nematode populations and for the production of compositions with pesticidal activity.
[00015] By "pesticide toxin" or "pesticide 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. Pesticide proteins have been isolated from organisms including, for example, Bacillus sp., Clostridium bifermentans and Paenibacillus popilliae. Pesticide proteins include amino acid sequences deduced from the full-length nucleotide sequences disclosed herein, and amino acid sequences that are shorter than the full-length 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 ingestion of the protein.
[00016] In various embodiments, the sequences disclosed herein have homology to delta-endotoxin proteins. Delta-endotoxins include proteins identified as cry1 through cry53, cyt1 and cyt2, and Cyt-like toxin. There are currently over 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 toxinnomenclature," at www.biols.susx.ac.uk/Home/Neil_Crickmore/Bt/index. In some embodiments, the delta-endotoxin sequences disclosed herein include the nucleotide sequences shown in any of SEQ ID NO:1-27, the amino acid sequences shown in any of SEQ ID NO:28-62, as well as variants and fragments thereof. .
[00017] Thus, novel isolated or recombinant nucleotide sequences that confer pesticidal activity are provided herein. Also provided are the amino acid sequences of 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
[00018] One aspect of the invention pertains to isolated or recombinant nucleic acid molecules comprising nucleotide sequences encoding pesticidal proteins and polypeptides or biologically active portions thereof, as well as nucleic acid molecules sufficient for use as hybridization probes to identify acids. nucleic acids encoding proteins with regions of sequence homology. As used herein, by the term "nucleic acid molecule" is meant to include DNA molecules (e.g. recombinant DNA, cDNA or genomic DNA) and RNA molecules (e.g. mRNA) and analogs of the generated DNA or RNA using nucleotide analogues. The nucleic acid molecule may be single-stranded or double-stranded, but is preferably double-stranded DNA.
[00019] An "isolated" nucleic acid (or DNA) sequence is used herein to refer to a nucleic acid (or DNA) sequence that is no longer in its natural environment, for example in vitro or in a bacterial recombinant host cell or plant. In some embodiments, an "isolated" nucleic acid is free of sequences (preferably protein-coding sequences) that naturally flank the nucleic acid (i.e., 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 purposes of the invention, "isolated" when used to refer to nucleic acid molecules excludes isolated chromosomes. For example, in various embodiments, the isolated nucleic acid molecule encoding the pesticidal protein 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. A pesticidal protein that is substantially free of cellular material includes protein preparations with less than about 30%, 20%, 10%, or 5% (by dry weight) of non-pesticidal protein (also referred to herein as "contaminating protein") .
[00020] Nucleotide sequences encoding the proteins of the present invention include the sequence shown in SEQ ID NO:1-27, and variants, fragments and complements thereof. By "complement" is meant a nucleotide sequence that is sufficiently complementary to a given nucleotide sequence such that it can hybridize to the given nucleotide sequence to thereby form a stable duplex. The corresponding amino acid sequences for the toxin protein for which this nucleotide sequence encodes are shown in SEQ ID NO:28-62.
[00021] Nucleic acid molecules that are fragments of these nucleotide sequences that encode pesticidal proteins are also encompassed by the present invention. By "fragment" is meant a portion of the nucleotide sequence that encodes a pesticidal protein. A fragment of a nucleotide sequence can encode 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 which 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, 1500, 1600, 1700, 1800, 1900, 2000, 2500, 3000, 3500, 4000, 4500, 5000 contiguous nucleotides, or up to 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 code for protein fragments that retain the biological activity of the pesticidal protein and thus retain pesticidal activity. By "retain 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, the pesticidal activity is coleoptericidal activity. In another embodiment, the pesticidal activity is lepidoptericidal activity. In another embodiment, the pesticidal activity is nematocidal activity. In another embodiment, the pesticidal activity is diptericidal 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 EconomicEntomology 78:290-293; and the U.S. patent. No. 5,743,477, all of which are incorporated herein by reference in their entirety.
[00022] A fragment of a nucleotide sequence encoding a biologically active portion of a pesticidal protein of the invention will code for at least about 15, 25, 30, 50, 75, 100, 125, 150, 175, 200, 250 , 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100 contiguous amino acids, or up to the total number of amino acids present in a complete pesticidal protein of the invention . In some embodiments, the fragment is the product of C-terminal truncation of at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 450, 500, 650, 600 or more amino acids relative to the amino acid sequences of the invention.
[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:1-27. 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. sequence identity, about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity compared to a reference sequence using a of the alignment programs described herein 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 degeneracy, amino acid similarity, reading frame positioning, and the like.
[00024] To determine the percent identity of two amino acid or nucleic acid sequences, the sequences are aligned for optimal comparison. The percent identity between two sequences is a function of the number of identical positions shared by the sequences (ie, percent identity = number of identical positions/number of total positions (eg, overlapping positions) x 100). In another embodiment, the comparison is performed over the entire reference sequence (i.e., over the entire sequence of any of SEQ ID NO:1-61). The percent identity between two sequences can be determined using techniques similar to those described below, with or without gaps. In calculating percent identity, exact matches are typically counted.
[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 of Karlin and Altschul (1990) Proc. natl. academy Sci. USA 87:2264, modified according to Karlin and Altschul (1993) Proc. natl. academy 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, score = 100, word length = 12, to obtain nucleotide sequences homologous to pesticide-like nucleic acid molecules of the invention. BLAST protein searches can be performed with the BLASTX program, score = 50, word length = 3, to obtain amino acid sequences homologous to pesticidal protein molecules of the invention. To obtain gapped alignments for comparison purposes, one can use Gapped BLAST (in BLAST 2.0) as described in Altschul et al. (1997) Nucleic Acids Res. 25:3389. Alternatively, PSI-Blast 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 default parameters of the respective programs (eg, BLASTX and BLASTN) can be used. Alignment can also be performed manually by inspection.
[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 sequence of amino acids or DNA, and can thus provide data on the sequence conservation of 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 percent amino acid identity can be determined. A non-limiting example of a useful computer program for analyzing ClustalW alignments is GENEDOCTM. GENEDOCTM (Karl Nicholas) allows the assessment of amino acid (or DNA) similarity and identity between multiple proteins. Another non-limiting example of a mathematical algorithm used for sequence comparison is the algorithm of 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 (available from 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.
[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 identical nucleotide residue matches and an identical percent sequence identity when compared to the corresponding alignment generated by the GAP version. 10.
[00028] The invention also encompasses variant nucleic acid molecules. "Variants" of nucleotide sequences encoding pesticidal protein include those sequences encoding pesticidal proteins presented herein but which differ conservatively due to degeneracy of the genetic code as well as those which are sufficiently identical as discussed above. Naturally occurring allelic variants can be identified using well known molecular biology techniques, such as the polymerase chain reaction (PCR) and hybridization techniques as presented below. Variant nucleotide sequences also include synthetically derived nucleotide sequences that have been generated, for example, using site-directed mutagenesis but that still code for the pesticidal proteins disclosed in the present invention as discussed below. Variant proteins encompassed by the present invention are biologically active, i.e., they continue to possess the desired biological activity of the native protein, i.e., retaining pesticidal activity. By "retain 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 the U.S. patent. No. 5,743,477, all of which are incorporated herein by reference in their entirety.
[00029] One of ordinary skill in the art will appreciate that changes can be introduced by mutation of the nucleotide sequences of the invention thus leading to changes in the amino acid sequence of the encoded pesticidal proteins, without altering the biological activity of the proteins. Thus, variant isolated 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 through conventional techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis. Such variant nucleotide sequences are also encompassed by the present invention.
[00030] For example, conservative amino acid substitutions may be made at one or more predicted non-essential amino acid residues. A "non-essential" amino acid is a residue that can be altered from the native sequence of a pesticidal protein without altering biological activity, whereas an "essential" amino acid residue is required for biological activity. A "conservative amino acid substitution" is one in which the amino acid residue is replaced by an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g. lysine, arginine, histidine), acidic side chains (e.g. aspartic acid, glutamic acid), uncharged polar side chains (e.g. 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 aromatics (eg tyrosine, phenylalanine, tryptophan, histidine).
[00031] Delta-endotoxins generally have five conserved sequence domains, and three conserved structural domains (see, for example, from 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 receptor recognition and binding and are therefore considered to be determinants of toxin specificity.
[00032] Amino acid substitutions can be performed 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 with respect to the sequences of the invention (e.g., residues that are identical in an alignment of homologous proteins). Examples of residues that are conserved but allow for 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 (e.g., residues that only contain conservative substitutions between all proteins contained in the homologous protein alignment). However, one of skill in the art will understand that functional variants may have small conserved or non-conserved changes at conserved residues.
[00033] Alternatively, variant nucleotide sequences can be produced by randomly introducing mutations throughout all or part of the coding sequence, such as by saturation mutagenesis, and the resulting mutants can be screened for their ability to confer pesticidal activity to identify mutants that retain activity. After mutagenesis, the encoded protein can be expressed recombinantly, and the activity of the protein can be determined using conventional testing techniques.
[00034] Using methods such as PCR, hybridization and the like, corresponding pesticidal sequences can be identified, isolated or recovered from a sample (for example, a sample containing nucleic acid sequences, such as a biological sample), provided that such sequences have substantial identity to the sequences of the invention (e.g., at least about 70%, at least about 75%, 80%, 85%, 90%, 95% or more identity over the entirety of the sequence of reference) and possess or confer pesticidal activity. See, for example, Sambrook and Russell (2001) Molecular Cloning: A Laboratory Manual. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY) and Innis, et al. (1990) PCR Protocols: A Guide to Methods and Applications (Academic Press, NI).
[00035] In a hybridization method, all or part of the pesticidal 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 Russell, 2001, supra. So-called hybridization probes may be genomic DNA fragments, cDNA fragments, RNA fragments, or other oligonucleotides, and may 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 encoding the pesticidal protein disclosed herein. Degenerate primers designed based on the conserved nucleotides or amino acid residues in the nucleotide sequence or encoded amino acid sequence may additionally be used. The probe typically comprises a region of nucleotide sequence that hybridizes under stringent conditions to at least about 12, at least about 25, at least about 50, 75, 100, 125, 150, 175, or 200 consecutive nucleotides of the probe 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 disclosed in Sambrook and Russell, 2001, supra incorporated by reference.
[00036] For example, a complete pesticidal protein sequence 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 nucleotides in length, or at least 20 nucleotides in length. Such probes can be used to PCR amplify corresponding pesticidal sequences from a chosen sample or 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 hybridization screening of plated DNA libraries (either plates 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).
[00037] 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 it can detect to be higher than to other sequences (e.g., at least 2-fold relative to to the background signal). Stringent conditions are sequence dependent and will differ depending on the circumstances. By controlling the stringency of hybridization and/or washing conditions, target sequences that are 100% complementary to the probe (homologous probing) can be identified. Alternatively, stringent conditions can be identified. 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.
[00038] Typically, stringent conditions will be chosen in which the salt concentration is less than about 1.5M Na ion, typically about 0.01 to 1.0M Na ion (or other salts) concentration at pH 7.0 to 8.3 and the temperature is at least 30°C for short probes (eg 10 to 50 nucleotides) and at least 60°C for longer probes (eg longer than 50 nucleotides). Stringent 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, 1M 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 in 40 to 45% formamide, 1.0M 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 in 50% formamide, 1M NaCl, 1% SDS at 37°C, and a wash in 0.1X SSC at 60 to 65°C. Optionally, wash buffers can comprise from 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.
