Insecticidal proteins and uses thereof

Abstract

Disclosed herein are novel plant insecticidal proteins (Vip) from Bacillus thuringiensis, nucleotide sequences encoding the proteins, and primers for identifying genes encoding the proteins that are active against pests. The Vip proteins disclosed herein exhibit activity against a broad range of pests, including but not limited to pests belonging to the order Lepidoptera. The nucleotide sequences encoding the Vip proteins can be used to transform various prokaryotes and eukaryotes, including plants, to express one or more Vip proteins. These recombinant organisms can be used to control a broad range of pests, including but not limited to Lepidoptera.

Description

[Technical field]

[0001] Reference to Electronically Submitted Sequence Listing An official copy of the sequence listing file submitted electronically contemporaneously herewith, entitled "PD045378IN-SC_SEQUENCE LISTING.xml", created on January 5, 2023 and having a size of 36 kb, is hereby incorporated by reference.

[0002] Field The present disclosure relates to novel pesticidal toxin proteins isolated from Bacillus thuringiensis (Bt). The proteins disclosed herein exhibit activity against a wide range of pests, including but not limited to pests belonging to the order Lepidoptera. The present disclosure also relates to DNA sequences encoding said proteins. The DNA sequences encoding said pesticidal toxin proteins can be used to transform various prokaryotic and eukaryotic organisms, including plants, to express said pesticidal toxin proteins. These recombinant organisms can be used to control lepidopteran insects in various environments. [Background technology]

[0003] Climate change and human activities are increasingly threatening plant health. Climate change has altered ecosystems, reduced biodiversity, and created new niches where pests can thrive. Recent studies have shown that a warmer climate will increase the metabolic rate of insects, the rate at which they digest food. This means that climate change will make insects hungrier and their numbers will increase significantly, causing greater crop losses. Rising temperatures will also lead to an increase in the numbers of these pests.

[0004] Crop-damaging pests and pathogens are a global challenge in the agricultural sector. Crop productivity is severely affected by various pests, resulting in economic hardship for farmers and nutritional deficiencies for local populations around the world. The Food and Agriculture Organization of the United Nations (FAO) estimates that approximately 40% of food crops are lost annually due to plant pests and diseases. Every year, plant diseases cost the world economy approximately $220 billion, and invasive insects cause losses of approximately $70 billion. This leaves millions of people without enough food and severely damages agriculture, which is the main source of income for poor rural communities.

[0005] Plant pests and diseases are often impossible to eradicate once established, and dealing with them is time-consuming and costly. Therefore, protecting plants from pests and diseases is an important aspect to reduce crop damage and thereby increase crop productivity. Although chemical pesticides are currently used to control plant pests, the excessive use of chemicals in agriculture has led to many impacts, including increased residues in plants, insect resistance, and soil, water, and air pollution. Disruption of the food chain due to accumulation of pesticide residues has a significant impact on ecosystems. As an alternative to chemical pesticides, bio-pesticides have also been used in agriculture. However, these methods are not sufficient, and there is great interest in developing effective alternative methods.

[0006] Control of various pests through the use of biological molecules has only been possible in a limited number of cases. The δ-endotoxin of Bacillus thuringiensis (Bt), a Gram-positive spore-forming microorganism, is the best-known example of a biological molecule. Various Bt strains are known that produce more than 25 different but related δ-endotoxins. Bt strains produce δ-endotoxins during sporulation. The majority of δ-endotoxins made by Bt are toxic to certain insect larvae of the orders Lepidoptera, Diptera, and Coleoptera. Some of these δ-endotoxins have useful insecticidal properties against various pests. However, their use is limited because these δ-endotoxins are active only against a small number of insect pests. The limited specificity of Bt endotoxins depends, at least in part, on both the activation of this toxin in the insect gut and its ability to bind to specific receptors present in the insect's midgut epithelial cells. Factors that prevent the activity of certain delta-endotoxins against certain insects include the lack of suitable receptors in the insect's gut or the lack of affinity of delta-endotoxins for receptors that may be present, resulting in the lack of delta-endotoxin binding to the brush border membrane. Thus, the ability to use delta-endotoxins to control specific pests currently relies on the ability to discover suitable delta-endotoxins with the desired spectrum of activity. However, the successful use of these molecules in many crop-pathogen systems has been limited in duration due to the rapid evolution of pests to overcome resistance genes.

[0007] Another family of insecticidal proteins (vegetative insecticidal proteins (Vips)) produced by Bacillus species during plant development has also been identified.

[0008] Therefore, scientists must constantly search for new molecules, compositions, or methods that can protect crops from plant pests. In the present invention, novel insecticidal toxin proteins have been discovered that can be used to control plant pests. The toxin proteins of the present invention and the nucleotide sequences encoding them are different from the toxin proteins and nucleotide sequences known in the art. Summary of the Invention

[0009] The present invention relates to materials and methods useful for controlling plant pests. In one embodiment, the present invention provides novel Bt toxins useful for controlling pests. In a preferred embodiment, these pests include, but are not limited to, Lepidoptera and Coleoptera. The toxin protein of the present invention is a plant insecticidal protein (Vip) toxin, particularly the Vip3 protein.

[0010] The present invention further relates to nucleotide sequences encoding the insecticidal proteins disclosed in the present invention.The present invention further provides nucleotide sequences and methods useful for identifying and characterizing the nucleotide sequences encoding said insecticidal proteins.

[0011] The present invention specifically relates to insecticidal proteins having an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:7, and SEQ ID NO:11, which exert insecticidal activity when expressed in plants.

[0012] The present invention also relates to a recombinant nucleic acid molecule comprising a nucleotide sequence encoding a protein of the present invention, said nucleotide sequence being selected from the group consisting of SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:13, and SEQ ID NO:14.

[0013] Furthermore, the present invention relates to expression cassettes, vectors, host cells and microorganisms comprising the nucleotide sequences disclosed in the present invention.

[0014] The present invention also relates to a method for producing a plant exhibiting insecticidal activity, said method comprising at least the step of introducing a nucleic acid molecule as disclosed in the present invention.The present invention further relates to a transgenic plant comprising a recombinant nucleic acid molecule comprising a nucleotide sequence encoding a protein as disclosed herein, said plant exhibiting insecticidal activity.

[0015] The present invention further relates to recombinant microorganisms comprising a recombinant nucleotide sequence encoding the insecticidal protein disclosed in the present invention, and compositions comprising the recombinant microorganisms of the present invention.

[0016] The present invention further relates to the use of the insecticidal proteins, recombinant nucleic acid molecules disclosed in the present invention for producing insecticidal compositions.

[0017] The present invention further relates to the use of the recombinant nucleic acid molecules, expression cassettes, and vectors disclosed herein for producing insect-resistant transgenic plants.

[0018] The present invention further relates to the use of the recombinant microorganisms and compositions disclosed herein to control pests.

[0019] Brief Description of the Sequence Listing SEQ ID NO:1 shows the amino acid sequence of the novel Vip protein 1 disclosed herein. SEQ ID NO:2 shows the native DNA sequence encoding the Vip protein 1 having the amino acid sequence shown in SEQ ID NO:1. SEQ ID NO:3 shows the DNA sequence of the forward degenerate primer. SEQ ID NO: 4 shows the DNA sequence of the reverse degenerate primer. SEQ ID NO:5 shows a modified DNA sequence encoding the Vip protein 1 having the amino acid sequence shown in SEQ ID NO:1. SEQ ID NO:6 shows a modified DNA sequence encoding the Vip protein 1 having the amino acid sequence shown in SEQ ID NO:1. SEQ ID NO: 7 shows the amino acid sequence of novel Vip protein 2. SEQ ID NO:8 shows a native DNA sequence encoding the novel Vip protein 2 having the amino acid sequence shown in SEQ ID NO:7. SEQ ID NO:9 shows a modified DNA sequence encoding the novel Vip protein 2 having the amino acid sequence shown in SEQ ID NO:7. SEQ ID NO:10 shows a modified DNA sequence encoding the novel Vip protein 2 having the amino acid sequence shown in SEQ ID NO:7. SEQ ID NO: 11 shows the amino acid sequence of novel Vip protein 3. SEQ ID NO:12 shows the native DNA sequence encoding the novel Vip protein 3 having the amino acid sequence shown in SEQ ID NO:11. SEQ ID NO:13 shows a modified DNA sequence encoding the novel Vip protein 3 having the amino acid sequence shown in SEQ ID NO:11. SEQ ID NO:14 shows a modified DNA sequence encoding the novel Vip protein 3 having the amino acid sequence shown in SEQ ID NO:11. [Brief description of the drawings]

[0020] [Figure 1] Figure 1 shows the detached leaf bioassay for Spodoptera litura. A: transgenic tobacco leaves expressing Vip protein 1; B: transgenic tobacco leaves expressing Vip protein 2; C: transgenic tobacco leaves expressing Vip protein 3; D: non-transgenic control sample-1; E: non-transgenic control sample-2. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0021] The detailed description provided herein is intended to assist those skilled in the art in practicing the present invention, and the detailed description should not be construed as unduly limiting the scope of the invention, since modifications and variations of the embodiments discussed herein may be made by those skilled in the art without departing from the spirit or scope of the present invention. The present invention will now be described in more detail with reference to the accompanying drawings and / or sequence listings, in which some, but not all, embodiments of the present invention are shown and / or described. The present invention may be embodied in many different forms and should not be construed as being limited to the embodiments depicted herein.

[0022] Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation. The following definitions are provided to facilitate an understanding of the embodiments.

[0023] It should be noted that, as used herein and in the appended claims, the singular forms "a," "an," and "the" are used herein to refer to one or to more than one (i.e., at least one) of the grammatical object of the article, unless the context clearly dictates otherwise. That is, for example, reference to "a probe" means that more than one such probe may be included in the composition. Similarly, reference to "an element" means one or more elements.

[0024] Throughout this specification, the words "comprise" or "comprising" are understood to mean the inclusion of a stated element, integer, or step, or group of elements, integers, or steps, but not the exclusion of any other element, integer, or step, or group of elements, integers, or steps.

[0025] Units, prefixes, and symbols may be represented in the form accepted in the SI. Unless otherwise indicated, nucleic acids are written left to right in 5' to 3' orientation, and amino acid sequences are written left to right in amino to carboxy orientation, respectively. Numeric ranges are inclusive of the numbers defining the range. Amino acids may be represented herein by either their commonly known three letter symbols or the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides may similarly be represented by their commonly accepted one-letter codes. The above defined terms are more fully defined by reference to the specification as a whole.

[0026] The term "nucleic acid" (DNA and RNA) typically refers to large polynucleotides. The terms "nucleic acid" and "nucleotide sequence" are used interchangeably herein. The term includes reference to deoxyribonucleotide or ribonucleotide polymers in either single-stranded or double-stranded form, and, unless otherwise limited, encompasses known analogs (e.g., peptide nucleic acids) that have the essential property of natural nucleotides to hybridize to single-stranded nucleic acids in a manner similar to naturally occurring nucleotides. Nucleotides are the subunits that polymerize (combine into long chains) to make nucleic acids (DNA and RNA). Nucleotides consist of three smaller building blocks: a ribose sugar, a nitrogenous base, and a phosphate group.

[0027] "Polynucleotide" refers to a strand of nucleic acid that is single-stranded, parallel, or anti-parallel, i.e., a polynucleotide can be either a single-stranded or double-stranded nucleic acid.

[0028] The term "oligonucleotide" typically refers to short polynucleotides, generally less than about 50 nucleotides. Where a nucleotide sequence is represented by a DNA sequence (i.e., A, T, G, C), this will be understood to also include RNA sequences in which "T" is replaced by "U" (i.e., A, U, G, C).

[0029] In the context of the present invention, the following abbreviations for commonly occurring nucleobases are used: "A" refers to adenosine, "C" refers to cytidine, "G" refers to guanosine, "T" refers to thymidine, and "U" refers to uridine.

[0030] In some embodiments, the non-genomic nucleic acid molecule is a cDNA. In some embodiments, the non-genomic nucleic acid molecule is a synthetic nucleotide sequence. Methods known in the art can be used to prepare codon-optimized nucleotide sequences for any target organism. Optimized nucleotide sequences are useful for increasing the expression of pesticidal proteins in plants, plant cells, tissues, or any part thereof, such as monocotyledonous and dicotyledonous plants, for example, rice, tomato, and cotton plants.

[0031] The terms "DNA construct," "nucleotide construct," and "DNA expression cassette" are used interchangeably herein and are not intended to limit embodiments to nucleotide constructs that contain DNA. Those skilled in the art will recognize that nucleotide constructs, particularly polynucleotides and oligonucleotides composed of ribonucleotides and combinations of ribonucleotides and deoxyribonucleotides, may also be employed in the methods disclosed herein.

[0032] A "recombinant" nucleic acid molecule or DNA or polynucleotide is used herein to refer to a nucleic acid molecule or DNA polynucleotide that has been artificially modified or produced and is contained in a recombinant bacterial or recombinant plant host cell. For example, a recombinant polynucleotide may be a polynucleotide isolated from a genome, a cDNA produced by reverse transcription of RNA, a synthetic nucleic acid molecule, or an artificial combination of two separate sequence segments, for example, by chemical synthesis or by genetic engineering manipulation of segments of an isolated polynucleotide.

[0033] The term "homology" as used herein refers to the similarity of nucleotide sequences between two regions of the same nucleic acid strand or between regions of two different nucleic acid strands. If a nucleotide residue position of both regions is occupied by the same nucleotide residue, the regions are homologous at that position. A first region is homologous to a second region if at least one nucleotide residue position of each region is occupied by the same residue. The homology between two regions is expressed as the percentage of nucleotide residue positions of the two regions that are occupied by the same nucleotide residue. As an example, a region having the nucleotide sequence 5'-ATTGCC-3' and a region having the nucleotide sequence 5'-TATGGC-3' share 50% homology. Preferably, the first region comprises a first portion and the second region comprises a second portion, whereby at least about 50%, and preferably at least about 75%, at least about 90%, or at least about 95% of the nucleotide residue positions of each portion are occupied by the same nucleotide residue. More preferably, all nucleotide residue positions of each portion are occupied by the same nucleotide residue.

