Synthetic crystal protein and uses thereof

Abstract

The present disclosure provides synthetic insecticidal crystal protein and the nucleotide sequences encoding the said protein. The present disclosure also provides a method for expression of the said protein for conferring insecticidal activity in to plants, plant cells, tissues and seeds and bacteria. The disclosure further provides a DNA construct, a vector, and a host cell comprising the nucleotide sequences of the invention. Further, use of the nucleotide sequences encoding the said protein for production of insect resistant transgenic plants, compositions and methods for conferring pesticidal activity to bacteria, plants, plant cells, tissues and seeds are provided.

Description

[0001] SYNTHETIC CRYSTAL PROTEIN AND USES THEREOF

[0002] REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

[0003] An official copy of the Sequence Listing a file named "PD059678IN-SC_sequence_listing.xml" created on November 07, 2024, having a size of 19 kb filed electronically concurrently with the Specification is part of the Specification.

[0004] FIELD

[0005] The present disclosure is related to synthetic crystal protein and the nucleotide sequence encoding the said protein. The present disclosure also relates to expression of the said protein in various crop plants.

[0006] BACKGROUND

[0007] Insect pests are a major factor in the loss of the world's agricultural crops and said to be responsible for destroying one fifth of the world’s total crop production annually. In the process of artificially selecting suitable crops for human consumption, highly susceptible plants for infestations by insects are also selected that ultimately reduced its economic value and increased production cost.

[0008] Traditionally, the insect pests are controlled by application of chemical and / or biological pesticides. There are certain concerns of using chemical pesticides due to the environmental hazards associated with the production and use of chemical pesticides. Because of such concerns, regulators have banned or limited the use of some of the more hazardous pesticides. Further, it is well known fact that insect pests are capable of evolving overtime as a process of natural selection that can adapt to new situations, for example, overcome the effect of toxic materials or bypass natural or artificial plant resistant, which further adds to the problem. Biological pesticide is an environmentally and commercially acceptable alternative to the chemical pesticides. It presents a lower risk of pollution and environmental hazards, and provides greater target specificity than the traditional broad- spectrum chemical insecticides. Certain species of microorganisms of the genus Bacillus for example Bacillus thuringiensis (B.t.) are known to possess insecticidal activity against a broad range of insect pests. The insecticidal activity appears to be concentrated in parasporal crystalline protein inclusions bodies, although insecticidal proteins have also been isolated from the vegetative growth stage of Bacillus thuringiensis.

[0009] In order to protect various crops furthermore, it is desired to control various insect pests belonging to the different group of insects including lepidoptera, diptera, and coleoptera. Also Expression of Bacillus thuringiensis (Bt) insecticidal crystal (cry) protein genes in plants is known in the art however it was found that it is extremely difficult to express the native Bt gene in plants. Attempts have been made to express Bt cry protein gene in plants in combinations with various promoters functional in plants. However, only low levels of protein have been obtained in transgenic plants. One of the reasons for low level expression of the Bt cry gene in plant is high A / T content in Bt DNA sequence than plant genes in which G / C ratio is higher than A / T. The overall value of A / T for bacterial genes is 60-70% and plant genes with 40-50%. As a consequence, GC ratio in cry genes codon usage is significantly insufficient to express at optimal level. Moreover, the A / T rich region may also contain transcriptional termination sites (AATAAA poly adenylation), mRNA instability motif (ATTTA) and cryptic mRNA splicing sites. It has been observed that the codon usage of a native Bt cry gene is significantly different from that of a plant gene. As a result, the mRNA from this gene may not be efficiently utilized. Codon usage might influence the expression of genes at the level of translation or transcription or mRNA processing. To optimize an insecticidal gene for expression in plants, attempts have been made to alter the gene to resemble, as much as possible, genes naturally contained within the host plant to be transformed.

[0010] The Bt pesticidal proteins have been known in the art and most of them require protein engineering to meet the high activity standard for commercialization. Genes encoding Bt pesticidal proteins were expressed in crop plants such as cotton and corn to protect these crops from insect damage. The first Bt protein used in transgenic cotton was Cry 1 Ac to control Heliothis armigera (pink bollworm) Heliothis virescens (tobacco budworm). Cry 1 Ab was applied to com to control Ostrinia nubilalis (European corn borer). Since these insects have developed resistance to Cry 1 Ac and Cry 1 Ab, new Bt proteins are required to overcome the resistance.

[0011] Also, development of crop plant varieties expressing high / optimum level of Bt cry protein conferring resistance to certain insect pests is still a major problem in agricultural field. Increased expression of insect-control protein genes has been critical to the development of genetically improved plants with agronomically acceptable levels of insect resistance. Various attempts to control or prevent insect infestation of crop plants have been made, yet certain insect pests remain to be a significant problem in agriculture. Therefore, there remains a need for insect resistant transgenic crop plants with desired expression levels of insecticidal proteins in the transgenic plants.

[0012] The present invention provides herein a solution to the existing problem of insect pest infestation by providing engineered Bacillus thuringiensis (Bt) crystal protein having pesticidal activity against insect pests and the nucleotide sequences encoding the same.

[0013] BRIEF SUMMARY

[0014] Disclosed herein is synthetic crystal protein having insecticidal activity against insect pests and the nucleotide sequences encoding the same protein. The disclosure is further drawn to methods for expression of the crystal protein in plant cells.

[0015] The synthetic crystal protein disclosed herein was engineered for enhanced insect resistance in various crop plants. The nucleotide sequence(s) encoding the crystal protein of the present invention upon expression in a plant showed enhance insects resistance in transgenic plants. Such nucleotide sequences can be operably linked to promoter capable of directing expression of the sequences in a plant cell. Transformed host cells and transgenic plants comprising the nucleotide sequence encoding the crystal protein disclosed in the present invention are also aspects of the present disclosure.

[0016] One of the objects of the present disclosure is to provide synthetic crystal protein and the nucleotide sequences encoding the said pesticidal protein. It is another object of the present disclosure to nucleotide sequences encoding the engineered Bt pesticidal crystal protein for enhanced insect resistance in a plant, preferably in a plant selected from a group consisting of eggplant, cotton, rice, tomato, wheat, corn, sorghum, oat, millet, legume, cabbage, cauliflower, broccoli, Brassica sp., beans, pea, pigeon-pea, potato, pepper, cucurbit, lettuce, sweet potato canola, soybean, alfalfa, peanuts, and sunflower.

[0017] According to the present disclosure, the inventors have engineered the Bt pesticidal crystal protein in order to increase the insect resistance in plants. The engineered Bt pesticidal crystal protein is effective in plants including dicotyledonous and monocotyledonous plants such as cotton, eggplant, tomato, rice, com, chickpea and pigeon pea.

[0018] Another object of the present invention is to provide a nucleic acid molecule comprising a nucleotide sequence encoding a protein having the amino acid sequence as set forth in SEQ ID NO: 4.

[0019] Another object of the present invention is to provide an engineered Bt pesticidal crystal protein having the amino acid sequence as set forth in SEQ ID NO: 4.

[0020] Another object of the the present invention is to provide the nucleotide sequence is as set forth in SEQ ID. NO: 3 encoding the engineered Bt pesticidal crystal protein having the amino acid sequence as set forth in SEQ ID NO: 4.

[0021] Another object of the present invention is to provide a recombinant DNA molecule comprising the nucleic acid molecule as claimed in claim 1, wherein the nucleotide sequence is operably linked to a heterologous regulatory element.

[0022] Another object of the present invention is to provide a DNA construct for expression of an insecticidal protein in plant comprising the nucleic acid molecule as disclosed herein.

[0023] Another object of the present invention is to provide a plasmid vector comprising the nucleic acid molecule of the present invention.

[0024] Another object of the present invention is to provide a host cell comprising the nucleic acid molecule as disclosed herein.

[0025] Yet another object of the present invention is to provide a method for conferring an insect resistance in a plant comprising:

[0026] (a) inserting into a plant cell the nucleic acid molecule as disclosed herein, wherein the nucleic acid molecule is operably linked to a (i) promoter functional in a plant cell and (ii) a terminator;

[0027] (b) obtaining a transformed plant cell from the plant cell of step (a), wherein said transformed plant cell comprises the nucleic acid molecule as disclosed herein; and (c) generating a transgenic plant from said transformed plant cell of step (b), wherein said transgenic plant comprises the nucleic acid molecule as disclosed herein.

[0028] Yet another object of the present invention is to provide a composition comprising Bacillus thuringiensis and an acceptable excipient, diluent, or carrier, wherein said Bacillus thuringiensis comprising the nucleic acid molecule as disclosed herein. Further object of the present invention is to provide the composition as disclosed herein, wherein said composition further comprises an insecticidal / pesticidal agent.

[0029] Yet another object of the present invention is to provide a method of controlling insect infestation in a crop plant and providing insect resistance management, wherein said method comprises contacting said crop plant with a pesticidally effective amount of the composition as claimed disclosed herein.

[0030] BRIEF DESCRIPTION OF THE SEQUENCES SEQ ID NO: 1 shows DNA Sequence of native Cry gene encoding the protein having amino acid sequence as set forth in SEQ ID NO: 2.

[0031] SEQ ID NO: 2 shows amino acid sequence of native cry protein.

[0032] SEQ ID NO: 3 shows DNA sequence of Modified Cry gene designated as X24A 1 DM encoding the protein having amino acid sequence as set forth in SEQ ID NO: 4.

[0033] SEQ ID NO: 4 shows the amino acid sequence of protein encoded by the DNA sequence of as set forth in SEQ ID NO: 3.

[0034] SEQ ID NO: 5 shows DNA sequence of X24A1DM specific forward primer.

[0035] SEQ ID NO: 6 shows X24A1DM specific reverse primer

[0036] SEQ ID NO: 7 shows nptll specific forward primer

[0037] SEQ ID NO: 8 shows nptll specific reverse primer

[0038] DETAILED DESCRIPTION

[0039] The detailed description provided herein is to assist person skilled in the art in practicing the present invention and should not be construed to unduly limit scope of the invention as modifications and variations in the embodiments discussed herein may be made by those of person skilled in the art without departing from the spirit or scope of the invention. The present inventions will be described more fully hereinafter with reference to the accompanying drawings and / or sequence listing, in which some, but not all embodiments of the inventions are shown and / or described. The invention may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein.

[0040] 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 understanding of the embodiments.

[0041] It must be noted that, as used in the specification and the appended claims, the singular forms "a," "an" and "the" are used herein to refer to one or more than one (i.e., to at least one) of the grammatical object of the article unless the context clearly dictates otherwise. Thus, for example, reference to "a probe" means that more than one such probe can be present in the composition. Similarly, reference to "an element" means one or more element.

[0042] Throughout the specification the word "comprises" or "comprising," will be understood to imply 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.

[0043] Units, prefixes, and symbols may be denoted in their SI accepted form. 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 referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes. The above-defined terms are more fully defined by reference to the specification as a whole.

[0044] The term "nucleic acid" typically refers to large polynucleotides. The term "nucleic acid" and “nucleotide sequence” are used interchangeably herein. It includes reference to a deoxyribonucleotide or ribonucleotide polymer in either single- or double- stranded form, and unless otherwise limited, encompasses known analogues (e.g., peptide nucleic acids) having the essential nature of natural nucleotides in that they hybridize to single-stranded nucleic acids in a manner similar to that of naturally occurring nucleotides. Nucleotides are the subunit that is polymerized (connected into a long chain) to make nucleic acids (DNA and RNA). Nucleotides consist of three smaller components a ribose sugar, a nitrogenous base, and phosphate group(s).

[0045] A "polynucleotide" means a single strand or parallel and anti-parallel strands of a nucleic acid. Thus, a polynucleotide may be either a single-stranded or a double- stranded nucleic acid. The term "oligonucleotide" typically refers to short polynucleotides, generally no greater than about 50 nucleotides. It will be understood that when a nucleotide sequence is represented by a DNA sequence (i.e., A, T, G, C), this also includes an RNA sequence (i.e., A, U, G, C) in which "U" replaces "T."

[0046] In some embodiments the change to a native or genomic nucleic acid molecule includes but is not limited to changes in the nucleic acid sequence due to codon optimization of the nucleic acid sequence for expression in a particular organism, for example a plant, the degeneracy of the genetic code, changes in the nucleic acid sequence to introduce at least one amino acid substitution, insertion, deletion and / or addition compared to the native or genomic sequence, changes in the nucleic acid sequence to introduce restriction enzyme sites, removal of one or more intron associated with the genomic nucleic acid sequence, insertion of one or more heterologous introns, deletion of one or more upstream or downstream regulatory regions associated with the genomic nucleic acid sequence, insertion of one or more heterologous upstream or downstream regulatory regions, deletion of the 5' and / or 3' un-translated region associated with the genomic nucleic acid sequence, insertion of a heterologous 5' and / or 3' untranslated region, and modification of a polyadenylation site.

[0047] In the context of the present invention, the following abbreviations for the commonly occurring nucleic acid bases are used. "A" refers to adenosine, "C" refers to cytidine, "G" refers to guanosine, "T" refers to thymidine, and "U" refers to uridine.

