RU2799821C2 - CODON-OPTIMIZED SYNTHETIC NUCLEOTIDE SEQUENCES ENCODING Cry2Ai PROTEIN AND THEIR USE - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to a polynucleotide encoding the Cry2Ai insecticidal protein, as well as to a construct, a vector, a cell, a plant, a plant tissue, a plant seed, a biological sample of a plant, as well as to a commercial product and a composition containing it. Also a method of producing a transgenic plant, which involves introducing the above polynucleotide into a plant cell is disclosed.

EFFECT: invention is effective for control of Scirpophaga incertulas, Spodoptera Mauritia and H. Armigera insects.

23 cl, 7dwg, 6 tbl, 7 ex

Description

LINK TO THE SEQUENCE LIST ELECTRONICALLY SUBMITTED

[001] The official copy of the sequence listing file called "PD033499IN-SC sequence listing.txt", created on April 23, 2019 and having a size of 25 kilobytes, which was electronically submitted at the same time as the specification, is part of the specification.

FIELD OF TECHNOLOGY TO WHICH THE INVENTION RELATES

[002] The present invention relates to codon-optimized synthetic nucleotide sequences encoding a Bacillus thuringiensis (Bt) insecticidal crystal protein having insecticidal activity against insect pests, including, but not limited to, pests belonging to the order Lepidoptera. The present invention also relates to the expression of these sequences in plants.

BACKGROUND OF THE INVENTION

[003] Insect pests are one of the main causes of crop losses in the world. It is believed that they annually destroy a fifth of all agricultural crops produced in the world. The process of artificial selection of crops suitable for human consumption also selects plants that are highly susceptible to insect infestation, which ultimately reduces their economic value and increases the cost of production.

[004] Traditionally, insect pests have been controlled by the use of chemical and/or biological pesticides. There are some concerns about the use of chemical pesticides due to the environmental hazards associated with the production and use of chemical pesticides. Due to these concerns, regulators have banned or restricted the use of some of the most hazardous pesticides.

[005] Further, it is a well-known fact that insect pests are able to evolve over time through natural selection, to adapt to new situations, for example, to overcome the action of toxic materials or bypass plants that are naturally or artificially resistant, which further exacerbates the problem.

[006] A biological pesticide is an environmentally and commercially acceptable alternative to chemical pesticides. It poses a lower risk of pollution and environmental hazard, and is more specific in terms of target than traditional broad-spectrum chemical insecticides.

[007] It is known that some species of microorganisms of the genus Bacillus, for example, Bacillus thuringiensis (Bt), have insecticidal activity against a wide range of insect pests. The insecticidal activity appears to be concentrated in the parasporal inclusion bodies of the crystalline protein, although insecticidal proteins have also been isolated from Bacillus thuringiensis during the vegetative growth stage.

[008] Expression of Bacillus thuringiensis (Bt) insecticidal crystal (cry) protein genes in plants has been described in the art, however, it has been found that it is extremely difficult to express the natural Bt gene in plants. Attempts have been made to express the cry Bt protein gene in plants in combination with various plant-functional promoters. However, in transgenic plants, the protein was obtained only at low levels.

[009] One of the reasons for the low expression of the Bt gene in plants is the high content of A/T in the Bt DNA sequence, in contrast to plant genes, in which the content of G/C is higher than A/T. The overall A/T for bacterial genes is 60-70%, and for plant genes 40-50%. As a consequence, the ratio of GC when using codons of the cry genes is largely insufficient for expression at the optimal level. In addition, the A/T rich region may also contain transcription termination (AATAAA polyadenylation) sites, an mRNA instability motif (ATTTA), and hidden mRNA splicing sites. The codon usage for the native cry Bt gene was found to be significantly different from that of plant genes. As a result, the mRNA of this gene can be used inefficiently. The use of codons can influence gene expression at the level of translation or transcription, or mRNA processing. In order to optimize the insecticidal gene for expression in plants, attempts have been made to modify the gene to resemble as closely as possible the genes naturally present in the transformed host plant.

[0010] However, the development of crop varieties expressing high/optimal levels of the cry Bt protein conferring resistance to certain insect pests, such as rice leafworm, rice stem borer, is still a major problem in agriculture. Various attempts have been made to control or prevent insect infestation of plants, however some insect pests remain a significant problem in agriculture. As a consequence, there remains a need to develop insect-resistant transgenic crops with desired levels of expression of insecticidal proteins in transgenic plants.

[0011] The present invention provides a solution to the present problem of infestation by insect pests by providing a codon-optimized for plants synthetic DNA sequence encoding a Cry2Ai Bt protein having insecticidal activity against insect pests, including, but not limited to, insect pests belonging to the order Lepidoptera.

SUMMARY OF THE INVENTION

[0012] Disclosed herein are codon-optimized synthetic nucleotide sequences encoding the B. thuringiensis Cry2Ai protein with insecticidal activity against insect pests. The present invention relates to methods for increasing the expression of heterologous genes in plant cells. The cry2Ai gene, or gene coding region, is designed to provide a plant-specific preferred codon sequence. Thus, the codon usage for the Cry2Ai protein has been modified to increase expression in plants. Such plant-optimized coding sequences can be operably linked to a promoter capable of directing expression of the coding sequences in a plant cell. Transformed host cells and transgenic plants containing codon-optimized B. thuringiensis synthetic nucleotide sequences are also aspects of the present invention.

[0013] One of the objectives of the present invention is to provide codon-optimized synthetic nucleotide sequences encoding an insecticidal protein that have been optimized for expression in plants.

[0014] Another object of the present invention is to provide codon-optimized nucleotide sequences encoding a Bt insecticidal protein to achieve maximum expression of Bt proteins in a plant, preferably a plant selected from the group consisting of eggplant, cotton, rice, tomato, wheat, corn, sorghum, oats, millet, legumes, cabbage, cauliflower, broccoli, Brassica species, beans, okha, pigeon peas, potatoes, peppers, cucurbits, lettuce, sweet potatoes, canola, soybeans, alfalfa, peanuts and sunflowers. One feature of the present invention is that the codon-optimized cry2Ai Bt synthetic genes are designed using the most preferred codons in eggplant, rice and legumes.

[0015] In accordance with the present invention, we have synthesized insecticidal Bt crystal protein Cry2Ai genes in which the use of codons has been changed to increase expression in plants, in particular eggplant, rice and legumes. However, instead of changing codon usage to resemble eggplant, rice, or legume genes in terms of overall codon distribution, the inventors optimized codon usage by using codons that are most preferred in plants such as rice, eggplant, tomato, corn, and legumes in the synthesis of the nucleotide sequences of the invention. Optimized, preferential plant codon usage is effective for expressing the insecticidal Bt protein at high levels in dicotyledonous plants such as eggplant and tomato; in monocots such as rice; and in legumes such as chickpeas and pigeonpeas.

[0016] The codon-optimized synthetic nucleotide sequences of the present invention are derived from the Bacillus thuringiensis Cry2Ai protein having the amino acid sequence shown in SEQ ID NO: 1 (NCBI GenBank: ACV97158.1). The protein having the amino acid sequence shown in SEQ ID NO: 1 is active against various Lepidoptera insects, including Helicoverpa armigera - cotton cutworm and corn cutworm, Cnaphalocrocis medinalis - rice leafworm, and Scirpophaga incertulas - rice stem borer and Pectinophora gossypiella .