[0034] Optimal sequence alignment for comparison may be performed by computer-implemented algorithms known in the art (GAP, BESTFIT, BLAST, PASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group (GCG), 575 Science Dr., Madison, WI) or by inspection.

[0035] As used herein, "percentage of sequence identity" is determined by comparing two optimally aligned sequences over a comparison window, where a fragment of a polynucleotide sequence or an amino acid sequence within the comparison window may contain insertions or deletions (e.g., gaps or overhangs) compared to a reference sequence (not including insertions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which identical nucleic acid bases or identical amino acid residues occur in both sequences to obtain the number of matching positions, dividing the number of matching positions by the total number of positions within the comparison window, and multiplying the quotient by 100 to obtain the percentage of sequence identity.

[0036] Optimal sequence alignment for comparison may be performed by computer-implemented algorithms known in the art (GAP, BESTFIT, BLAST, PASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group (GCG), 575 Science Dr., Madison, WI) or by inspection.

[0037] As used herein, the term "substantial sequence identity" between nucleotide sequences refers to a polynucleotide comprising a sequence having at least 65% sequence identity, preferably at least 69% to 77% sequence identity, compared to a reference sequence.

[0038] As used herein, "heterologous DNA coding sequence" or "heterologous nucleic acid" or "heterologous polynucleotide" refers to any coding sequence other than the sequence that naturally encodes a Vip protein or any homologue of a Vip protein.

[0039] As used herein, "coding region" refers to the portion of a gene, DNA, or nucleotide sequence that codes for a protein. The term "non-coding region" refers to the portion of a gene, DNA, or nucleotide sequence that is not the coding region.

[0040] As used herein in the context of a particular nucleic acid, the term "encode" or "encoded" means that the nucleic acid contains the information necessary to directly translate its nucleotide sequence into a specified protein. The information for encoding a protein is specified by the use of codons. A nucleic acid that encodes a protein may contain non-translated sequences (e.g., introns) within the translated region of the nucleic acid sequence, or may lack such intervening non-translated sequences (e.g., in cDNA).

[0041] The terms "polypeptide", "peptide" and "protein" are used interchangeably herein to refer to polymers composed of amino acid residues linked by peptide bonds, with respect to naturally occurring structural variants and synthetic non-naturally occurring analogs thereof. Synthetic polypeptides may be synthesized, for example, using an automated polypeptide synthesizer. These terms apply to amino acid polymers in which one or more amino acid residues are artificial chemical analogs of the corresponding naturally occurring amino acids, as well as to naturally occurring amino acid polymers. The terms "residue" or "amino acid residue" or "amino acid" are used interchangeably herein to refer to an amino acid that is incorporated into a protein, polypeptide, or peptide (collectively, "protein"). The amino acids may be naturally occurring amino acids and, unless otherwise limited, may include known analogs of natural amino acids that may function similarly to the naturally occurring amino acids.

[0042] The term "protein" typically refers to a large polypeptide. The term "peptide" typically refers to a short polypeptide. However, as used herein, the term "polypeptide" refers to any amino acid polymer that contains two or more amino acid residues linked via peptide bonds.

[0043] As used herein, "expression cassette" means a genetic module containing a gene and the regulatory regions required for its expression, which may be incorporated into a vector.

[0044] A "vector" is a composition of matter that contains a nucleic acid molecule and can be used to deliver an isolated nucleic acid inside a cell. Numerous vectors are known in the art, including, but not limited to, linear polynucleotides, circular polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. That is, the term "vector" includes an autonomously replicating plasmid or virus. The term should also be construed to include non-plasmid and non-viral compounds that facilitate the transfer of nucleic acid into cells, such as, for example, polylysine compounds, liposomes, etc. Examples of viral vectors include, but are not limited to, adenoviral vectors, adeno-associated viral vectors, retroviral vectors, etc.

[0045] The term "expression vector" refers to a vector that contains a recombinant nucleic acid, which comprises an expression control sequence operably linked to a nucleotide sequence to be expressed. An expression vector contains sufficient cis-acting elements for expression; other elements for expression can be supplied by the host cell or an in vitro expression system. Expression vectors include all expression vectors known in the art, such as cosmids, plasmids (e.g., naked plasmids or plasmids contained in liposomes) and viruses that incorporate recombinant nucleic acids.

[0046] As used herein, "promoter sequence / regulatory sequence" refers to a nucleic acid sequence required for expression of a gene product operably linked to the promoter sequence / regulatory sequence. In some cases, this sequence may be a core promoter sequence, and in other cases, this sequence may also include enhancer sequences and other regulatory elements required for expression of the gene product. The promoter sequence / regulatory sequence may be, for example, a sequence that expresses the gene product in a tissue-specific manner.

[0047] A "constitutive" promoter is a promoter that constitutively drives expression of a gene to which it is operably linked in a cell. By way of example, a promoter that drives expression of a cellular housekeeping gene is considered to be a constitutive promoter.

[0048] An "inducible" promoter is a nucleotide sequence that, when operably linked to a polynucleotide encoding or specifying a gene product, causes the gene product to be produced in a living cell only when an inducer corresponding to the promoter is substantially present in the cell.

[0049] A "tissue-specific" promoter is a nucleotide sequence that, when operably linked to a polynucleotide encoding or specifying a gene product, causes the gene product to be produced in a living cell substantially only if the cell is a cell of the tissue type corresponding to the promoter.

[0050] As used herein, "operably linked" refers to any linkage between a regulatory sequence and a coding sequence, regardless of orientation or distance, where the linkage allows the regulatory sequence to control the expression of the coding sequence. The term "operably linked" means that the linked nucleic acid sequences are contiguous, and further means that, where necessary to link two protein coding regions, the linked nucleic acid sequences are contiguous and in the same reading frame. The term "operably linked" also refers to a functional linkage between a promoter and a second sequence, where the promoter sequence initiates and mediates the transcription of the DNA sequence corresponding to the second sequence.

[0051] The term "host cell" as used herein is intended to refer to a cell that contains a vector and supports the replication and / or expression of an expression vector. A host cell may be a prokaryotic cell, such as E. coli, or a eukaryotic cell, such as yeast, insect cell, amphibian cell, or mammalian cell, or a monocotyledonous or dicotyledonous cell. An example of a monocotyledonous host cell is a rice host cell, and an example of a dicotyledonous host cell is an eggplant host cell or a tomato host cell. Where possible, the sequence may be modified to avoid predicted hairpin secondary structures of mRNA.

[0052] As used herein, "toxin" or "insecticidal protein" refers to a polypeptide that exhibits pesticidal or insecticidal activity. "Bt" toxins or "Bacillus thuringiensis" toxins are intended to include the broad class of plant insecticidal protein (Vip) toxins found in various strains of Bacillus species that contain such toxins.

[0053] The term "probe" or "sample probe" refers to a molecule that is recognized by its complement or a particular microarray element. Examples of probes that may be contemplated by the present invention include, but are not limited to, DNA, RNA, oligonucleotides, oligosaccharides, polysaccharides, sugars, proteins, peptides, monoclonal antibodies, toxins, viral epitopes, hormones, hormone receptors, enzymes, enzyme substrates, cofactors, and drugs, including agonists and antagonists of cell surface receptors.

[0054] As used herein, the term "complementary" or "complementary sequence" refers to the pairing of bases, purines, and pyrimidines that associate through hydrogen bonds in a double-stranded nucleic acid. The following base pairs are complementary: guanine and cytosine; adenine and thymidine; and adenine and uracil. As used herein, these terms include complete complementarity and partial complementarity.

[0055] The term "hybridization" as used herein refers to the process of nucleic acid strand binding to complementary strand through base pairing.The conditions adopted in hybridization of two non-identical but highly similar complementary nucleic acids vary according to the degree of complementarity of the two strands and the length of the strands.Thus, this term contemplates partial hybridization and complete hybridization.Such methods and conditions are well known to those skilled in the art.

[0056] The terms "insecticidal activity" and "pesticidal activity" are used interchangeably herein and refer to the activity of an organism or substance (such as a protein) that can be measured, but is not limited to, by pest mortality, pest weight loss, pest repellency, and other behavioral and physical changes of a pest after a suitable period of feeding and exposure. Thus, an organism or substance with pesticidal activity adversely affects at least one measurable parameter of the fitness of a pest. For example, an "insecticidal protein" is a protein that exhibits insecticidal activity by itself or in combination with other proteins.

[0057] As used herein, the term "affecting pests" refers to controlling feeding, growth, and / or altering behavior of insects at any developmental stage, including, but not limited to, killing insects, retarding growth, preventing reproduction, anti-feeding activity, and the like.

[0058] As used herein, "pesticidally effective amount" refers to the amount of a substance or organism that has pesticidal activity when present in the environment of a pest. For each substance or organism, the pesticidal amount is empirically determined for each affected pest in a particular environment. Similarly, "pesticidally effective amount" may be used to refer to a "pesticidally effective amount" when the pest is an insect pest.

[0059] A "control plant" as referred to herein is a plant that does not contain the protein and / or nucleic acid of the invention.

[0060] When the test plant is a plant containing the protein and / or nucleic acid of the present invention.

[0061] Preferably, the control plants are grown under the same conditions as the test plants containing the proteins and / or nucleic acids of the invention.

[0062] As used herein, "transformed plant" and "transgenic plant" refer to a plant that contains one or more heterologous polynucleotides in its genome. The heterologous polynucleotide is stably integrated into the genome of the transgenic or transformed plant so that the polynucleotide is passed on to subsequent generations. The heterologous polynucleotide may be integrated into the genome alone or as part of a recombinant DNA molecule.

[0063] The term "transgenic" as used herein should be understood to include any plant cell, plant cell line, callus, tissue, plant part, or plant whose genotype has been altered by the presence of one or more heterologous nucleic acids. The term includes those transgenics originally obtained using genetic transformation techniques known in the art, as well as those produced from the original transgenics by sexual cross, conventional breeding, or asexual propagation.

[0064] As used herein, the term "original genetic modification" does not encompass modifications of the genome (chromosomal or extrachromosomal) by conventional plant breeding methods or by naturally occurring events such as random cross-fertilization, non-recombinant viral infection, non-recombinant bacterial transformation, non-recombinant translocation, or spontaneous mutation.

[0065] The term "plant" as used herein includes whole plants, plant cells, plant protoplasts, plant cell culture tissues from which plants can be regenerated, plant callus, plant clumps, and intact plant cells in a plant, or parts of a plant such as embryos, pollen, ovaries, seeds, leaves, flowers, branches, fruits, grains, ears, cobs, pericarp, stems, roots, root tips, anthers, and their progeny, etc. Parts of transgenic plants are within the scope of the embodiments and include, for example, plant cells, protoplasts, tissues, callus, embryos, as well as flowers, stems, fruits, leaves, and roots that originate from a transgenic plant or their progeny that have been previously transformed with a DNA molecule of the embodiments and therefore consist at least in part of the transgenic cells.

[0066] It will be apparent to those skilled in the art that to achieve improved expression of a Bt insecticidal protein in a plant, it is first necessary to prepare a nucleotide sequence encoding the Bt protein, or an active variant or fragment of said protein. The nucleotide sequence encoding said protein or a fragment thereof will then be placed in an expression cassette that functions in the plant to cause transcription of the coding sequence into messenger RNA. The messenger RNA is then translated in the plant's cells such that an insecticidally effective amount of the insecticidal protein is produced in the plant tissue. It will also be clear to one skilled in the art to transform plant cells, preferably cotton, corn, brinjal, tomato, rice, wheat, oat, grass, forage plant, crucifer, fruit tree, ornamental flower, potato, carrot, kale, Arabidopsis, and tobacco plant cells, etc., with a nucleotide sequence incorporated into an expression cassette functional in plants, and to select cells that contain said sequence and express an insecticidally effective amount of an insecticidal protein, preferably a Vip protein or related protein or an insecticidal fragment thereof disclosed herein, and to produce plants from such transformed cells. It will also be clear to one skilled in the art to use electroporation, injection, ballistic, or Agrobacterium tumefaciens-mediated methods, etc., to introduce the nucleotide sequence of the present invention or a variant thereof into plant cells.

[0067] The term "mutant or modified" referring to a nucleotide sequence is intended to refer to a nucleotide sequence that encodes the same toxin or an equivalent toxin with similar pesticidal activity. The word "equivalent toxin" refers to a toxin that exhibits the same, substantially the same, or improved biological activity against a target pest as the claimed native or reference toxin. A mutant or modified nucleotide sequence intended for use in a dicotyledonous plant will encode substantially the same amino acid sequence as the native coding sequence, i.e., the coding sequence found in nature, but will contain a total GC composition of about 49 to about 58 percent and will utilize substantially the codon preference and codon usage as determined by collecting codon preferences and codon usage from a consortium of coding sequences from one or more individual dicotyledonous plant species intended to be transformed with the mutant or modified nucleotide. A mutant or modified nucleotide sequence intended for use in a monocotyledonous plant will also code for substantially the same amino acid sequence as the native coding sequence, but will have a total GC composition of about 52 to about 59 percent, and will utilize substantially the codon preference and codon usage frequency determined by collecting codon preference and codon usage frequency from a consortium of coding sequences from one or more individual monocotyledonous plants intended to be transformed with the mutant or modified nucleotide. A mutant or modified nucleotide sequence intended for use in a legume will also code for substantially the same amino acid sequence as the native coding sequence, but will have a total GC composition of about 52 to about 59 percent, and will utilize substantially the codon preference and codon usage frequency determined by collecting codon preference and codon usage frequency from a consortium of coding sequences from one or more individual legumes intended to be transformed with the mutant or modified nucleotide. Codon usage frequency is intended to refer to the average number of times a particular codon is used in a coding sequence.For a particular plant species, the codons intended to incorporate a particular amino acid sequence into the nascent amino acid sequence will, on average, be utilized at a certain relative frequency. For amino acids that utilize only two codons, this frequency will generally be about 50 / 50, i.e., each codon will be used about half the time, unless one of the codons utilizes a substantially higher number of purines or pyrimidines that are not typical of the GC content of the particular plant species.