[0048] 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. Codon-optimized nucleotide sequences may be prepared for any organism of interest using methods known in the art. Optimized nucleotide sequences find use in increasing expression of a pesticidal protein in a plant, for example monocot and dicot plants such as, rice, tomato, and cotton plant. The terms “DNA construct”, "nucleotide constructs", and “DNA expression cassette” are used interchangeably herein and is not intended to limit the embodiments to nucleotide constructs comprising DNA. Those of ordinary skill 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.

[0049] 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 altered or produced by the hand of man and is in a recombinant bacterial or plant host cell. For example, a recombinant polynucleotide may be a polynucleotide isolated from a genome, a cDNA produced by the reverse transcription of an RNA, a synthetic nucleic acid molecule or an artificial combination of two otherwise separated segments of sequence, e.g., by chemical synthesis or by the manipulation of isolated segments of polynucleotides by genetic engineering techniques. The term "Homologous" as used herein, refers to nucleotide sequence similarity between two regions of the same nucleic acid strand or between regions of two different nucleic acid strands. When a nucleotide residue position in both regions is occupied by the same nucleotide residue, then 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. Homology between two regions is expressed in terms of the proportion of nucleotide residue positions of the two regions that are occupied by the same nucleotide residue. By way of 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 of the portions are occupied by the same nucleotide residue. More preferably, all nucleotide residue positions of each of the portions are occupied by the same nucleotide residue.

[0050] Optimal alignment of sequences for comparison may be conducted by computerized implementations of algorithms known in the art.

[0051] "Percentage of sequence identity," as used herein, is determined by comparing two optimally aligned sequences over a comparison window, where the fragment of the polynucleotide or amino acid sequence in the comparison window may comprise additions or deletions (e.g., gaps or overhangs) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity.

[0052] Optimal alignment of sequences for comparison may be conducted by computerized implementations of algorithms known in the art. The term "substantial sequence identity" between nucleotide sequences used herein refers to polynucleotide comprising a sequence that has at least 65% sequence identity, preferably at least 69% to 77% sequence identity compared to the reference sequence.

[0053] As used herein, the term "promoter / regulatory sequence" means a nucleic acid sequence which is required for expression of a gene product operably linked to the promoter / regulatory sequence. In some instances, this sequence may be the core promoter sequence and in other instances, this sequence may also include an enhancer sequence and other regulatory elements which are required for expression of the gene product. The promoter / regulatory sequence may, for example, be one which expresses the gene product in a tissue specific manner.

[0054] A "constitutive” promoter is a promoter which drives expression of a gene to which it is operably linked, in a constant manner in a cell. By way of example, promoters which drive expression of cellular housekeeping genes are considered to be constitutive promoters.

[0055] An "inducible" promoter is a nucleotide sequence which, when operably linked with a polynucleotide which encodes or specifies a gene product, causes the gene product to be produced in a living cell substantially only when an inducer which corresponds to the promoter is present in the cell.

[0056] A "tissue-specific" promoter is a nucleotide sequence which, when operably linked with a polynucleotide which encodes or specifies 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.

[0057] As used herein, “Operably linked” means any linkage, irrespective of orientation or distance, between a regulatory sequence and coding sequence, where the linkage permits the regulatory sequence to control expression of the coding sequence. The term “operably linked” further means that the nucleic acid sequences being linked are contiguous and, where necessary to join two protein coding regions, contiguous and in the same reading frame. The term "operably linked" also refers to a functional linkage between a promoter and a second sequence, wherein the promoter sequence initiates and mediates transcription of the DNA sequence corresponding to the second sequence.

[0058] As used herein, the terms "encoding" or "encoded" when used in the context of a specified nucleic acid mean that the nucleic acid comprises the requisite information to direct translation of the nucleotide sequence into a specified protein. The information by which a protein is encoded is specified by the use of codons. A nucleic acid encoding a protein may comprise non-translated sequences (e.g., introns) within translated regions of the nucleic acid or may lack such intervening non-translated sequences (e.g., as in cDNA).

[0059] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to a polymer composed of amino acid residues related naturally occurring structural variants, and synthetic non-naturally occurring analogs thereof linked via peptide bonds. Synthetic polypeptides can be synthesized, for example, using an automated polypeptide synthesizer. The terms apply to amino acid polymers in which one or more amino acid residues is an artificial chemical analogue of a corresponding naturally occurring amino acid, 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 acid may be a naturally occurring amino acid and, unless otherwise limited, may encompass known analogues of natural amino acids that can function in a similar manner as naturally occurring amino acids.

[0060] The term "protein" typically refers to large polypeptides. The term "peptide" typically refers to short polypeptides. However, the term "polypeptide" is used herein to refer to any amino acid polymer comprised of two or more amino acid residues linked via peptide bonds.

[0061] As used herein, “expression cassette” means a genetic module comprising a gene and the regulatory regions necessary for its expression, which may be incorporated into a vector. A "vector" is a composition of matter which comprises nucleic acid molecule and which can be used to deliver the isolated nucleic acid to the interior of a cell. Numerous vectors are known in the art including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term "vector" includes an autonomously replicating plasmid or a virus. The term should also be construed to include nonplasmid and non- viral compounds which facilitate transfer of nucleic acid into cells, such as, for example, polylysine compounds, liposomes, and the like. Examples of viral vectors include, but are not limited to, adenoviral vectors, adeno-associated virus vectors, retroviral vectors, and the like.

[0062] The term "Expression vector" refers to a vector comprising a recombinant nucleic acid comprising expression control sequences operatively linked to a nucleotide sequence to be expressed. An expression vector comprises sufficient cis-acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes) and viruses that incorporate the recombinant nucleic acid.

[0063] The term "host cell" as used herein refers to a cell which contains a vector and supports the replication and / or expression of the expression vector is intended. Host cells may be prokaryotic cells such as E. coli, or eukaryotic cells such as yeast, insect, amphibian, or mammalian cells, or monocotyledonous or dicotyledonous plant cells. An example of a monocotyledonous host cell is a rice host cell and an example of a dicotyledonous host cell is eggplant or tomato host cell. When possible, the sequence is modified to avoid predicted hairpin secondary mRNA structures.

[0064] The present disclosure provides engineered Bacillus thuringiensis (Bt) pesticidal crystal protein and the nucleotide sequences encoding the said protein. The present disclosure also provides a method for expression of the said protein for conferring pesticidal activity in to bacteria, plants, plant cells, tissues and seeds. The disclosure further provides a DNA construct, a vector, and a host cell comprising the nucleotide sequences of the invention. Further, use of the nucleotide sequences encoding the said protein for production of insect resistant transgenic plants, compositions and methods for conferring pesticidal activity to bacteria, plants, plant cells, tissues and seeds are provided.

[0065] The nucleotide sequence disclosed herein can be used in DNA constructs or expression cassettes for transformation and expression in plants and bacteria. Compositions also comprise transformed bacteria, plants, plant cells, tissues, and seeds. In particular, isolated deltaendotoxin nucleic acid molecules are provided. Additionally, amino acid sequences corresponding to the polynucleotides are encompassed, and antibodies specifically binding to those amino acid sequences.

[0066] 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 can be investigated by this 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 for cell surface receptors.

[0067] As used herein, the term “complementary” or “complement” refer to the pairing of bases, purines and pyrimidines that associate through hydrogen bonding in double stranded nucleic acid. The following base pairs are complementary: guanine and cytosine; adenine and thymidine; and adenine and uracil. The terms as used herein include complete and partial complementarity.

[0068] As used herein, the term “hybridization” refers to a process in which a strand of nucleic acid joins with a complementary strand through base pairing. The conditions employed in the hybridization of two non-identical, but very similar, complementary nucleic acids vary with the degree of complementarity of the two strands and the length of the strands. Thus the term contemplates partial as well as complete hybridization. Such techniques and conditions are well known to practitioners in this field.

[0069] The terms "insecticidal activity" and "pesticidal activity" are used interchangeably herein to refer to activity of an organism or a substance (such as, for example, a protein) that can be measured by, but is not limited to, pest mortality, pest weight loss, pest repellency, and other behavioural and physical changes of a pest after feeding and exposure for an appropriate length of time. Thus, an organism or substance having pesticidal activity adversely impacts at least one measurable parameter of pest fitness. For example, "insecticidal proteins" are proteins that display insecticidal activity by themselves or in combination with other proteins.

[0070] As used herein, the term "affecting insect pests" refers to controlling changes in insect feeding, growth, and / or behaviour at any stage of development, including but not limited to killing the insect, retarding growth, preventing reproductive capability, antifeedant activity, and the like. As used herein, the term "pesticidally effective amount" connotes a quantity of a substance or organism that has pesticidal activity when present in the environment of a pest. For each substance or organism, the pesticidally effective amount is determined empirically for each pest affected in a specific environment. Similarly, an "insecticidally effective amount" may be used to refer to a "pesticidally effective amount" when the pest is an insect pest.

[0071] As used herein, the terms "transformed plant" and "transgenic plant" refer to a plant that comprises one or more heterologous polynucleotide within its genome. The heterologous polynucleotide(s) is stably integrated within the genome of a transgenic or transformed plant such that the polynucleotide is passed on to successive generations. The heterologous polynucleotide may be integrated into the genome alone or as part of a recombinant DNA molecule. It is to be understood that as used herein the term "transgenic" includes any plant cell, plant cell line, callus, tissue, a plant part, or a plant the genotype of which has been altered by the presence of one or more heterologous nucleic acid. The term includes those transgenics initially obtained using genetic transformation method known in the art as well as those created by sexual crosses or asexual propagation from the initial transgenic.

[0072] The term “initial transgenic" as used herein does not encompass the alteration of the genome (chromosomal or extra-chromosomal) by conventional plant breeding methods or by naturally occurring events such as random cross-fertilization, non-recombinant viral infection, nonrecombinant bacterial transformation, non-recombinant transposition, or spontaneous mutation.

[0073] As used herein, the term "plant" includes whole plants, plant cells, plant protoplasts, plant cell tissue cultures from which plants can be regenerated, plant calli, plant clumps, and plant cells that are intact in plants or parts of plants such as embryos, pollen, ovules, seeds, leaves, flowers, branches, fruit, kernels, ears, cobs, husks, stalks, roots, root tips, anthers, and the like and progeny thereof. Parts of transgenic plants are within the scope of the embodiments and comprise, for example, plant cells, protoplasts, tissues, callus, embryos as well as flowers, stems, fruits, leaves, and roots originating in transgenic plants or their progeny previously transformed with a DNA molecule of the embodiments and therefore consisting at least in part of transgenic cells.

[0074] Regulatory sequences

[0075] Transcriptional and translational regulatory signals include, but are not limited to, promoters, transcriptional initiation start sites, operators, activators, enhancers, other regulatory elements, ribosomal binding sites, an initiation codon, termination signals, and the like.

[0076] The polynucleotide / DNA construct will include in the 5' to 3' direction of transcription: a transcriptional and translational initiation region (i.e., a promoter), a DNA sequence of the embodiments, and a transcriptional and translational termination region (i.e., termination region) functional in the organism serving as a host. The transcriptional initiation region (i.e., the promoter) may be native, analogous, foreign or heterologous to the host organism and / or to the sequence of the embodiments. Additionally, the promoter may be the natural sequence or alternatively a synthetic sequence. The term "foreign" as used herein indicates that the promoter is not found in the native organism into which the promoter is introduced. Where the promoter is "foreign" or "heterologous" to the sequence of the embodiments, it is intended that the promoter is not the native or naturally occurring promoter for the operably linked sequence of the embodiments.

[0077] A number of promoters can be used in the practice of the embodiments. The promoters can be selected based on the desired outcome. The codon optimized nucleotide sequence of the invention can be combined with constitutive, tissue-preferred, inducible, or other promoters for expression in the host organism. Suitable constitutive promoters for use in a plant host cell include, for example, the core CaMV 35S promoter; rice actin; ubiquitin; ALS promoter etc. 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 sequences of the embodiments in plants are wound-inducible promoters. Such wound-inducible promoters, may respond to damage caused by insect feeding, and include potato proteinase inhibitor (pin II) gene; wunl and wun2, winl and win2; WIP1; MPI gene etc.

[0078] Additionally, pathogen-inducible promoters may be employed in the methods and nucleotide constructs of the embodiments. Such pathogen-inducible promoters include those from pathogenesis-related proteins (PR proteins), which are induced following infection by a pathogen; e.g., PR proteins, SAR proteins, P-l,3-glucanase, chitinase, etc.

[0079] Chemical-regulated promoters can be used to modulate the expression of a gene in a plant through the application of an exogenous chemical regulator. Depending upon the objective, the promoter may be a chemical-inducible promoter, where application of the chemical induces gene expression, or a chemical-repressible promoter, where application of the chemical represses gene expression. Chemical-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 that are used as pre-emergent herbicides, and the tobacco PR- la promoter, which is activated by salicylic acid. Other chemical-regulated promoters of interest include steroidresponsive promoters.