[0017] While the present invention has been described in terms of synthesizing eggplant, rice, and legume optimized cry2Ai Bt nucleotides for expression in rice, eggplant, and chickpea, respectively, it is understood that eggplant optimized cry2Ai Bt nucleotides can be used to optimize protein expression in other plants as well, such as tomato, corn, and cotton. In addition, it is understood that rice-optimized cry2Ai Bt nucleotides can be used to optimize protein expression in other plants as well. Similarly, it is understood that the legume-optimized cry2Ai Bt nucleotides can be used to optimize protein expression in other plants as well.

[0018] Accordingly, one aspect of the present invention relates to a codon-optimized synthetic nucleotide sequence encoding a protein having the amino acid sequence shown in SEQ ID NO: 1, where the specified nucleotide sequence is

a. the sequence shown in SEQ ID NO: 2, or its complementary nucleotide sequence, or

b. a nucleotide sequence that specifically hybridizes to at least 10 nucleotides of the nucleotide sequence shown in SEQ ID NO: 2 at nucleotide positions 251 to 402 and/or 1456 to 1628, or its complementary sequence.

[0019] Another aspect of the present invention relates to recombinant DNA containing the codon-optimized synthetic nucleotide sequence disclosed herein, where the nucleotide sequence is operably linked to a heterologous regulatory element.

[0020] Another aspect of the present invention relates to a DNA construct for expressing an insecticidal protein of interest, comprising a 5'-untranslated sequence encoding a sequence encoding a Cry2Ai insecticidal protein comprising the amino acid sequence of SEQ ID NO: 1, or an insecticidal fragment thereof, and a 3'-untranslated region, wherein said 5'-untranslated sequence contains a promoter functional in a plant cell, where indicated The coding sequence is the codon-optimized synthetic nucleotide sequence disclosed herein, and wherein said 3'-untranslated sequence contains a transcription termination sequence and a polyadenylation signal.

[0021] Another aspect of the present invention relates to a plasmid vector containing the recombinant DNA disclosed herein, or the DNA construct disclosed herein.

[0022] Another aspect of the present invention relates to a host cell containing a codon-optimized synthetic nucleotide sequence disclosed herein.

[0023] Another aspect of the present invention relates to a method for imparting insect resistance to a plant, comprising

a. introducing into a plant cell a codon-optimized synthetic nucleotide sequence disclosed herein, wherein the nucleotide sequence is operably linked to (i) a plant cell-functional promoter and (ii) a terminator;

b. obtaining a transformed plant cell from a plant cell of step (a), wherein said transformed plant cell contains said codon-optimized synthetic nucleotide sequence disclosed herein; And

c. obtaining a transgenic plant from said transformed plant cell of step (b), wherein said transgenic plant contains said codon-optimized synthetic nucleotide sequence disclosed herein.

[0024] Another aspect of the present invention relates to a transgenic plant containing a codon-optimized synthetic nucleotide sequence disclosed herein.

[0025] Another aspect of the present invention relates to a composition containing Bacillus thuringiensis containing the codon-optimized synthetic nucleotide sequence disclosed herein, encoding a Cry2Ai protein having the amino acid sequence shown in SEQ ID NO: 1.

[0026] Another aspect of the present invention relates to a method for controlling insects that attack crops and providing insect resistance, which method includes contacting said crop with an insecticidal effective amount of the composition disclosed herein.

[0027] Another aspect of the present invention relates to the use of a codon-optimized synthetic nucleotide sequence, DNA construct or plasmid disclosed herein for the production of insect-resistant transgenic plants.

[0028] Another aspect of the present invention relates to the use of a codon-optimized synthetic nucleotide sequence disclosed herein for the production of an insecticidal composition that contains cells of Bacillus thuringiensis containing these nucleotide sequences.

BRIEF DESCRIPTION OF THE ACCOMPANYING FIGURES

[0029] FIGURE 1 shows a map of the pGreen0179-CaMV35S-201M1 T-DNA construct.

[0030] FIGURE 2 shows the genetic transformation and regeneration of transgenic rice plants.

[0031] FIGURE 3 is a map of the pGreen0029-CaMV35S-201M1 T-DNA construct.

[0032] FIGURE 4 shows genetic transformation and regeneration of transgenic tomato plants.

[0033] FIGURE 5 shows the results of a bioassay of the effect of a separated stem fragment of a transgenic rice plant containing the 201M1 nucleotide sequence against larvae of the rice stem borer, S. incertulas .

[0034] FIGURE 6 shows the results of a bioassay of the effect of a separated leaf fragment of a transgenic rice plant containing the 201M1 nucleotide sequence against the larvae of the Rice Fall Armyworm, S. mauritia .

[0035] FIGURE 7 shows the results of a bioassay of the effect of a leaf disk of a transgenic tomato plant containing the 201M1 nucleotide sequence against H. armigera larvae.

BRIEF DESCRIPTION OF SEQUENCES

[0036] SEQ ID NO: 1 is the sequence of the Cry2Ai protein (NCBI GenBank: ACV97158.1).

[0037] SEQ ID NO: 2 is the full length DNA sequence of the monocot codon-optimized synthetic cry2Ai gene (201M1) encoding the Cry2Ai protein (SEQ ID NO: 1).

[0038] SEQ ID NO: 3 is the full length DNA sequence of the monocot codon-optimized synthetic cry2Ai gene (201M2) encoding the Cry2Ai protein (SEQ ID NO: 1).

[0039] SEQ ID NO: 4 is the full length DNA sequence of the monocot codon-optimized synthetic cry2Ai gene (201M3) encoding the Cry2Ai protein (SEQ ID NO: 1).

[0040] SEQ ID NO: 5 is the full length DNA sequence of the monocot codon-optimized synthetic cry2Ai gene (201M4) encoding the Cry2Ai protein (SEQ ID NO: 1).

[0041] SEQ ID NO: 6 is the full length DNA sequence of the monocot codon-optimized synthetic cry2Ai gene (201M5) encoding the Cry2Ai protein (SEQ ID NO: 1).

[0042] SEQ ID NO: 7 is the full length DNA sequence of the monocot codon-optimized synthetic cry2Ai gene (201M6) encoding the Cry2Ai protein (SEQ ID NO: 1).

[0043] SEQ ID NO: 8 is the sequence of a forward primer for amplifying the 201M1 DNA sequence (SEQ ID NO: 2).

[0044] SEQ ID NO: 9 is the reverse primer sequence for amplifying the 201M1 DNA sequence (SEQ ID NO: 2).

[0045] SEQ ID NO: 10 is the sequence of the forward primer for DNA amplification of the nptII gene.

[0046] SEQ ID NO: 11 is the reverse primer sequence for DNA amplification of the nptII gene.

[0047] SEQ ID NO: 12 is the sequence of a forward primer for amplifying the hptII gene.

[0048] SEQ ID NO: 13 is the reverse primer sequence for amplifying the hptII gene.

DETAILED DESCRIPTION OF THE INVENTION

[0049] The detailed description provided herein is intended to assist a person skilled in the art in practicing the present invention and should not be construed as unduly limiting the scope of the invention, since modifications and variations of the embodiments described herein may be applied by those skilled in the art without deviating from the spirit or scope of the invention. Hereinafter, the present invention is described in more detail with reference to the accompanying drawings and/or sequence listing, this description presents and/or describes some, but not all, embodiments of the invention. The invention may be embodied in many different forms and should not be construed as being limited to the embodiments provided herein.