[0068] For Bacillus species, for example, coding sequences are typically about 60 to about 70 percent AT. Codon usage in Bacillus species is biased towards the use of certain codons that are rich in the presence of A or T. Thus, codons that utilize primarily G or C are used less frequently than codons containing A or T in native and / or naturally occurring Bacillus coding sequences. Thus, when producing mutant or modified nucleotide sequences intended for use in a particular plant, monocot, dicot, or legume, it is important to ensure that, where possible, appropriate care is taken in the use of codons that are not rich in A and T, particularly, and to avoid the incorporation of sequences suspected of polyadenylation.

[0069] As used herein, a "synthetic coding sequence" or "non-naturally occurring coding sequence" that encodes the B. thuringiensis Vip protein or homologs or derivatives thereof disclosed herein as insecticidal toxins of the invention is one that has been prepared by any method involving the isolation or manipulation of a gene, including isolating said coding sequence from its naturally occurring state, modifying the coding sequence with nucleotides as disclosed herein, chemically synthesizing all or part of the coding sequence using phosphoramidate chemistry or the like, or manipulating said coding sequence by site-directed mutagenesis, truncating the coding sequence, or any other method of manipulation or isolation, such that the amino acid sequence encoded by the non-naturally occurring coding sequence encodes substantially the same insecticidal protein as the naturally occurring coding sequence, and further exhibits substantially the same or an improved level of insecticidal bioactivity as the naturally occurring insecticidal protein.

[0070] The phrase "percentage of sequence identity" as used herein is determined by comparing two optimally aligned sequences over a comparison window, where a portion of the sequence within the comparison window may contain insertions or deletions (i.e., gaps) compared to the reference sequence (not including insertions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which identical nucleic acid bases or amino acid residues occur in both sequences to obtain the number of matching positions, dividing the number of matching positions by the total number of positions within the comparison window, and multiplying the quotient by 100 to obtain the percentage of sequence identity. A sequence that is identical at all positions compared to a reference sequence is said to be identical to the reference sequence, and vice versa. If a first nucleotide sequence exhibits complete complementarity to a second or reference sequence, the first nucleotide sequence viewed in the 5' to 3' direction is said to be the "complementary sequence" of the second or reference nucleotide sequence viewed in the 3' to 5' direction. As used herein, a nucleic acid sequence molecule is said to exhibit "perfect complementarity" when every nucleotide of one sequence, read 5' to 3', is complementary to every nucleotide of the other sequence, read 3' to 5'. A nucleotide sequence that is identical at every position, read 5' to 3', compared to a reference nucleotide sequence, read 5' to 3', is said to be identical to the reference sequence, and vice versa. A nucleotide sequence that is complementary to a reference nucleotide sequence will exhibit a sequence that is identical to the reverse complement sequence of the reference nucleotide sequence. These terms and descriptions are well defined in the art and are easily understood by those skilled in the art.

[0071] "Substantially complementary" as used herein in reference to a nucleotide sequence refers to a nucleotide sequence that hybridizes under stringent conditions to the sequence encoding Vip as shown in SEQ ID NO:2 or its complementary sequence. A sequence that hybridizes under stringent conditions to SEQ ID NO:2 or its complementary sequence. Such a homologous sequence is about 67% identical to about 70% identical to about 80% identical to about 85% identical to about 90% identical to about 95% identical to about 99% identical or more to the reference nucleotide sequence as shown in SEQ ID NO:2 or its complementary sequence, where the nucleotide sequence encodes the Vip protein disclosed herein. In addition, it is assumed that the nucleotide sequence encoding an insecticidal protein that hybridizes under stringent conditions to SEQ ID NO:2 exhibits substantial homology to the reference nucleotide sequence that hybridizes under stringent conditions to the sequence encoding Vip as shown in SEQ ID NO:2 or its complementary sequence. Such nucleotide sequences are referred to herein as homologues of SEQ ID NO:2, etc.

[0072] The term "substantial homology" when referring to a polypeptide sequence refers to a polypeptide that is about 70% homologous, about 80% homologous, about 86% homologous, about 90% homologous, about 95% homologous, about 99% homologous to a reference polypeptide sequence. More specifically, the inventors contemplate that a substantial homolog is about 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, and 99 percent homologous to the reference polypeptide sequence represented herein as SEQ ID NO:1.

[0073] When referring to the proteins of the present application, "mutated amino acid sequence" or "amino acid sequence variant" or "modified amino acid sequence variant" is intended to refer to an amino acid sequence that is substantially equivalent to the amino acid sequence of the present invention. For example, a protein produced by introducing a restriction site for easy molecular manipulation into the coding sequence of the present invention, resulting in the addition or deletion of one or more codons without otherwise (1) disrupting the native coding sequence, (2) disrupting the native open reading frame, and (3) interfering with the insecticidal bioactivity of the protein, may constitute (a) a mutant amino acid sequence compared to the native insecticidal toxin, (b) an amino acid sequence variant compared to the native insecticidal toxin, or (c) an altered amino acid sequence variant compared to the native insecticidal toxin. Those skilled in the art will recognize that there are other types of modifications that can be made to the amino acid sequence of the present invention without interfering with the bioactivity of the protein. Insertions, deletions, and substitutions are within the scope of the present disclosure, so long as the resulting amino acid sequence variant exhibits insecticidal activity equal to or greater than that of the native insecticidal protein. Chimeras of the proteins disclosed herein, fusions of the proteins or portions of the proteins disclosed herein, and permuteins of the proteins disclosed herein are specifically contemplated.

[0074] Bacillus thuringiensis insecticidal Vip protein and Vip gene Bacillus thuringiensis (Bt) is a Gram-positive bacterium that produces insecticidal proteins known as Cry or Cyt toxins as crystal inclusions during the sporulation stage of development. The insecticidal proteins have proven effective against major crop pests and also against mosquitoes. Bt also produces a phyto-insecticidal protein (Vip) that is a secreted protein and shares no sequence homology with known Cry proteins, and that exhibits insecticidal activity against a wide variety of lepidopteran and coleopteran pests and some sap-sucking pests of plants.

[0075] Vip proteins are divided into four families according to their amino acid identity. The Vip1 and Vip2 proteins act as binary toxins and are toxic to some members of the orders Coleoptera and Hemiptera. The Vip1 component is thought to bind to receptors in the insect midgut membrane, and the Vip2 component enters the cell, where it exerts its ADP-ribosyltransferase activity on actin and prevents the formation of microfilaments. Vip3 has no sequence similarity to Vip1 or Vip2 and is toxic to a wide variety of members of the order Lepidoptera. The Vip3A protein does not share a binding site with Cry proteins, although its mechanism of action has been shown to resemble that of Cry proteins in terms of activation of proteolysis, binding to midgut epithelial membranes, and pore formation.

[0076] The present invention encompasses the pesticidal proteins disclosed herein and their components and fragments. That is, it is recognized that component promoters, polypeptides, or fragments of the proteins may be produced that retain pesticidal activity. These fragments include truncated sequences, and amino acid sequences with N-terminal, C-terminal, internal, and internal deletions of the protein.

[0077] The present invention encompasses any deletion, insertion and substitution of the protein sequence that does not significantly change the properties of the pesticidal protein.Those skilled in the art will understand that the effect of such deletion, insertion and substitution of the protein will be evaluated by routine screening assay.The proteins and their components and fragments described herein may be used alone or in combination.That is, multiple proteins may be used to control different pests.

[0078] The pesticidal protein of the present invention can be used in combination with Bt endotoxin or other insecticidal proteins to increase the target range of insects.In addition, the use of the Vip protein of the present invention in combination with Bt δ-endotoxin or other insecticidal principles of different nature is particularly useful to prevent and / or manage insect resistance.Other insecticidal principles include protease inhibitors (both serine and cysteine ​​type), lectins, α-amylase and peroxidase.

[0079] A preferred embodiment includes expressing the VIP disclosed herein alone or in combination with one or more Bt δ-endotoxins in a transgenic plant. This co-expression of more than one insecticidal principle in the same transgenic plant can be achieved by genetic transformation and / or traditional breeding methods. Those skilled in the art will know how to implement these methods to achieve the desired expression of the Vip protein described herein in combination with one or more Bt δ-endotoxins using these methods.

[0080] For example, a transgenic plant expressing a Vip protein disclosed herein in combination with one or more Bt delta-endotoxins or any other insecticidal proteins can be obtained by crossing a transgenic plant expressing a Vip as described herein, obtained using genetic transformation methods or any other methods known in the art, with a second plant expressing one or more Bt delta-endotoxins or any other insecticidal proteins, obtained using genetic transformation methods or any other methods known in the art.

[0081] The present invention also encompasses nucleotide sequences from organisms other than Bacillus thuringiensis, which can be isolated by hybridization with the nucleotide sequences of the present invention. Proteins encoded by such nucleotide sequences can be tested for pesticidal activity. The present invention also encompasses proteins encoded by the nucleotide sequences. Additionally, the present invention encompasses proteins obtained from organisms other than Bacillus thuringiensis that cross-react with antibodies raised against the proteins of the present invention. Again, the isolated proteins can be assayed for pesticidal activity by the methods disclosed herein or other methods well known in the art.

[0082] Once the nucleotide sequences encoding the pesticidal proteins of the present invention have been isolated, they can be manipulated and used to express the proteins in a variety of hosts, including microorganisms and other organisms, including plants.

[0083] The nucleotide sequence encoding the Vip of the present disclosure may be optimized to enhance expression in plants. For example, the nucleotide sequence may be synthesized utilizing plant-preferred codons, which are codons preferred to a particular plant, such as monocot-preferred codons, dicot-preferred codons, and / or legume-preferred codons. A synthetic gene may be created based on the distribution of codons used by a particular host for a particular amino acid. In this way, the nucleotide sequence may be optimized for expression in any plant. It is recognized that all or any part of the gene sequence may be optimized or synthetic. That is, synthetic or partially optimized sequences may also be used.

[0084] In this way, the nucleotide sequence can be optimized for expression in any microorganism.

[0085] The methodology for constructing plant expression cassettes and introducing foreign DNA into plants is well known in the art.Such expression cassettes may comprise one or more regulatory sequences, such as promoters, terminators, enhancers, leader sequences, introns, plant translation consensus sequences and other regulatory sequences, operably linked to the coding sequence that codes for the pesticidal protein described herein.Furthermore, it is recognized that the promoter or terminator of Vip gene can be used in the expression cassette.

[0086] Generally, introduction of foreign DNA into plants has utilized Ti plasmid vectors for delivery of foreign DNA, as well as direct DNA uptake, liposomes, electroporation, microinjection, and biolistics. Such methods have been published in the art. It is understood that the method of transformation will depend on the plant cell to be transformed.

[0087] Regulatory Sequences It is further recognized that components of the expression cassette may be modified to increase expression. For example, sequence truncations, nucleotide substitutions, or other modifications may be employed. Transcriptional and translational regulatory signals include, but are not limited to, promoters, transcription initiation sites, operators, activators, enhancers, other regulatory elements, ribosome binding sites, initiation codons, termination signals, and the like.

[0088] It may be beneficial to include a 5' leader sequence in the expression cassette construct. Such a leader sequence may serve to enhance translation. Translation leaders are known in the art and include, but are not limited to, Picomavirus leaders, Polyvirus leaders, such as the TEV leader (Tobacco Etch Virus); MDMV leader (Maize Dwarf Mosaic Virus), the non-translated leader of the coat protein mRNA of Alfalfa Mosaic Virus (AMV RNA 4), Tobacco Mosaic Virus leader (TMV), and Maize Chlorotic Mottle Virus leader (MCMV).

[0089] The polynucleotide / DNA construct will include, in the 5' to 3' transcriptional direction, a transcriptional and translational initiation region (i.e., promoter), the DNA sequence of the embodiment, and a transcriptional and translational termination region (i.e., termination region) that functions in the host organism. The transcriptional initiation region (i.e., promoter) may be native, analogous, foreign, or heterologous to the host organism and / or the sequence of the embodiment. In addition, the promoter may be a natural sequence or may be a synthetic sequence. The term "foreign" as used herein refers to the promoter not being found in the native organism into which it is introduced. When a promoter is "foreign" or "heterologous" to the sequence of the embodiment, it is intended that the promoter is not a native or naturally occurring promoter to the sequence of the embodiment to which it is operably linked.

[0090] Numerous promoters may be used in the practice of the embodiments. The promoter may be selected based on the desired outcome. The nucleotide sequence of the present invention may be combined with a constitutive promoter, a tissue-preferred promoter, an inducible promoter, or other promoter for expression in a host organism. Constitutive promoters suitable for use in plant host cells include, for example, the core CaMV 35S promoter, the rice actin promoter, the ubiquitin promoter, the ALS promoter, and the like.

[0091] Depending on the desired outcome, it may be beneficial to express the gene from an inducible promoter. Of particular interest for regulating the expression of the nucleotide sequence of the embodiment in plants are wound-inducible promoters. Such wound-inducible promoters may respond to damage caused by insect feeding and include the potato proteinase inhibitor (pin II) gene, wun1 and wun2, win1 and win2, WIP1, MPI gene, etc.

[0092] In addition, pathogen-inducible promoters may be employed in the methods and nucleotide constructs of the embodiments. Such pathogen-inducible promoters include promoters of pathogenesis-related proteins (PR proteins) that are induced following infection by a pathogen, such as PR proteins, SAR proteins, β-1,3-glucanase, chitinase, and the like.