[0080] A promoter that has "preferred" expression in a particular tissue is expressed in that tissue to a greater degree than in at least one other plant tissue. Some tissue-preferred promoters show expression almost exclusively in the particular tissue. Tissue-preferred promoters can be utilized to target enhanced pesticidal protein expression within a particular plant tissue. Such promoters can be modified, if necessary, for weak expression.

[0081] Example of some of the tissue specific promoters includes but is not limited to leaf-pref erred promoters, root specific or root preferred promoters, seed specific or seed preferred promoters, pollen specific promoters, and pith specific promoters.

[0082] Root-preferred or root-specific promoters are known and can be selected from the available from the literature or isolated de novo from various compatible species.

[0083] "Seed-preferred" promoters include both "seed- specific" promoters (those promoters active during seed development such as promoters of seed storage proteins) as well as "seedgerminating" promoters (those promoters active during seed germination). Gamma-zein and Glob-1 are endosperm- specific promoters. For dicots, seed-specific promoters include, but are not limited to p.-phaseolin, p.-conglycinin, soybean lectin, cruciferin, and the like. For monocots, 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, etc.

[0084] Where low level expression is desired, weak promoters will be used. Generally, the term "weak promoter" as used herein refers to a promoter that drives 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.

[0085] Termination regions are available from the Ti-plasmid of A. tumefaciens, such as the octopine synthase (OCS) and nopaline synthase (NOS) termination regions.

[0086] The DNA expression cassettes may additionally contain 5' leader sequences. Such leader sequences can act to enhance translation. Translation leaders are known in the art and include: picomavirus leaders, for example, EMCV leader (Encephalomyocarditis 5' noncoding region); potyvirus leaders, for example, TEV leader (Tobacco Etch Virus), MDMV leader (Maize Dwarf Mosaic Virus), untranslated leader from the coat protein mRNA of alfalfa mosaic virus (AMV RNA 4); tobacco mosaic virus leader (TMV); and maize chlorotic mottle virus leader (MCMV).

[0087] In one specific embodiment of the invention disclosed and claimed herein, the tissue-preferred or tissue-specific promoter is operably linked to a synthetic DNA sequence of the disclosure encoding the insecticidal protein, and a transgenic plant stably transformed with at least one such recombinant molecule. The resultant plant will be resistant to particular insects which feed on those parts of the plant in which the DNA(s) is (are) expressed.

[0088] Selectable marker gene

[0089] Generally, the expression cassette will comprise a selectable marker gene for the selection of transformed cells. Selectable marker genes are utilized for the selection of transformed cells or tissues. Marker genes include genes encoding antibiotic resistance, such as those encoding neomycin phosphotransferase II (nptll) and hygromycin phosphotransferase (hptll) protein, as well as genes conferring resistance to herbicidal compounds, such as glufosinate ammonium, bromoxynil, imidazolinones, and 2,4-dichlorophenoxyacetate (2,4-D). Additional examples of suitable selectable marker genes include, but are not limited to, genes encoding resistance to chloramphenicol, methotrexate, streptomycin, spectinomycin, bleomycin, sulphonamide, bromoxynil, glyphosate, phosphinothricin.

[0090] The above list of selectable marker genes is not meant to be limiting. Any selectable marker gene can be used in the embodiments.

[0091] DNA constructs and vectors

[0092] The codon optimized synthetic nucleotide sequences of the inventions are provided in DNA constructs for expression in the organism of interest. The construct includes 5' and 3' regulatory sequences operably linked to a sequence of the invention.

[0093] Such a polynucleotide construct is provided with a plurality of restriction sites for insertion of the DNA sequences encoding pesticidal crystal protein sequence to be under the transcriptional regulation of the regulatory regions. The polynucleotide construct may additionally contain selectable marker genes. The construct may additionally contain at least one additional gene to be co-transformed into the desired organism. Alternatively, the additional gene(s) can be provided on multiple polynucleotide constructs.

[0094] In preparing the DNA construct / expression cassette, the various DNA fragments may be manipulated so as to provide for the DNA sequences in the proper orientation and, as appropriate, in the proper reading frame. Toward this end, adapters or linkers may be employed to join the DNA fragments or other manipulations may be involved to provide for convenient restriction sites, removal of superfluous DNA, removal of restriction sites, or the like. For this purpose, in vitro mutagenesis, primer repair, restriction, annealing, resubstitutions, e.g., transitions and transversions, may be involved.

[0095] According to the present invention, the DNA construct / expression cassette disclosed herein may be inserted to the recombinant expression vector. The expression "recombinant expression vector" means a bacteria plasmid, a phage, a yeast plasmid, a plant cell virus, a mammalian cell virus, or other vector. In general, as long as it can be replicated and stabilized in a host, any plasmid or vector can be used. Important characteristic of the expression vector is that it has a replication origin, a promoter, a marker gene, and a translation control element.

[0096] A large number of cloning vectors comprising a replication system in E. coli and a marker that permits selection of the transformed cells are available for preparation for the insertion of foreign genes into higher plants. The vectors comprise, for example, pBR322, pUC series, M13mp series, pACYC184, inter alia. Accordingly, the DNA fragment having the sequence encoding the Bt toxin protein can be inserted into the vector at a suitable restriction site. The resulting plasmid is used for transformation into E. coli. The E. coli cells are cultivated in a suitable nutrient medium, then harvested and lysed. The plasmid is recovered. Sequence analysis, restriction analysis, electrophoresis, and other biochemical-molecular biological methods are generally carried out as methods of analysis. After each manipulation, the DNA sequence used can be cleaved and joined to the next DNA sequence. Each plasmid sequence can be cloned in the same or other plasmids. Depending on the method of inserting desired genes into the plant, other DNA sequences may be necessary. If, for example, the Ti or Ri plasmid is used for the transformation of the plant cell, then at least the right border, but often the right and the left border of the Ti or Ri plasmid T-DNA, has to be joined as the flanking region of the genes to be inserted.

[0097] The expression vector comprising the codon optimized nucleotide sequence of the disclosure and a suitable signal for regulating transcription / translation can be constructed by a method which is well known to a person in the art. Examples of such method include an in vitro recombination DNA technique, a DNA synthesis technique, and an in vivo recombination technique. The DNA sequence can be effectively linked to a suitable promoter in the expression vector in order to induce synthesis of mRNA. Furthermore, the expression vector may contain, as a site for translation initiation, a ribosome binding site and a transcription terminator.

[0098] A preferred example of the recombinant vector of the present invention is Ti-plasmid vector which can transfer a part of itself, i.e., so called T-region, to a plant cell when the vector is present in an appropriate host such as Agrobacterium tumefaciens. Other types of Ti-plasmid vector are currently used for transferring a hybrid gene to protoplasts that can produce a new plant by appropriately inserting a plant cell or hybrid DNA to a genome of a plant.

[0099] Expression vector may comprise at least one selectable marker gene. The selectable marker gene is a nucleotide sequence having a property based on that it can be selected by a common chemical method. Every gene which can be used for the differentiation of transformed cells from non-transformed cell can be a selective marker. Example includes, a gene resistant to herbicide such as glyphosate and phosphintricin, and a gene resistant to antibiotics such as kanamycin, hygromycin, G418, bleomycin, and chloramphenicol, but not limited thereto. For the recombinant vector of the present invention, a promoter can be any of CaMV 35S, actin, or ubiquitin promoter, but not limited thereto. Since a transformant can be selected with various mechanisms at various stages, a constitutive promoter can be preferable for the present invention. Therefore, a possibility for choosing a constitutive promoter is not limited herein. For the recombinant vector of the present invention, any conventional terminator can be used. Examples thereof include nopaline synthase (NOS), rice a-amylase RAmyl A terminator, phaseoline terminator, and a terminator for optopine gene of Agrobacterium tumefaciens, etc., but are not limited thereto. Regarding the necessity of terminator, it is generally known that such region can increase reliability and an efficiency of transcription in plant cells. Therefore, the use of terminator is highly preferable in view of the contexts of the present invention. One skilled in the art will know that the DNA construct and vector disclosed herein can be used for production of insect resistant transgenic plants and / or production of pesticidal composition, wherein the composition comprises may comprise Bacillus thuringiensis cells comprising the said nucleotide sequence or any other microorganism capable of expressing the nucleotide sequence disclosed herein to produce the pesticidal crystal protein.

[0100] Recombinant cell

[0101] The embodiments further encompass a microorganism that is transformed with at least one nucleotide sequence of the invention, with an expression cassette comprising the nucleotide sequence, or with a vector comprising the expression cassette. In some embodiments, the microorganism is one that multiplies on plants. An embodiment of the invention relates to an encapsulated pesticidal protein which comprises a transformed microorganism capable of expressing the pesticidal crystal protein of the invention.

[0102] A further embodiment relates to a transformed organism such as an organism selected from the group consisting of plant and insect cells, bacteria, yeast, baculoviruses, protozoa, nematodes, and algae. The transformed organism comprises a codon optimized synthetic DNA molecule of the invention, an expression cassette comprising the said DNA molecule, or a vector comprising the said expression cassette, which may be stably incorporated into the genome of the transformed organism.

[0103] It is recognized that the nucleotide sequence encoding the pesticidal crystal protein as disclosed herein can be used to transform insect pathogenic organisms. Such organisms include baculoviruses, fungi, protozoa, bacteria, and nematodes.

[0104] The nucleotide sequence(s) encoding the pesticidal crystal protein of the embodiments may be introduced via a suitable vector into a microbial host, and said host applied to the environment, or to plants or animals. The term "introduced" 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 eukaryotic or prokaryotic cell where the nucleic acid may be incorporated into the genome of the cell (e.g., chromosome, plasmid, plastid, or mitochondrial DNA), converted into an autonomous replicon, or transiently expressed (e.g., transfected mRNA).

[0105] A number of ways are available for introducing a foreign DNA expressing the pesticidal protein into the microorganism host under conditions that allow for stable maintenance and expression of the DNA. For example, expression cassettes can be constructed which include the nucleotide constructs of interest operably linked with the transcriptional and translational regulatory signals for expression of the nucleotide constructs, and a nucleotide sequence homologous with a sequence in the host organism, whereby integration will occur, and / or a replication system that is functional in the host, whereby integration or stable maintenance will occur. Plant transformation Methods and Production of Transgenic plants

[0106] The nucleotide sequence of the present invention encoding synthetic Bt crystal protein can be inserted into plant cells using a variety of techniques which are well known in the art. Once the inserted DNA has been integrated in the plant genome, it is relatively stable. The transformation vector normally contains a selectable marker that confers on the transformed plant cells resistance to a biocide or an antibiotic, such as Kanamycin, Bialaphos, G418, Bleomycin, or Hygromycin. The individually employed marker should accordingly permit the selection of transformed cells rather than cells that do not contain the inserted DNA.

[0107] A large number of techniques are available for inserting DNA into a plant host cell. Those techniques include transformation with T-DNA using Agrobacterium tumefaciens or Agrobacterium rhizogenes as transformation agent, fusion, injection, biolistics (microparticle bombardment), or electroporation as well as other possible methods. If Agrobacteria are used for the transformation, the DNA to be inserted has to be cloned into special plasmids, namely either into an intermediate vector or into a binary vector. The intermediate vectors can be integrated into the Ti or Ri plasmid by homologous recombination owing to sequences that are homologous to sequences in the T-DNA. The Ti or Ri plasmid also comprises the vir region necessary for the transfer of the T-DNA.

[0108] Intermediate vectors cannot replicate themselves in Agrobacteria. The intermediate vector can be transferred into Agrobacterium tumefaciens by means of a helper plasmid (conjugation). Binary vectors can replicate themselves both in E. coli and in Agrobacteria. They comprise a selection marker gene and a linker or poly linker which are framed by the Right and Left T-DNA border regions. They can be transformed directly into Agrobacteria. The Agrobacterium used as host cell is to comprise a plasmid carrying a virulence (vzr) region. The vir region is necessary for the transfer of the T-DNA into the plant cell. Additional T-DNA may be contained. The bacterium so transformed is used for the transformation of plant cells. Plant explants can advantageously be cultivated with Agrobacterium tumefaciens or Agrobacterium rhizogenes for the transfer of the DNA into the plant cell. Whole plants can then be regenerated from the infected plant material (for example, leaf pieces, segments of stalk, roots, but also protoplasts or suspension- cultivated cells) in a suitable medium, which may contain antibiotics or biocides for selection. The plants so obtained can then be tested for the presence of the inserted DNA. No special demands are made of the plasmids in the case of injection and electroporation. It is possible to use ordinary plasmids, such as, for example, pUC derivatives. The cells that have been transformed may be grown into plants in accordance with conventional ways. These plants may then be grown, and either pollinated with the same transformed strain or different strains, and the resulting hybrid having constitutive or inducible expression of the desired phenotypic characteristic identified. Two or more generations may be grown to ensure that expression of the desired phenotypic characteristic is stably maintained and inherited and then seeds harvested to ensure that expression of the desired phenotypic characteristic has been achieved. They can form germ cells and transmit the transformed trait(s) to progeny plants. Such plants can be grown in the normal manner and crossed with plants that have the same transformed hereditary factors or other hereditary factors. The resulting hybrid individuals have the corresponding phenotypic properties. The plant transformation methods of the present invention involve introducing the polynucleotide(s) of the invention into a plant and do not depend on a particular method for introducing a polynucleotide(s) into a plant. Methods for introducing polynucleotide(s) into plants are known in the art including, but not limited to, stable transformation methods, transient transformation methods, and virus-mediated methods.