[00039] Specificity is typically a function of post-hybridization washes, with critical factors being the ionic strength and temperature of the final wash solution. For DNA-DNA hybrids, the Tm can be approximated by the equation of Meinkoth and Wahl (1984) Anal.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 the DNA, % form is the percentage of formamide in the hybridization solution, and L is the length of the hybrid in base pairs. Tm is the temperature (under defined ionic strength and pH) at which 50% of a complementary target sequence hybridizes to a perfectly matched probe. Tm is reduced by about 1°C for every 1% discrepancy; thus, the Tm, hybridization, and/or washing conditions can be adjusted to hybridize to sequences of desired identity. For example, if sequences with an identity > 90% are sought, the T m can be lowered by 10 °C. Stringent conditions are usually 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 utilize hybridization and/or washing at 1, 2, 3, or 4°C lower than the thermal melting point (Tm); moderately stringent conditions may utilize hybridization and/or washing at 6, 7, 8, 9, or 10°C below the thermal melting point (Tm); low stringency conditions may utilize 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 wash compositions, and the desired Tm, those skilled in the art will understand that variations in the stringency of the hybridization and/or wash 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 SSC concentration 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
[00040] Pesticide proteins are also encompassed within the scope of the present invention. By "pesticide protein" is meant a protein containing the amino acid sequence shown in SEQ ID NO:28-62. 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 which is no longer in its natural environment, for example in vitro or in a bacterial or plant recombinant host cell.
[00041] "Fragments" or "biologically active portions" include polypeptide fragments comprising amino acid sequences sufficiently identical to the amino acid sequence shown in any of SEQ ID NO:28-62 and which exhibit pesticidal activity. A biologically active portion of a pesticidal protein may be a polypeptide that is, for example, 10, 25, 50, 100 or more amino acids in length. Such biologically active portions may be prepared by 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 the 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 NOs:28-62. The invention encompasses other fragments, however, such as any fragment in the protein greater than about 10, 20, 30, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650 , 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, or 1300 amino acids.
[00042] By "variants" is 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:28-62. Variants further include polypeptides encoded by a nucleic acid molecule that hybridizes to the nucleic acid molecules of SEQ ID NOs: 1-27, or its complement, under stringent conditions. Variants include polypeptides that differ in amino acid sequence due to mutagenesis. Variant proteins encompassed by the present invention are biologically active, i.e., they continue to possess the desired biological activity of the native protein, i.e., retaining pesticidal activity. In some modalities, variants show improved 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 the U.S. patent. No. 5,743,477, all of which are incorporated herein by reference in their entirety.
[00043] Bacterial genes, such as the axmi genes of this invention, very often have multiple methionine initiation codons in the vicinity of the start of the open reading frame. Often, translation initiation at one or more of these initiation codons will lead to the generation of a functional protein. These initiation codons may include ATG codons. However, bacteria such as Bacillus sp. also recognize the GTG codon as the initiation codon, and proteins that initiate translation at the GTG codons contain a methionine at the first amino acid. On rare occasions, translation in bacterial systems can start at a TTG codon, through which TTG encodes a methionine. Furthermore, it is not often 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 may also lead to the generation of pesticidal proteins. These pesticidal proteins are encompassed by the present invention and can be used in the methods of the present invention. It will be understood that when expressed in plants it will be necessary to change the alternative start codon to ATG for proper translation.
[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, NY; U.S. Patent No. 4,196,265). Changed or Improved Variants
[00045] It is recognized that the DNA sequences of a pesticidal protein can be altered by various methods, and that these alterations can result in DNA sequences encoding different amino acid sequences than those encoded by a pesticidal protein of the present invention. invention. This protein can be altered in a number of ways including amino acid substitutions, deletions, truncations, and insertions of one or more amino acids of SEQ ID NO:28-62, 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 about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 100, about 105, about 110, about 115 , about 120, about 125, about 130 or more amino acid substitutions, deletions or insertions.
[00046] Methods for such manipulations are generally known in the prior art. For example, variant amino acid sequences of a pesticidal protein can be prepared through mutations in the DNA. This can also be accomplished by one of several forms of mutagenesis and/or evolution. directed. In some respects, 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 the use of such techniques in the compositions of this invention. For example, the pesticidal protein can be expressed in host cells that show high rates of incorrect base incorporation during DNA replication, such as XL-1 Red (Stratagene). After propagation in such strains, the toxin DNA can be isolated (e.g. by preparing plasmid DNA, or by PCR amplification and cloning the resulting PCR fragment into a vector), culturing the toxin mutations in a non-mutagenic strain, and identifying mutated toxin genes with pesticidal activity, for example by performing an assay to test for insecticidal activity. Generally, the protein is mixed and used in ingestion trials. See, for example, Marrone et al. (1985) J. of Economic Entomology 78:290-293. Such assays may include placing the plants in contact with one or more pests and determining the plant's ability to survive and/or cause the pests to die. Examples of mutations that result in increased toxicity are found in Schnepf et al. (1998) Microbiol.Mol. Biol. Rev. 62:775-806.
[00047] 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 alterations 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 amplification by PCR Alternatively, added protein sequences may include full-length sequences encoding proteins, such as those commonly used in the art to generate protein fusions. Such fusion proteins are often used to (1) increase expression of a protein of interest, (2) introduce a binding domain, enzyme activity, or epitope to facilitate either protein purification, protein detection, or other experimental uses known in the art, (3) directing 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.
[00048] Variant nucleotide and amino acid sequences of the present invention also encompass sequences derived from mutagenesis and recombination procedures such as "DNA shuffling". With such a procedure, one or more regions coding 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 sequence-related 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. academy Sci. USA 91:1074710751; Stemmer (1994) Nature 370:389-391; Crameri et al. (1997) Nature Biotech. 15:436-438; Moore et al. (1997) J. Mol. Biol. 272:336-347; Zhanget al. (1997) Proc. natl. academy Sci. USA94:4504-4509; Crameri et al. (1998) Nature 391: 288-291; and U.S. Patent No. 5,605,793 and 5,837,458.
[00049] Domain switching or rearrangement is another mechanism for generating altered delta-endotoxin proteins. Domains can be switched between delta-endotoxin 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 265:20923-20930 Rang et al. (91999) Appl. Environ. Microbiol. 65:2918-2925 ).
[00050] In an additional embodiment, variant nucleotide and/or amino acid sequences may be obtained by one or more of error-prone PCR, oligonucleotide-directed mutagenesis, PCA ("Polymerase Cycling Assembly") reaction, homologous PCR recombination, mutagenesis in vivo, cassette mutagenesis, recursive ensemble mutagenesis, exponential ensemble mutagenesis, site-specific mutagenesis, gene reassembly, GSSM-type mutagenesis ("gene site saturation mutagenesis" ), permutation mutagenesis, synthetic ligation reassembly (SLR), recombination, recursive sequence recombination, phosphorothioate-modified DNA mutagenesis, uracil-containing template mutagenesis, gapped duplex mutagenesis (gapped duplex mutagenesis "), point mismatch repair mutagenesis, mutagenesis of host strains of deficient parity, chemical mutagenesis, radiogenic mutagenesis, deletion mutagenesis, restriction and selection mutagenesis, restriction and purification mutagenesis, artificial gene synthesis, ensemble mutagenesis, chimeric multimer nucleic acid creation, and the like . vectors
[00051] 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 which is capable of resulting in the expression of a protein from an open reading frame in a plant cell. Typically these contain a promoter and a coding sequence. Often such constructs will also contain a 3' untranslated region. Such constructs may contain a "signal sequence" or a "leader sequence" to facilitate translational or post-translational targeting of the peptide to certain intracellular structures such as the chloroplast (or other plastids), endoplasmic reticulum, or Golgi apparatus.
[00052] By "signal sequence" is meant a sequence that is known or suspected to result in translational or post-translational transport of the peptide across the cell membrane. In eukaryotes, this typically involves secretion into the Golgi apparatus, with some resulting in glycosylation. Bacterial insecticidal toxins are often synthesized as protoxins, which are proteolytically activated in the gut 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 may be derived from a sequence of the invention. By "leader sequence" is meant any sequence which, when translated, results in an amino acid sequence sufficient to initiate targeting during translation of the peptide chain into a subcellular organelle. Thus, this includes leader sequences directed at transport and/or glycosylation by passage into the endoplasmic reticulum, passage into vacuoles, plastids including chloroplasts, mitochondria, and the like.
[00053] By "plant transformation vector" is meant a DNA molecule that is necessary for the efficient transformation of a plant cell. Such a molecule may consist of one or more plant expression cassettes, and may 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 of the 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 5' and 3' regulatory sequences operably linked to a sequence of the invention. By "operably linked" is meant a functional linkage between a promoter and a second sequence, wherein the promoter sequence initiates and mediates 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 coding for the protein, contiguous and in the same open reading frame. The cassette may additionally contain at least one additional gene to be co-transformed in the organism. Alternatively, the additional gene(s) may be provided in multiple expression cassettes.
[00054] "Promoter" refers to a nucleic acid sequence that functions to direct transcription of a downstream coding sequence. The promoter in conjunction with other transcriptional and translational regulatory nucleic acid sequences (also called "control sequences") are required for the expression of a DNA sequence of interest.
[00055] Such an expression cassette is provided with a plurality of restriction sites for insertion of the pesticidal sequence which will be under the transcriptional regulation of regulatory regions.
[00056] The expression cassette will include in the 5'-3' direction of transcription, a transcriptional and translational initiation region (i.e., a promoter), a DNA sequence of the invention, and a transcription and transcription termination region. translation (ie, termination region) functional in plants. The promoter may be native, or analogous, or foreign or heterologous, with respect to the host plant and/or the DNA sequence of the invention. Additionally, the promoter may 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. Where the promoter is "foreign" or "heterologous" to the DNA sequence of the invention, the promoter is intended to be not the native or naturally occurring promoter for the operably linked DNA sequence of the invention.
[00057] The termination region may be native to the transcription initiation region, may be native to the operably linked DNA sequence of interest, may be native to the host plant, or may be derived from another source (i.e., 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. tumefaciens Ti plasmid, such as the octopine synthase and nopaline synthase termination regions. See also Guerineau et al. (1991) Mol. Gen. Gene 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.
[00058] Where appropriate, the gene(s) may 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 enhanced expression, or they can be synthesized using codons at a host cell's preferred codon usage frequency. 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-preferred codon usage. Methods are available in the art for the synthesis of plant-preferred genes. See, for example, U.S. Patents. Nos. 5,380,831 and 5,436,391 and Murray et al. (1989) Nucleic Acids Res. 17:477-498, incorporated herein by reference.
[00059] In one embodiment, the pesticidal sequence is targeted to the chloroplast for expression. Thus, in cases where the pesticidal sequence is not inserted directly into the chloroplast, the expression cassette will additionally contain a nucleic acid encoding a peptide carrier to direct the pesticidal sequence to the chloroplasts. Such carrier peptides are known in the state of 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. Res. common 196:1414-1421; and Shah et al. (1986) Science 233:478-481.
[00060] The pesticide gene to be targeted to the chloroplast can be optimized for expression in the chloroplast to account for differences in codon usage between the plant nucleus and this organelle. In this way, nucleic acids of interest can be synthesized using chloroplast-preferred codons. See, for example, the U.S. patent. No. 5,380,831, incorporated herein by reference. Plant Transformation
[00061] 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 gain 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.
[00062] By "plant" is meant whole plants, plant organs (eg leaves, stems, roots, etc.), seeds, plant cells, propagules, embryos and progeny thereof. Plant cells can be differentiated or undifferentiated (eg callus, cells in suspension culture, protoplasts, leaf cells, root cells, phloem cells, pollen).
[00063] "Transgenic plants" or "transformed plants" or "stably transformed" plants or cells or tissues refer to plants that have incorporated or integrated exogenous nucleic acid sequences or 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. "Heterologous" 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.
[00064] The transgenic plants of the invention express one or more of the pesticidal sequences disclosed herein. In various embodiments, the transgenic plant additionally comprises one or more additional genes for insect resistance, for example one or more additional genes for controlling Coleopteran, Lepidoptera, Heteropteran or nematode pests. One skilled in the art will understand that the transgenic plant can contain any gene that confers an agronomic trait of interest.