[0050] Although specific terms are used herein, they are used in a general and descriptive sense only, and not for purposes of limitation. The following definitions are provided to facilitate understanding of the embodiments.

[0051] It should be noted that when used in the specification and the appended claims, the singular form of the terms means "one or more than one" (i.e., at least one) grammatical entity, unless the context clearly indicates otherwise. Thus, for example, reference to "probe" means that more than one such probe may be present in a composition. Likewise, the reference to "element" means "one or more elements".

[0052] In the text of the specification, the words "comprises" or "comprising" should be understood to mean the inclusion of a specified 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.

[0053] Units, prefixes, and symbols may be given in conventional SI form. Unless otherwise indicated, nucleic acids are written from left to right in the 5'-3' orientation, and amino acid sequences are written from left to right in the amino- to carboxyl-terminal orientation, respectively. Numeric ranges include numbers that define the range. In this document, amino acids may be designated either by their well-known three-letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Commission on Biochemical Nomenclature. Likewise, nucleotides may be designated by their common single letter codes. The above terms are more fully defined with reference to the specification as a whole.

[0054] The term "nucleic acid" generally refers to large polynucleotides. In this document, the terms "nucleic acid" and "nucleotide sequence" are used interchangeably. They mean a deoxyribonucleotide or ribonucleotide polymer in single-stranded or double-stranded form and, unless otherwise limited, encompass known analogues (e.g., peptido-nucleic acids) having the essential nature of naturally occurring nucleotides in that they hybridize to single-stranded nucleic acids in a manner similar to naturally occurring nucleotides. Nucleotides are subunits that are polymerized (linked in a long chain) to form nucleic acids (DNA and RNA). Nucleotides are composed of three smaller components: ribose sugar, nitrogenous base and phosphate group(s).

[0055] The term "polynucleotide" means a single strand, or parallel and antiparallel strands of a nucleic acid. Thus, a polynucleotide can be either a single-stranded or double-stranded nucleic acid.

[0056] The term "oligonucleotide" generally refers to short polynucleotides, typically no more than about 50 nucleotides in length. It should be understood that when a nucleotide sequence is represented by a DNA sequence (i.e., A, T, G, C), it also includes an RNA sequence (i.e., A, U, G, C) in which "U" replaces "T".

[0057] The term "codon-optimized synthetic nucleotide sequences" refers to non-genomic nucleotide sequences and is used interchangeably herein to refer to synthetic nucleotide sequences or a nucleic acid molecule that has one or more changes in the nucleotide sequence from a natural or genomic nucleotide sequence. In some embodiments, a change in a natural 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, e.g. inoic acid for introducing restriction enzyme sites, deleting one or more introns associated with a genomic nucleic acid sequence, inserting one or more heterologous introns, deleting one or more regulatory regions located upstream or downstream of transcription and associated with a genomic nucleic acid sequence, inserting one or more heterologous regulatory regions located upstream or downstream of transcription, deletions of a 5' and/or 3' untranslated region, linked genomic nucleic acid sequence, insertion of a heterologous 5' and/or 3' untranslated region, and modification of the polyadenylation site.

[0058] In the context of the present invention, the following abbreviations are used for commonly occurring nucleotide bases. "A" means adenosine, "C" means cytidine, "G" means guanosine, "T" means thymidine and "U" means uridine.

[0059] In some embodiments, the non-genomic nucleic acid molecule is cDNA. In some embodiments, the non-genomic nucleic acid molecule is a synthetic nucleotide sequence. Codon-optimized nucleotide sequences can be generated for any organism of interest using methods known in the art, such as those described in Murray et al. (1989) Nucleic Acids Res. 17:477-498. Optimized nucleotide sequences are used to increase the expression of a pesticidal protein in a plant such as monocots and dicots such as rice, tomato and cotton plants.

[0060] The newly developed cry2Ai DNA sequences disclosed herein are referred to as "codon-optimized cry2Ai synthetic nucleotide sequences".

[0061] The terms "DNA construct", "nucleotide constructs", and "DNA expression cassette" are used interchangeably herein and should not limit the embodiments to DNA nucleotide constructs. Those skilled in the art understand that nucleotide constructs, in particular polynucleotides and oligonucleotides consisting of ribonucleotides and combinations of ribonucleotides and deoxyribonucleotides, can also be used in the methods disclosed herein.

[0062] As used herein, the term "recombinant" nucleic acid molecule, or DNA, or polynucleotide, means a nucleic acid molecule, or DNA, polynucleotide that has been modified or produced by human hand and is in a recombinant bacterial or plant host cell. For example, a recombinant polynucleotide can be a polynucleotide isolated from a genome, cDNA obtained by reverse transcription of RNA, a synthetic nucleic acid molecule, or an artificial combination of two otherwise separated sequence segments, for example, obtained by chemical synthesis or by manipulation of isolated polynucleotide segments by genetic engineering methods.

[0063] As used herein, the term "homologous" refers to the similarity of nucleotide sequences between two regions of the same nucleic acid strand or between regions of two different nucleic acid strands. When a nucleotide position in both regions is occupied by the same nucleotide, the regions are homologous at that position. The first region is homologous to the second region if at least one nucleotide position in each region is occupied by the same nucleotide. Homology between two regions is expressed as the relative proportion of nucleotide positions in the two regions that are occupied by the same nucleotide. As an example, the region having the nucleotide sequence 5'-ATTGCC-3' and the region having the nucleotide sequence 5'-TATGGC-3' have 50% homology. Preferably, the first region contains the first fragment and the second region contains the second fragment, wherein at least about 50% and preferably at least about 75%, at least about 90%, or at least about 95% of the nucleotide positions of each of the fragments are occupied by the same nucleotide. More preferably, all nucleotide positions of each of the fragments are occupied by the same nucleotide.

[0064] Optimal sequence alignment for comparison can be performed using computerized implementations of algorithms known in the art (GAP, BESTFIT, BLAST, PASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group (GCG), 575 Science Dr., Madison, WI), or by visual inspection.

[0065] As used herein, "percent sequence identity" is determined by comparing two optimally aligned sequences in a comparison window, where a fragment of a polynucleotide or amino acid sequence in the comparison window may have additions or deletions (e.g., gaps or overhangs) compared to a reference sequence (which has no addition or deletion) to optimally align the two sequences. The percentage is calculated by determining the number of positions at which identical nucleotide bases or amino acid residues are present in both sequences to obtain the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window, and multiplying the result by 100 to obtain percent sequence identity.

[0066] Optimal sequence alignment for comparison can be performed using computerized implementations of algorithms known in the art (GAP, BESTFIT, BLAST, PASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group (GCG), 575 Science Dr., Madison, WI), or by visual inspection.

[0067] As used herein, the term "substantial sequence identity" between two nucleotide sequences refers to a polynucleotide containing a sequence that has at least 65% sequence identity, preferably at least 69%-77% sequence identity, compared to a reference sequence.

[0068] As used herein, the term "promoter/regulatory sequence" means a nucleotide sequence necessary for the expression of a gene product operably linked to a promoter/regulatory sequence. In some cases, this sequence may be the main promoter sequence, and in other cases, this sequence may also include the enhancer sequence and other regulatory elements necessary for the expression of the gene product. A promoter/regulatory sequence may, for example, be a sequence that allows the gene product to be expressed in a tissue-specific manner.