[0093] Chemically regulated promoters can be used to regulate the expression of genes in plants through the application of exogenous chemical regulators. Depending on the purpose, the promoter may be a chemically inducible promoter, in which gene expression is induced by application of a chemical, or a chemically repressible promoter, in which gene expression is repressed by application of a chemical. Chemically inducible promoters are known in the art and include, but are not limited to, the maize In2-2 promoter, which is activated by benzenesulfonamide herbicide safeners, the maize GST promoter, which is activated by hydrophobic electrophilic compounds used as pre-emergent herbicide, and the tobacco PR-1a promoter, which is activated by salicylic acid. Other interesting chemically regulated promoters include steroid-responsive promoters.

[0094] A promoter that has "preferential" expression in a particular tissue is expressed to a higher extent in that tissue than in at least one other plant tissue. Some tissue-preferential promoters are expressed almost exclusively in a particular tissue. Tissue-preferential promoters can be used to enhance the expression of pesticidal proteins in a particular plant tissue. Such promoters can be modified for weak expression, if necessary.

[0095] Some examples of tissue-specific promoters include, but are not limited to, leaf-preferred promoters, root-specific or root-preferred promoters, seed-specific or seed-preferred promoters, pollen-specific promoters, and pith-specific promoters.

[0096] Root-preferred or root-specific promoters are known and can be selected from those described in the literature or can be isolated de novo from a variety of compatible species.

[0097] "Seed-preferential" promoters include both "seed-specific" promoters (promoters active during seed development, such as promoters of seed storage proteins) and "seed-germinating" promoters (promoters active during seed germination). Gamma-zein and Glob-1 are endosperm-specific promoters. For dicotyledons, seed-specific promoters include, but are not limited to, β-phaseolin, β-conglycinin, soybean lectin, cruciferin, and the like. For monocotyledons, seed-specific promoters include, but are not limited to, maize 15 kDa zein, 22 kDa zein, 27 kDa zein, g-zein, waxy, shrunken-1, shrunken-2, globulin 1, and the like.

[0098] When low level expression is desired, a weak promoter will be used. Generally, the term "weak promoter" as used herein refers to a promoter that drives the expression of a coding sequence at a low level. Such weak constitutive promoters include, for example, the core promoter of the Rsyn7 promoter, the core 35S CaMV promoter, etc.

[0099] Termination regions are available from the Ti-plasmid of A. tumefaciens and include the termination regions for octopine synthase (OCS) and nopaline synthase (NOS).

[0100] The DNA expression cassette may additionally contain a 5' leader sequence. Such a leader sequence serves to enhance translation. Translation leaders are known in the art and include picornavirus leaders, such as the EMCV leader (encephalomyocarditis virus 5' non-coding region); potyvirus leaders, such as the TEV leader (tobacco etch virus), the MDMV leader (maize dwarf mosaic virus), the non-translated leader of the coat protein mRNA of alfalfa mosaic virus (AMV RNA 4); the tobacco mosaic virus leader (TMV); and the corn chlorotic mottle virus leader (MCMV).

[0101] In certain embodiments of the invention disclosed and claimed herein, the tissue-preferential or tissue-specific promoter is operably linked to a synthetic DNA sequence of the present disclosure encoding an insecticidal protein, and a transgenic plant is stably transformed with at least one such recombinant molecule. The resulting plant will be resistant to the particular insect that feeds on the part of the plant in which the DNA is expressed.

[0102] It will be appreciated that genes encoding said pesticidal proteins may be used to transform insect pathogenic organisms, including baculoviruses, fungi, protists, bacteria, and nematodes.

[0103] Numerous methods are available for introducing the gene expressing the pesticidal protein into the microbial host under conditions that allow the gene to be stably maintained and expressed.For example, an expression cassette can be constructed that includes the desired DNA construct, which is operably linked with transcriptional and translational regulatory signals for the expression of the DNA construct, and a DNA sequence that is homologous to the sequence of the host organism, thereby allowing integration, and / or that includes a replication system that functions in the host, thereby allowing integration or stable maintenance.

[0104] Selectable marker genes Typically, the expression cassette will include a selectable marker gene for the selection of transformed cells. The selectable marker gene is utilized for the selection of transformed cells or tissues. Marker genes include genes encoding antibiotic resistance, such as genes encoding neomycin phosphotransferase II (nptII) and hygromycin phosphotransferase (hptII), as well as genes conferring resistance to herbicidal compounds, such as glufosinate ammonium, bromoxynil, imidazolinone, and 2,4-dichlorophenoxyacetic acid (2,4-D). Further examples of suitable selectable marker genes include, but are not limited to, genes encoding resistance to chloramphenicol, methotrexate, streptomycin, spectinomycin, bleomycin, sulfonamides, bromoxynil, glyphosate, and phosphinothricin.

[0105] The above list of selectable markers is not meant to be limiting. Any selectable marker gene may be used in the embodiments.

[0106] DNA constructs and vectors The DNA / nucleotide sequences of the invention are provided in a DNA construct for expression in an organism of interest, said construct will include 5' and 3' regulatory sequences operably linked to the sequences of the invention.

[0107] Such polynucleotide constructs are provided with multiple restriction sites for inserting a DNA sequence encoding a Vip toxin protein under the transcriptional control of a regulatory region. The polynucleotide construct may additionally contain a selectable marker gene. The construct may additionally contain at least one additional gene that is co-transformed into a desired organism. Alternatively, the additional genes may be provided on multiple polynucleotide constructs.

[0108] In preparing the DNA construct / expression cassette, various DNA fragments may be manipulated to provide DNA sequences that are in the correct orientation and, if necessary, in the correct reading frame. For this purpose, adapters or linkers may be employed to join the DNA fragments, or other manipulations may be involved to provide convenient restriction sites, removal of excess DNA, removal of restriction sites, etc. For this purpose, in vitro mutagenesis, primer repair, restriction, annealing, resubstitutions, such as transitions and transversions, may be involved.

[0109] According to the present invention, the DNA construct / expression cassette disclosed herein may be inserted into a recombinant expression vector. The term "recombinant expression vector" refers to bacterial plasmid, phage, yeast plasmid, plant cell virus, mammalian cell virus, or other vector. Generally, any plasmid or vector may be used as long as it can be replicated and stabilized in the host. The important features of an expression vector are that it has a replication origin, a promoter, a marker gene, and a translation control element.

[0110] In preparation for inserting a foreign gene into a higher plant, numerous cloning vectors are available that contain the E. coli replication system and a marker that allows the selection of transformed cells. The vectors include, for example, pBR322, pUC series, M13mp series, pACYC184, etc. Thus, a DNA fragment carrying the sequence coding for the Bt toxin protein can be inserted into the vector at a suitable restriction site. The resulting plasmid is used for transformation of E. coli. The E. coli cells are cultivated in a suitable nutrient medium, then harvested and lysed. The plasmid is recovered. As a method of analysis, generally, sequence analysis, restriction analysis, electrophoresis, and other biochemical-molecular biological methods are performed. After each manipulation, the DNA sequence used can be cut and ligated to the next DNA sequence. Each plasmid sequence can be cloned into the same plasmid or other plasmids. Depending on the method of inserting the desired gene into the plant, other DNA sequences may be required. For example, when a Ti or Ri plasmid is used to transform a plant cell, at least the right border, but often both the right and left borders, of the T-DNA of the Ti or Ri plasmid must be linked as flanking regions to the gene to be inserted.

[0111] The expression vector containing the nucleotide sequence of the present invention and suitable signals for regulating transcription / translation can be constructed by methods well known to those skilled in the art. Examples of such methods include in vitro recombinant DNA techniques, DNA synthesis techniques, and in vivo recombinant techniques. The DNA sequence can be effectively linked to a suitable promoter in the expression vector to induce the synthesis of mRNA. In addition, the expression vector can contain a ribosome binding site and a transcription terminator as a translation initiation site.

[0112] A preferred example of a recombinant vector according to the invention is a Ti-plasmid vector, which can transfer a part of it, the so-called T-region, into a plant cell when this vector is present in a suitable host, such as Agrobacterium tumefaciens. Other types of Ti-plasmid vectors are currently used to transfer hybrid genes into protoplasts, which can produce new plants by appropriate insertion of plant cell DNA or hybrid DNA into the plant genome.

[0113] The expression vector may contain at least one selectable marker gene. The selectable marker gene is a nucleotide sequence that has a characteristic based on which it can be selected by common chemical methods. Any gene that can be used to distinguish transformed cells from untransformed cells can be a selective marker. Examples include, but are not limited to, resistance genes to herbicides such as glyphosate and phosphinothricin, and resistance genes to antibiotics such as kanamycin, hygromycin, G418, bleomycin, and chloramphenicol.

[0114] For the recombinant vector of the present invention, the promoter can be, but is not limited to, CaMV 35S promoter, actin promoter, or ubiquitin promoter. Because transformants can be selected by different mechanisms at different stages, constitutive promoters may be preferred for the present invention. Therefore, the possibility of selecting constitutive promoters is not limited herein.

[0115] Any conventional terminator can be used for the recombinant vector of the present invention. Examples include, but are not limited to, nopaline synthase (NOS), rice α-amylase RAmy1 A terminator, phaseoline terminator, and the terminator of the octopine gene of Agrobacterium tumefaciens. Regarding the necessity of a terminator, it is generally known that such a region can increase the reliability and efficiency of transcription in plant cells. Therefore, the use of a terminator is highly preferred in the context of the present invention.

[0116] It will be apparent to one skilled in the art that the DNA constructs and vectors disclosed herein can be used for the production of insect-resistant transgenic plants and / or for the production of insecticidal compositions, which may comprise Bacillus thuringiensis cells comprising the nucleotide sequences disclosed herein, or any other microorganism capable of expressing the nucleotide sequences disclosed herein, to produce the Vip insecticidal proteins disclosed herein.

[0117] Host Cells and Recombinant Cells Suitable host cells may include prokaryotes and / or eukaryotes, and are usually limited to cells that do not produce substances that are toxic to higher organisms, such as mammals. However, organisms that produce substances that are toxic to higher organisms may also be used if the toxin is unstable or the amount applied is low enough to avoid potential toxicity to the mammalian host. Of particular interest as hosts would be prokaryotes and lower eukaryotes, such as fungi. Examples of both gram-negative and positive prokaryotic cells include Enterobacteriaceae, such as Escherichia, Erwinia, Shigella, Salmonella, Proteus; Bacillaceae; Rhizobium; Spirillumaceae, such as Photobacterium, Zymomonas, Serratia, Aeromonas, Vibrio, Desulfovibrio, Spirillum; Lactobacillaceae; Pseudomonadaceae, such as Pseudomonas, Acetobacter; Azotobacteraceae, and Nitrobacteraceae. Eukaryotes include fungi such as Phycomycota and Ascomycota, which include yeasts such as Saccharomyces and Schizosaccharomyces; and basidiomycete yeasts such as Rhodotorula, Aureobasidium, and Sporobolomyces.

[0118] Host organisms of particular interest include yeasts such as Rhodotorula spp., Aureobasidium spp., Saccharomyces spp., and Sporobolomyces spp.; phylloplane organisms such as Pseudomonas spp., Erwinia spp., and Flavobacterium spp.; or other organisms such as Escherichia, Lactobacillus spp., Bacillus spp., etc. Specific organisms include Pseudomonas aeurginosa, Pseudomonas fluorescens, Saccharomyces cerevisiae, Bacillus thuringiensis, Escherichia coli, Bacillus subtilis, etc.

[0119] To deliver the VIP protein to a potential target pest, the nucleotide sequence encoding the Vip protein disclosed herein can be introduced into a microorganism (epiphyte) that grows on a plant. The epiphyte can be a gram-positive or gram-negative bacterium.

[0120] Various expression systems can be designed to allow the Vip protein disclosed herein to be secreted outside the cytoplasm of Gram-negative bacteria, such as E. coli. Secretion of the protein outside the cytoplasm avoids the potential toxic effects of Vip protein expressed in the cytoplasm. This allows the level of expressed Vip protein to be increased, and can aid in the efficient purification of Vip protein.

[0121] The Vip proteins disclosed herein can be secreted in E. coli, for example, by fusing an appropriate E. coli signal peptide to the amino-terminal side of the Vip protein signal peptide or by replacing the Vip signal peptide with an E. coli signal peptide. Signal peptides recognized by E. coli can be found in proteins already known to be secreted by E. coli.

[0122] The embodiments further include a microorganism transformed with a nucleotide sequence encoding the insecticidal Vip protein of the present invention, with an expression cassette comprising said nucleotide sequence, or with a vector comprising said expression cassette. In some embodiments, the microorganism is a microorganism that grows on a plant. Some embodiments of the present invention relate to an encapsulated pesticidal protein comprising a transformed microorganism capable of expressing the insecticidal Vip protein of the present invention.

[0123] Further embodiments relate to transformed organisms, such as organisms selected from the group consisting of plant and insect cells, bacteria, yeast, baculovirus, protozoa, nematodes, and algae, which contain the DNA sequence of the invention, an expression cassette comprising said DNA sequence, or a vector comprising said expression cassette, which may be stably introduced into the genome of the transformed organism.

[0124] It is recognized that the genes encoding the insecticidal Vip proteins of the present invention can be used to transform insect pathogenic organisms, including baculoviruses, fungi, protozoa, bacteria, and nematodes.

[0125] The nucleotide sequence encoding the insecticidal Vip protein of the present invention may be introduced into a microbial host via a suitable vector, and the host may be applied to the environment or to a plant or animal. The term "introduction" in the context of inserting a nucleic acid into a cell means "transfection" or "transformation" or "transduction" and includes reference to the incorporation of a nucleic acid into a prokaryotic or eukaryotic cell, where the nucleic acid may be integrated into the genome of the cell (e.g., chromosome, plasmid, plastid, or mitochondrial DNA), converted into an autonomous replicon, or expressed transiently (e.g., transfected mRNA).