[00065] Transformation of plant cells can be performed by one of several techniques known in the prior art. The pesticidal gene of the invention can be modified so as to promote or increase expression in plant cells. Typically a construct expressing such a protein would contain a promoter to drive transcription of the gene, as well as a 3' untranslated region to allow for transcription termination and polyadenylation. The organization of such constructs is well known in the prior art. In some cases, it may be useful to modify the gene such 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 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 intron mRNA processing is required for expression.
[00066] Typically, such a "plant expression cassette" will be introduced into a "plant transformation vector". This plant transformation vector may be comprised of one or more DNA vectors necessary to achieve plant transformation. For example, it is common practice in the art to use plant transformation vectors that are comprised of more than one contiguous DNA segment. 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, where the size and complexity of the DNA segments required to achieve efficient transformation are significantly high, and it is advantageous to separate functions into separate DNA molecules. Binary vectors typically contain a plasmid vector that contains the cis-acting sequences necessary for T-DNA transfer (such as left margin and right margin), 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 so as to allow efficient transfer into plant cells and expression therein. For example, the selectable genetic marker and the pesticide gene are located between the left and right margins. Often, a second plasmid vector contains the trans-acting factors that mediate the transfer of T-DNA from Agrobacterium to plant cells. This plasmid often contains virulence functions (Vir genes) that allow for the infection of plant cells by Agrobacterium, and DNA transfer by cleavage at flanking sequences and vir-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 (eg LBA4404, GV3101, EHA101, EHA105, etc.) can be used for plant transformation. The second plasmid vector is not needed to transform plants by other methods such as microprojection, microinjection, electroporation, polyethylene glycol, etc.
[00067] In general, plant transformation methods involve transferring heterologous DNA into target plant cells (eg immature or mature embryos, suspension cultures, undifferentiated callus, protoplasts, etc.), followed by application a threshold level of appropriate 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 routinely cultured. Subsequently, the transformed cells are differentiated into shoots after being placed in regeneration medium supplemented with a maximum threshold 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 (eg, 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 routinely cultured. A general description of 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 piece of callus or tissue or group of subject target cells. The ability to kill untransformed cells and allow transformed cells to proliferate results in transformed plant cultures. Often, the ability to remove untransformed cells is a limitation to the rapid recovery of transformed plant cells and the successful generation of transgenic plants. .
[00068] Transformation protocols as well as protocols for introducing nucleotide sequences into plants may vary depending on the type of plant or plant cell, ie monocot or dicot, which is the target of the transformation. The generation of transgenic plants can be accomplished by one of several methods, including, but not limited to, microinjection, electroporation, direct gene transfer, introduction of heterologous DNA by Agrobacterium into plant cells (Agrobacterium-mediated transformation), bombardment from plant cells with heterologous foreign DNA attached 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 N° 2002015066), transformation with Lec1, and various other particle-free direct mediation methods to transfer DNA.
[00069] Methods for the transformation of chloroplasts are well known in the prior art. See, for example, Svab et al. (1990) Proc. natl. academy Sci. USA 87:8526-8530; Svab and Maliga (1993) Proc. natl. academy Sci. USA 90:913-917; Svab and Maliga (1993) EMBO J. 12:601-606. The method is based on delivering DNA containing a selection marker with a particle weapon and targeting the DNA to the plastid genome by homologous recombination. Furthermore, plastid transformation can be accomplished by transactivating an inactive plastid transgene by preferential expression in tissue of a plastid-targeted RNA polymerase encoded by nuclear genes. Such a system was presented in McBride et al. (1994) Proc. natl. academy Sci. USA. 91:7301-7305.
[00070] After the integration of heterologous foreign DNA into plant cells, it is then necessary to apply a threshold level of appropriate selection in the medium to kill the untransformed cells and separate and proliferate the putatively transformed cells that survive this selection treatment by transferring regularly to a cool medium. 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 into the genome of the transgenic plant.
[00071] Cells which have been transformed can be grown to form 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 either pollinated with the same transformed strain or different strains, and the resulting hybrid with constitutive expression of the desired phenotypic trait can be identified. Two or more generations may be grown to ensure that expression of the desired phenotypic trait is stably maintained and inherited and then seeds collected to ensure that expression of the desired phenotypic trait has been achieved. Thus, the present invention provides transformed seeds (also referred to as "transgenic seeds") with a nucleotide construct of the invention, e.g., an expression cassette of the invention, stably incorporated into their genome. Plant Transformation Assessment
[00072] 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 various methods such as the analysis of nucleic acids, proteins and metabolites associated with the integrated gene.
[00073] PCR analysis is a rapid method to screen 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, NY). The PCR reaction is performed using oligonucleotide primers specific for the gene of interest or a region of the Agrobacterium vector, etc.
[00074] Plant transformation can be confirmed by Southern blot analysis of genomic DNA (Sambrook and Russell, 2001, supra). In general, total DNA is extracted from the transformant, digested with appropriate restriction enzymes, resolved into a agarose gel and transferred to a nitrocellulose or nylon membrane. The membrane or "blot" is then probed with, for example, a 32P-radiolabeled target DNA fragment to confirm integration of the introduced gene into the plant genome according to standard techniques (Sambrook and Russell, 2001, supra).
[00075] In Northern blot analysis, RNA is isolated from specific tissues of a transformant, fractionated on an agarose formaldehyde gel, and transferred to a nylon filter according to standard procedures that are routinely used in the art (Sambrook and Russell, 2001, supra). The expression of RNA encoded by the pesticide gene is then tested by hybridizing the filter to a radioactive probe derived from a toxin, by methods known in the art (Sambrook and Russell, 2001, supra).
[00076] Western blot, biochemical assays and the like 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 pesticide protein. Pesticide Activity in Plants
[00077] In another aspect of the invention, transgenic plants can be generated that express a toxin that has pesticidal activity. The methods described above can be used by way of example to generate transgenic plants, but the manner in which transgenic plants are generated is not critical to this invention. Methods known or described in the art such as Agrobacterium-mediated transformation, biolistic transformation, and non-particle-mediated methods may be used at the discretion of the experimenter. Plants expressing a pesticidal sequence can be isolated by conventional methods described in the art, for example, by callus transformation, selection of the transformed callus, and regeneration of fertile plants from such transgenic callus. 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.
[00078] Various 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 chloroplast metabolism can also be used as selection markers. For example, genes that provide resistance to plant herbicides such as glyphosate, bromoxynil, or imidazolinone may be particularly useful. Such genes have been reported (Stalker et al. (1985) J. Biol. Chem. (1985) J. Biol. Chem. 263:6310-6314 (bromoxynil resistance nitrilase gene); and Sathasivan et al. (1990) Nucl Acids Res. 18:2188 (imidazolinone resistance gene AHAS) Additionally, the genes disclosed herein are useful as markers for evaluating bacterial or plant cell transformation Methods for detecting the presence of a transgene in a plant , plant organ (e.g., 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 of its pesticidal activity.
[00079] Fertile plants that express a pesticidal sequence can be tested for pesticidal activity, and plants that show optimal activity selected for further breeding. Methods for testing pest activity are available in the art. Generally, the protein is mixed and used in ingestion trials. See, for example, Marrone et al. (1985) J. of Economic Entomology 78:290-293.
[00080] The present invention can be used for the transformation of any plant species, including, but not limited to, monocots and dicots. Examples of plants of interest include, but are not limited to, corn, sorghum, wheat, sunflower, tomato, crucifers, peppers, potatoes, cotton, rice, soybeans, sugar beet, 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.
[00081] Vegetables include, but are not limited to, tomatoes, lettuce, green beans, lima beans, peas, and members of the Curcumis genus such as cucumber, cantaloupe melon, and oak bark melon. 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, crucifers, peppers, potato, cotton, rice, soybean, beet, sugar cane, tobacco, barley, rapeseed, etc.) . Use in Pesticide Control
[00082] General methods are known in the art for using strains comprising a nucleotide sequence of the present invention, or a variant thereof, in pesticidal control or modification of other organisms as pesticidal agents. See, for example, U.S. No. 5,039,523 and EP 0480762A2.
[00083] 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, i.e. unlysed, cells of a toxin (pesticide) producing organism are treated with reagents that prolong the activity of the pesticidal protein produced in the cell when the cell is applied to the pest(s) environment. (saved.
[00084] Alternatively, the pesticide is produced by introducing a pesticide gene into a cellular host. Expression of the pesticide gene results, directly or indirectly, in 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 pesticide produced in the cell when the cell is applied to the environment of the target pest(s). The resulting product retains the toxicity of the pesticidal protein. These naturally encapsulated pesticides can then be formulated according to conventional techniques for application to the environment harboring the target pest, for example, soil, water, and plant foliage. See, for example, EPA 0192319, and the references cited therein. Alternatively, cells expressing a gene of this invention may be formulated to, for example, allow application of the resulting material as a pesticide. pesticidal compositions
[00085] The active ingredients of the present invention are normally applied in the form of compositions and can be applied to the growing area or 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 controlled-release or biodegradable vehicle formulations that allow prolonged dosing of the target area after a single application of the formulation. They may 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 further agriculturally acceptable carriers, surfactants or application-promoting adjuvants. normally employed in the art of formulation. Suitable carriers and adjuvants can be solid or liquid and correspond to substances commonly used in formulation technology, for example natural or regenerated mineral substances, solvents, dispersants, wetting agents, tackifiers, binders or fertilizers. Likewise, the formulations may be prepared into edible "baits" or processed into pest "traps" to allow feeding or ingestion by a target pest of the pesticide formulation.
[00086] Methods for applying an active ingredient of the present invention or an agrochemical composition of the present invention that contains at least one of the pesticidal proteins produced by the bacterial strains of the present invention include leaf application, seed coating and soil application. . The number of applications and the application rate depend on the intensity of infestation by the corresponding pest.
[00087] The composition may be formulated in the form of a powder, dust, grain, granule, spray, emulsion, colloid, solution or the like, and may 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 such compositions which contain at least one of these pesticidal polypeptides, the polypeptide may be present in a concentration of from about 1% to about 99% by weight.
[00088] Lepidoptera, dipteran, heteropteran or coleopteran pests 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 into contact with, a pesticidally effective amount of the polypeptide. By "pesticidally effective amount" is meant an amount of pesticide that is capable of causing the death of at least one pest, or of significantly reducing the pest's growth, feeding, or normal physiological development. This amount will vary depending on factors such as, for example, the specific target pests to be controlled, the specific environment, location, plant, crop, or agricultural site being treated, environmental conditions, and the method, rate, concentration, stability and amount of application of the polypeptide composition with effective pesticidal activity. The formulations may also vary with respect to climatic conditions, environmental considerations and/or frequency of application and/or severity of pest infestation.
[00089] The described pesticidal compositions can be prepared by formulating either the bacterial cells, suspension of crystals and/or spores, or the isolated protein component with the desired agriculturally acceptable carrier. The compositions may be formulated prior to administration by an appropriate means such as lyophilized, dried, or in a suitable aqueous vehicle, medium or diluent, such as saline or other buffer. The formulated compositions may 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 material. vehicle suitable for agricultural application. Carriers suitable for agricultural application can be solid or liquid and are well known in the art. The term "acceptable carrier for agricultural application" covers all adjuvants, inert components, dispersants, surfactants, tackifiers, binders, etc. which are commonly used in pesticide formulation technology; these are well known to those skilled in pesticide formulation. The formulations may be mixed with one or more solid or liquid adjuvants and prepared by various means, for example, by mixing, blending and/or milling the pesticidal composition with suitable adjuvants using conventional formulation techniques. Suitable formulations and application methods are described in the U.S. patent. No. 6,468,523, incorporated herein by reference.
[00090] Plants may 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, Fluazifop, Glufosinate, Halosulfuron Gowan, Paraquat, Propizamide, Sethoxidime, Butaphenacyl, Halosulfuron, Indaziflame; Fruit/Vegetable Insecticides: Aldicarb, Bacillus thuringiensis, Carbaryl, Carbofuran, Chlorpyrifos, Cypermethrin, Deltamethrin, Diazinon, Malathion, Abamectin, Cyfluthrin/beta-cyfluthrin, Sfenvalerate, Lambda-cyhalothrin, Acequinocil, Biphenazate, Methoxyfenozide, Novaluron, Chromafenozide , Tiacloprid, Dinotefuron, Fluacripyrim, Tolfenpirad, Clothianidin, Spirodiclofen, Gamma-cyhalothrin, Spiromesifen, Spinosad, Rinaxipir, Ciazipyr, Spinoteram, Triflumuron, Spirotetramate, Imidacloprid, Flubendiamide, Thiodicarb, Metaflumizone, Sulfoxaflor, Cyflumethophen, Cyanopyrafen, Imidacloprid, Thiamethian , Spinotoram, Thiodicarb, Flonicamid, Methiocarb, Emamectin Benzoate, Indoxacarb, Fozthiazate, Fenamiphos, Cadusaphos, Pyriproxyfen, Fenbutatin Oxide, Hexthiazox, Methomyl, 4-[[(6-Chloropyridin-3-yl)methyl](2,2 -difluoroethyl)amino]furan-2(5H)-one; Fungicides for Fruits/Vegetables and Vegetables:
[00091] Carbendazime, Chlorothalonil, EBDCs, Sulfur, Thiophanate-methyl, Azoxystrobin, Cymoxanil, Fluazinam, Fosetyl, Iprodione, Kresoxime-methyl, Metalaxyl/mefenoxam, Trifloxystrobin, Ethaboxam, Iprovalicarb, Trifloxystrobin, Phenhexamide, Oxpoconazole Fumarate, Cyazofamide, Phenamidone , Zoxamide, Picoxystrobin, Pyraclostrobin, Cyflufenamide, Boscalide; Cereal Herbicides: Isoproturon, Bromoxynil, Ioxynil, Phenoxys, Chlorosulfuron, Clodinafop, Diclofop, Diflufenican, Fenoxaprop, Florasulam, Fluroxypyr, Metsulfuron, Triasulfuron, Flucarbazone, Iodosulfuron, Propoxycarbazone, Picolinafen, Mesosulfuron, Beflubutamide, Pinoxaden, Amidosulfuron, Trifenuron, Flupyrsulfuron, Sulfosulfuron, Pyrasulfotole, Pyroxsulam, Flufenacet, Tralkoxydime, Pyroxasulfone; Cereal Fungicides: Carbendazime, Chlorothalonil, Azoxystrobin, Epoxiconazole, Trifloxystrobin, Dimoxystrobin, Cyproconazole, Kresoxime-methyl, Simeconazole, Prothioconazole, Cyprodinil, Quinoxyfen, Picoxystrobin, Fluoxastrobin; Phenpropimorph, Tebuconazole, Pyraclostrobin, Cereal Insecticides: Dimethoate, Lambda-cialthrin , Deltamethrin, alpha-Cypermethrin, β-Cyfluthrin, Bifenthrin, Imidacloprid, Clothianidin, Thiamethoxam, Thiacloprid, Acetamiprid, Dinetofuron, Chlorphyrifs, Methamidophos, Oxidemeton-methyl, Pyrimicarb, Methiocarb; Herbicides for Corn: Atrazine, Alachlor, Bromoxynil, Acetochlor, Dicamba, Clopyralide, (S-)Dimethenamid, Glufosinate, Glyphosate, Isoxaflutole, (S-)Metolachlor, Mesotrione, Nicosulfuron, Primisulfuron, Rimsulfuron, Sulcotrione,Foramsulfuron, Topramezone, Tembotrione, Saflufenacil, Thiencarbazone, Flufenacet, Pyroxasulfone; Insecticides for Corn: Carbofuran, Chlorpyrifos, Bifenthrin, Fipronil, Imidacloprid, Lambda-Cyhalothrin, Tefluthrin, Terbufos, Thiamethoxam, Clothianidin, Spiromesifen, Flubendiamide, Triflumuron, Rinaxipyr, Deltamethrin, Thiodicarb, β-Cyfluthrin, Cypermethrin, Bifenthrin, Lufenuron, Triflumoron, Tefluthrin, Tebupyrimphos, Ethiprole, Ciazipyr, Thiacloprid, Acetamiprid, Dinetofuran, Avermectin, Methiocarb, Spirodiclofen, Spirotetramate; Corn Fungicides: Phenitropane, Thiram, Prothioconazole, Tebuconazole, Trifloxystrobin; Rice Herbicides: Butachlor, Propanil, Azimsulfuron, Bensulfuron, Cyalofop, Daimuron, Fentrazamide, Imazosulfuron, Mephenacet, Oxaziclomefon, Pyrazosulfuron, Pyributicarb, Quinchlorac, Thiobencarb, Indaphane, Flufenacet, Fentrazamide, Halosulfuron, Oxaziclomefon, Benzosulfuron, Pyributicarb, Quinchlorac, Thiobencarb, Indaphane, Flufenacet, Fentrazamide, Halosulfuron, Oxaziclomefon, Benzobicyclome, Pyrifhalide, Pen, Pyxpyribac Oxadiargyl, Ethoxysulfuron, Pretilachlor, Mesotrione, Tefuryltrione, Oxadiazone, Fenoxaprop, Pyrimisulfan; Insecticides for Rice: Diazinon, Phenitrothione, Fenobucarb, Monocrotophos, Benfuracarb, Buprofezin, Dinotefuran, Fipronil, Imidacloprid, Isoprocarb, Thiacloprid, Chromafenozide, Thiacloprid, Dinotefuran, Clothianidin, Ethiprole, Flubendiamide, Rinaxipyr, Deltamethrin, Acetamiprid, Thiamethoxam, Spinozadezide, Spinotoram, Emamectin-Benzoate, Cypermethrin, Chlorpyrifos, Cartap, Methamidophos, Etofenprox, Triazophos, 4-[[(6-Chloropyridin-3-yl)methyl](2,2-difluoroethyl)amino]furan-2(5H)-one , Carbofuron, Benfuracarb; Rice Fungicides: Thiophanate-methyl, Azoxystrobin, Carpropamide, Edifenphos, Ferimzone, Iprobenphos, Isoprothiolane, Pencicuron, Probenazole, Pyroquilone, Tricyclazole, Trifloxystrobin, Diclocymet, Phenoxanil, Simeconazole, Thiadinil; Cotton Herbicides: Diuron, Fluometuron, MSMA, Oxyfluorfen, Promethrin, Trifluralin, Carfentrazone, Clethodime, Fluazifop-butyl, Glyphosate, Norflurazone, Pendimethalin, Pyrithiobac-sodium, Trifloxysulfuron, Tepraloxidime, Glufosinate, Flumioxazin, Thidiazuron; Cotton Insecticides: Acephate, Aldicarb, Chlorpyrifos, Cypermethrin, Deltamethrin, Malathion, Monocrotophos, Abamectin, Acetamiprid, Emamectin Benzoate, Imidacloprid, Indoxacarb, Lambda-Cyhalothrin, Spinosad, Thiocarb, Gamma-cyhalothrin, Spiromesifen, Pyridalyl, Flonicamide, Flubendiamide, Triflumuron, Rinaxipir, beta-cyfluthrin, Spirotetramate,
[00092] Clothianidin, Thiamethoxam, Thiacloprid, Dinetofuran, Flubendiamide, Ciazipyr, Spinosad, Spinotoram, gamma Cyhalothrin, 4-[[(6-Chloropyridin-3-yl)methyl](2,2-difluoroethyl)amino]furan-2( 5H)-one, Thiocarb, Avermectin, Flonicamid, Pyridalyl, Spiromesifen, Sulfoxaflor, Profenofos, Triazophos, Endosulfan; Cotton Fungicides: Etridiazole, Metalaxyl, Quintozene; Soy Herbicides: Alachlor, Bentazone, Trifluralin, Chlorimuron-Ethyl, Chloransulam-Methyl, Fenoxaprop, Fomesafene, Fluazifop, Glyphosate, Imazamox, Imazaquin, Imazetapyr, (S-)Metolachlor, Metribuzin, Pendimethalin, Tepraloxydime, Glufosinate; Soy insecticides: Lambda-cyhalothrin, Methomyl, Parathion, Thiocarb, Imidacloprid, Clothianidin, Thiamethoxam, Thiamethoxam, Thiacloprid, Acetamiprid, Dinetofuran, Flubendiamide, Rinaxipir, Ciazipyr, Spinosad, Spinotoram, Emamectin Benzoate, Fipronil, Ethiprole, Deltamethrin, β-Cyfluthrin, gamma and lambda Cyhalothrin, 4-[[(6-Chloropyridin-3-yl)methyl](2,2-difluoroethyl)amino]furan-2(5H)-one, Spirotetramate, Spinodiclofen, Triflumuron, Flonicamide, Thiodicarb, beta- Cyfluthrin; Soy Fungicides: Azoxystrobin, Cyproconazole, Epoxiconazole, Flutriafole, Pyraclostrobin, Tebuconazole, Trifloxystrobin, Prothioconazole, Tetraconazole; Beet Herbicides: Chloridazone, Desmedipham, Ethofumesate, Phenmedipham, Trialate, Clopyralid, Fluazifop, Lenacil, Metamitron, Quinmerac, Cycloxydim, Triflusulfuron, Tepraloxidim, Kizalofop; Beet Insecticides: Imidacloprid, Clothianidin, Thiamethoxam, Thiacloprid, Acetamiprid, Dinetofuran, Deltamethrin, β-Cyfluthrin, gamma/lambda Cyhalothrin, 4-[[(6-Chloropyridin-3-yl)methyl](2,2-difluoroethyl)amino ]furan-2(5H)-one, Tefluthrin, Rinaxipyr, Cyaxipyr, Fipronil, Carbofuron; Herbicides for Canola: Clopyralid, Diclofop, Fluazifop, Glufosinate, Glyphosate, Metazachlor, Trifluralin Ethametsulfuron, Quinmerac, Kizalofop, Clethodime, Tepraloxidim; Canola Fungicides: Azoxystrobin, Carbendazim, Fludioxonil, Iprodione, Prochloraz, Vinclozolin; Insecticides for Canola:
4- [[(6-Chloropyridin-3-yl)methyl](2,2-difluoroethyl)amino]furan-2(5H)-one.
[00094] "Pest" includes, but is not limited to, insects, fungi, bacteria, nematodes, mites, ticks, and the like. Insect pests include selected insects from the orders Coleoptera, Diptera, Hymenoptera, Lepidoptera, Mallophaga, Homoptera, Hemiptera, Orthroptera, Thysanoptera, Dermaptera, Isoptera, Anoplura, Siphonaptera, Trichoptera, etc., particularly Coleoptera, Lepidoptera and Diptera.
[00095] 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, Mordelloidea, Tenebrionoidea, Bostrichoidea, Scarabaeoidea, Cerambycoidea, and Curryionsomeoidea. . The superfamily Caraboidea includes the families Cicindelidae, Carabidae and Dytiscidae.The superfamily Gyrinoidea includes the family Gyrinidae.The superfamily Hydrophiloidea includes the family Hydrophilidae.The superfamily Staphylinoidea includes the families Silphidae and Staphylinidae.The superfamily Cantharoidea includes the families Cantharidae and Lampyridae.The superfamily Cleroidea includes the families Cleridae and Dermestidae.The superfamily Elateroidea 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 superfamily Scarabaeoidea includes the families Passalidae and Scarabaeidae. The superfamily Cerambycoidea includes the family Cerambycidae. The superfamily Chrysomeloidea includes the family Chrysomelidae. The superfamily Curculionoidea includes the families Curculionidae and Scolytidae.
[00096] 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 suborder Cychlorrhapha includes the divisions Aschiza and Aschiza.The division Aschiza includes the families Phoridae, Syrphidae, and Conopidae. .The Acalyptratae section includes the families Ottidae, Tephritidae, Agromyzidae and Drosophilidae. The Calyptratae section includes the families Hippoboscidae, Oestridae, Tachinidae, Anthomyiidae, Muscidae, Calliphoridae and Sarcophagidae.
[00097] The order Lepidoptera includes the families Papilionidae, Pieridae, Lycaenidae, Nymphalidae, Danaidae, Satyridae, Hesperiidae, Sphingidae, Saturniidae, Geometridae, Arctiidae, Noctuidae, Lymantriidae, Sesiidae and Tineidae.
[00098] Nematodes include parasitic nematodes such as root nodule, cyst and lesion nematodes, including Heterodera spp., Meloidogyne spp. and Globodera spp.; particularly members of the cyst nematode, including, but not limited to, Heterodera glycines (soybean cyst nematode); Heterodera schachtii (beet cyst nematode); Heterodera avenae (cereal cyst nematode); and Globodera rostochiensis and Globodera pailida (potato cyst nematode). Wound nematodes include Pratylenchus spp., eg Pratylenchus penetrans.
[00099] Insect pests of the invention for major crops include: Corn: Ostrinia nubilalis, European corn borer; Agrotis ipsilon, black agrotis; Helicoverpa zea, corn gordium; Spodoptera frugiperda, fall armyworm; Diatraea grandiosella, southwestern corn borer; Elasmopalpus lignosellus, corn cane borer; Diatraea saccharalis, sugar cane borer; Diabrotica virgifera, western pinworm; Diabrotica longicornis barberi, northern pinworm larva; Diabrotica undecimpunctata howardi, southern pinworm; Melanotus spp., elaterid larvae; 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, grass bug; Melanoplus femurrubrum, red-legged grasshopper; Melanoplus sanguinipes, migratory grasshopper; Hylemya platura, maize seed fly larvae; Agromyza parvicornis, corn leafminer; Anaphothrips obscrurus, herbs thrips; Solenopsis milesta, thief ant; Tetranychus urticae, two-spotted spider mite; Sorghum: Chilo partellus, sorghum borer; Spodoptera frugiperda, fall armyworm; Helicoverpa zea, corn gordium; Elasmopalpus lignosellus, corn cane borer; Underground feltia, thread; Phyllophaga crinita, coró; 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, grass bug; Contarinia sorghicola, sorghum fly; Tetranychus cinnabarinus, red mite; Tetranychus urticae, two-spotted spider mite; Wheat: Pseudaletia unipunctata, military caterpillar; Spodoptera frugiperda, fall armyworm; Elasmopalpus lignosellus, lesser cornstalk borer; Agrotis orthogonia, western cutworm; Elasmopalpus lignosellus, corn cane borer; Oulema melanopus, cereal leaf beetle; Hypera punctata, tree leaf weevil; Diabrotica undecimpunctata howardi, southern pinworm; Russian wheat aphid; Schizaphis graminum, green aphid; Macrosiphum avenae, ear aphid; Melanoplus femurrubrum, red-legged grasshopper; Melanoplus differentialis, differential locust; Melanoplus sanguinipes, migratory grasshopper; Mayetiola destructor, Hessian fly; Sitodiplosis mosellana, wheat mosquito; American Meromyza, wheat stalk larva; Hylemya coarctata, wheat bulb fly; Frankliniella fusca, tobacco thrips; Cephus cinctus, wheat stalk hornet; Aceria tulipae, Blossom mite; Sunflower: Suleima helianthana, sunflower seedling 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, fall armyworm; Pectinophora gossypiella, pink cotton bollworm; Anthonomus grandis, cotton boll weevil; Aphis gossypii, cotton aphid; Pseudatomoscelis seriatus, cotton flea; Trialeurodes abutilonea, whitefly; Lygus lineolaris, bed bug; Melanoplus femurrubrum, red-legged grasshopper; Melanoplus differentialis, differential locust; Thrips tabaci, onion thrips; Franklinkiella fusca, tobacco thrips; Tetranychus cinnabarinus, red mite; Tetranychus urticae, two-spotted spider mite; Rice: Diatraea saccharalis, sugar cane borer; Spodoptera frugiperda, military caterpillar; Helicoverpa zea, corn gordium; Colaspis brunnea, grape beetle; Lissorhoptrus oryzophilus, North American rootworm; Sitophilus oryzae, rice weevil; Nephotettix nigropictus, rice leafhopper; Blissus leucopterus leucopterus, grass bug; Acrosternum hilare, green bed bug; Soybean: Pseudoplusia includens, false caterpillar; Anticarsia gemmatalis, soybean caterpillar; Plathypena scabra, green clover larva; Ostrinia nubilalis, European corn borer; Agrotis ipsilon, threaded caterpillar; Spodoptera exigua, fall armyworm; 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 bed bug; Melanoplus femurrubrum, red-legged grasshopper; Melanoplus differentialis, differential locust; Hylemya platura, maize seed fly larvae; Sericothrips variabilis, soybean thrips; Thrips tabaci, onion thrips; Tetranychus turkestani, strawberry mite; Tetranychus urticae, two-spotted spider mite; Barley: Ostrinia nubilalis, European corn borer; Agrotis ipsilon, black agrotis; Schizaphis graminum, green aphid; Blissus leucopterus leucopterus, grass bug; Acrosternum hilare, green bed bug; Euschistus servus, Neartic brown stink bug; Delia platura, maize 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., root larvae.Methods to increase plant yield
[000100] Methods for increasing plant yield are provided. The methods comprise providing a plant or plant cell that expresses a polynucleotide encoding the pesticidal polypeptide sequence disclosed herein, and growing the plant or seed thereof in a field infested with a pest against which said polypeptide has pesticidal activity. In some embodiments, the polypeptide has pesticidal activity against a lepidopteran, coleopteran, dipteran, hemipteran or nematode pest, and said field is infested with a lepidopteran, coleopteran, dipteran, hemipterans or nematodes.
[000101] As defined herein, plant "yield" refers to the quality and/or quantity of biomass produced by the plant. By "biomass" is meant any measured plant product. An increase in biomass production is any improvement in measured plant product yield. Increasing plant yield has several commercial applications. For example, increasing the leaf biomass of the plant can increase the yield of leafy vegetables for human or animal consumption. In addition, increasing the leaf biomass can be used to increase the production of pharmaceutical or industrial products derived from the plant. An increase in yield 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 a 20%, at least a 30%, at least 50%, at least 70%, at least 100% or greater increase in yield compared to a plant not expressing the pesticide sequence.
[000102] In specific methods, plant yield is increased as a result of an improvement in pest resistance of the plant expressing a pesticidal protein disclosed herein. The expression of the pesticidal protein results in a reduced ability of a pest to infest or feed on the plant, thus improving plant yield.
[000103] The following examples are presented for purposes of illustration and not by way of limitation.EXPERIMENTALExample 1. Discovery of new pesticidal genes from Bacillus thuringiensis with homology to pesticidal genes
[000104] Novel pesticidal genes were identified from the bacterial strains listed in Table 1 through the following steps:• Preparation of extrachromosomal DNA from the strain, including plasmids that typically carry delta-endotoxin genes.• Mechanical cutting of extrachromosomal DNA to generate fragments with a length distribution.• Cloning of fragments of approximately 2 kb to 10 kb of extrachromosomal DNA.• Multiplication of approximately 1500 extrachromosomal DNA clones.• Partial sequencing of the 1500 clones using specific primers for the cloning vector (reading of the ends).• Identification of putative toxin genes by homology analysis by the MiDAS method (as described in US Patent Publication No. 20040014091, which is hereby fully incorporated by reference).• Acquisition of unknown sequences (by "sequence walking") from clones containing fragments of the putative toxin genes of interest.