[0069] A "constitutive" promoter is a promoter that directs the expression of a gene to which it is operatively linked on a permanent basis in a cell. As an example, promoters that drive the expression of cellular housekeeping genes are considered constitutive promoters.

[0070] An "inducible" promoter is a nucleotide sequence that, when operably linked to a polynucleotide that encodes or defines a gene product, causes the gene product to be formed in a living cell substantially only when an inducer corresponding to the promoter is present in the cell.

[0071] A "tissue-specific" promoter is a nucleotide sequence that, when operably linked to a polynucleotide that encodes or defines a gene product, causes the gene product to form in a living cell substantially only if the cell is a cell of the tissue type that corresponds to the promoter.

[0072] As used herein, the term "operably linked" refers to any link, regardless of orientation or distance, between a regulatory sequence and a coding sequence, where the link allows the regulatory sequence to control expression of the coding sequence. The term "operably linked" also means that the linked nucleotide sequences are contiguous and, if necessary, linking two protein-coding regions, contiguous and in the same reading frame. The term "operably linked" also refers to a functional relationship between a promoter and a second sequence, where the promoter sequence drives and mediates transcription of the DNA sequence corresponding to the second sequence.

[0073] As used herein, the term "heterologous DNA coding sequence" or "heterologous nucleic acid" or "heterologous polynucleotide" means any coding sequence other than that which naturally encodes a Cry2Ai protein or any homologue of a Cry2Ai protein.

[0074] As used herein, the term "coding region" refers to that portion of a gene, DNA, or nucleotide sequence that codes for a protein. The term "non-coding region" means that fragment of a gene, DNA or nucleotide sequence that is not a coding region.

[0075] As used herein, the terms "coding" or "encoded", when used in the context of a specific nucleic acid, means that the nucleic acid contains the necessary information for direct translation of the nucleotide sequence into a specific protein. The information by which a protein is encoded is determined by the use of codons. The nucleic acid encoding the protein may contain non-translated sequences (eg, introns) in the translated regions of the nucleic acid, or may not contain such intermediate non-translated sequences (eg, as in cDNA).

[0076] The terms "polypeptide", "peptide", and "protein" are used interchangeably herein to refer to a polymer composed of amino acid residues, both natural structural variants and synthetic, non-naturally occurring counterparts linked by 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 are artificial chemical analogues of the corresponding natural amino acids, as well as to natural 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 (commonly referred to as "protein"). The amino acid may be a naturally occurring amino acid and, unless otherwise limited, may be known analogs of naturally occurring amino acids that may act similarly to naturally occurring amino acids.

[0077] The term "protein" generally refers to large polypeptides. The term "peptide" generally refers to short polypeptides. However, the term "polypeptide" as used herein refers to any amino acid polymer consisting of two or more amino acid residues linked by peptide bonds.

[0078] As used herein, the term "expression cassette" means a genetic module that includes a gene and the regulatory regions necessary for its expression, which can be included in a vector.

[0079] A "vector" is a chemical compound containing a nucleic acid molecule that can be used to deliver an isolated nucleic acid into a cell. Many vectors are known in the art, including, but not limited to, linear polynucleotides, polynucleotides linked to ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term "vector" includes an autonomously replicating plasmid or virus. The term should also be construed to include non-plasmid and non-viral compounds that facilitate the transfer of the nucleic acid into cells, such as, for example, polylysine compounds, liposomes, and the like. Examples of viral vectors include, but are not limited to, adenovirus vectors, adeno-associated viral vectors, retroviral vectors, and the like.

[0080] The term "expression vector" means a vector containing a recombinant nucleic acid containing expression control sequences operably linked to an expressed nucleotide sequence. The expression vector contains sufficient cis-acting elements for expression; other elements for expression may be provided by the host cell or be in an in vitro expression system. Expression vectors include all expression vectors known in the art, such as cosmids, plasmids (eg, "naked" or enclosed in liposomes), and viruses that contain a recombinant nucleic acid.

[0081] As used herein, the term "host cell" means a cell that contains the vector and maintains the replication and/or expression of the desired expression vector. Host cells can be prokaryotic cells such as E. coli or eukaryotic cells such as yeast, insect, amphibian or mammalian cells, or monocot or dicot plant cells. An example of a monocot plant host cell is a rice host cell, and an example of a dicot plant host cell is an eggplant or tomato host cell. When possible, the sequence is modified to avoid predicted mRNA hairpin secondary structures.

[0082] As used herein, the term "toxin" means a polypeptide exhibiting pesticidal activity or insecticidal activity. The "Bt" or " Bacillus thuringiensis " toxin would include the broad class of Cry toxins found in the various strains of Bt containing such toxins.

[0083] The term "probe" or "sample probe" means a molecule that its complement or specific microchip element recognizes. Examples of probes that can be used in the present invention include, but are not limited to, DNA, RNA, oligonucleotides, oligosaccharides, polysaccharides, sugars, proteins, peptides, monoclonal antibodies, toxins, viral epitopes, hormones, hormone receptors, enzymes, enzyme substrates, cofactors, and drugs, including cell surface receptor agonists and antagonists.

[0084] As used herein, the term "complementary" or "complement" refers to the pairing of bases, purines and pyrimidines, which are linked by hydrogen bonds in a double-stranded nucleic acid. The following base pairs are complementary: guanine and cytosine; adenine and thymidine; as well as adenine and uracil. As used herein, the terms include complete and partial complementarity.

[0085] As used herein, the term "hybridization" refers to a process in which a nucleic acid strand is joined to a complementary strand by base pairing. The conditions used to hybridize two non-identical, but very similar, complementary nucleic acids vary depending on the degree of complementarity of the two strands and the length of the strands. Thus, the term covers partial as well as full hybridization. Such methods and conditions are well known to those of skill in the art.

[0086] The terms "insecticidal activity" and "pesticidal activity" are used interchangeably herein to refer to the activity of a microorganism or substance (such as, for example, a protein) that can be measured, for example, but not limited to, pest mortality, pest reduction, pest repellency, and other behavioral and physical changes in pests after ingestion and exposure for an appropriate period of time. Thus, a microorganism or substance with pesticidal activity adversely affects at least one measurable parameter of pest fitness. For example, "insecticidal proteins" are proteins that exhibit insecticidal activity alone or in combination with other proteins.

[0087] As used herein, the term "influencing insect pests" means insect deterrent changes in feeding, growth and/or behavior of insects at any stage of development, including, but not limited to, insect destruction, growth retardation, prevention of reproductive ability, anti-feeding activity, and the like.

[0088] As used herein, the term "pesticidal effective amount" means the amount of a substance or microorganism that has pesticidal activity when present in a pest habitat. For each substance or microorganism, the effective pesticidal amount is determined empirically for each pest affected in a particular environment. Similarly, the term "insecticidal effective amount" can be used instead of the term "pesticidal effective amount" when the pest is an insect pest.

[0089] As used herein, the terms "transformed plant" and "transgenic plant" mean a plant that contains one or more heterologous polynucleotides in its genome. The heterologous polynucleotide(s) is stably integrated into the genome of the transgenic or transformed plant such that the polynucleotide is passed on to subsequent generations. The heterologous polynucleotide may be integrated into the genome alone or as part of a recombinant DNA molecule.