[0126] Numerous methods are available for introducing the foreign DNA expressing the pesticidal protein into the microbial host under conditions that allow the stable maintenance and expression of the DNA.For example, an expression cassette can be constructed that includes the nucleotide construct of interest, which is operably linked with transcriptional and translational regulatory signals for the expression of the nucleotide construct, and a nucleotide sequence that is homologous to the sequence of the host organism, thereby allowing integration, and / or that includes a replication system that functions in the host, thereby allowing integration or stable maintenance.

[0127] pest For the purposes of this invention, pests include, but are not limited to, insects, fungi, bacteria, nematodes, mites, ticks, protozoan pathogens, animal parasitic liver flukes, etc. Pests include insects selected from the orders Lepidoptera, Coleoptera, Diptera, Homoptera, Hymenoptera, Mallophaga, Hemiptera, Orthoptera, Thripida, Dermaptera, Isoptera, Gnathagona, Crypoptera, Trichoptera, etc., particularly insects of the orders Coleoptera and Lepidoptera.

[0128] Proteins with insecticidal activity against a broad spectrum of lepidopteran pests, including, but not limited to, black cutworm (BCW, Agrotis ipsilon), fall armyworm (FAW, Spodoptera frugiperda), tobacco budworm (TBW, Heliothis virescens), sugarcane borer (SCB, Diatraea saccharalis), lesser cornstalk borer (LCB, Elasmopalpus lignosellus), and corn earworm (CEW, Helicoverpa zea).

[0129] Lepidoptera (moths and butterflies) are the second most numerous and diverse order of pest insects after beetles. Nearly every cultivated plant is attacked by at least one species of lepidopteran pest. As pollinators of many plants, adult moths and butterflies are usually beneficial insects that feed on nectar using their suction proboscis. However, nearly all caterpillars and caterpillars have chewing mouthparts adapted to feed on various parts of plants. Most caterpillars and caterpillars either eat leaves or burrow into juicy plant tissues.

[0130] Examples of Lepidoptera pests are Agrotis ipsilon, Agrotis orthogonia, Agrotis segetum, Anticarsia gemmatalis, Artogeia rapae, Ostrinia nubilalis, Ostrinia furnacalis, Helicoverpa zea, Helicoverpa armigera, Heliothis virescens, Spodoptera frugiperda, Spodoptera exigua, Spodoptera exigua, Spodoptera ornithogalli, Spodoptera praefica, Spodoptera eridania, Diatraea grandiosella, Diatraea saccharalis, Elasmopalpus lignosellus, Sesamia nonagroides, Chilo partellus, Chilo suppressalis, Feltia subterranean, Pseudaletia unipunctata, Suleima heliantha helianthana, Homoeosoma electellum, Pectinophora gossypiella, Scirpophaga sp.), Pseudoplusia includens, Plathypena scabra, Peridroma saucia, Papaipema nebris, Phthorimaea operculella, Pieris brassicae, Plutella xylostella, Platynota idaeusalis, Trichoplusia ni, Keiferia lycopersicella, Manduca sexta, Manduca quinquemaculata, Mamestra configura configura, Endopiza viteana, and Cydia pomonella.

[0131] Coleoptera (beetles) Examples of Coleoptera pests are Diabrotica virgifera virgifera, Diabrotica longicornis barberi, Diabrotica undecimpunctata howardi, Melanotus spp., Cyclocephala borealis, Cyclocephala immaculata, Popillia japonica, Chaetocnema pulicaria, Sphenophorus maidis, Phyllophaga crinita, Eleodes spp. spp.), Conoderus spp., Aeolus spp.), Oulema melanopus, Chaetocnema pulicaria, Sphenophorus maidis, Oulema melanopus, Hypera punctata, Diabrotica undecimpunctata howardi, Zygogramma exclamationis, Bothyrus gibbosus, Anthonomus grandis, Colaspis brunnea, colaspis, Lissorhoptrus oryzopyhilus, Sitophilus oryzae, Oulema oryzae, Epilachna varivestis, Leptinotarsa ​​decemlineata, Epitrix hirtipennis, Phyllotreta cruciferae, Phyllotreta pusilla, Anthonomus eugenii, Leptinotarsa ​​decemlineata, Epitrix cucumeris, Hemicrepidus memnonius memnonius, Melanpotus spp., and Ceutorhychus assimilis.

[0132] Homoptera (whiteflies, aphids) Examples of homopteran pests are Rhopalosiphum maidis, Anuraphis maidiradicis, Sipha flava, Schizaphis graminum, Macrosiphum avenae, Macrosiphum euphorbiae, Aphis gossypii, Pseudatomoscelis seriatus, Trialeurodes abutilonea, Nephotettix nigropictus, Nilaparvata lugens, and others. lugens, Sogatella furcifera, Laodelphaax striatellus, Myzus persicae, Empoasca fabae, Schizaphis graminum, Brevicoryne brassicae, Trileurodes vaporariorum, Bemisia tabaci, Bemisia argentifolii, Paratrioza cockerelli, Cavariella egopodii aegopodii, Brevicoryne brassicae, Pemphigus popullivenae, Dysaphis plantaginea, and Saccharosydne saccharivora.

[0133] Bioassay A wide variety of bioassay procedures are known to those skilled in the art. A typical procedure involves adding the compound or organism to be tested to the food in a closed container. Pesticidal activity can be measured by, but is not limited to, changes in mortality, weight loss, attraction, repellency, and other behavioral and physical changes after feeding and exposure for an appropriate length of time. The bioassays described herein can be used with any feeding pest, at the larval or adult stage.

[0134] Insecticidal Composition The compositions of the embodiments are useful in various ways to protect plants, seeds, and plant products. For example, the compositions can be used in a method that involves placing an effective amount of the pesticidal composition in the environment surrounding the pest by a procedure selected from the group consisting of spraying, dusting, broadcasting, or seed coating.

[0135] Before plant propagation material (fruits, tubers, bulbs, bulbs, grains, seeds), but especially seeds, is sold commercially, it is customarily treated with a protective coating containing a herbicide, insecticide, fungicide, bactericide, nematicide, molluscicide, or a mixture of a plurality of formulations thereof, together with, if necessary, further carriers, surfactants, or application-facilitating adjuvants customarily employed in the formulation industry, which provide protection against damage caused by harmful bacteria, fungi, or animals. For treating seeds, said protective coating may be applied to the seeds either by impregnating the tubers or grains with a liquid formulation or by coating them with a combination of wet or dry formulations. In addition, in special cases, other methods of application to the plants are possible, for example treatment on the buds or fruits.

[0136] Seeds of the plants of the embodiments comprising a nucleotide sequence encoding a pesticidal protein of the embodiments may be treated with a seed protection coating comprising a seed treatment compound, such as captan, carboxin, thiram, metalaxyl, pirimiphos-methyl, and other compounds commonly used in seed treatment. In some embodiments, the seed protection coating comprising the pesticidal composition of the embodiments is used alone or in combination with one of the seed protection coatings conventionally used in seed treatment. The compositions of the embodiments may be in a form suitable for direct application or a concentrate of a primary composition that requires dilution with an appropriate amount of water or other diluent before application. The concentration of the pesticide will vary depending on the nature of the particular formulation, in particular whether it is a concentrate or for direct use. The compositions contain 1 to 98% of a solid or liquid inert carrier, and 0 to 50% or 0.1 to 50% of a surfactant. These compositions are administered at the rate indicated on the product.

[0137] In some embodiments, the transformed microorganisms (including whole organisms, cells, spores, such as Bacillus thuringiensis transformed with a nucleotide sequence disclosed herein, pesticidal proteins, pesticidal components, pest-affected components, mutants, live or dead cells and cellular components (including mixtures of live and dead cells and cellular components, and including disrupted cells and cellular components) or isolated pesticidal proteins may be formulated with an acceptable carrier into pesticidal compositions, such as suspensions, solutions, emulsions, dusting powders, dispersible granules or pellets, wettable powders, and concentrates for emulsification, aerosols or sprays, impregnated granules, adjuvants, coating pastes, colloids, and encapsulations, for example, in polymeric materials. Such formulated compositions may be prepared by conventional methods, such as drying, lyophilization, homogenization, extraction, filtration, centrifugation, sedimentation, or concentration of cultured cells containing the polypeptide.

[0138] The compositions as disclosed above may be obtained by adding surfactants, inert carriers, preservatives, water-retaining materials, feeding stimulants, attractants, encapsulants, binders, emulsifiers, dyes, UV protectants, buffers, fluidizing agents or fertilizers, micronutrient donors, or other formulations that affect plant growth. One or more agricultural chemicals, including but not limited to herbicides, insecticides, fungicides, bactericides, nematicides, molluscicides, acaricides, plant growth regulators, harvest aids and fertilizers, may be combined with carriers, surfactants or adjuvants conventionally employed in the formulation industry, or other components that facilitate product handling and application to specific target pests. Suitable carriers and adjuvants may be solid or liquid and may correspond to substances commonly employed in formulation technology, such as natural or regenerated mineral substances, solvents, dispersants, wettable powders, tackifiers, binders, or fertilizers. The active ingredients of the embodiments are usually applied in the form of a composition and can be applied to the crop area, plants, or seeds to be treated. For example, the compositions of the embodiments can be applied to grains in preparation for storage or during storage, such as in grain bins or silos. The compositions of the embodiments can be applied simultaneously or sequentially with other compounds. Methods of applying the agricultural chemical compositions of the embodiments containing the active ingredients of the embodiments or at least one pesticidal protein produced by the bacterial strains of the embodiments include, but are not limited to, foliar application, seed coating, and soil application. The number of applications and frequency of application depend on the intensity of the infestation of the corresponding pest.

[0139] In further embodiments, the compositions of the embodiments and the transformed microorganisms and pesticidal proteins may be treated prior to formulation to extend pesticidal activity when applied to the environment of the target pest, so long as the pretreatment does not adversely affect the pesticidal activity. Such treatment may be by chemical and / or physical means, so long as the treatment does not adversely affect the properties of the composition. Examples of chemical agents include, but are not limited to, halogenated agents, aldehydes such as formaldehyde and glutaraldehyde, anti-infective agents such as zephiran chloride, and alcohols such as isopropanol and ethanol.

[0140] In other embodiments, it may be advantageous to treat the toxin polypeptide with a protease, such as trypsin, to activate the protein prior to application of the pesticidal protein composition of the embodiments to the environment of the target pest. Methods for activating protoxins with serine proteases are well known in the art.

[0141] The composition (comprising the transformed microorganism and pesticidal protein of the embodiment) may be applied to the pest's surroundings, for example, by spraying, atomizing, dusting, spreading, coating, or pouring, by introduction into or onto the soil, by introduction into irrigation, by treating seeds, or as a protective measure, by general application or dusting when the pest begins to appear or before the pest appears. For example, the pesticidal protein and / or transformed microorganism of the embodiment may be mixed with grain to protect the grain during storage. In general, it is important to control pests well in the early stages of plant growth, when the plant can be most seriously damaged. If deemed necessary, the composition of the embodiment may conveniently contain another pesticide. In some embodiments, the composition is applied directly to the soil at the time of planting in the form of a granule of a composition comprising a carrier and dead cells of the Bacillus strain or the transformed microorganism of the embodiment. Another embodiment is a granular form of the composition comprising an agricultural chemical, such as, for example, a herbicide, a pesticide, a fertilizer, an inert carrier, and killed cells of the Bacillus strain or transformed microorganism of the embodiments.

[0142] Those skilled in the art will recognize that not all compounds are equally effective against all pests.The compounds of the embodiments exhibit activity against pests, which may include economically important agricultural pests, forest pests, greenhouse pests, nursery pests, ornamental plant pests, food pests and fiber pests, public health pests and animal health pests, domestic and commercial pests, household pests, and stored product pests.Pests include lepidopteran insects. Lepidoptera larvae include armyworms, cutworms, loopers, and heliothines of the noctuid moths Spodoptera frugiperda J.E. Smith (spodoptera fall armyworm); S. exigua Hubner (beet armyworm); S. litura Fabricius (tobacco cutworm, cluster caterpillar); Mamestra configurata Walker (bertha armyworm); M. brassicae Linnaeus (cabbage moth); Agrostis epsilon Hufnagel (black cutworm); A. orthogonia Morrison (western cutworm); A. subterranea Fabricius(granulate cutworm);Alabama argillacea Hubner(cotton leaf worm);Trichoplusia ni Hubner(cabbage looper);Pseudoplusia includens Walker(soybean looper);Anticalcia gemataris Hubner(velvetbean caterpillar);Hypena scabra Fabricius(green cloverworm);Heliotis virescens Fabricius(false tobacco moth);Pseudaletia unipuncta Haworth (army worm); Athetis mindara Barnes and Mcdunnough (rough skinned cutworm); Euxoa messoria Harris (darksided cutworm); Earias insulana Boisduval (spiny bollworm); E. vittella Fabricius (spotted bollworm); Helicoverpa armigera Hubner (American bollworm); H. zea Boddie (corn earworm or cotton bollworm); Melanchra picta Harris (zebra caterpillar) caterpillar);Egira curialis (Xylomyges curialis) Grote (citrus cutworm);borer, casebearer, webworm, coneworm, and skeletonizer of the family Pyralidae Ostrinia nubilalis Hubner (European corn borer);Amyelois transitella Walker (naval orangeworm);Anagasta kuehniella Zeller (Mediterranean flour moth);Cadra cautella Walker (almond moth);Kiro supressalis Walker (rice stem borer);C. partellus (sorghum borer);Corcyra cephalonica Stainton (rice moth);Crambus caliginosellus Clemens (corn root webworm);C. teterrellus Zincken (bluegrass webworm);Cnaphalocrocis medinalis Guenee (rice leaf roller);Desmia funeralis Hubner (grape leaffolder);Diaphania hyalinata Linnaeus (melon worm);D. nitidalis Stoll(pickleworm); Diatrea grandiosera Dyar(southwestern corn borer), D. saccharalis Fabricius(sugarcane borer); Eoreuma loftini Dyar(Mexican rice borer); Ephestia elutella Hubner(tobacco (cacao) moth); Galleria mellonella Linnaeus(greater wax moth); Herpetogramma licarsisalis Walker(sod webworm); Homeosoma erectum Hulst(sunflower moth) Elasmopalpus lignocellus Zeller (corn moth);Achroia grisella Fabricius (lesser wax moth); Loxostege sticticalis Linnaeus (beet webworm); Orthaga thyrisalis Walker (tea tree web moth); Maruca testulalis Geyer (bean pod borer); Plodia interpunctella Hubner (Indian meal moth); Scirpophaga incertulas Walker (yellow stem borer); Udea rubigalis Guenee (celery leaftier); and leafrollers, budworms, seed worms, and fruit worms of the Tortricidae Acleris gloverana Walsingham (Western blackheaded budworm); A. variana Fernald (Eastern blackheaded budworm); Archips argyrospila Walker (fruit tree leaf roller); A. rosana Linnaeus (European leaf roller); and other Archips species, Adoxophyes orana Fischer von Rosslerstamm (summer fruit tortrix moth));Cochylis hospes Walsingham (banded sunflower moth); Cydia latiferreana Walsingham (filbertworm); C. pomonella Linnaeus (codling moth); Platynota flavedana Clemens (variegated leafroller); P. stultana Walsingham (omnivorous leafroller); Lobesia botrana Denis & Schiffermuller (European grape vine moth); Spironota ocellana Denis & Schiffermuller (eyespotted bud moth);Endopizza vitaeana Clemens(grape berry moth);Eupoecilia ambiguella Hubner(vine moth);Bonagota salubricola Meyrick(Brazilian apple leafroller);Grapholita molesta Busck(oriental fruit moth);Suleima heliantsana Riley(sunflower bud moth);Argyrotaenia spp.;Choristoneura spp.;