1pair with axmi2012pair with axmi2003pair with axmi2144pair with axmi2135By analyzing the sequence of ATX24031, two overlapping open reading frames (ORF) were identified, each having homology to endotoxin-like genes. Upon inspection of these ORFs and the proteins they code for, it became apparent that these two ORFs likely originated from a single ORF that underwent a single nucleotide insertion (or larger insertion creating a reading frame shift due to a single nucleotide) into the region comprised between nucleotides 224 and 309 from the beginning of the first ORF. These ORFs are designated herein as ATX24031_contig4_orf1 (SEQ ID NO:63) and ATX424031_contig4_orf2 (SEQ ID NO:64). The complete sequence is disclosed in SEQ ID NO:65. A composite ORF that has homology to endotoxins along its entire length can be assembled by "arranging" the insert to create a single ORF. While it is understood that multiple solutions could be developed to provide such an ORF, and that these solutions would differ in terms of the region of overlap between the ORFs, one solution is provided here, which is designated as axmi196(SEQ ID NO:7 ).6 A gene similar to p19/CryBP1 was identified immediately upstream of axmi211. The nucleotide sequence of this gene is disclosed in SEQ ID NO:66 and the amino acid sequence is disclosed in SEQ ID NO:67.6 A p19-like gene was identified immediately upstream of axmi211. The nucleotide sequence of this gene is disclosed in SEQ ID NO:68 and the amino acid sequence is disclosed in SEQ ID NO:69.Example 2. Expression in Bacillus
[000105] The pesticidal gene disclosed herein is amplified by PCR from pAX980 and the product of the PCR reaction is cloned into the Bacillus expression vector pAX916, or other 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 Bactocasitone; 3 g/l yeast extract; 6 g/l of KH2PO4; 14 g/l K2HPO4; MgSO4 0.5 mM; MnCl2 0.05 mM; FeSO4 0.05 mM) until spore formation is evident by microscopic analysis. Samples are prepared and tested for activity in bioassays.Example 3. Insecticidal activity of Axmi-191 and Axmi-192Gene expression and purification
[000106] The DNA regions encoding the toxin domains of axmi-191 and axmi-192 were cloned separately into an E. coli expression vector pMAL-C4x behind the malE gene encoding the maltose-binding protein ( MBP). These fusions, which are in the same reading frame, resulted in the expression of MBP-Axmi fusion proteins in E. coli.
[000107] For expression in E.coli, BL21*DE3 were transformed with individual plasmids. An isolated colony was inoculated into LB supplemented with carbenicillin and glucose, and grown overnight at 37°C. The following day, fresh medium was inoculated with 1% of the overnight culture and grown at 37°C to log phase. Subsequently, cultures were induced with 0.3 mM IPTG overnight at 20°C. Each cell pellet was resuspended in 20 mM Tris-Cl buffer, pH 7.4 + 200 mM NaCl + 1 mM DTT + protease inhibitors, and sonicated. An SDS-PAGE analysis confirmed the expression of fusion proteins.
[000108] Total cell-free extracts were passed through an amylose column linked to an FPLC for affinity purification of the MBP-axmi fusion proteins. The resin-bound fusion protein was eluted with 10 mM maltose solution. The purified proteins were then cleaved with factor Xa to remove the amino terminal MBP tag from the Axmi protein. Cleavage and solubility of the proteins were determined by SDS-PAGE. Bioassays with insects
[000109] Cleaved proteins were tested in insect assays with appropriate controls. A 5-day reading of the plates showed activity of Axmi191 and Axmi192 against the diamondback moth species. Axmi191 led to inhibition of growth and Axmi192 led to more severe inhibition of growth and mortality by 100%.Example 4. Construction of synthetic sequences
[000110] In one aspect of the invention, synthetic toxin sequences are generated. These synthetic sequences have an altered DNA sequence from the sequence of the parent toxin, and code for a protein that is collinear to the corresponding parent toxin protein, but which lacks the C-terminal "crystal domain" that is present in many proteins. of delta-endotoxin.
[000111] In another aspect of the invention, modified versions of the synthetic genes are designed such that the resulting peptide is targeted to a plant organelle, such as the endoplasmic reticulum or the apoplast. Peptide sequences known to result in targeting of fusion proteins to plant organelles are known in the art. For example, the N-terminal region of the acid phosphatase gene from White LupinLupinus albus (Genebank ID GI:14276838; Miller et al. (2001) Plant Physiology 127:594-606) is known in the art to result in heterologous protein targeting. to the endoplasmic reticulum. If the resulting fusion protein also has a sequence for retention in the endoplasmic reticulum that comprises the peptide N-terminal lysine-aspartic acid-glutamic acid-leucine (i.e., the "KDEL" motif (SEQ ID NO: 70) at the C-terminus -terminal, the fusion protein will target the endoplasmic reticulum.If the fusion protein does not have an endoplasmic reticulum targeting sequence at the C-terminal end, the protein will target the endoplasmic reticulum, but will eventually be sequestered in the apoplast. Example 5. Tests for pesticidal activity
[000112] The ability of a pesticide protein to act as a pesticide on a pest is often assessed in several ways. One way well known in the art is to perform an ingestion test. In one of these ingestion tests, the pest is exposed to a sample containing either the compounds to be tested, or control samples. This is often accomplished by placing the material to be tested, or a suitable dilution of such material in a material that the pest will ingest, such as an artificial diet. The material to be tested may 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 cup, a plate, or a well of a microplate.
[000113] Testing for sucking pests (e.g. aphids) may involve separating the test material from the insect by means of a partition, ideally a portion that can be pierced by the sucking parts of the sucking insect's mouth, to allow ingestion of the material topping up. Test material is often mixed with an ingestion enhancer, such as sucrose, to promote ingestion of the test compound.
[000114] Other types of tests may include microinjection of the material to be tested in the mouth or intestine of the pest, as well as the development of transgenic plants, followed by a test for the ability of the pest to feed on the transgenic plant. Plant testing may involve isolating the parts of plants normally consumed, for example small cages attached to a leaf, or isolating whole plants in cages containing insects.
[000115] Other methods and approaches for testing pests are known in the art, and can be found, for example, in Robertson, J.L. and H.K. Preisler. 1992. Pesticide bioassays with arthropods. CRC, Boca Raton, FL. Alternatively, assays are commonly described in the journals "Arthropod Management Tests" and "Journal of Economic Entomology" or discussed with members of the "Entomological Society of America" (ESA).Example 6. Expression of pMal fusion proteins
[000116] For expression in E. coli, select genes of the invention were cloned into a pMal expression vector (New England Biolabs) so that the protein was expressed in E. coli fused to the maltose binding protein (MBP) at its end N-terminal. A nucleotide sequence encoding a truncated variant of Axmi207 (corresponding to positions 15 to 647 of SEQ ID NO:47) was used in bioactivity tests. The sequence of the truncated variant of Axmi207 is disclosed in SEQ ID NO:62. In the case of Axmi196, Axmi204 and Axmi209, the complete native sequence was used.
[000117] These fusion proteins were then purified by affinity chromatography as is known in the prior art. The purified proteins were then protease cleaved as is known in the art to separate MBP from the protein of the invention. The resulting proteins were then tested in bioassays against selected pests. The results are shown in table 2.