[0109] In one embodiment of the present invention, plants were transformed with the nucleotide sequence(s) disclosed herein. Some non-limiting example of transformed plants is fertile transgenic plant comprising the nucleotide sequence(s) of the invention encoding the pesticidal protein. Methods of transformation of various plants are known in the art. Various other plants can also be transformed using the nucleotide sequence(s) disclosed herein.

[0110] "Stable transformation" is intended to mean that the nucleotide construct introduced into a plant integrates into the genome of the plant and is capable of being inherited by the progeny thereof. "Transient transformation" is intended to mean that a polynucleotide is introduced into the plant and does not integrate into the genome of the plant or a polypeptide is introduced into a plant. Transformation protocols as well as protocols for introducing nucleotide sequences into plants may vary depending on the type of plant or plant cell, i.e., monocot or dicot, targeted for transformation. Suitable methods of introducing nucleotide sequences into plant cells and subsequent insertion into the plant genome include microinjection, electroporation, AgroZ?acterzMm-mediated transformation and ballistic particle acceleration.

[0111] In one embodiment of the invention, the nucleotide sequence(s) of the invention encoding the engineered Bt pesticidal crystal protein is expressed in a higher organism, e.g., a plant using the nucleotide sequence of the disclosure. In this case, transgenic plants expressing effective amounts of the protein protect themselves from insect pests. When the insect starts feeding on such a transgenic plant, it also ingests the expressed protein. This will deter the insect from further biting into the plant tissue or may even harm or kill the insect. The nucleotide sequence of the invention is inserted into an expression cassette, which is then stably integrated in the genome of the plant.

[0112] The embodiments also encompass transformed or transgenic plants comprising at least one nucleotide sequence of the embodiments. In some embodiments, the plant is stably transformed with a nucleotide construct comprising at least one nucleotide sequence of the embodiments operably linked to a promoter that drives expression in a plant cell.

[0113] While the embodiments do not depend on a particular biological mechanism for increasing the resistance of a plant to a plant pest, expression of the nucleotide sequences of the embodiments in a plant can result in the production of the pesticidal proteins of the embodiments and in an increase in the resistance of the plant to a plant pest. The plants of the embodiments find use in agriculture methods for affecting insect pests.

[0114] A "subject plant or plant cell" is one in which genetic alteration, such as transformation, has been affected as to a gene of interest, or is a plant or plant cell which is descended from a plant or cell so altered and which comprises the alteration. A "control" or "control plant" or "control plant cell" provides a reference point for measuring changes in phenotype of the subject plant or plant cell. A control plant or plant cell may comprise, for example: (a) a wild-type plant or cell, i.e., of the same genotype as the starting material for the genetic alteration which resulted in the subject plant or cell; (b) a plant or plant cell of the same genotype as the starting material but which has been transformed with a null construct (i.e., with a construct which has no known effect on the trait of interest, such as a construct comprising a marker gene); (c) a plant or plant cell which is a non-transformed segregant among progeny of a subject plant or plant cell; (d) a plant or plant cell genetically identical to the subject plant or plant cell but which is not exposed to conditions or stimuli that would induce expression of the gene of interest; or (e) the subject plant or plant cell itself, under conditions in which the gene of interest is not expressed.

[0115] Transfer (or introgression) of the nucleotides disclosed herein determined trait into inbred plants such as cotton, rice, eggplant (brinjal), tomato and legume lines can be achieved by recurrent selection breeding, for example by backcrossing. In this case, a desired recurrent parent is first crossed to a donor inbred (the non-recurrent parent) that carries the nucleotide sequence disclosed herein for determined traits. The progeny of this cross is then backcrossed with the recurrent parent followed by selection in the resultant progeny for the desired trait(s) to be transferred from the non-recurrent parent. After three, preferably four, more preferably five or more generations of backcrosses with the recurrent parent with selection for the desired trait(s), the progeny will be heterozygous for loci controlling the trait(s) being transferred, but will be like the recurrent parent for most or almost all other genes.

[0116] The embodiments further relate to plant-propagating material of a transformed plant of the embodiments including, but not limited to, seeds, tubers, corms, bulbs, leaves, and cuttings of roots and shoots.

[0117] The class of plants that can be used in the methods of the embodiments is generally as broad as the class of higher plants amenable to transformation techniques, including but not limited to monocotyledonous and dicotyledonous plants. Examples of plants of interest include, but are not limited to grains, cereal, vegetable, oil, fruits, ornamentals, Turfgrasses and others. For example, rice (Oryza sativa, Oryza spp.), corn (Zea mays), rye (Secale cereale), sorghum (Sorghum bicolor, Sorghum vulgare), millet (e.g., pearl millet (Pennisetum glaucum), proso millet (Panicum miliaceum). foxtail millet (Setaria ilalica). finger millet (Eleusine coracana)), wheat (Triticum aestivum, Triticum sp.), oats (Avena sativa), barley (Hordeum vulgare L), sugarcane (Saccharum spp.), cotton (Gossypium hirsutum, Gossypium barbadense, Gossypium sp.), tomatoes (Lycopersicon esculentum), brinjal / eggplant (Solanum melongena), potato (Solanum tuberosum), sugar beets (Beta vulgaris), sweet potato (Ipomoea batatus), cassava (Manihot esculenta), lettuce (Lactuca sativa), cabbage (Brassica oleracea var. capitata), cauliflower (Brassica oleracea var. botrytis), broccoli (Brassica oleracea var. indica), Brassica sp. (e.g., B. napus, B. rapa, B. juncea), particularly those Brassica species useful as sources of seed oil, soybean (Glycine max), sunflower (Helianthus annuus), safflower (Carthamus tinctorius), peanuts (Arachis hypogaea), pigeon-pea (Cajanus cajan), Chickpea (Cicer arietinum), green beans (Phaseolus vulgaris), lima beans (Phaseolus limensis), pea (Pisum sativum), peas (Lathyrus spp.), cucumber (Cucumis sativus), cantaloupe (Cucumis cantalupensis), and musk melon (Cucumis melo), alfalfa (Medicago sativa), tobacco (Nicotiana tabacum) and Arabidopsis (Arabidopsis thaliana).

[0118] Plants of interest include grain plants that provide seeds of interest, oil-seed plants, and leguminous plants. Seeds of interest include grain seeds, such as corn, wheat, barley, rice, sorghum, rye, millet, etc. Oil-seed plants include cotton, soybean, safflower, sunflower, Brassica, maize, alfalfa, palm, coconut, flax, castor, olive etc. Leguminous plants include beans and peas. Beans include guar, locust bean, fenugreek, soybean, garden beans, cowpea, mungbean, lima bean, fava bean, lentils, chickpea, etc.

[0119] In certain embodiments at least one codon optimized synthetic nucleotide sequence(s) of the invention can be stacked with any combination of polynucleotide sequences of interest in order to create plants with a desired phenotype. For example, the codon optimized synthetic nucleotide sequence may be stacked with any other polynucleotide encoding polypeptide having pesticidal and / or insecticidal activity, such as other Bt toxic proteins and the like. The combinations generated can also include multiple copies of any one of the polynucleotide of interest. The nucleotide sequences of the embodiments can also be stacked with any other gene or combination of genes to produce plants with a variety of desired trait combinations including but not limited to traits desirable for animal feed such as high oil genes, balanced amino acids, abiotic stress resistance etc.

[0120] The nucleotide sequences of the invention can also be stacked with traits desirable for disease or herbicide resistance, avirulence and disease resistance genes, acetolactate synthase (ALS), inhibitors of glutamine synthase such as phosphinothricin or basta (e.g., bar gene), glyphosate resistance, traits desirable for processing or process products such as high oil, modified oils, modified starches (e.g., ADPG pyrophosphorylases (AGPase), starch synthases (SS), starch branching enzymes (SBE) and starch debranching enzymes (SDBE)). One could also combine the polynucleotides of the embodiments with polynucleotides providing agronomic traits such as male sterility, stalk strength, flowering time, or transformation technology traits such as cell cycle regulation or gene targeting.

[0121] These stacked combinations can be created by any method including but not limited to cross breeding plants by any conventional, genetic transformation or any other method known in the art. If the traits are stacked by genetically transforming the plants, the polynucleotide sequences of interest can be combined at any time and in any order. For example, a transgenic plant comprising one or more desired traits can be used as the target to introduce further traits by subsequent transformation. The traits can be introduced simultaneously in a co-transformation protocol with the polynucleotides of interest provided by any combination of transformation cassettes. For example, if two sequences will be introduced, the two sequences can be contained in separate transformation cassettes (trans) or contained on the same transformation cassette (cis). Expression of the sequences can be driven by the same promoter or by different promoters. In certain cases, it may be desirable to introduce a transformation cassette that will suppress the expression of the polynucleotide of interest. This may be combined with any combination of other suppression cassettes or over-expression cassettes to generate the desired combination of traits in the plant. It is further recognized that polynucleotide sequences can be stacked at a desired genomic location using a site-specific recombination system.

[0122] Analysis of Transgenic plants

[0123] Polymerase Chain Reaction ( PCR )

[0124] The present invention provides a method for PCR amplification of a fragment of the nucleotide sequence disclosed in the invention encoding the synthetic Bt crystal protein, comprising amplifying DNA by PCR in presence of the primer set capable of amplifying the desired fragment of the nucleotide sequence disclosed such as the primer set as set forth in the SEQ ID NO: 5 and SEQ ID NO: 6. Similarly a fragment of the nucleotide sequence disclosed in the invention can be amplified using the primers specific to the said nucleotide sequence. A person skilled in the art can design the primer set for amplification of the said nucleotides. Protocols and conditions for the PCR amplification of a DNA fragment from template DNA are described elsewhere herein or are otherwise known in the art.

[0125] Oligonucleotide primers can be designed for use in PCR reactions to amplify corresponding DNA sequences from the modified DNA sequences of the invention. Methods for designing PCR primers and PCR cloning are generally known in the art. Known methods of PCR include, but are not limited to, methods using paired primers, nested primers, single specific primers, degenerate primers, gene-specific primers, vector- specific primers, partially-mismatched primers, and the like.

[0126] In one embodiment, the present invention provides a primer set suitable for the PCR amplification of a fragment of the nucleotide sequence(s) of the present invention that encode a pesticidal polypeptide and methods of using the primer set in the PCR amplification of the DNA. The primer sets comprise forward (SEQ ID NO: 50 and reverse primers (SEQ ID NO: 6) that have been designed to anneal to the nucleotide sequences of the present invention. Southern Hybridization

[0127] In hybridization techniques, all or part of a known nucleotide sequence is used as a probe that selectively hybridizes to other corresponding nucleotide sequences present in a population of cloned genomic DNA fragments or from a chosen organism. The hybridization probes may be PCR amplified DNA fragments of the codon optimised DNA sequence(s) of the present invention, or linerarized plasmid containing the nucleotide sequence(s) or other oligonucleotides capable of hybridizing to the corresponding sequences of the synthetic nucleotide disclosed herein, and may be labelled with a detectable group such32P or any other detectable marker. Thus, for example, probes for hybridization can be made by labelling synthetic oligonucleotides based on the sequences of the embodiments. Methods for preparation of probes for hybridization are generally known in the art.

[0128] For example, an entire sequence disclosed herein, or one or more portions thereof, may be used as a probe capable of specifically hybridizing to corresponding sequences. To achieve specific hybridization under a variety of conditions, such probes include sequences that are unique to the sequences of the embodiments and are generally at least about 10 or 20 nucleotides in length. Such probes may be used to amplify corresponding nucleotide sequence(s) of the nucleotide sequences of the present invention by PCR.

[0129] Hybridization of such sequences may be carried out under stringent conditions. The term "stringent conditions" or "stringent hybridization conditions" as used herein refers to conditions under which a probe will hybridize to its target sequence to a detectably greater degree than to other sequences (e.g., at least 2-fold, 5-fold, or 10-fold over background). Stringent conditions are sequence-dependent and will be different in different circumstances. By controlling the stringency of the hybridization and / or washing conditions, target sequences that are 100% complementary to the probe can be identified (homologous probing). Alternatively, stringency conditions can be adjusted to allow some mismatching in sequences so that lower degrees of similarity are detected (heterologous probing).