[0143] Other agricultural pests of the Lepidoptera order that are selected are Alsophila pometaria Harris (fall cankerworm); Anarsia lineatella Zeller (peach twig borer); Anisota senatoria J.E. Smith (orange striped oakworm); Antheraea pernyi Guerin-Meneville (Chinese Oak Silkmoth); Bombyx mori Linnaeus (Silkworm); Bucculatrix thurberiella Busck (cotton leaf perforator); Collas eurytheme Boisduval (alfalfa caterpillar); Datana integerrima Grote & Robinson (walnut caterpillar); Dendrolimus sibiricus Tschetwerikov (Siberian silk moth); Ennomos subsignaria Hubner (elm spanworm); Erannis tiliaria Harris (linden looper); Euproctis chtysorrhoea Linnaeus (browntail moth); Harrisina americana Guerin-Meneville (grapeleaf skeletonizer);Hemileuca oliviae Cockrell (range caterpillar); Hyphantria cunea Drury (fall webworm); Keiferia lycopersicella Walsingham (tomato pinworm); Lambdina fiscellaria fiscellaria Hulst (Eastern hemlock looper); L. fiscellaria lugubrosa Hulst (Western hemlock looper); Leucoma salicis Linnaeus (satin tussock moth) moth);Lymantria dispar Linnaeus(gypsy moth);Manduka cinchemaculata Haworth(five spotted hawk moth, tomato hornworm);M. sexta Haworth(tomato hornworm, tobacco hornworm);Operophtera brumata Linnaeus(winter moth);Paleacrita vemata Peck(spring cankerworm);Papilio cresphontes Cramer(giant swallowtail, orange dog);Phryganidia californica Packard(California oakworm);Phyllocnistis citrella Stainton (citrus leafminer); Phyllonotycter blancardella Fabricius (spotted tentiform leafminer); Pieris brassicae Linnaeus (large white butterfly); P. rapae Linnaeus (small white butterfly); P. napi Linnaeus (green veined white butterfly); Platyptilia carduidactyla Riley (artichoke plume moth); Plutella xylostella Linnaeus(diamondback moth);Pectinophora gossypiella Saunders(pink bollworm);Pontia protodice Boisduval & Leconte(southern cabbageworm);Sabulodes aegrotata Guenee(omnivorous looper);Schizura concinna JE Smith(red humped caterpillar);Sitotroga cerealella Olivier(Angoumois grain moth);Thaumetopoea pityocampa Schiffermuller(pine processionary moth) caterpillar);including, but not limited to, Tineola bisselliella Hummel (webbing clothesmoth); Tuta absolute Meyrick (tomato leafminer); Yponomeuta padella Linnaeus (ermine moth); Heliothis subflexa Guenee; Malacosoma spp. and Orgyia spp.;

[0144] Pests may be tested for pesticidal activity of the compositions of the embodiments at an early developmental stage, such as larvae or other immature forms. The pests may be reared in complete darkness at about 20° C. to about 30° C. and a relative humidity of about 50% to about 70%. Methods for rearing insect larvae and performing bioassays are well known to those skilled in the art.

[0145] Methods for controlling insects, particularly lepidoptera, according to the invention may include applying (e.g., spraying) an insecticidal / pesticidal composition disclosed herein, comprising a host cell transformed with a nucleotide sequence of the invention, to the area or plant to be protected, to the location (area) to be protected. The location to be protected may include, for example, the habitat of the pest or growing vegetation, or the area where vegetation is to be grown.

[0146] The present disclosure further relates to a method for controlling lepidopteran corn pests, comprising applying an insecticidal / pesticidal composition disclosed herein to the area or plant to be protected by planting a corn plant transformed with a nucleotide sequence of the present invention or by spraying a composition containing a Vip protein of the present invention. The present invention also relates to the use of the compositions disclosed herein against lepidopteran corn pests to minimize damage to corn plants.

[0147] The present disclosure relates to a method for controlling lepidopteran eggplant pests, which comprises applying an insecticidal / pesticidal composition as disclosed herein to the area or plant to be protected, either by planting an eggplant plant transformed with a nucleotide sequence of the present invention or by spraying a composition containing a protein of the present invention. The present invention also relates to the use of the compositions of the present invention against lepidopteran eggplant pests to minimize damage to eggplant plants.

[0148] The present disclosure relates to a method for controlling rice pests of the order Lepidoptera, in particular the order Lepidoptera rice stem borer, rice leaffoloder, rice skipper, rice cutworm, rice armyworm or rice caseworm, preferably Cyro spressalis, Cyro partellus, Cyrophaga incertulus, Sesamia inferens, Cnaphalocrosis medinalis, Marasmia patnalis, Marasmia exigua, Marasmia ruralis, Scirpophaga innotata, and the like. innotata), which comprises applying an insecticidal / pesticidal composition disclosed herein to the area or plant to be protected by planting a rice plant transformed with a nucleotide sequence of the present invention or by spraying a composition containing a Vip protein of the present invention. The present invention also relates to the use of the compositions disclosed herein against lepidopteran rice pests to minimize damage to rice plants.

[0149] The present disclosure further relates to a method for controlling lepidopteran tomato pests, particularly the lepidopteran Helicoverpa armigera, comprising applying an insecticidal / pesticidal composition disclosed herein to the area or plant to be protected, either by planting a tomato plant transformed with a nucleotide sequence of the present invention or by spraying a composition containing a Vip protein of the present invention. The present invention also relates to the use of the compositions disclosed herein against lepidopteran tomato pests to minimize damage to tomato plants.

[0150] The present disclosure further relates to a method for controlling lepidopteran cotton pests, comprising applying an insecticidal / pesticidal composition disclosed herein to the area or plant to be protected by planting a cotton plant transformed with a nucleotide sequence of the present invention or by spraying a composition containing a Vip protein of the present invention. The present invention also relates to the use of the compositions disclosed herein against lepidopteran cotton pests to minimize damage to cotton plants.

[0151] To obtain the insecticidal proteins (SEQ ID NO:1, SEQ ID NO:7, and SEQ ID NO:11), recombinant host cells expressing the Vip protein can be conventionally grown in an appropriate culture medium. The toxins can then be isolated and purified by standard techniques such as chromatography, extraction, electrophoresis, and the like.

[0152] Thus, the present invention provides compositions and methods for affecting pests, particularly plant pests. More specifically, the present invention provides polynucleotides encoding the Vip proteins disclosed herein against pests such as, but not limited to, lepidopteran pests such as Spodoptera frugiperda (Fallen armyworm), Helicoverpa armigera (Cotton bollworm and cotton earworm), Cnaphalochrosis medinalis (Rice leafroller), and Cyrpophaga incertulus (Rice yellow stem borer), and Pectinophora gossypiella.

[0153] According to the present invention, a nucleotide sequence encoding the Vip protein from Bacillus thuringiensis has been identified. A preferred embodiment of the present invention provides an insecticidal protein having an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:7, and SEQ ID NO:11, which exerts insecticidal activity when expressed in a plant.

[0154] In another embodiment, a recombinant nucleic acid molecule is provided comprising a nucleotide sequence encoding a protein having an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:7, and SEQ ID NO:11. In another embodiment, a recombinant nucleic acid molecule is provided comprising a nucleotide sequence encoding a protein disclosed herein, said nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:13, and SEQ ID NO:14. In yet another embodiment, a recombinant nucleic acid molecule is provided comprising a nucleotide sequence encoding a protein disclosed herein, said nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:13, and SEQ ID NO:14.

[0155] In a further embodiment, there is provided a recombinant nucleic acid molecule as disclosed herein, wherein said nucleotide sequence is a synthetic sequence designed for optimal expression in plants.

[0156] Yet another embodiment of the present invention relates to a nucleotide sequence encoding a protein disclosed in the present invention, said sequence having a complementary sequence that hybridizes to a coding sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:13, and SEQ ID NO:14 under hybridization conditions of 68°C followed by washing at 68°C in 2xSSC containing 0.1% SDS.

[0157] Yet another embodiment of the present invention relates to a nucleotide sequence encoding a protein disclosed in the present invention, said sequence having a complementary sequence that hybridizes to a coding sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:5, and SEQ ID NO:6 under hybridization conditions of 37°C to 68°C followed by washing at 37°C to 68°C in 2xSSC containing 0.1% SDS.

[0158] Yet another embodiment of the present invention relates to a nucleotide sequence encoding a protein disclosed in the present invention, said sequence having a complementary sequence that hybridizes to a coding sequence selected from the group consisting of SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10 under hybridization conditions of 37°C to 68°C followed by washing at 37°C to 68°C in 2xSSC containing 0.1% SDS.

[0159] Yet another embodiment of the present invention relates to a nucleotide sequence encoding a protein disclosed in the present invention, said sequence having a complementary sequence that hybridizes to a coding sequence selected from the group consisting of SEQ ID NO:12, SEQ ID NO:13, and SEQ ID NO:14 under hybridization conditions of 37°C to 68°C followed by washing at 37°C to 68°C in 2xSSC containing 0.1% SDS.

[0160] The present invention further provides a nucleotide sequence encoding the protein disclosed in the present invention, which hybridizes to a completely complementary sequence of a nucleotide sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:13, and SEQ ID NO:14 under conditions of 0.1×SSPE and 65° C.

[0161] The present invention further provides a nucleotide sequence encoding the protein disclosed in the present invention, which hybridizes to a completely complementary sequence of a nucleotide sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:13, and SEQ ID NO:14 under conditions of 0.1×SSPE and 55° C.

[0162] In another embodiment, a recombinant nucleic acid molecule as disclosed herein is provided, wherein said nucleic acid molecule comprises a promoter operably linked to said nucleotide sequence encoding the insecticidal protein of the present invention.

[0163] In another embodiment, an expression cassette is provided, comprising a heterologous promoter operably linked to at least one nucleotide sequence disclosed in the present invention. Another embodiment of the present invention relates to an expression cassette disclosed herein, wherein the promoter functional in plants is selected from the group consisting of an inducible promoter, a constitutive promoter, a tissue-preferred promoter, and a tissue-specific promoter.

[0164] A further embodiment of the present invention provides a vector comprising a recombinant nucleic acid molecule disclosed in the present invention. Furthermore, in another embodiment, a host cell is provided comprising at least one protein disclosed in the present invention. In a further embodiment, a host cell is provided comprising at least one recombinant nucleic acid molecule disclosed in the present invention. In a further embodiment, a host cell is provided comprising a vector disclosed and claimed in the present invention. The host cell may be a bacterium, a cyanobacterium, a virus, a fungus, an insect, or a yeast. Another embodiment of the present invention relates to a host cell disclosed in the present invention, wherein the host cell is an Agrobacterium or an E. coli.

[0165] In another embodiment of the present invention, a method for producing a plant exhibiting insecticidal activity is provided, said method comprising at least the step of introducing at least one nucleic acid molecule of the present invention. Another embodiment of the present invention relates to a method for producing a plant exhibiting insecticidal activity, said method comprising at least the step of introducing at least one nucleic acid molecule of the present invention, said introduction being carried out by introgressing into the plant a nucleic acid molecule encoding a protein having an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:7, and SEQ ID NO:11.

[0166] Another embodiment of the present invention relates to a method for producing a plant exhibiting insecticidal activity, said method comprising at least the step of introducing at least one nucleic acid molecule of the present invention, said introduction being (a) inserting the recombinant nucleic acid molecule of claim 2 into a plant cell, wherein the nucleic acid molecule further comprises a promoter and a terminator functional in the plant cell, the promoter and terminator being operably linked to a nucleotide sequence encoding a protein having an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:7, and SEQ ID NO:11; (b) obtaining a transformed plant cell from said plant cell of step (a), wherein said transformed plant cell comprises a nucleotide sequence encoding at least one of said proteins; and (c) generating a transgenic plant from the transformed plant cell of step (b), wherein the transgenic plant comprises a nucleotide sequence encoding at least one of the proteins. This is carried out by.