DBM - diamondback moth CPB - potato beetle SWCB - southwestern corn borer VBC - soybean caterpillarECB - European corn borer Hz - Helicoverpa zeaFAW - fall armyworm SCB - sugar cane borer SCN - cereal cyst nematodeExample 7. Gene placement of toxin of the invention in a Vector for expression in plants
[000118] Each of the coding regions of the genes of the invention is independently linked with promoter and terminator sequences appropriate for expression in plants. Such sequences are well known in the art and may include the rice actin promoter or the maize ubiquitin promoter for expression in monocots, the UBQ3 promoter or the Arabidopsis CaMV 35S promoter for expression in dicots, and the nos or PinII terminators. Techniques for producing and confirming promoter-gene-terminator constructs are also well known in the art.Example 8. Transformation of the genes of the invention in plant cells by Agrobacterium-mediated transformation
[000119] Ears are harvested 8-12 days after pollination. Embryos are isolated from the spikes, and these embryos with a size of 0.8-1.5 mm are used for transformation. Embryos are plated scutellum facing up in a suitable incubation medium, and incubated overnight at 25°C in the dark. However, it is not necessary per se to incubate the embryos overnight. Embryos are contacted with an Agrobacterium strain containing the appropriate vectors for Ti-plasmid-mediated transfer 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 recovery period medium for 5 days (at 25°C in the dark). Explants are incubated in 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 maturation medium, until the formation of mature somatic embryos is observed. The resulting mature somatic embryos are then placed under 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.Example 9. Transformation of Corn Cells with the Toxin Genes of the Invention
[000120] Ears are harvested 8-12 days after pollination. Embryos are isolated from the spikes, and these embryos with a size of 0.8-1.5 mm are used for transformation. Embryos are plated with the scutellum facing up in a suitable incubation medium, such as DN62A5S medium (3.98 g/l N6 salts; 1 ml/l (1000x stock solution) N6 vitamins; 800 mg/l L-Asparagine; 100 mg/l of myo-inositol; 1.4 g/l of L-Proline; 100 mg/l of casamino acids; 50 g/l of sucrose; 1 ml/l (of stock solution at 1 mg/l ml) of 2,4-D), and incubated overnight at 25°C in the dark.
[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).
[000122] DNA constructs designed to express the genes of the invention in plant cells are accelerated into plant tissue using an aerosol accelerator, using conditions essentially as described in PCT Publication No. WO/0138514. After bombardment, embryos are incubated for 30 min in osmotic medium, then placed in incubation medium overnight at 25°C in the dark. To avoid unduly damaged bombed explants, they are incubated for at least 24 hours prior to transfer to recovery medium. The embryos are then spread on a recovery period medium for 5 days at 25°C in the dark and then transferred to a selection medium. Explants are incubated in 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 maturation medium, until the formation of mature somatic embryos is observed. The resulting mature somatic embryos are then placed under 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 Medium DN62A5S