[0090] As used herein, the term "transgenic" should be understood to include any plant cell, plant cell line, callus, tissue, plant part, or plant whose genotype has been altered by the presence of one or more heterologous nucleic acids. The term encompasses transgenic events originally obtained using a genetic transformation method known in the art, as well as those resulting from sexual interbreeding or asexual reproduction from the original transgenic event.

[0091] As used herein, the term "original transgenic entity" does not refer to a change in the genome (chromosomal or extrachromosomal) by conventional plant breeding methods or as a result of a natural event such as accidental cross-fertilization, non-recombinant viral infection, non-recombinant bacterial transformation, non-recombinant transposition, or spontaneous mutation.

[0092] As used herein, the term "plant" includes whole plants, plant cells, plant protoplasts, tissue cultures of plant cells from which plants can be regenerated, plant calli, plant uterine roots, and plant cells that are intact in plants or plant parts such as embryos, pollen, ovules, seeds, leaves, flowers, branches, fruits, kernels, ears, cobs, husks, stems, roots. , root tips, anthers and the like, as well as their descendants. Parts of transgenic plants are included within the scope of the embodiments and are, for example, plant cells, protoplasts, tissues, callus, embryos, as well as flowers, stems, fruits, leaves and roots of transgenic plants, or their descendants, previously transformed with the DNA molecule of the embodiments and thus consisting at least in part of the transgenic cells.

Gene cry Bacillus thuringiensis and codon optimization

[0093] Approximately 400 cry genes encoding δ-endotoxins have been sequenced to date (Crickmore, N. 2005. Using worms to better understand how Bacillus thuringiensis kills insects. Trends in Microbiology, 13(8): 347-350). Various δ-endotoxins have been classified into classes (Cry 1, 2, 3, 4, and so on) based on similarity in amino acid sequences. These classes consist of several subclasses (Cry1A, Cry1B, Cry1C, and so on), which are themselves subdivided into subfamilies or variants (Cry1Aa, Cry1Ab, Cry1Ac, and so on). The genes of each class are more than 45% identical to each other. The product of each individual cry gene generally has a limited spectrum of activity, which is limited to the larval stages of a small number of species. However, it was not possible to establish a correlation between the degree of identity of Cry proteins and their activity spectrum. Proteins Cry1Aa and Cry1Ac are 84% identical, but only Cry1Aa is toxic to Bombyx mori (L.). Conversely, Cry3Aa and Cry7Aa, which are only 33% identical, are both active against the Colorado potato beetle, Leptinotarsa decemlineata . Other Cry toxins are completely inactive against insects, but are active against other invertebrates. For example, proteins of the Cry5 and Cry6 classes are active against nematodes. More recently, binary toxins from Bt, designated Cry34Ab1/Cry35Ab1, have also been characterized and are active against various beetle pests of the family Chrysomelidae. They have been given the name Cry, although they share little homology with other members of the Cry toxin family.

[0094] In order to achieve desired levels of expression of heterologous proteins in transgenic plants, it has been found useful to alter the natural, sometimes referred to as "wild type" or original, genomic DNA coding sequence in various ways so that codon usage more closely matches codon usage in host plants, and likewise so that the G+C content of the coding sequence more closely matches the G+C content of the host plant species.

[0095] One of skill in the field of plant molecular biology understands that multiple DNA sequences can be designed to encode a single amino acid sequence. A common way to increase the expression of a coding region for a protein of interest is to modify the coding region so that its codon composition resembles the general codon composition of the host in which the gene is to be expressed.

[0096] The genomic/natural nucleic acid can be optimized for increased expression in the host organism. Thus, when the host organism is a plant, synthetic nucleic acids can be synthesized using plant-preferred codons to increase expression. For example, although the nucleotide sequences of the embodiments can be expressed in both monocots and dicots, the sequences can be altered to account for specific codon preferences and preferred GC content in monocots or dicots, as these preferences have been shown to differ (Murray et al. (1989) Nucleic Acids Res. 17:477-498). Thus, the preferred codon for a particular amino acid in rice can be determined from the known gene sequences of rice, and the preferred codon for a particular amino acid in eggplant can be determined from the known gene sequences of eggplant.

[0097] Additional sequence modifications are known to increase gene expression in the cellular host. These modifications include deletion of sequences encoding false polyadenylation signals, exon-intron splicing site signals, transposon-like repeats, and other well-studied sequences that may be unfavorable for gene expression. The content of GC in the sequence can be adjusted to the average levels in a particular cellular host, which is calculated based on the known genes expressed in the host cell.

[0098] In the Vaeck articleet al., ((Vaeck M, Reynaerts A, Höfte H, Jansens S, De Beukeleer M, Dean C, Zabeau M, Van Montagu M, Leemans J (1987) Transgenic plants protected from insect attack. Nature 327:33-37) describes the production of insect-resistant transgenic tobacco plants expressing the genecry1Ab Bt, to protect against the European grain grinder, one of the main pests attacking corn in the US and Europe. However, despite the use of strong promoters, toxin production in plants was initially too weak to be effectively used in agriculture (Koziel G M, Beland G L, Bowman C, Carozzi N B, Crenshaw R, Crossland L, Dawson J, Desai N, Hill M, Kadwell S, Launis K, Maddox D, McPherson K, Heghji M, Merlin E, Rhodes R, Warren G, Wright M, Evola S (1993) Field performance of elite transgenic maize plants expressing an insecticidal protein derived fromBacillus thuringiensis. Biotechnology 11:194-200). Unlike plant genes, Bt genes have a high A+T content (66%), which is a suboptimal codon usage for plants and potentially leads to missplicing or premature termination of transcription (De la Riva and Adang, 1996).

[0099] Perlak et al (Perlak FJ, Fuchs RL, Dean DA, McPherson SL, Fishhoff DA (1991) Modification of the coding sequences enhances plant expression of insect control protein genes, Proc. Natl. Acad. Sci. (USA) 88:3324-3328) modified the cry gene coding sequence without modifying the encoded peptide sequences to ensure optimal codon usage for plants, resulting in a doubling of toxin production in plants compared to the natural gene. This strategy has been successfully used in many plants such as cotton, rice and maize transformed with modified cry1 genes and potatoes transformed with modified cry3A genes. Bt corn and Bt cotton are grown on a large scale around the world.

[00100] Thus, naturally existing codon preference in different organisms leads to suboptimal gene expression in a heterologous organism. In the present invention, the native cry2Ai gene from Bacillus thuringiensis has been modified in-silico for optimal expression of the recombinant protein in plants, including dicots and monocots. When designing with multivariate analysis, rare and very rare codons were changed to the most preferred codons in dicots/monocots. The modified synthetic gene, constructed using a gene constructor, was manually checked for the use of rare codons, the stability of the secondary structure of the mRNA, the presence of the beginning of the secondary transcription of the gene to avoid the expression of truncated proteins. The structure and stability of the optimized mRNA was verified and validated using an mRNA optimization program.

[00101] A specific aspect of the invention relates to a codon-optimized synthetic nucleic acid encoding Cry2Ai insecticidal proteins, insecticidal compositions, polynucleotide constructs, a recombinant nucleotide sequence, a recombinant vector, transformed microorganisms, and plants containing the nucleic acid molecule of the invention. These compositions are used in methods for controlling insect pests, in particular insect pests of crops.