[0167] In another embodiment, a transgenic plant is provided comprising a recombinant nucleic acid molecule comprising a nucleotide sequence encoding at least one protein disclosed in the present invention, said plant exhibiting insecticidal activity. In a further embodiment of the present invention, progeny and seeds obtained from the transgenic plant disclosed in the present invention are provided.

[0168] The plant is a monocotyledonous plant, a dicotyledonous plant, and / or a legume. The monocotyledonous plants include, but are not limited to, maize, wheat, rice, oats, sorghum, millets, and grasses. The dicotyledonous plants include, but are not limited to, cotton, eggplant, tomato, canola, soybean, tobacco, fruit trees, cruciferous plants, pepper plants, ornamentals, sunflowers, cucurbits, pigeon peas, peas, chickpeas, and melons.

[0169] In one embodiment of the present invention, a recombinant microorganism is provided comprising a recombinant nucleotide sequence encoding an insecticidal protein of the present invention. Another embodiment provides a recombinant microorganism comprising a recombinant nucleotide sequence encoding an insecticidal protein of the present invention, said nucleotide sequence being selected from the group consisting of SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:13, and SEQ ID NO:14.

[0170] Another embodiment relates to a recombinant microorganism comprising a recombinant nucleotide sequence encoding an insecticidal protein of the present invention, said nucleotide sequence being selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 6. Another embodiment relates to a recombinant microorganism comprising a recombinant nucleotide sequence encoding an insecticidal protein of the present invention, said nucleotide sequence being selected from the group consisting of SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10. Another embodiment relates to a recombinant microorganism comprising a recombinant nucleotide sequence encoding an insecticidal protein of the present invention, said nucleotide sequence being selected from the group consisting of SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14.

[0171] A further embodiment of the present invention relates to a recombinant microorganism comprising a recombinant nucleotide sequence encoding an insecticidal protein of the present invention, said microorganism being selected from the group consisting of bacteria, viruses, algae, and fungi. A further embodiment of the present invention provides a recombinant microorganism comprising a recombinant nucleotide sequence encoding an insecticidal protein of the present invention and a second nucleotide sequence encoding a second insecticidal protein. The second insecticidal protein may be a delta-endotoxin or a plant insecticidal protein, and the second nucleotide may code for a delta-endotoxin protein or a plant insecticidal protein. Another embodiment of the present invention provides a recombinant microorganism comprising a second nucleotide sequence encoding a delta-endotoxin or a plant insecticidal protein.

[0172] A further embodiment of the present invention provides a composition comprising a nucleotide sequence encoding an insecticidal protein of the present invention. Another embodiment of the present invention provides a composition comprising a recombinant nucleotide sequence encoding an insecticidal protein of the present invention, said nucleotide sequence being selected from the group consisting of SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:13, and SEQ ID NO:14.

[0173] It is contemplated that the insecticidal Vip proteins disclosed in the present invention may be used for agricultural purposes, i.e., to protect plants from pest infestation, more specifically to protect plants from lepidopteran pest infestation.

[0174] Another embodiment of the present invention provides a nucleotide sequence encoding the insecticidal toxin protein disclosed herein, wherein said nucleotide sequence is isolated from Bacillus thuringiensis.

[0175] Another embodiment of the present invention provides a nucleotide sequence encoding an insecticidal toxin protein disclosed herein, said nucleotide sequence being optimized for expression in a plant. Another embodiment of the present invention provides a nucleotide sequence encoding an insecticidal toxin protein disclosed herein, said nucleotide sequence being optimized for expression in a plant, preferably said nucleotide sequence being optimized for expression in a monocotyledonous plant. Another embodiment of the present invention provides a nucleotide sequence encoding an insecticidal toxin protein disclosed herein, said nucleotide sequence being optimized for expression in a plant, preferably said nucleotide sequence being optimized for expression in a dicotyledonous plant. Another embodiment of the present invention provides a nucleotide sequence encoding an insecticidal toxin protein disclosed herein, said nucleotide sequence being optimized for expression in a plant, preferably said nucleotide sequence being optimized for expression in a legume.

[0176] Another embodiment of the present invention provides a nucleotide sequence encoding an insecticidal protein disclosed herein, wherein said nucleotide sequence has been optimized for expression in a plant, said optimization comprising the steps of: (i) removing polyadenylation sequences; (ii) adjusting the A and T content of said nucleotide sequence to between 40% and 49% without modifying the amino acid sequence of the protein; and (iii) modifying the codons of the coding sequence consistent with steps (i) and (ii).

[0177] A further embodiment of the present invention provides a method of controlling a pest comprising contacting the pest with a pesticidal amount of a Vip protein disclosed herein.

[0178] Yet another embodiment of the present invention provides a method for controlling insect infestations and providing insect resistance management in crop plants, the method comprising contacting the crop plants with an insecticidally effective amount of a composition comprising a Vip protein as disclosed herein.

[0179] Another embodiment of the present invention provides a method for expressing an insecticidal protein toxin disclosed herein in a plant, comprising: (a) inserting into the genome of a plant cell a nucleotide sequence operably linked in a 5' to 3' direction: a recombinant double-stranded DNA molecule, wherein said recombinant double-stranded DNA molecule comprises a promoter functional in a plant cell; a nucleotide sequence encoding an insecticidal amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:7, and SEQ ID NO:11; and a 3' untranslated nucleotide sequence functional to terminate transcription in a cell of said plant; (b) obtaining a transformed plant cell containing the nucleic acid sequence of step (a); and (c) generating from said transformed plant cell a transformed plant that expresses said insecticidal protein in the plant.

[0180] Another embodiment of the present invention provides a seed of the transgenic plant described herein or a progeny of said seed, wherein said seed and said progeny comprise a nucleic acid sequence encoding an insecticidal Vip protein having an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:7, and SEQ ID NO:11.

[0181] Another embodiment of the present invention provides a method for expressing an insecticidal protein disclosed herein in a plant, wherein said plant is a monocotyledonous plant, including but not limited to corn, wheat, rice, oat, sorghum, millet, and grasses, or a dicotyledonous plant, including but not limited to cotton, eggplant, tomato, canola, soybean, tobacco, fruit trees, cruciferous plants, pepper plants, ornamentals, sunflower, cucurbits, pigeon pea, pea, chickpea, and melon.

[0182] Yet another embodiment of the present invention provides a biological sample derived from a plant cell, tissue, or seed obtainable by the methods disclosed herein, said sample comprising a detectable amount of a nucleotide sequence of the present invention.

[0183] Yet another embodiment of the present invention provides an application of a protein in controlling pests, wherein said protein has an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:7, and SEQ ID NO:11.

[0184] Yet another embodiment of the present invention provides the use of a nucleotide sequence disclosed herein for producing an insecticidal composition, said composition comprising a Bacillus thuringiensis cell comprising said nucleotide sequence.

[0185] Yet another embodiment of the present invention provides the use of the nucleotide sequences, DNA constructs and / or DNA expression cassettes or plasmids disclosed herein for producing insect-resistant transgenic plants.

[0186] Yet another embodiment of the present invention provides the use of a composition comprising the insecticidal Vip protein disclosed herein for controlling pests.

[0187] Yet another embodiment of the present invention provides the use of a microorganism comprising an insecticidal Vip protein as disclosed herein for controlling pests, said microorganism being Escherichia coli and / or Bacillus thuringiensis.

[0188] Yet another embodiment of the present invention provides the use of a microorganism comprising an insecticidal Vip protein as disclosed herein for controlling pests, said microorganism comprising a gene encoding said protein.

[0189] Yet another embodiment of the present invention provides the use of a microorganism comprising an insecticidal Vip protein as disclosed herein for controlling pests, said microorganism comprising a gene encoding said protein having an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:7, and SEQ ID NO:11.

[0190] These and / or other embodiments of the invention are reflected in the claims which form a part of the description of the invention.

[0191] Various modifications and other embodiments of the invention may be suggested by those skilled in the art having the benefit of the descriptions set forth in the description and associated drawings pertaining to those inventions. It is to be understood, therefore, that the invention is not to be limited to the specific embodiments disclosed herein, and that modifications and other embodiments are intended to be included within the scope of the appended claims.

[0192] The following examples are illustrative of the present invention and are not offered to limit the invention or the protection sought. EXAMPLES

[0193] The following examples are presented so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors perceive as their invention, nor are they intended to represent that the experiments described below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should be accounted for.

[0194] Common methods DNA manipulations were performed using standard procedures in the art. These procedures can often be modified and / or substituted without substantially changing the results. Most of these procedures are described in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, second edition, 1989, unless other references are specified.

[0195] Example 1: Isolation and characterization of the Vip gene Degenerate PCR primers (SEQ ID NO: 3 and SEQ ID NO: 4) were designed to identify the gene encoding the plant insecticidal protein (Vip) protein disclosed herein. DNA isolated from Bacillus thuringiensis (isolated from a soil sample obtained from Telangana, India) was subjected to PCR using these primers. The amplified product obtained after PCR amplification was ligated into pCR2.1-TOPO TA vector (ThermoFisher, USA) and TOP10 E. coli chemically competent cells were transformed with the recombinant pCR2.1-TOPO plasmid. Approximately 198 colonies were screened by PCR using M13 universal primers. Restriction enzyme digestion analysis was performed to confirm the Vip gene amplified using the degenerate primers. The amplified product was then sequenced using Sanger sequencing (Eurofins Genomics, India). Three nucleotide sequences having the nucleotide sequences depicted in SEQ ID NO: 2, SEQ ID NO: 8, and SEQ ID NO: 12 could be amplified. The amino acid sequences deduced from the nucleotide sequences are shown in SEQ ID NO:1, SEQ ID NO:7, and SEQ ID NO:11, respectively.

[0196] For ease of understanding, the three Vip proteins are named as follows: Vip protein 1 (SEQ ID NO:1), Vip protein 2 (SEQ ID NO:7), and Vip protein 3 (SEQ ID NO:11) SEQ ID NO:3 - Degenerate forward primer: ATGAMYAWGAATAATRCTAARTTAA SEQ ID NO:4 - Degenerate reverse primer: YTAHYTAATAGAGAMATCRTAAAA

[0197] Example 2: Cloning and expression of nucleotide sequences encoding Vip proteins To obtain a recombinant vector containing the nucleotide sequence shown in SEQ ID NO:2, the nucleotide sequence shown in SEQ ID NO:2 was cloned into a pET-32a(+) expression vector. E. coli cells (BL21(DE3)) were then transformed with the recombinant vector to obtain recombinant E. coli cells containing the recombinant vector containing the nucleotide sequence shown in SEQ ID NO:2. The BL21-cells were transformed with 10-50 ng of an expression vector having the nucleotide sequence shown in SEQ ID NO:2, and 50 μl aliquots of the transformed cells were plated onto agar plates containing 100 μg / ml ampicillin. The agar plates were incubated overnight at 37° C. A single colony from the plate was inoculated into 10 mL of LB medium in a flask containing 100 μg / ml ampicillin. The culture was grown overnight at 37° C. with shaking at 250 rpm.

[0198] Similarly, to obtain recombinant vectors containing the nucleotide sequences shown in SEQ ID NO:8 and SEQ ID NO:12, the nucleotide sequences were separately cloned into pET-32a(+) expression vectors, and then E. coli cells (BL21(DE3)) were separately transformed with these recombinant vectors to obtain recombinant E. coli cells.

[0199] Example 3: Expression of Vip protein The cultures of Example 2 (Vip protein 1, Vip protein 2, and Vip protein 3) were subcultured by inoculating 10 mL of LB medium in a 250 ml flask with 0.1 ml of the overnight culture. The cells were grown at 37° C. with shaking at 250 rpm until the OD600 reached 0.5-0.6. IPTG (1 mM final concentration) was added and the culture was incubated at 30° C. with shaking at 150 rpm. The overnight culture was centrifuged at 6,000 rpm for 10 min to harvest the cells at 4° C. The cells were resuspended in 0.4 ml of lysis buffer and the suspension was transferred to a 1.5 ml microcentrifuge tube. The tube was vortexed briefly for 5-10 min to completely resuspend the cells and ensure complete extraction of the soluble proteins. The cell lysate was centrifuged at 14000 rpm for 5 min to pellet the insoluble material. The soluble protein fraction was carefully removed from the cell debris. To assess the isolation efficiency and identity of the Vip protein, the cleared supernatants were analyzed by SDS-PAGE.

[0200] Example 4: Purification of Vip protein The recombinant Vip protein obtained in Example 3 was purified by column filtration using a kit from BioRad, USA. For purification of the recombinant Vip protein, the required amount of Ni-NTA agarose was transferred to the column, and the storage buffer was drained by gravity flow. The column was equilibrated by adding lysis buffer, and the excess lysis buffer was drained by gravity flow. The soluble protein fraction was loaded onto the column, and the flow-through was collected. A washing buffer was added to the column, and it was allowed to drain by gravity flow. The column was washed twice. An elution buffer containing a required amount of imidazole was used to run the column, and the eluate containing the His-tagged recombinant Vip protein was collected. The eluted protein was dialyzed to remove imidazole to obtain pure protein.

[0201] Example 5: Insect diet bioassay Bioassays with recombinant Vip proteins (SEQ ID NO: 1, SEQ ID NO: 7, and SEQ ID NO: 11) To determine the toxicity of the Vip protein, an insect diet bioassay was performed on first instar larvae of Spodoptera frugiperda and Helicoverpa amygella using the diet incorporation method.