[000123] Adjust the pH of the solution to pH 5.8 with 1 N KOH/1 N KCl, add Gelrite (Sigma) at 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). This recipe makes about 20 plates.
[000124] All publications and patent applications mentioned in the specification are indicative of the skill level of those skilled in the art to which this invention pertains. All publications and patent applications are hereby incorporated by reference to the same extent as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference.
[000125] While the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be obvious that certain changes and modifications may be practiced within the scope of the appended claims.

权利要求:
Claims (13)
[0001]
1. An isolated recombinant nucleic acid molecule, characterized in that it comprises a nucleotide sequence operably linked to a heterologous promoter, wherein said nucleotide sequence is selected from the group consisting of: (a) the nucleotide sequence of SEQ ID NO : 7; and (b) a nucleotide sequence of SEQ ID NO: 7 and degenerate sequences thereof that encode a polypeptide consisting of the amino acid sequence of SEQ ID NO: 34 or 35.
[0002]
2. Isolated recombinant nucleic acid molecule, according to claim 1, characterized in that said nucleotide sequence is a synthetic sequence that was designed for expression in a plant.
[0003]
3. Isolated recombinant nucleic acid molecule, according to claim 1, characterized in that said heterologous promoter is a promoter capable of directing the expression of said nucleotide sequence in a plant cell.
[0004]
4. Isolated expression cassette, characterized by the fact that it comprises the recombinant nucleotide sequence, as defined in claim 3.
[0005]
5. Isolated expression cassette, according to claim 4, characterized in that it further comprises a nucleic acid molecule that encodes a heterologous polypeptide.
[0006]
6. Isolated bacterial host cell, characterized in that it contains the expression cassette, as defined in claim 4.
[0007]
7. An isolated recombinant polypeptide with pesticidal activity, characterized in that it is selected from the group consisting of: (a) a polypeptide consisting of the amino acid sequence of SEQ ID NO: 34 or 35; (b) a polypeptide comprising an amino acid sequence of SEQ ID NO: 34 or 35, wherein said amino acid sequence has pesticidal activity; and (c) a polypeptide that is encoded by the nucleotide sequence of SEQ ID NO: 7; wherein said polypeptide further comprises heterologous amino acid sequences.
[0008]
8. Composition, characterized in that it comprises the polypeptide, as defined in claim 7, wherein the composition is selected from the group consisting of a powder, dust, grain, granule, spray, emulsion, colloid and solution.
[0009]
9. Composition according to claim 8, characterized in that it is prepared by desiccation, lyophilization, homogenization, extraction, filtration, centrifugation, sedimentation or concentration of a Bacillus thuringiensis cell culture.
[0010]
10. Composition, according to claim 8, characterized in that it comprises from 1% to 99% by weight of said polypeptide.
[0011]
11. Method for controlling a population of lepidopteran, coleopteran or nematode pests, characterized in that it comprises placing said population in contact with a pesticidally effective amount of the polypeptide, as defined in claim 7, wherein said lepidopterans , Coleoptera or nematodes are selected from the group consisting of: soybean caterpillar, Helicoverpa zea, the cereal cyst nematode, C. elegans, Pratylenchus and Pene-trans.
[0012]
12. Method for killing a lepidopteran, coleopteran or nematode pest, characterized in that it comprises bringing said pest into contact with or feeding said pest with a pesticidally effective amount of the polypeptide, as defined in claim 7, wherein said Lepidoptera, Coleoptera or nematodes are selected from the group consisting of: soybean caterpillar, Helicoverpa zea, the cereal cyst nematode, C. elegans, Pratylenchus and Pene-trans.
[0013]
13. Method for producing a polypeptide with pesticidal activity, characterized in that it comprises culturing the host cell, as defined in claim 6, under conditions in which the nucleic acid molecule encoding the polypeptide is expressed.