[00102] The codon-optimized synthetic nucleotide sequence disclosed herein can be fused to various promoters, including constitutive, inducible, transiently regulated, developmentally regulated, tissue-preferred, and tissue-specific promoters, to produce recombinant DNA molecules. The codon-optimized cry2Ai synthetic nucleotide sequences disclosed herein (coding sequences) provide much higher levels of expression in the transformed plant than the natural cry2Ai gene. Accordingly, plants resistant to Lepidoptera pests such as Helicoverpa armigera - cotton bollworm and corn cutworm, Cnaphalocrocis medinalis - rice leafworm, as well as Scirpophaga incertulas - rice stem borer and Pectinophora gossypiella can be obtained.

[00103] One embodiment of the present invention relates to synthetic codon-optimized cry2Ai nucleotide sequences with plant-preferred codons. Another embodiment of the present invention relates to the expression of synthetic codon-optimized cry2Ai nucleotide sequence(s) in plants such as rice, tomato and eggplant. Another embodiment of the present invention relates to DNA expression cassettes, plant transformation vectors containing synthetic cry2Ai nucleotide sequence(s) of the invention. Another embodiment of the present invention provides compositions comprising the cry2Ai synthetic codon-optimized nucleotide sequence(s) disclosed herein or an insecticidal polypeptide encoded by the cry2Ai synthetic codon-optimized nucleotide sequence(s) disclosed herein. The composition disclosed herein may be pesticidal and/or insecticidal compositions containing the pesticidal and/or insecticidal proteins/polypeptides of the invention. Another embodiment relates to transgenic plants containing the codon-optimized cry2Ai synthetic nucleotide sequence(s) of the invention and expressing the Cry2Ai protein toxin.

[00104] In particular, the present invention relates to a codon-optimized synthetic nucleotide sequence set forth in SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7, said nucleotide encoding the Cry2Ai insecticidal protein having the amino acid sequence shown in SEQ ID NO: 1.

[00105] In some embodiments, the invention also relates to plants and microorganisms transformed with the codon-optimized cry2Ai nucleotide sequence(s) disclosed herein, and methods including the use of such nucleotide sequence(s), pesticide compositions, transformed organisms, and their products in the control of insect pests.

[00106] The polynucleotide sequences of the embodiments can be used to transform any organism, such as plants and microorganisms such as Bacillus thuringiensis , to produce the encoded insecticidal and/or pesticidal proteins. Methods are provided that include the use of such transformed organisms to control or control plant pests. The nucleic acids and nucleotide sequences of the embodiments can also be used to transform organelles such as chloroplasts. A method of transforming a desired organism is well known in the art, allowing a person skilled in the art to perform transformation using the polynucleotide sequences disclosed herein.

[00107] Nucleic acids of embodiments include nucleic acid or nucleotide sequences that have been optimized for expression by cells of a particular organism, for example, nucleotide sequences that have been reverse-translated (i.e., peptide-reconstituted) using plant-preferred codons based on the amino acid sequence of the polypeptide having pesticidal activity.

[00108] The invention relates to codon-optimized synthetic nucleic acids/nucleotide sequences encoding Cry2ai insecticidal proteins. The synthetic coding sequences are specifically adapted for use in protein expression in dicots and monocots such as rice, tomato, eggplant, corn, cotton and legumes.

[00109] The invention relates to synthetic nucleotide sequences encoding the Cry2Ai protein that are specifically adapted for high expression in plants. The disclosed codon-optimized synthetic nucleotide sequences use plant-optimized codons at about the same frequency as they are used on average in genes naturally present in a given plant species. The invention also relates to a codon-optimized synthetic nucleotide sequence for conferring insect resistance to plants. The present invention uses selectable marker genes to transform plants. DNA constructs and transgenic plants containing the synthetic sequences disclosed herein are described, as well as methods and compositions for use with agriculturally important plants.

[00110] Each of the proteins encoded by the codon-optimized synthetic nucleotide sequences exhibits inhibitory biological activity against Lepidoptera insect species. Dicot and/or monocot plants can be transformed with each of the nucleotide sequences disclosed herein, alone or in combination with other nucleotide sequences encoding insecticidal agents, such as proteins, crystalline proteins, toxins, and/or pest-specific double-stranded RNAs, designed to suppress genes in the body of one or more target pests, and the like, to achieve insect resistance controls in the field that were not previously possible. by simply using known Lepidoptera insecticidal proteins derived from strains of Bacillus thuringiensis .

[00111] The codon-optimized synthetic nucleotide sequences of the present invention can also be used in plants in combination with other kinds of nucleotide sequences encoding insecticidal toxins to produce plants transformed to contain at least one agent for controlling one or more common plant pests selected from the group consisting of Lepidoptera insect pests, Coleoptera pests, plant piercing insects, and sucking insect pests, and the like.

Regulatory sequences

[00112] Transcriptional and translational regulatory signals include, but are not limited to, promoters, transcription start sites, operators, activators, enhancers, other regulatory elements, ribosome binding sites, start codon, termination signals, and the like.

[00113] The polynucleotide/DNA construct will include, in the 5' to 3' direction of transcription: a transcription and translation initiation region (i.e., a promoter), a DNA sequence of embodiments, and a transcription and translation termination region (i.e., a termination region) functional in the host organism. The transcriptional initiation region (ie, promoter) may be natural, analogous, foreign, or heterologous to the host organism and/or to the sequence of the embodiments. In addition, the promoter may be a natural sequence or, alternatively, a synthetic sequence. As used herein, the term "foreign" means that the promoter does not occur naturally in the organism into which the promoter is introduced. If a promoter is "foreign" or "heterologous" to the sequence of the embodiments, it is understood that the promoter is not a natural or native promoter to the operably linked sequence of the embodiments.

[00114] In the practice of the embodiments, a variety of promoters can be used. Promoters can be selected based on the desired result. The codon-optimized nucleotide sequence of the invention can be combined with a constitutive, tissue-preferred, inducible, or other promoter for expression in a host organism. Suitable constitutive promoters for use in a plant cell host include, for example, the CaMV 35S core promoter; rice actin; ubiquitin; ALS promoter and so on.

[00115] Depending on the desired result, it may be useful to express the gene from an inducible promoter. Damage-inducible promoters are of particular interest in regulating the expression of the nucleotide sequences of the embodiments in plants. Such damage inducible promoters can respond to damage caused by eating insects and include promoters of the potato protease inhibitor gene (pin II); wun1 and wun2, win1 and win2; WIP1; MPI and so on.

[00116] In addition, pathogen-inducible promoters can be used in the methods and nucleotide constructs of the embodiments. Such pathogen-inducible promoters include pathogenesis-associated protein (PR) promoters that are inducible after infection with a pathogen; for example, PR proteins, SAR proteins, β-1,3-glucanases, chitinases, and so on.

[00117] Chemically regulated promoters can be used to modulate gene expression in a plant through the use of an exogenous chemical regulator. Depending on the purpose, the promoter may be a chemically inducible promoter, where the application of the chemical induces gene expression, or a chemically repressible promoter, where the application of the chemical suppresses gene expression. Chemically inducible promoters are known in the art and include, but are not limited to, the maize In2-2 promoter activated by benzenesulfonamide herbicide safeners, the maize GST promoter activated by hydrophobic electrophiles used as pre-emergence herbicides, and the tobacco PR-1a promoter activated by salicylic acid. Other chemically regulated promoters of interest include steroid dependent promoters.