[0202] Preparation of artificial diet All components listed in Table 1 (except dry yeast and agar) were weighed and mixed in a large bowl containing 500 ml of pre-boiled warm water. In a separate beaker, 53 g of active dry yeast was mixed with 350 ml of water. Agar (16 g) was dissolved in 350 ml of water and boiled for 5 minutes. The yeast and agar suspension was transferred to the bowl containing the other feed components previously prepared. The slurry was boiled for 5 minutes and then mixed thoroughly. 10% formaldehyde (13.5 ml) was added to the slurry and mixed with a spatula. The slurry was then cooled to 50°C. [Table 1]

[0203] Three artificial diets containing one of the purified recombinant Vip proteins, Vip protein 1, Vip protein 2, and Vip protein 3, at various concentrations (10 μg / ml, 15 μg / ml, 20 μg / ml, and 25 μg / ml), respectively, were prepared by adding the Vip proteins separately to the lukewarm artificial diet (approximately 50° C.) and mixing thoroughly. A negative control (without any Vip protein) was also prepared by mixing the diet with buffer. A positive control diet containing Vip protein (accession number DQ539887.1) at various concentrations (10 μg / ml, 15 μg / ml, 20 μg / ml, and 25 μg / ml) was prepared. The samples were then spread on Petri dishes, and the Petri dishes were exposed to UV light in a laminar flow for 1 hour to sterilize the surfaces of the samples. The samples were cut into equal-sized pieces and placed in labeled sterile cups.

[0204] First instar larvae were tested for toxicity of the Vip protein. The insects were placed on artificial diet containing recombinant Vip protein, positive control diet, and negative control diet. One larva was placed in each cup and the lid was closed. Each bioassay was repeated five times, using one first instar larva per replicate for each concentration. The insects were incubated at 28±2°C, 65±5% relative humidity, and a 14:10 L:D photoperiod. Observations were recorded after 7 days.

[0205] After 7 days, lethal concentration (LC50) values ​​were estimated using standard statistical analysis methods and the LC(50) for the insects was observed to range from 5μg / ml to 6.5μg / ml of the recombinant Vip protein. Surprisingly, all three recombinant Vip proteins (Vip protein 1, Vip protein 2, and Vip protein 3) were observed to show similar LC(50) values ​​ranging from 5μg / ml to 6.5μg / ml, whereas the LC(50) value for the positive control Vip protein (accession number DQ539887.1) was observed to be 8.23μg / ml.

[0206] Example 6: Construction of plant transformation vectors A pUC57 vector having a DNA sequence shown in SEQ ID NO:5 encoding Vip protein 1 was digested with restriction enzymes to release the DNA fragment of Vip1 protein 1 (reaction volume-20 μl, plasmid DNA-8.0 μl, 10X buffer-2.0 μl, restriction enzymes Bam HI and EcoRI-0.5 μl, distilled water-9.5 μl). All the reagents were mixed and the mixture was incubated at 37° C. for 30 minutes. The products obtained after restriction digestion were analyzed by gel electrophoresis and further purified.

[0207] The released fragment was subcloned into pUC57 carrying the CaMv35S cassette. Ligation was performed using T4 DNA ligase enzyme (reaction volume-30 μl, 10X ligation buffer-3.0 μl, vector DNA-5.0 μl, insert DNA-15.0 μl, T4 DNA ligase enzyme-1.0 μl, distilled water-6.0 μl). The reagents were mixed well and the resulting reaction mixture was incubated at 16° C. for 2 hours. Competent cells of E. coli DH5 alpha strain were then transformed with the ligation mixture containing pUC57 carrying the CaMv 35S cassette having the DNA sequence shown in SEQ ID NO:5 by adding 10 μl of the ligation mixture to 100 μl of E. coli DH5 alpha competent cells. The cell mixture thus obtained was placed on ice for 30 minutes and incubated in a water bath at 42° C. for 60 seconds for heat shock, and the cell mixture was returned to ice for 5-10 minutes. Then 1 ml of LB broth was added to the mixture and further incubated at 37° C. for 1 hour in an incubator shaker at 200 rpm. The cell suspension (100 μl) was evenly spread on LB agar medium containing 30 μg / ml kanamycin. The plate was incubated overnight at 37° C. Positive clones were confirmed by restriction digestion analysis. Plasmids were isolated from the positive clones and digested with Eco RV to release the fragment for cloning in pGreen0029.

[0208] Ti plasmid pGreen0029 vector was prepared by restriction digestion with EcoRV enzyme (reaction volume-20 μl, plasmid DNA-8.0 μl, 10X buffer-2.0 μl, restriction enzyme EcoRV-0.5 μl, distilled water-9.5 μl). All reagents were mixed and the mixture was incubated at 37° C. for 30 minutes. The products obtained after restriction digestion were analyzed by gel electrophoresis.

[0209] The purified Vip protein 1 DNA fragment (SEQ ID NO: 5) was ligated into the linearized pGreen0029 vector. The ligation was performed using T4 DNA ligase enzyme (reaction volume-30 μl, 10X ligation buffer-3.0 μl, vector DNA-5.0 μl, insert DNA-15.0 μl, T4 DNA ligase enzyme-1.0 μl, distilled water-6.0 μl). The reagents were mixed well and the resulting reaction mixture was incubated at 16° C. for 2 hours. Competent cells of E. coli DH5 alpha strain were then transformed with the ligation mixture containing the pGreen0029 vector having the DNA sequence (SEQ ID NO: 5) by adding 10 μl of the ligation mixture to 100 μl of E. coli DH5 alpha competent cells. The cell mixture thus obtained was placed on ice for 30 minutes and incubated in a water bath at 42° C. for 60 seconds for heat shock and the cell mixture was returned to ice for 5-10 minutes. Then 1 ml of LB broth was added to the mixture and further incubated at 37°C for 1 hour in an incubator shaker at 200 rpm. The cell suspension (100 μl) was evenly spread on LB agar medium containing 30 μg / ml kanamycin. The plate was incubated overnight at 37°C. Positive clones were confirmed by restriction digestion analysis. The resulting recombinant vector was named pGreen0029-CaMV35S-Vip protein 1.

[0210] Thus, the recombinant plasmid pGreen0029-CaMV35S-Vip protein 1 contains the DNA sequence (SEQ ID NO: 5) of Vip protein 1 optimized for plants under the transcriptional control of the 35SCaMV promoter. In addition, pGreen0029-CaMV35S-Vip protein 1 contains the nptII gene, a plant selection marker gene, under the transcriptional control of the NOS promoter. The physical arrangement of the components of the T-region of pGreen0029-CaMV35S-Vip protein 1 is illustrated below: RB>35SCaMV:Vip protein 1 CDS:CaMV polyA>NOS promoter:nptII CDS:NOS polyA>LB

[0211] Next, recombinant pGreen0029 vectors having the DNA sequences depicted in SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:3, and SEQ ID NO:14 were constructed as described above.

[0212] Example 6(A): Transformation of Agrobacterium tumefaciens Agrobacterium tumefaciens strain LBA4404 was transformed with the recombinant pGreen0029-CaMV35S vector obtained in Example 6. 200ng of plasmid DNA was added to an aliquot of 100μl of A. tumefaciens strain LBA4404 competent cells. The mixture was incubated on ice for 30 minutes and transferred to liquid nitrogen for 20 minutes, then thawed at room temperature. The Agrobacterium cells were transferred to 1ml of LB broth and incubated at 28℃ for 24 hours in a water bath shaker at 200rpm. The cell suspension was evenly spread on LB agar medium containing 50μg / ml rifampicin, 30μg / ml kanamycin, and 5μg / ml tetracycline. The plate was incubated at 28℃ overnight. The transformed Agrobacterium cells were analyzed by plasmid extraction and restriction digestion, and the positive colonies of A. tumefaciens were selected and stored for further use.

[0213] Example 7: Agrobacterium-mediated tobacco transformation Experimental details for tobacco transformation are described below. Tobacco transformation practitioners will appreciate that other methods are available for cotton transformation and selection of transformed plants when using selectable marker genes that can be expressed in other plants.

[0214] material plant material Tobacco (Nicotiana tabacum): Accession number PI105952

[0215] Agrobacterium-mediated transformation of tobacco leaf discs Tobacco seeds were sterilized using 25% (v / v) bleach + 0.1% (v / v) Tween-20 for 20 min, and the seeds were washed five times with sterile water, and then inoculated onto MS (Murashige and Skoog) medium for germination.

[0216] Leaf explants (0.5 cm) from healthy green tobacco plants aged 4-6 weeks. 2 ) were prepared, pre-cultured abaxial side down on tobacco regeneration medium (TRM) containing MS medium, 2 mg / L BAP, and 1 mg / L NAA, and incubated at 25°C overnight to 3 days with a 16:8 light:dark photoperiod.

[0217] Transformed Agrobacterium tumefaciens containing the recombinant plasmid pGreen0029-CaMV35S-Vip protein 1 was grown overnight at 28 °C and 250 rpm. The overnight culture was centrifuged and cultured in liquid 1 / 2 MS medium until a final OD 600The explants were then resuspended to a pH of 0.5. Precultured tobacco leaf explants were then co-cultured with the Agrobacterium suspension for 30 minutes at room temperature with occasional gentle shaking. The explants were then placed on sterile filter paper to remove excess Agrobacterium, and the infected explants were co-cultured with the abaxial surface in contact with the medium (TRM) at 25°C under a 16:8 light:dark photoperiod for 2-3 days. The explants were then washed and placed on TRM containing 300 mg / L timentin and 100 mg / L kanamycin for culture. The explants were subcultured every two weeks until shoots emerged. The regenerated shoots were then transferred to MS medium containing 300 mg / L timentin and 100 mg / L kanamycin for rooting. The rooted tobacco plantlets were then transferred to pots under controlled conditions. Putative transgenic tobacco plants were confirmed by standard PCR analysis methods known in the art, and PCR-positive plants were then utilized in a defoliation bioassay.

[0218] Those skilled in the art of tobacco transformation and regeneration will appreciate that other methods are available for the transformation and regeneration of tobacco, and selectable marker genes that can be expressed in other plants may be used for the selection of transformed plants.

[0219] Example 8: Leaf-based bioassay Defoliation bioassay A defoliation bioassay against Spodoptera litura was performed using transgenic tobacco plants expressing Vip protein 1, Vip protein 2, and Vip protein 3 individually and non-transgenic (control) tobacco leaf samples. Defoliated leaves of transgenic tobacco plants expressing the Vip proteins of the present invention were placed on Petri dishes and then inoculated with one first-instar larva of Spodoptera litura. The assay was performed in triplicate. After 7 days, the leaf damage area was observed to be almost nonexistent in transgenic tobacco leaves compared to the untransformed control plants (Figure 1).

[0220] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be apparent that certain changes and modifications may be practiced within the scope of the disclosure.

Claims

1. An insecticidal protein having an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:7, and SEQ ID NO:11, which exerts insecticidal activity when expressed in a plant.

2. A recombinant nucleic acid molecule comprising a nucleotide sequence encoding the protein of claim 1.

3. 3. The recombinant nucleic acid molecule of claim 2, comprising a nucleotide sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:13, and SEQ ID NO:

14.

4. 3. The recombinant nucleic acid molecule of claim 2, comprising a nucleotide sequence selected from the group consisting of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:13, and SEQ ID NO:

14.

5. 5. The recombinant nucleic acid molecule of claim 4, wherein the nucleotide sequence is a synthetic sequence designed for optimal expression in plants.

6. 3. The recombinant nucleic acid molecule of claim 2, comprising a promoter operably linked to the nucleotide sequence encoding the protein of claim 1.

7. 3. An expression cassette comprising a heterologous promoter operably linked to the nucleotide sequence of claim 2.

8. 8. The expression cassette of claim 7, wherein the promoter that functions in plants is selected from the group consisting of an inducible promoter, a constitutive promoter, a tissue-preferred promoter, and a tissue-specific promoter.

9. A vector comprising a recombinant nucleic acid molecule according to any one of the preceding claims.

10. A host cell comprising a protein according to claim 1, a recombinant nucleic acid molecule according to any one of the preceding claims, or a vector according to claim 9.

11. The host cell of claim 10 , which is a bacterium, a cyanobacterium, a virus, a fungus, an insect, or a yeast.

12. 13. A method for producing a plant exhibiting insecticidal activity, comprising at least the step of introducing a nucleic acid molecule according to any one of the preceding claims.

13. 13. The method of claim 12, wherein the introducing is carried out by introgressing into the plant a nucleic acid molecule encoding a protein having an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:7, and SEQ ID NO:

11.

14. The introduction, (a) inserting the recombinant nucleic acid molecule of claim 2 into a plant cell, wherein said nucleic acid molecule further comprises a promoter and a terminator functional in the plant cell, wherein said promoter and terminator are operably linked to a nucleotide sequence encoding a protein having an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:7, and SEQ ID NO:11; (b) obtaining a transformed plant cell from said plant cell of step (a), wherein said transformed plant cell comprises said nucleotide sequence encoding at least one of said proteins; and (c) generating a transgenic plant from the transformed plant cell of step (b), wherein the transgenic plant comprises the nucleotide sequence encoding at least one of the proteins. The method of claim 13, wherein the method is carried out by

15. A transgenic plant exhibiting insecticidal activity, comprising a recombinant nucleic acid molecule comprising a nucleotide sequence encoding the protein of claim 1.

16. A recombinant microorganism comprising a recombinant nucleotide sequence encoding the insecticidal protein of claim 1.

17. 17. The recombinant microorganism of claim 16, wherein the nucleotide sequence is selected from the group consisting of SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:13, and SEQ ID NO:

14.

18. 17. The recombinant microorganism of claim 16, selected from the group consisting of bacteria, viruses, algae, and fungi.

19. 17. The recombinant microorganism of claim 16, further comprising a second nucleotide sequence encoding a second insecticidal protein.

20. 20. The recombinant microorganism of claim 19, wherein the second nucleotide sequence encodes a delta-endotoxin or a plant insecticidal protein.

21. A composition comprising the recombinant microorganism of claim 16.

22. 10. Use of an insecticidal protein according to claim 1 or a recombinant nucleic acid molecule according to claim 2 for producing an insecticidal composition.

23. 10. Use of a recombinant nucleic acid molecule according to claim 2, an expression cassette according to claim 7, or a vector according to claim 9 for producing an insect-resistant transgenic plant.

24. 27. Use of a recombinant microorganism according to claim 16 or a composition according to claim 21 for controlling pests.

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