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WO2021076455A1|2019-10-14|2021-04-22|BASF Agricultural Solutions Seed US LLC|Novel insect resistant genes and methods of use|

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WO2021168309A1|2020-02-21|2021-08-26|BASF Agricultural Solutions Seed US LLC|Toxin gene and methods for its use|

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WO2022015619A2|2020-07-14|2022-01-20|Pioneer Hi-Bred International, Inc.|Insecticidal proteins and methods for their use|

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CN112210506B|2020-07-17|2021-05-28|江西省农业科学院农业应用微生物研究所（江西省农村能源研究中心）|Bacillus thuringiensis JXBT-0350 with insecticidal activity on ganoderma lucidum and rice moth and application thereof|

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

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2018-10-16| B07D| Technical examination (opinion) related to article 229 of industrial property law [chapter 7.4 patent gazette]|Free format text: DE ACORDO COM O ARTIGO 229-C DA LEI NO 10196/2001, QUE MODIFICOU A LEI NO 9279/96, A CONCESSAO DA PATENTE ESTA CONDICIONADA A ANUENCIA PREVIA DA ANVISA. CONSIDERANDO A APROVACAO DOS TERMOS DO PARECER NO 337/PGF/EA/2010, BEM COMO A PORTARIA INTERMINISTERIAL NO 1065 DE 24/05/2012, ENCAMINHA-SE O PRESENTE PEDIDO PARA AS PROVIDENCIAS CABIVEIS. |

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2019-12-03| B07G| Grant request does not fulfill article 229-c lpi (prior consent of anvisa) [chapter 7.7 patent gazette]|

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2020-02-27| B06U| Preliminary requirement: requests with searches performed by other patent offices: procedure suspended [chapter 6.21 patent gazette]|

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2021-07-06| B06A| Patent application procedure suspended [chapter 6.1 patent gazette]|

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2021-11-30| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|

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2022-02-01| B16A| Patent or certificate of addition of invention granted [chapter 16.1 patent gazette]|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 30/07/2010, OBSERVADAS AS CONDICOES LEGAIS. PATENTE CONCEDIDA CONFORME ADI 5.529/DF, QUE DETERMINA A ALTERACAO DO PRAZO DE CONCESSAO. |

优先权:

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申请号 | 申请日 | 专利标题

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US23065909P| true| 2009-07-31|2009-07-31|

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US61/230,659|2009-07-31|

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PCT/US2010/043871|WO2011014749A1|2009-07-31|2010-07-30|Axmi-192 family of pesticidal genes and methods for their use|

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