[00118] A promoter that is "preferred" expressed in a particular tissue provides a greater degree of expression in that tissue than in at least one other plant tissue. Some tissue-preferred promoters show expression almost exclusively in a particular tissue. Tissue-preferred promoters can be used to target increased expression of the pesticidal protein in a particular plant tissue. Such promoters can be modified, if necessary, for weak expression.

[00119] Examples of some of the tissue-specific promoters include, but are not limited to, leaf-preferred promoters, root-specific or root-preferred promoters, seed-specific or seed-preferred promoters, pollen-specific promoters, and parenchyma-specific promoters.

[00120] Root-preferred or root-specific promoters are known and may be selected from promoters described in the literature or isolated de novo from various compatible species.

[00121] "Seed-preferred" promoters include both "seed-specific" promoters (these promoters are active during seed development, e.g., seed storage protein promoters) and "germinating seed" promoters (these promoters are active during seed germination). Gamma-zein and Glob-1 promoters are endosperm-specific promoters. In the case of dicotyledonous plants, seed-specific promoters include, but are not limited to, promoters for β-phaseolin, β-conglycinin, soy lectin, cruciferin, and the like. In the case of monocots, seed-specific promoters include, but are not limited to, corn 15-kDa zein, 22-kDa zein, 27-kDa zein, g-zein, waxy, shrunken 1, shrunken 2, globulin 1, and so on.

[00122] Where a low level of expression is required, weak promoters can be used. Generally, the term "weak promoter" as used herein means a promoter that drives the expression of a coding sequence at a low level. Such weak constitutive promoters include, for example, the Rsyn7 core promoter, the CaMV 35S core promoter, and so on.

[00123] Termination regions can be derived from the A. tumefaciens Ti plasmid, such as octopine synthase (OCS) and nopaline synthase (NOS) termination regions.

[00124] DNA expression cassettes may also contain 5' leader sequences. Such leader sequences can help increase the level of translation. Translational leaders are known in the art and include: picornavirus leaders, such as the EMCV (5' non-coding region of encephalomyocarditis virus) leader; potyvirus leaders, eg TEV (tobacco etch virus) leader, MDMV (maize dwarf mosaic virus) leader, untranslated leader from alfalfa mosaic virus coat protein mRNA (AMV RNA 4); tobacco mosaic virus (TMV) leader; and maize chlorotic mottle virus (MCMV) leader.

[00125] In one specific embodiment of the invention disclosed and claimed herein, a tissue-preferred or tissue-specific promoter is operably linked to a synthetic DNA sequence of the invention encoding an insecticidal protein, and the transgenic plant is stably transformed with at least one such recombinant molecule. The resulting plant will be resistant to specific insects feeding on those parts of the plant in which the DNA molecule(s) is expressed.

Selectable marker gene

[00126] Typically, the expression cassette will contain a selectable marker gene for selecting transformed cells. Selectable marker genes are used to select transformed cells or tissues. Marker genes include genes conferring antibiotic resistance, such as genes encoding neomycin phosphotransferase II ( nptII ) and hygromycin phosphotransferase ( hpt II), as well as genes conferring resistance to herbicides such as ammonium glufosinate, bromoxynil, imidazolinones, and 2,4-dichlorophenoxyacetate (2,4-D). Additional examples of suitable selectable marker genes include, but are not limited to, chloramphenicol, methotrexate, streptomycin, spectinomycin, bleomycin, sulfonamide, bromoxynil, glyphosate, phosphinothricin resistance genes.

[00127] The above list of selectable marker genes should not be limiting. In embodiments, any selectable marker gene can be used.

DNA constructs and vectors

[00128] The codon-optimized DNA/nucleotide sequences of the invention are provided in DNA constructs for expression in an organism of interest. The construct will include 5' and 3' regulatory sequences operably linked to the sequence of the invention.

[00129] Such a polynucleotide construct is provided with multiple restriction sites for the insertion of DNA sequences encoding the Cry2Ai toxin protein sequence under transcriptional regulation of regulatory regions. The polynucleotide construct may also contain selectable marker genes. In addition, the construct may contain at least one additional gene for co-introduction by transformation into the desired organism. Alternatively, additional gene(s) may be provided on multiple polynucleotide constructs.

[00130] When creating a DNA/expression cassette construct, various DNA fragments can be manipulated so as to obtain DNA sequences in the correct orientation, as needed, in the correct reading frame. To this end, adapters or linkers can be used to connect DNA fragments, or other manipulations can be performed to create convenient restriction sites, remove excess DNA, remove restriction sites, or the like. For these purposes, you can use in vitro mutagenesis, restoration due to primers, restriction, annealing, repeated substitutions, for example, transpositions and transversions.

[00131] In accordance with the present invention, the DNA construct/expression cassette disclosed herein can be inserted into a recombinant expression vector. The expression "recombinant expression vector" means a bacterial plasmid, phage, yeast plasmid, plant cell virus, mammalian cell virus, or other vector. Generally, any plasmid or vector can be used, provided that it(s) can replicate and stabilize in the host. An important characteristic of an expression vector is that it has an origin of replication, a promoter, a marker gene, and a translation control element.

[00132] A variety of cloning vectors containing the replication system in E. coli and a marker allowing selection of transformed cells are available in preparation for the insertion of foreign genes into higher plants. Vectors include, for example, pBR322, pUC series, M13 mp series, pACYC184, among others. Accordingly, a DNA fragment having a sequence encoding a Bt toxin protein can be inserted into the vector at a suitable restriction site. The resulting plasmid is used to introduce transformation into E. coli. E. coli cells are cultured in an appropriate nutrient medium, then harvested and lysed. The plasmid is removed. As analysis methods, as a rule, sequence analysis, restriction analysis, electrophoresis and other biochemical and molecular biological methods are used. After each manipulation, the used DNA sequence can be cleaved and joined to the next DNA sequence. All plasmid sequences can be cloned into the same or different plasmids. Depending on how the desired genes are inserted into plants, other DNA sequences may be needed. When using, for example, a Ti or Ri plasmid to transform a plant cell, at least the right border, but often the right and left border, the Ti or Ri plasmid T-DNA should be added as a flanking region of the inserted genes.

[00133] An expression vector containing a codon-optimized nucleotide sequence of the invention and an appropriate signal to regulate transcription/translation can be constructed in a manner well known to those skilled in the art. Examples of such methods include an in vitro DNA recombination method, a DNA synthesis method, and an in vivo recombination method. The DNA sequence can be efficiently linked to an appropriate promoter in an expression vector to induce mRNA synthesis. In addition, the expression vector may contain, as a translation initiation site, a ribosome binding site and a transcription terminator.

[00134] A preferred example of a recombinant vector of the present invention is a Ti plasmid vector that can transfer a portion of itself, ie, a so-called T region, into 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 to transfer the hybrid gene into protoplasts capable of forming a new plant by appropriately inserting the plant cell DNA or hybrid DNA into the plant genome.

[00135] The expression vector may contain at least one selectable marker gene. A selectable marker gene is a nucleotide sequence having a capability on the basis of which it can be selected by a conventional chemical method. Each gene that can be used to distinguish between transformed cells and non-transformed cells can be a selectable marker gene. An example includes, but is not limited to, a herbicide resistance gene such as glyphosate and phosphinothricin and an antibiotic resistance gene such as kanamycin, hygromycin, G418, bleomycin, and chloramphenicol.