[0130] Typically, stringent conditions will be those in which the salt concentration is less than about 1.5 M Na ion, typically about 0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30°C for short probes (e.g., 10 to 50 nucleotides) and at least about 60°C for long probes (e.g., greater than 50 nucleotides). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. Exemplary low stringency conditions include hybridization with a buffer solution of 30% to 35% formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulfate) at 37°C and a wash in lx to 2x SSC (20x SSC=3.0 M NaCl / 0.3 M trisodium citrate) at 50°C to 55°C. Exemplary moderate stringency conditions include hybridization in 40% to 45% formamide, 1.0 M NaCl, 1% SDS at 37°C, and a wash in 0.5x to lx SSC at 55 to 60°C. Exemplary high stringency conditions include hybridization in 50% formamide, 1 M NaCl, 1% SDS at 37°C, and a final wash in O.lx SSC at 60°C to 65°C for at least about 20 minutes. Optionally, wash buffers may comprise about 0.1% to about 1% SDS. The duration of hybridization is generally less than about 24 hours, usually about 4 to about 12 hours.

[0131] Specificity is typically the function of post-hybridization washes, the critical factors being the ionic strength and temperature of the final wash solution. Tm =81.5°C +16.6 (log M)+0.41 (% GC)-0.61 (% form)-500 / L; where M is the molarity of monovalent cations, % GC is the percentage of guanosine and cytosine nucleotides in the DNA, "% form" is the percentage of formamide in the hybridization solution, and L is the length of the hybrid in base pairs. The Tmis the temperature (under defined ionic strength and pH) at which 50% of a complementary target sequence hybridizes to a perfectly matched probe. Washes are typically performed at least until equilibrium is reached and a low background level of hybridization is achieved, such as for 2 hours, 1 hour, or 30 minutes. Tmis reduced by about 1°C for each 1% of mismatching; thus, Tmhybridization, and / or wash conditions can be adjusted to hybridize to sequences of the desired identity. For example, if sequences with 90% identity are sought, the Tmcan be decreased 10°C Generally, stringent conditions are selected to be about 5°C lower than the Tmfor the specific sequence and its complement at a defined ionic strength and pH. However, severely stringent conditions can utilize a hybridization and / or wash at 1, 2, 3, or 4°C lower than the Tm; moderately stringent conditions can utilize a hybridization and / or wash at 6°C, 7°C, 8°C, 9°C, or 10°C lower than the Tm; low stringency conditions can utilize a hybridization and / or wash at 11°C, 12°C, 13°C, 14°C, 15°C, or 20°C lower than the Tm.

[0132] Using the equation, hybridization and wash compositions, and desired Tm, those of ordinary skill will understand that variations in the stringency of hybridization and / or wash solutions are inherently described. If the desired degree of mismatching results in a Tmof less than 45°C (aqueous solution) or 32°C (formamide solution), the SSC concentration can be increased so that a higher temperature can be used.

[0133] In the present invention the synthetic Bt crystal protein has been engineered from the Bacillus thuringiensis protein having amino acid sequence of NCBI GenBank: WP-086406662.1. Recombinant Bt Protein Bacillus thuringiensis Bacillus thuringiensis is called for short Bl) insecticidal crystalline gene anti insect gene has obtained very successful application in cotton, com and other crops. According to the insecticidal spectrum of the homology of coding gene sequence and encoding proteins, Bt insecticidal crystal protein is divided into cry family and cryt family, is divided into again the subclasses. The Bt gene expressing in plant at present has Bt crylAa, crylAb, cry 1 Ac, cry2Aa, crySBb, cry9c.

[0134] The present invention discloses the synthetic crystal protein designated as X24A1DM having amino acid sequence as set forth in SEQ ID NO: 4 and the nucleotide sequence (SEQ ID NO: 3; designated as X24A1 DM) encoding the same. In the present invention the toxicity of the X24A1DM protein was evaluated against various lepidopteran insect pests, including Helicoverpa armigera, Spodoptera litura, Spodoptera frugiperda, Plutella xylostella, and Leucinodes orbonalis, to determine the gene's insecticidal spectrum. The results showed that X24A1DM caused mortality in Helicoverpa armigera, Spodoptera litura, Plutella xylostella, and Leucinodes orbonalis, but exhibited no toxicity against Spodoptera frugiperda larvae In one embodiment there is provided a nucleic acid molecule comprising a nucleotide sequence as set forth in SEQ ID NO: 1 encoding a protein having the amino acid sequence as set forth in SEQ ID NO: 2.

[0135] In one embodiment there is provided a nucleic acid molecule comprising a nucleotide sequence encoding a protein having the amino acid sequence as set forth in SEQ ID NO: 2.

[0136] In an embodiment of the present invention, there is provided a Bt crystal protein having the amino acid sequence as set forth in SEQ ID NO: 2.

[0137] In one embodiment there is provided a nucleic acid molecule comprising a nucleotide sequence as set forth in SEQ ID NO: 3 encoding a synthetic Bt crystal protein having the amino acid sequence as set forth in SEQ ID NO: 4.

[0138] In an embodiment of the present invention, there is provided a synthetic Bt crystal protein having the amino acid sequence as set forth in SEQ ID NO: 4.

[0139] In another embodiment there is provided a recombinant DNA molecule comprising the nucleic acid molecule comprising a nucleotide sequence encoding a synthetic Bt crystal protein having the amino acid sequence as set forth in SEQ ID NO: 4, wherein the nucleotide sequence is operably linked to a heterologous regulatory element.

[0140] In another embodiment of the present invention, there is provided a recombinant DNA molecule comprising the nucleic acid molecule comprising a nucleotide sequence encoding a protein having the amino acid sequence as set forth in SEQ ID NO: 4, wherein said nucleic acid molecule optionally comprises a selectable marker gene, a reporter gene or a combination thereof.

[0141] In another embodiment, there is provided a recombinant DNA molecule comprising the nucleic acid molecule comprising a nucleotide sequence encoding a protein having the amino acid sequence as set forth in SEQ ID NO: 4, wherein said nucleic acid molecule optionally comprises a DNA sequence encoding a targeting or transit peptide for secretion or for targeting to the vacuole, mitochondrium, chloroplast, or plastid. In another embodiment, there is provided a DNA construct for expression of an insecticidal protein in plant comprising the nucleic acid molecule comprising a nucleotide sequence encoding a protein having the amino acid sequence as set forth in SEQ ID NO: 4.

[0142] In another embodiment there is provided a plasmid vector comprising the nucleic acid molecule comprising a nucleotide sequence encoding a protein having the amino acid sequence as set forth in SEQ ID NO: 4.

[0143] In another embodiment there is provided a host cell comprising the nucleic acid molecule comprising a nucleotide sequence encoding a synthetic Bt crystal protein having the amino acid sequence as set forth in SEQ ID NO: 4. The host cell of the embodiment is a plant, bacterial, virus, fungi or a yeast cell. Further, the host cell is Agrobacterium or E. coli.

[0144] In another embodiment there is provided a method for conferring an insect resistance in a plant comprising:

[0145] (d) inserting into a plant cell the nucleic acid molecule comprising a nucleotide sequence encoding a synthetic Bt crystal protein having the amino acid sequence as set forth in SEQ ID NO: 4, wherein the nucleic acid molecule is operably linked to a (i) promoter functional in a plant cell and (ii) a terminator;

[0146] (e) obtaining a transformed plant cell from the plant cell of step (a), wherein said transformed plant cell comprises the nucleic acid molecule comprising a nucleotide sequence encoding a synthetic Bt crystal protein having the amino acid sequence as set forth in SEQ ID NO: 4; and

[0147] (f) generating a transgenic plant from said transformed plant cell of step (b), wherein said transgenic plant comprises the nucleic acid molecule comprising a nucleotide sequence encoding a synthetic Bt crystal protein having the amino acid sequence as set forth in SEQ ID NO: 4.

[0148] In another embodiment there is provided a method for conferring an insect resistance in a plant comprising:

[0149] (a) inserting into a plant cell the nucleic acid molecule comprising a nucleotide sequence as set forth in SEQ ID NO: 3 encoding a synthetic Bt crystal protein having the amino acid sequence as set forth in SEQ ID NO: 4, wherein the nucleic acid molecule is operably linked to a (i) promoter functional in a plant cell and (ii) a terminator; (b) obtaining a transformed plant cell from the plant cell of step (a), wherein said transformed plant cell comprises the nucleic acid molecule comprising a nucleotide sequence as set forth in SEQ ID NO: 3 encoding a synthetic Bt crystal protein having the amino acid sequence as set forth in SEQ ID NO: 4; and

[0150] (c) generating a transgenic plant from said transformed plant cell of step (b), wherein said transgenic plant comprises the nucleic acid molecule comprising a nucleotide sequence as set forth in SEQ ID NO: 3 encoding a synthetic Bt crystal protein having the amino acid sequence as set forth in SEQ ID NO: 4.

[0151] In another embodiment there is provided a transgenic plant obtained by the method as described herein. In another embodiment there is provided a transgenic plant comprising the nucleic acid molecule comprising a nucleotide sequence encoding a synthetic Bt crystal protein having the amino acid sequence as set forth in SEQ ID NO: 4. The transgenic plant disclosed herein is selected from a group consisting of rice, wheat, corn, sorghum, oat, millet, legume, cotton, tomato, eggplant, cabbage, cauliflower, broccoli, Brassica sp., beans, pea, pigeonpea, potato, pepper, cucurbit, lettuce, sweet potato canola, soybean, alfalfa, peanuts, sunflower, safflower, tobacco, sugarcane, cassava, coffee, pineapple, citrus, cocoa, tea, banana and melon.

[0152] In another embodiment there is provided a transgenic plant comprising the nucleic acid molecule comprising a nucleotide sequence as set forth in SEQ ID NO: 3 encoding a synthetic Bt crystal protein having the amino acid sequence as set forth in SEQ ID NO: 4. The transgenic plant disclosed herein is selected from a group consisting of rice, wheat, corn, sorghum, oat, millet, legume, cotton, tomato, eggplant, cabbage, cauliflower, broccoli, Brassica sp., beans, pea, pigeonpea, potato, pepper, cucurbit, lettuce, sweet potato canola, soybean, alfalfa, peanuts, sunflower, safflower, tobacco, sugarcane, cassava, coffee, pineapple, citrus, cocoa, tea, banana and melon

[0153] In another embodiment of the present invention there is provided a tissue, seed or a progeny obtained from the transgenic plant as disclosed herein, wherein said seed or progeny comprises the nucleic acid molecule of the present invention.

[0154] Another embodiment of the present invention relates to a biological sample derived from the tissues or seed or progeny disclosed herein, wherein said sample comprising a detectable amount of the nucleic acid molecule of the present invention.

[0155] Another embodiment of the present invention relates to a commodity product derived from the transgenic plant disclosed herein, wherein said product comprises a detectable amount of the nucleic acid molecule of the present invention.

[0156] Further embodiment of the present invention relates to a composition comprising Bacillus thuringiensis and an acceptable excipient, diluent, or carrier, said Bacillus thuringiensis comprising the nucleic acid molecule of the present invention.

[0157] Yet another embodiment of the present invention relates to the composition of the present invention, wherein said composition further comprises an insecticidal agent. Further, the composition of the present invention comprising the insecticidal agent, wherein said insecticidal agent is selected from the group consisting of a Bacillus toxin, a Xenorhabdus toxin, a Photorhabdus toxin, and a dsRNA specific for suppression of one or more essential genes in said insect pest.

[0158] Yet another embodiment of the present invention relates to a method of controlling insect infestation in a crop plant and providing insect resistance management, wherein said method comprises contacting said crop plant with an insecticidally effective amount of the composition of the present invention.

[0159] Yet another embodiment of the present invention relates to use of the nucleic acid molecule of the present invention for production of insect resistant transgenic plants.

[0160] Yet another embodiment of the present invention relates to use of the nucleic acid molecule of the present invention for production of a pesticidal composition. The use of the nucleic acid molecule of the present invention for production of a pesticidal composition comprising Bacillus thuringiensis cells comprising the nucleic acid molecule as disclosed herein.

[0161] These and / or other embodiments of this invention are reflected in the wordings of the claims that form part of the description of the invention.

[0162] Various modifications and other embodiments of the present invention can be presented by a person skilled in the art to which these inventions pertain having the benefit of the teachings presented in the descriptions. Therefore, it is to be understood that the present 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.

[0163] The following Examples illustrate the invention, and are not provided to limit the invention or the protection sought.

[0164] EXAMPLES

[0165] The following examples are put forth 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 regard as their invention nor are they intended to represent that the experiments 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 errors and deviations should be accounted for.

[0166] Example 1: Cry Gene Sequence Modification and production of modified synthetic nucleotide encoding the synthetic crystal protein

[0167] An insecticidal gene variant was generated by modifying the native Cry gene sequence (SEQ ID NO: 1) by nucleotide substitution in the receptor-binding and pore-forming domains of native crystal protein (SEQ ID NO: 2). The modified nucleotide sequence was further codon optimized to optimise expression in dicot plants. The modified synthetic variant thus created was designated as X24A1DM having nucleotide sequence as set forth in SEQ ID NO: 3. The synthetic variant thus created encodes a synthetic crystal protein designated as X24A1DM having amino acid sequence as set forth in SEQ ID NO: 4.

[0168] To facilitate cloning and sub-cloning restriction endonuclease recognition sites were added to the 5’ and 3’ ends of the X24A1DM DNA sequence. The optimized and modified DNA sequence was synthesized by GenScript (USA). The synthesized DNA sequence was subsequently cloned into the pUC57 vector at the EcoBN restriction sites and the recombinant pUC57 vector thus created was designated as pUC57_X24AlDM.

[0169] Example 2: Transformation of pUC57 X24A1DM Plasmid into E. coli TOPIC cells and Screening the Colonies for Positive Clones by Colony PCR

[0170] The plasmid DNA pUC5'l_X24A! DM was transformed into One Shot E. coli TOPIO cells (Invitrogen, USA #C404003) following the manufacturer's protocol. The transformed cells were then plated on LB agar containing 50pg / mL ampicillin. As a control, blank E. coli TOPIO cells were also spread on LB agar containing ampicillin. The plates were incubated overnight at 37°C. The colonies that appeared on the plates were screened for the presence of the nucleotide sequence as set forth in SEQ ID NO: 3 (X24A1DM) using the primers as set forth in the SEQ ID NO: 5 and SEQ ID NO: 6. The results indicate that the cells transformed with the pUC57 _X24A1 DM plasmid successfully grew on the LB agar plates containing ampicillin of 495 bp.

[0171] This suggests that the plasmid contains the ampicillin resistance gene, allowing the transformed cells to survive and form colonies. In contrast, no colonies were observed on the plate spread with the blank E. coli TOP 10 cells, which confirms that the cells without the plasmid lack ampicillin resistance and therefore could not grow in the presence of the antibiotic.

[0172] The reaction mix was set up at a final volume of 25 pL containing 0.5 pL (1.0 U) of Taq DNA Polymerase (Promega, USA), 2.5 pL (IX) of 10X Taq Polymerase Buffer, 1 pL (100 ng / pL) each primer, 0.2 pL of dNTPs (25 mM each), and 14.8 pL distilled water (dtbO). The colonies were picked using sterile toothpicks and suspended in the PCR reaction mix in the PCR tubes labelled with the respective colony number. The PCR amplification was carried out using genespecific primers. The amplification was carried out in the Eppendorf Master cycler with the program of initial denaturation at 94°C for 5 min, 30 thermal cycles of denaturation at 95°C for 45 s, annealing at 58°C for 45 s, and extension at 72°C for 30 s, with a final extension step of 20 min at 72°C. The PCR amplicons were resolved on a 1.5% agarose gel. Five colonies containing X24A1DM insert were selected and stocked up using glycerol.

[0173] The transformed colonies were screened using the primers as set forth in SEQ ID NO: 5 and SEQ ID NO: 5. These primers successfully amplified a 495 bp fragment in the positive colonies, confirming the presence of the target gene in the plasmid carried by these colonies. This PCR amplification result indicates that the gene of interest has been correctly inserted and maintained in the plasmid constructs within the transformed cells.

[0174] Plasmid DNA was extracted from five positive clones using the alkaline lysis method known in the art and subjected to restriction digestion using BamHI and 7 / zndIII enzymes to release the X24A1DM fragment from the pUC57 _X24A1 DM plasmid. A restriction digestion reaction was set up with a total volume of 30 pL, consisting of 10 pL (1 pg) of plasmid DNA, 3 pL of 10X fast digest restriction enzyme buffer, 1 pL (20 U / pL) of each fast digest restriction enzyme (BamHI and HmdIII), and 15 pL of nuclease-free water. The contents were mixed by vortexing the tubes at a low speed and then incubated at 37 °C for 15 minutes. The reaction was stopped by heat- inactivating the restriction endonucleases at 80°C following the manufacturer’s instructions. The restriction digestion was expected to release a 3501 bp fragment of the insert from the p JC57 _X24A1 DM plasmid. The digested products were then resolved on a 1.5% agarose gel to confirm the presence and size of the released gene fragments.

[0175] The restriction endonuclease digestion of the pJC57 _X24A1 DM using BamHI and Hfodlll released a 3.5 kb fragment from the pUC57 plasmid. This result matched the theoretical map of the pJC57 _X24A1 DM plasmid, confirming the correct insertion and integrity of the gene fragment in these plasmid constructs.

[0176] Example 3: Bacterial expression, and Purification of Recombinant synthetic bt crystal Protein (SEQ ID NO: 4)

[0177] The nucleotide sequence as set forth in SEQ IDNO: 3 (X24A1 DM) was cloned into the bacterial expression vector pET-32a(+) to facilitate gene expression and recombinant protein production. This vector choice allows for efficient protein expression in E. coli, enabling subsequent purification and characterization of the recombinant proteins. The resulting recombinant plasmid was then transformed into E. coli BL21(DE3) cells for protein expression. The colony PCR of the colonies that appeared on LB agar (ampicillin) was screened using the primers as set forth in SEQ ID NO: 5 and SEQ ID NO: 6. These primers successfully amplified a fragment of 495 bp in the positive clones of pJC57_X24AlDM.

[0178] The nucleotide sequence as set forth in SEQ IDNO: 3 (X24A1 DM) was released from pUC57 _X24A1 DM plasmid using BamHI and H dlll restriction endonucleases. A restriction digestion reaction with a total volume of 30 pL was set up as follows: 10 pL of pUC57 _X24A1 DM plasmid DNA (3 pg), 3 pL of 10X fast digest restriction enzyme buffer, 1 pL (20 U / pL) of each fast digest restriction enzyme (BamHI and HmdIII), and 15 pL of nuclease-free water in a 1.5 mL polypropylene tube. The contents were mixed by vortexing the tubes at low speed and incubated at 37°C for 15 minutes. The reaction was stopped by incubating at 80°C for 10 minutes. To verify the digestion, 5 pL of the digested sample along with a DNA marker were run on a 1.5% agarose gel. The digested product was then eluted and purified using the QIAquick PCR and Gel Extraction Kit (Qiagen #28506). This purified product was subsequently used for cloning into the pET-32a (+) vector for gene expression and recombinant protein production.

[0179] The restriction endonuclease digestion of the pET-32a(+)_,¥24A7DM using BamHl and Hindlll released a 3.5 kb gene fragment from the pUC57 plasmid. This result was as expected and matched the theoretical maps of pET-32a(+)_X24AlDM.

[0180] Example 3A: Preparation of Bacterial Expression Vector pET-32a (+) and Transformation into E. coli BL21 (DE3)

[0181] The bacterial expression vector, pET-32a(+), was used to clone the nucleotide sequence as set forth in SEQ IDNO: 3 X24A1DM). For the cloning process, a restriction digestion reaction was set up with a total volume of 30 pL, containing 10 pL of pET-32a(+) plasmid DNA (3 pg), 3 pL of 10X fast digest restriction enzyme buffer, 1 pL (20 LVpL) of each fast digest restriction enzyme (BamHl and Hzndlll), and 15 pL of nuclease-free water in a 1.5 mL Eppendorf tube. The contents were mixed by vortexing the tubes at low speed and incubated at 37°C for 15 minutes. The reaction was stopped by incubating at 80°C for 10 minutes. To verify the digestion, 5 pL of the digested product along with a DNA marker were run on a 1.5% agarose gel. The digested product was then eluted and purified using the QIAquick PCR and Gel Extraction Kit. This purified vector was subsequently used for the ligation of the nucleotide sequence as set forth in SEQ IDNO: 3 X24A1DM) facilitating gene expression and recombinant protein production in bacterial cells.

[0182] The ligation reaction was set up in a 200 pL PCR tube with a total volume of 50 pL. The components included the pET-32a(+) vector and the insert DNA (SEQ IDNO: 3 ) in a 1:3 molar ratio. The following were added:

[0183] • Vector pET-32a(+)

[0184] • Insert DNA

[0185] • 2 pL of 10X Ligase buffer

[0186] • 1 pL of T4 DNA ligase (0.2-0.4 U / pL)

[0187] • 1 pL of T4 DNA ligase (0.2-0.4 U / pL) The contents of the tube were gently mixed by pipetting and incubated overnight at 16°C. The ligated product was then used for bacterial transformation to facilitate the expression of the cloned nucleotide sequence as set forth in SEQ IDNO: 3.

[0188] The ligation product containing pET-32a (+)__X24A1DM was introduced aseptically into 100 pL of competent E. coli BL21 (DE3) cells into a 1.5 ml polypropylene tube. The mixture was gently combined and left to incubate on ice for 30 minutes. Subsequently, the bacterial cells underwent a heat shock procedure, being briefly exposed to a water bath set at 42°C for 90 seconds before being rapidly cooled on ice for 5 minutes. After this treatment, 900 pL of sterile LB liquid medium was added to the tube, followed by incubation at 37°C for 1 hour with gentle shaking at 175 rpm. The transformed cells were then plated onto LB agar supplemented with ampicillin (50pg / mL) and incubated overnight at 37°C. Positive clones were identified through colony PCR and restriction digestion of the plasmid DNA.

[0189] After the bacterial colonies emerged on LB agar, they were carefully collected using sterile toothpicks and transferred into PCR tubes (0.2 ml) containing the PCR reaction mixture. Each tube was labelled with the colony number for identification. PCR was carried out in a total reaction volume of 25 pL using gene-specific primers. The PCR reaction mixture for each amplification consisted of 0.5 pL (1.0 U) of Taq DNA Polymerase (Promega, USA), 2.5 pL of 10X Taq DNA Polymerase Buffer with MgC12, 1.0 pL (100 ng / pL) of each primer, 0.2 pL of dNTPs (25mM each), 1.0 pL of bacterial colony suspension as the DNA template, and 18.8 pL of distilled water. PCR amplifications were conducted using the following cycling program: initial denaturation at 95°C for 5 minutes, followed by 30 cycles of denaturation at 95°C for 45 seconds, annealing at 58°C for 45 seconds, and extension at 72°C for 30 seconds, with a final extension step of 20 minutes at 72°C. The PCR products were then separated by electrophoresis on a 1.5% agarose gel to visualize the amplified DNA fragments.

[0190] Following positive selection by colony PCR, plasmid DNA was isolated from the identified clones using the alkaline lysis method. Subsequently, the extracted plasmid was subjected to restriction digestion using the enzymes BamHI and HindSl to release the insert. For the restriction digestion, a 30 pL reaction mixture was prepared comprising 10 pL of each amplicon, 3 pL of 10X fast digest restriction enzyme buffer, 1 pL (20 U / pL) of each fast digest restriction endonuclease (BamHI and Hindlll), and 15 pL of nuclease-free water. The contents were gently mixed by vortexing at low speed and then incubated at 37°C for 15 minutes. The reaction was terminated by heat inactivation at 80°C as per the manufacturer’s instructions. Finally, the digested products were analyzed via electrophoresis on a 1.5% agarose gel to visualize the released inserts.

[0191] Example 3B: Expression of X24A1DM (SEQ ID NO: 3) in E. coli BL21 (DE3) Cells To produce the recombinant proteins, a single colony of the positive clone from Example 2A was inoculated into LB broth containing 50pg / mL ampicillin and incubated overnight at 37°C with continuous shaking. After overnight growth, 100 pL of the overnight culture was transferred into 100 mL of LB broth supplemented with ampicillin (50pg / mL) and incubated at 37°C with continuous shaking until reaching an optical density (OD) of 0.6-0.8 at 600 nm. Once the desired OD was attained, the culture was induced by adding 0.25mM IPTG and incubating at 25 °C for 18 hours. After induction, the culture was centrifuged at 5000 rpm for 10 minutes at 4°C. The resulting pellet was then re-suspended in 5 mL of celLytic buffer (Sigma #C8740) containing 50 pL of lysozyme (10 mg / mL) and incubated for 1 hour at 30°C. Following the incubation, the mixture was centrifuged at 14,000 rpm for 10 minutes at 4°C to separate the cellular debris. The recombinant proteins present in the supernatant were then analyzed using 12% SDS-PAGE method known in the art. Throughout the experiments, E. coli BL21 (DE3) cells transformed with the non-recombinant pET-32a(+) plasmid were used as a control.

[0192] The nucleotide sequence as set forth in SEQ IDNO: 3 (X24A1DM) was successfully expressed in E. coll B L21 (DE3) cells producing a protein of 149 kDa. Based on the coding sequence (CDS) of this nucleotide sequence, the expected molecular weight of the protein is 129.8 kDa. However, the presence of thioredoxin and histidine tags from the pET-32a(+) vector added 19.2 kDa, resulting in the observed protein size of 149 kDa. Control experiments with an induced blank pET-32a(+) vector showed a protein of 20.4 kDa, while no protein was observed in the un-induced blank pET-32a(+) vector (Figure: 1).

[0193] Example 3C: Purification of recombinant Protein (SEQ ID NO: 4) The recombinant protein as described in Example 3B was obtained from E. coli BL21 (DE3) using the method known in the art. Purification was conducted using Ni-charged resin (Profinity IMAC; #156-0135) following the manufacturer's guidelines. The Ni-resin, amounting to a 1 mL bed volume, was packed into the column. The resin was prepared by eliminating the storage buffer through gravity flow and subsequently equilibrated with celLytic buffer. The crude recombinant protein was introduced into the column and allowed to flow through. Following this, the resin underwent two washes with five-column volumes of wash buffer (150mM NaCl, 50mM Tris, and 25mM Imidazole, pH 8.0) before being drained. A 3 mL aliquot of elution buffer (150mM NaCl, 50mM Tris, and 250mM Imidazole; pH 8.0) was then added to the column, allowing the eluent, containing the His-tagged recombinant protein, to be collected in a sterile polypropylene tube. The protein was subsequently subjected to analysis using 12% SDS-PAGE and stored at 4°C until further usage. Quantification of the purified protein was done using a spectrophotometer (Eppendorf, Germany).

[0194] The recombinant protein as set forth in SEQ IDNO: 3 was purified using Ni-NTA column (BioRad). This purification method effectively eliminated unwanted cellular proteins, resulting in a protein purity of over 90%.

[0195] Example 4: Insect Toxicity Assay of Recombinant Protein (SEQ ID NO: 4) Toxicity assay of the recombinant protein having amino acid sequence as set forth in SEQ ID NO: 4 was carried out using artificial diet based assay method and leaf based assay method against Pectinophora gossypiella, Helicoverpa armigera, Spodoptera litura, Spodoptera frugiperda, Plutella xylostella, and Leucinoides orbonalis

[0196] Example 4A: Artificial Diet Based Bioassay Against Pectinophora gossypiella:

[0197] The semi-synthetic diet for Pectinophora gossypiella. (Pink Boll Worm; PBW) was prepared following the method outlined by Murali Mohan et al., 2009. Recombinant protein having amino acid sequence as set forth in SEQ ID NO: 4 dispensed into sterile 50 mL polypropylene tubes at desired concentrations (5, 10, 15, 20, and 25 pg / g diet). The tube was filled with approximately 30 g of semi-solid, lukewarm diet. The protein and diet were thoroughly mixed by shaking the tubes. Subsequently, the mixed diet was transferred to sterile Petri dishes labelled with the protein concentration and exposed to UV light for an hour within a laminar airflow environment. After solidification, the diet was cut into equal-sized pieces and placed in sterile cups, which were labelled with the protein concentration, and replication number. The same procedure was conducted for two control groups: one with CelLytic buffer and the other with water. Each bioassay was replicated five times, with one neonate larva per replication for each toxin concentration. The insects were then incubated under controlled conditions: 28 ±2°C temperature, 65 ± 5% relative humidity, and a 14: 10 L photoperiod. After 21 days, the bioassays were evaluated, and the LD50 (dose required to kill half the members of a tested insect population) values were calculated using probit analysis with SPSS Software. This methodology allowed for the assessment of the toxicity of the recombinant protein against P. gossypiella larvae.

[0198] The toxicity assessment involved testing five concentrations (5, 10, 15, 20, and 25 pg / g diet) of the recombinant protein (SEQ ID NO: 4), with five replicates per concentration. Controls consisted of an artificial diet containing only water and protein elution buffer. The recombinant protein has exhibited toxicity with an LD50 value of 2.23 pg / g diet. Figure: 2 along with Tables 1 & 2 provide detailed insights into the toxicity against PBW. The observations were recorded after 21 days of setting up the experiment. The PBW larvae were alive in both the water and protein elution buffer control cups, except in one cup containing the diet with water as control. However, the larvae in all replicates of 5 to 25 pg of the recombinant protein (SEQ ID NO: 4) per gram of the diet died.

[0199] Table 1 shows X24A1DM protein insect toxicity observation

[0200]

[0201] Table 2 shows LD50 calculation of X24A1DM protein

[0202]

[0203]

[0204] Example 4B: Bioassay Against Helicoverpa armigera, Spodoptera litura, Spodoptera f'ni iperda. Plutella xylostella. and Leucinoides orbonalis'.

[0205] Artificial diet based assay was setup for Helicoverpa armigera, Spodoptera litura, Spodoptera frugiperda, leaf based assay was setup for Plutella xylostella, and potatotes infused with X24A1DM protein was used for Leucinoides orbonalis.

[0206] A) Artificial Diet based Assay:

[0207] The ingredients listed in the Table 3 were added to a flask in the specified quantities. Subsequently, 500 ml of warm water was added to the flask, and the contents were thoroughly mixed with a glass rod. In a separate beaker, 53 g of yeast was suspended in 350 ml of water and boiled for 5 minutes. Meanwhile, 16 g of agar-agar was combined with 350 ml of water in another flask and boiled for 5 minutes.

[0208] Table 3: Primary ingredients for the diet preparation

[0209]

[0210] The yeast and agar suspensions were combined in a large flask, and the mixture was boiled for 5 minutes. While still hot, it was added to the flask containing the diet ingredients. The diet mixture was thoroughly blended using a blender. Subsequently, 13.5 ml of 10% formaldehyde and two multivitamin capsules were added to the mixture and blended again thoroughly. The hot diet was then transferred to glass beakers and allowed to cool to 50°C.

[0211] To incorporate the recombinant protein into the diet, different concentrations (5, 10, 15, 20, and 25 pg / ml) of the protein were prepared in 50 ml polypropylene tubes (protein concentrations were calculated based on the diet requirement per container). The lukewarm diet was poured into the tubes containing test protein, and after securely tightening the caps, the tubes were gently inverted several times to mix the protein with the diet. Each concentration was prepared in three replicates. The diet was poured into the petri-plates and the petri-plates were placed in a laminar airflow under UV light to allow the diet to solidify and to sterilize the surface. The solidified diet was cut into cubes and placed in the cups for bioassay.

[0212] B) Leaf based bioassay for Plutella xylostella

[0213] Fresh brassica leaves were collected and thoroughly washed with sterile distilled water. The washed leaves were then dried using tissue paper to absorb excess moisture. Sterile filter papers, cut to fit the size of the petri plates, were moistened with sterile distilled water and placed in sterile petri plates. The leaves were placed on the pre-wet filter paper inside the petri plates. The protein to be tested was prepared in concentrations of 5, 10, 15, 20, and 25 pg / cm2and applied to the leaves using a sterilized brush. Each concentration was repeated five times. Two first instar larvae per leaf were carefully placed on the treated leaves in the petri plates using a paint brush. The petri plates were sealed with micropore tape. Leaves without protein treatment were used as controls. The plates containing the larvae were incubated at 25 ± 1°C, with 70% relative humidity, and a 14-hour light / 10-hour dark photoperiod for 7 days.

[0214] C) Potato based bioassay for Leucinoides orbonalis

[0215] Fresh potatoes were peeled off and cut into 1 cm3pieces. The test protein was coated onto the cubes in the concentrations 5, 10, 15, 20, and 25 pg / cm3. Each concentration was repeated five times. Untreated cubes were used as controls. Followed by coating, the cubes were airdried and placed in the sterile petri plates. Two first instar larvae were placed per cube and the plates were sealed with micorpore tape and incubated at at 25 ±1°C, with 70% relative humidity, and a 14-hour light / 10-hour dark photoperiod for 7 days.

[0216] The toxicity of the X24A1DM protein was evaluated against various lepidopteran insect pests, including Helicoverpa armigera, Spodoptera litura, Spodoptera frugiperda, Plutella xylostella, and Leucinodes orbonalis, to determine the protein’s insecticidal spectrum. The results showed that X24A1DM caused mortality in Helicoverpa armigera, Spodoptera litura, Plutella xylostella, and Leucinodes orbonalis, but exhibited insignificant toxicity against Spodoptera frugiperda larvae (Table 4 & 5).

[0217] Table 4 shows Insect toxicity Assay of the Recombinant protein against Helicoverpa armigera, Spodoptera litura, Spodoptera frugiperda (5 replicates; 1 larvae / Replicate)

[0218]

[0219] Table 5 shows Insect toxicity Assay of X24A1DM Recombinant protein Against Plutella xylostella, and Leucinoides orbonalis (5 replicates; 1 larvae / Replicate).

[0220]

[0221] Example 5: Cloning of X24A1DM DNA Sequence (SEO ID NO: 3) into Binary Vector pGreen0029

[0222] The DNA fragment X24A1DM (SEQ ID NO: 3) from pUC57 _X24A1DM was released using BamHI and EcoRI restriction endonucleases. A total reaction volume of 30 pL was set up with 10 pL of pUC57 _X24A1 DM plasmid DNA (3pg), 3 pL of 10X fast digest restriction enzyme buffer, 1 pL (20U / pL) of each fast digest restriction enzyme (BamHI and EcoRI) following the cloning strategy, and 15 pL of nuclease-free water in a 1.5 mL polypropylene tube. After gentle vortexing, the tubes were incubated at 37 °C for 15 minutes. Subsequently, the reaction was halted by incubating at 80°C for 10 minutes. The resulting digested product underwent elution and purification using a QIAquick PCR and Gel Extraction Kit. The restriction digestion of pUC51 _X24A1 DM plasmid using BamHI and EcoRI resulted in the release of a 3.5 kb fragment. This fragment was then purified using a Gel Extraction Kit.

[0223] Subsequently, the purified DNA fragment (X24A1 DM) was sub-cloned into the CaMV35S promoter cassette of the pUC57_CaMV35S plasmid. The resulting ligated product was transformed into E. coli TOPIO cells and positive clones were identified through colony PCR using the primers as set forth in SEQ ID NO: 5 and SEQ ID NO: 6 that amplified a 495 bp fragment in the positive clones.

[0224] The pUC57 plasmid carrying the CaMV-35S promoter cassette was digested using BamHI and EcoRI restriction endonucleases. The restriction digestion was performed in a total volume of 30 pL, consisting of 10 pL of pUC57_CaMV 35S plasmid DNA (5 pg), 5 pL of 10X fast digest restriction enzyme buffer, 2 pL (20 U / pL) of each fast digest restriction enzyme (BamHI and EcoRI), and 15 pL of nuclease-free water in a 1.5 mL polypropylene tube. The mixture was gently vortexed at low speed and incubated at 37°C for 15 minutes. The reaction was terminated by incubating at 80°C for 10 minutes. To verify the digestion, 5 pL of the digested sample along with a DNA marker were run on a 1.5% agarose gel. The remaining digested product was eluted and purified using a QIAquick PCR and Gel Extraction Kit.

[0225] The ligation reaction was prepared in a 1.5 mL polypropylene tube with a total volume of 50 pL. The components included the pUC57_CaMV 35S vector and the insert DNA (X24A1 DM) in a 1:3 molar ratio, 2 pL of 10X Ligase buffer, pUC57_CaMV 35S vector, IpL of T4 DNA ligase (0.2-0.4 U / pL), the insert DNA and nuclease-free water to reach the final volume. The mixture was gently pipetted to mix the contents thoroughly and then incubated overnight at 16°C. The resulting ligated product was subsequently used for bacterial transformation.

[0226] These positive clones were further validated by restriction digestion analysis using EcoRV endonuclease, which released the DNA fragment X24A1DM (SEQ ID NO: 3) and promoter cassette from the pUC57_CaMV35S_X24AJ£> Af_CaMV poly(A) plasmid, yielding a 4.1 kb cassette.

[0227] The ligated product pUC57_CaMV 35S_X2 A7DM_CaMV poly(A) signal was added aseptically to 100 pL of E. coli TOPIO competent cells, mixed gently, and incubated on ice for 30 minutes. The bacterial cells were then subjected to heat shock by placing the tubes in a water bath set to 42°C for 90 seconds, followed by snap-cooling on ice for 5 minutes. Next, 900 pL of sterile LB liquid medium was added to the tube, and the mixture was incubated at 37°C for 1 hour with gentle shaking at 175 rpm. The transformed cells were then spread on LB agar plates containing ampicillin (50pg / mL) and incubated overnight at 37°C. Positive clones were confirmed by performing colony PCR and restriction digestion of the plasmid DNA.

[0228] The pUC57 plasmid carrying the CaMV 35S_X24A7DA7_CaMV poly(A) cassette was digested with the EcoRV restriction endonuclease to release the cassette comprising the X24A1DM DNA. The restriction digestion reaction of a total volume of 30pL was set up with 10 pL of pUC57 carrying CaMV 35S _X24 Al DM. _CaMV poly(A) plasmid DNA (5 pg), 5 pL of 10X fast digest restriction enzyme buffer, 2 pL (20U / pL) of EcoRV fast digest restriction endonuclease, 13 pL of nuclease-free water. The mixture was gently vortexed at low speed and incubated at 37°C for 15 minutes. To check the digestion, 5 pL of the digested sample and a DNA marker were run on a 1.5% agarose gel. The desired insert was then eluted and purified using a Gel Extraction Kit.

[0229] The plant transformation vector plasmid pGreen0029 was digested with EcoRV to clone the gene cassette CaMV 35S _X24A1 DM _CaMV poly(A) signal, which was also digested with the same enzyme. The restriction digestion reaction of a total volume of 30 pL was set up with 10 pL of pGreen0029 plasmid DNA (3 pg), 3 pL of 10X fast digest restriction enzyme buffer, 1 pL (20 U / pL) of EcoRV fast digest restriction endonuclease, 16 pL of nuclease-free water. The contents were gently vortexed at low speed and incubated at 37°C for 15 minutes. To verify the digestion, 5 pL of the digested sample and a DNA marker were run on a 1.5% agarose gel. The digested product was then eluted and purified using a QIAquick PCR and Gel Extraction Kit. The ligation reaction of a total volume of 50 pL was set up in a 200 pL PCR tube by adding the linearized pGreen0029 plasmid and the CaMV 35S _X24 Al DM _CaMN poly(A) signal insert DNA in a 1:3 ratio. The reaction mixture included: Linearized pGreen 0029 plasmid DNA, CaMV 35S _X24A1 DM _CaMV poly(A) signal insert DNA, 2 pL of 10X Ligase buffer, 1 pL of T4 DNA ligase (0.2-0.4 U / pL), Nuclease-free water to make up the final volume to 50 pL. The contents were gently mixed by pipetting and incubated overnight at 16°C. The ligated product was then used for bacterial transformation.

[0230] The ligated product, pGreen0029_CaM N35S_X24A J DM _CaM V poly(A) (hereafter referred to as BRI- 135), was added aseptically to 100 pL of E. coli TOP10 competent cells. The mixture was gently mixed and incubated on ice for 30 minutes. The bacterial cells were then subjected to heat shock by placing the tubes in a water bath set to 42°C for 90 seconds, followed by snapcooling on ice for 5 minutes. After the heat shock, 900 pL of sterile LB liquid medium was added to the tube, and the cells were incubated at 37°C for 1 hour with gentle shaking at 175 rpm. Subsequently, the cells were spread on LB agar plates containing kanamycin (50pg / mL). The plates were incubated overnight at 37°C. Positive clones were confirmed by colony PCR and restriction digestion of the plasmid DNA.

[0231] The pUC57 _CaMV35S_X24A7 DM _CaMV poly(A) plasmid underwent restriction endonuclease digestion using EcoRV, resulting in the release of a 4.1 kb fragment. This fragment was then eluted from agarose gel and purified using a Gel Extraction Kit. Subsequently, the purified gene fragment was ligated with the linearized pGreen()029 plasmid. The resulting ligated product was transformed into E. coli TOP10 cells, and positive clones were identified through colony PCR using the primers as set forth in SEQ ID NO: 5 and SEQ ID NO: 6 which amplified a 495 bp fragment in the positive clones. These positive clones were further validated by restriction digestion analysis of the plasmid using EcoRV endonuclease, which released the cassette comprising promoter and X24A1DM DNA from the pGreen0029_CaMV35S_X24Aj,£)Af_CaMV poly(A) plasmid, yielding a 4.1 kb cassette. Example 6: Transformation oi Agrobacterium tumefaciens strain LBA4404 with BRI- 135

[0232] Plasmid DN A

[0233] The plasmid BRI-135 (1 pg) was added aseptically to 100 pL of competent cells of Agrobacterium tumefaciens strain LBA4404 carrying the pSOUP plasmid DNA. The mixture was gently stirred and incubated on ice for 10 minutes. The bacterial cells were then subjected to a snap freeze in liquid nitrogen for 5 minutes, followed by incubation at 37°C for 5 minutes. Next, 900 pL of sterile LB liquid medium was added to the tubes, which were incubated at 28°C for 3 hours with gentle shaking at 200 rpm. The cells were then spread on LB agar containing Rifampicin (50pg / mL) Streptomycin (50 pg / mL) Kanamycin (30 pg / mL) Tetracycline (5pg / mL). The plates were incubated at 37°C for 2 days. The transformed positive colonies were confirmed by colony PCR using the primer sets as set forth in SEQ ID NO: 5 and SEQ ID NO: 6; and SEQ ID NO: 7 and SEQ ID NO: 8. The colony PCR targeting the X24A1DM DNA yielded a fragment of 495 bp, while the marker- specific nptll primers produced a fragment of 707 bp. These results validate the successful incorporation of the BRI- 135 plasmid into the Agrobacterium tumefaciens LBA4404 cells, affirming the presence of both the X24A1DM DNA and the selectable marker gene within the transformed bacterial colonies

[0234] Example 7: mediated Cotton transformation with BRI135 Plasmid

[0235]

[0236] Transgenic lines of cotton were developed by Agro / zacterzzzm-mediated transformation using cotylendonary leaf explants of in-vitro grown seedlings of Gossypium hirsutum L. cv. Coker 310. Neomycin phosphotransferase (nptll) gene was used as a selectable marker and kanamycin as a selection agent. Surface sterilize delinted cotton seeds were germinated on MS basal medium under photoperiod of 16 / 8 light / dark at 25°C. Approximately 1 cm2 cotyledon explants from 7 day old seedlings were pre-cultured on MT1 medium (Table 6) and incubated under 16 h / 8 hour light and 25 °C overnight. The explants from the pre-culture were transferred to 50 ml corning tube and approximately 30 ml Agrobacterium suspension was added to the explants and the tube was gently agitated to mix explants with the Agrobacterium. After 20 min incubation the explants were transferred on co cultivation medium (MT1 with 100 pM aceto syringone) medium. The plates were incubated at 25°C in dark for 2 days.

[0237] After 6 days the co-cultvated explants were collected in a 50 ml corning tube and washed thoroughly with sterile distilled water three times and soaked in sterile water with Augmentin 300 mg / L for 15 min. Washed explants were transferred to MT1 selection medium (Table 6). The plates were kept at 28 ± 1°C under lights at a 16-h light / 8-h dark photoperiod for 2 weeks. The explants were subcultured on MT1 selection medium under same culture conditions for 2 weeks. The developing calli were sub cultured on MT2 selection plates for 60 days with fresh subculture after every 21 days.

[0238] Table 6 shows Composition of culture medium

[0239]

[0240] Figure 3 shows different stages of in-vitro regeneration of transgenic cotton plants. The embryogenic calli (EC) was observed by the end of third subculture (Figure 3 D). The embryogenic calli was sub-cultured on MT3A medium (Figure 3E) and maintained on MT3A medium by sub-culturing at an interval of 21 days (Figure 3F). The differentiated embryos with distinct shoot and root poles were transferred to i MSD medium in bottles (Figure 3H). The germinated embryos formed plantlets in 14-25 days. The plantlets were acclimatized in plastic cups containing soilrite until new leaves emerged and the roots spread in the soil (Figure 31). The acclimatized plants were transferred to 25 cm pots in green house. The flowers were selfpollinated to collect TO seeds in 2-4 months.

[0241] Example 8A: Molecular analysis of TO, TE T2, and T3 transgenic cotton plants Polymerase Chain Reaction was used to detect the presence of transgenes in cotton transgenic lines carrying X24A1DM DNA and nptll genes in the transgenic event. Genomic DNA was isolated from TO plants. Fresh leaf tissue (20 mg) was collected in a 2 ml tube and homogenized with stainless steel 2 mm beads for 1 minute and using QIAGEN Tissue Eyser with extraction buffer containing 2% CT AB. Equal volume of chloroform was added to the extract and mixed well for 1 min. The mixture was centrifuged for 10 min at 10000 rpm at room temperature. The supernatant was transferred to a clean 2 ml tube and equal volume of chilled iso-propanol was added to it and mixed thoroughly and centrifuged at 14000 rpm for 10 min at 4°C to precipitate the DNA. Supernatant was discarded and pellet was washed with chilled 70% ethanol twice by centrifuging at 14000 rpm for 10 min at 4°C. Finally, the pellet was air dried for 30 min at room temperature and finally resuspended in 200 pl of TE buffer { 10 mM Tris (pH-8.0), 1 mM EDTA}. The purified DNA was quantified and quality of extracted DNA was checked by loading on a 1% agarose gel. Genomic DNA (200 ng) of from each sample was used for amplification of both X24A1DM as well as marker gene nptll. To confirm the presence of the genes PCR was carried out using the primers as set forth in SEQ ID NO: 5 and SEQ ID NO: 6; and SEQ ID NO: 7 and SEQ ID NO: 8.

[0242] The PCR genotyping of the cotton TO plants for the X24A1DM DNA (SEQ ID NO: 3) and the nptll marker gene revealed that all the plants were positive for the X24A1DM DNA and nptll. The primer as set forth in SEQ ID NO: 5 and SEQ ID NO: 6 amplified an expected amplicon of 495 bp and the primers as set forth in SEQ ID NO: 7 and SEQ ID NO: 8 amplified 795 bp amplicon in all the plants. No amplification was seen in the non-transgenic control cotton plants. The plasmid pBRI135 was used as a positive control. Thus, it was concluded that all TO transgenic cotton plants contained both X24A1DM DNA (SEQ ID NO: 3) as well as nptll genes. Subsequently T1 and T2 and T3 transgenic cotton progenies were obtained from the TO plants. PCR analysis of the said progenies was carried out to detect the presence of transgenes in cotton transgenic lines carrying X24A1DM DNA and nptll genes.

[0243] Example 8B: Insect bioassay with Tl, T2, and T3 plant tissues Comparison with NTC &

[0244] BGII

[0245] Using 1stinstar larvae of Pectinophora gossypiella (Pink Bollworm) lab cotton leaf bioassay was carried out on leaf collected from Tl, T2 and T3 generation 120-150 days old cotton transgenic plants carrying X24A1DM DNA (SEQ ID NO: 3) (Table 7). The leaf squares were placed in clean transparent assay containers containing a moist paper at the bottom. Newly hatched larvae (1stinstar) of Pectinophora gossypiella were released with the help of a brush on the leaf squares. Observations were taken after 7-9 days. The leaf squares were checked for the damage status and growth stage of larvae.

[0246] From the bioassay results it has been observed that none of the cotton leaf squares collected from Tl, T2, and T3 plants transformed with the plasmid BRI135 were infested by the PBW larvae, whereas 46 percentage leaf squares in non-transgenic cotton (negative control) and 50.43 percentage leaf squares in transgenic cotton variety BIO GHH029 (positive control) (propriety material of DCM Shriram Ltd.) expressing Cry 1 Ac and Cry2 Ab protein were damaged by the larvae (Table 7). The larvae fed on these leaf squares of negative control and positive control have become adults. This indicates that the insecticidal protein expressed by transgenic cotton plants comprising the nucleotide sequence as set forth in SEQ ID NO: 3 (X24A1 DM) inhibited the growth of PBW larvae, while Cry 1 Ac and Cry2Ab protein expressed in positive control plants did not exhibit any toxicity to the PBW larvae.

[0247] Table 7: Insect toxicity assay of Tl, T2, and T3 transgenic plants against PBW

[0248]

Claims

I / We Claim:

1. A nucleic acid molecule comprising a nucleotide sequence encoding a protein having the amino acid sequence as set forth in SEQ ID NO: 4.

2. The nucleic acid molecule as claimed in claim 1, wherein the nucleotide sequence is as set forth in SEQ ID. NO: 3.

3. A DNA construct comprising the nucleic acid molecule as claimed in claim 1.

4. A plasmid vector comprising the nucleic acid molecule as claimed in claim 1.

5. A host cell comprising the nucleic acid molecule as claimed in claim 1.

6. The host cell as claimed in claim 5, wherein the host cell is selected from a group consisting of bacterium, virus, insect, and plant cell.

7. A method for conferring an insect resistance in a plant comprising:(d) inserting into a plant cell the nucleic acid molecule as claimed in claim 1, wherein the nucleic acid molecule is operably linked to a (i) promoter functional in a plant cell and (ii) a terminator;(e) obtaining a transformed plant cell from the plant cell of step (a), wherein said transformed plant cell comprises the nucleic acid molecule of claim 1; and(f) generating a transgenic plant from said transformed plant cell of step (b), wherein said transgenic plant comprises the nucleic acid molecule of claim 1.

8. A transgenic plant comprising the the nucleic acid molecule as claimed in claim 1.

9. A tissue, seed or a progeny obtained from the transgenic plant as claimed in claim 8, wherein said seed or progeny comprises the the nucleic acid molecule as claimed in claim 1.

10. A biological sample derived from the tissues or seed or progeny as claimed in claim 9, wherein said sample comprising a detectable amount of said nucleic acid molecule as claimed in claim 1.

11. A commodity product derived from the transgenic plant as claimed in claim 8, wherein said product comprises a detectable amount of said nucleic acid molecule as claimed in claim 1.

12. A composition comprising Bacillus thuringiensis comprising nucleic acid molecule comprising a nucleotide sequence encoding a protein having the amino acid sequence as set forth in SEQ ID NO: 4.

13. The composition as claimed in claim 12, wherein said composition optionally comprises an additional insecticidal agent, wherein said insecticidal agent is selected from the group consisting of a Bacillus toxin, a Xenorhabdus toxin, a Photorhabdus toxin, and a dsRNA specific for suppression of one or more essential genes in said insect pest.

14. Use of the nucleic acid molecule comprising a nucleotide sequence encoding a protein having the amino acid sequence as set forth in SEQ ID NO: 4, the DNA construct as claimed in claim 3 or the plasmid as claimed in claim 4 for production of insect resistant transgenic plants.

15. Use of the nucleic acid molecule as claimed in claim 1 for production of insecticidal composition, wherein the composition comprises Bacillus thuringz'c / z.vz.s' cells comprising the said the nucleic acid molecule.

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