[00136] For the recombinant vector of the present invention, the promoter can be any of, but is not limited to, CaMV 35S, actin, or ubiquitin promoters. Since the transformant can be selected by various mechanisms at different stages, the constitutive promoter may be preferred in the present invention. Thus, the selection of a constitutive promoter is not limited herein.

[00137] Any conventional terminator may be used for the recombinant vector of the present invention. Examples include but are not limited to the nopaline synthase (NOS) terminator, the rice α-amylase RAmy1 A terminator, the phaseolin terminator and the Agrobacterium tumefaciens optopin gene terminator, and so on. With regard to the need for a terminator, it is well known that such a region can increase the reliability and efficiency of transcription in plant cells. Thus, the use of a terminator is highly preferred given the contexts of the present invention.

[00138] One of skill in the art is aware that the DNA construct and vector disclosed herein can be used to produce insect-resistant transgenic plants and/or produce an insecticidal composition that may contain Bacillus thuringiensis cells containing said nucleotide sequence, or any other microorganism capable of expressing the nucleotide sequence disclosed herein, to produce the Cry2Ai insecticidal protein.

recombinant cell

[00139] Embodiments also include a microorganism transformed with at least one codon-optimized nucleic acid of the invention, an expression cassette containing a nucleic acid, or a vector containing an expression cassette. In some embodiments, the microorganism is a microorganism that proliferates in plants. One embodiment of the invention relates to an encapsulated pesticidal protein that contains a transformed microorganism capable of expressing the Cry2Ai protein of the invention.

[00140] The following embodiment relates to a transformed organism, such as an organism selected from the group consisting of plant and insect cells, bacteria, yeasts, baculoviruses, protozoa, nematodes, and algae. The transformed organism contains a codon-optimized synthetic DNA molecule according to the invention, an expression cassette containing said DNA molecule, or a vector containing said expression cassette capable of stably integrating into the genome of the transformed organism.

[00141] It is recognized that the genes encoding the Cry2Ai protein can be used to transform insect pathogens. Such organisms include baculoviruses, fungi, protozoa, bacteria and nematodes.

[00142] The codon-optimized synthetic nucleotide sequence(s) encoding the Cry2Ai protein of the embodiments can be introduced via an appropriate vector into a microbial host, and said host introduced into the environment, applied to plants or animals. The term "introduction" in the context of inserting a nucleic acid into a cell means "transfection" or "transformation" or "transduction", and includes the introduction of the nucleic acid into a eukaryotic or prokaryotic cell, where the nucleic acid can be integrated into the cell's genome (e.g., chromosome, plasmid, plastid, or mitochondrial DNA), become an autonomous replicon, or be transiently expressed (e.g., transfected mRNA).

[00143] There are a number of methods for introducing foreign DNA expressing a pesticidal protein into a host microorganism under conditions that allow for stable preservation and expression of the DNA. For example, expression cassettes can be constructed that include nucleotide constructs of interest operably linked to transcriptional and translational regulatory signals for expression of the nucleotide constructs, and a nucleotide sequence that is homologous to sequences in the host organism such that integration will occur and/or a replication system that is functional in the host organism such that integration or stable maintenance will occur.

Methods for transforming plants and obtaining transgenic plants

[00144] The codon-optimized synthetic nucleotide sequence (DNA sequence) of the present invention encoding the Cry2Ai Bt toxin protein can be introduced into plant cells using various methods well known in the art. Once the introduced DNA is integrated into the plant genome, it is relatively stable. The transformation vector typically contains a selectable marker that renders the transformed plant cells resistant to a biocide or antibiotic such as kanamycin, bialaphos, G418, bleomycin, or hygromycin. A separately used marker should therefore allow the selection of transformed cells rather than cells lacking the inserted DNA.

[00145] There are a large number of methods for inserting DNA into a plant host cell. These methods include T-DNA transformation using Agrobacterium tumefaciens or Agrobacterium rhizogenes as the transformation agent, fusion, injection, biolistic methods (microparticle bombardment) or electroporation, among other possible methods. If used for the transformation of agrobacteria, the inserted DNA must be cloned into special plasmids, namely, either into an intermediate vector or into a binary vector. Intermediate vectors can be inserted into the Ti or Ri plasmid by a homologous recombination mechanism at the expense of sequences homologous to sequences in the T-DNA. The Ti or Ri plasmid also contains the vir region required for T-DNA transfer.

[00146] Intermediate vectors are unable to replicate in agrobacteria. The intermediate vector can be transferred into Agrobacterium tumefaciens using a helper plasmid (conjugation). Binary vectors are able to replicate both in E. coli and agrobacteria. They contain a selectable marker gene and a linker or polylinker that is delimited by the right and left T-DNA border regions. They can be introduced by transformation directly into agrobacteria. Agrobacterium used as a host cell must contain a plasmid carrying a virulence region ( vir ). The vir region is essential for the transfer of T-DNA into the plant cell. Additional T-DNA may also be contained. The bacterium thus transformed is used to transform plant cells. Plant explants can be cultured with Agrobacterium tumefaciens or Agrobacterium rhizogenes to transfer DNA into the plant cell. Whole plants can then be regenerated from infected plant material (eg, leaf fragments, stem segments, roots, as well as protoplasts or cells cultured in suspension) in an appropriate medium, which may contain antibiotics or selection biocides. The plants thus obtained can then be tested for the presence of the introduced DNA. In the case of injection and electroporation, no special requirements are imposed on plasmids. You can use conventional plasmids, such as, for example, derivatives of pUC.

[00147] Cells that have been transformed can be grown into plants by conventional methods. These plants can then be grown and pollinated with the same transformed strain, or with other strains, and the resulting hybrid will have constitutive or inducible expression of the particular desired phenotypic characteristic. One can grow two or more generations to ensure that the expression of the desired phenotypic trait is stably maintained and inherited, and then collect seeds to ensure that the expression of the desired phenotypic trait is achieved. They can form germ cells and transmit the trait(s) resulting from transformation to descendant plants. Such plants can be grown in the usual way and crossed with plants having the same hereditary factors or other hereditary factors resulting from transformation. The resulting hybrid plants have the appropriate phenotypic properties.

[00148] The methods of plant transformation of the present invention include the introduction of the polynucleotide(s) of the invention into the plant and are independent of the specific method of introducing the polynucleotide(s) into the 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.

[00149] In one embodiment of the present invention, plants have been transformed with the codon-optimized synthetic nucleotide sequence(s) disclosed herein. Some non-limiting examples of transformed plants are fertile transgenic rice and tomato plants containing the codon-optimized synthetic nucleotide sequence(s) of the invention encoding the Cry2Ai protein. Methods for transforming rice and tomato are known in the art. Various other plants can also be transformed using the codon-optimized synthetic nucleotide sequence(s) disclosed herein.

[00150] The term "stable transformation" should mean that the nucleotide construct introduced into the plant is integrated into the plant's genome and can be inherited by its descendants. The term "transient transformation" should mean that the polynucleotide is introduced into the plant and does not integrate into the plant's genome, or the polypeptide is introduced into the plant.

[00151] Transformation protocols, as well as protocols for introducing nucleotide sequences into plants, may vary depending on the type of plant, or plant cell, ie, monocot or dicot, intended for transformation. Appropriate methods for introducing nucleotide sequences into plant cells, followed by integration into the plant genome, include microinjection, electroporation, Agrobacterium-mediated transformation, and ballistic particle acceleration.

[00152] In one embodiment, the codon-optimized synthetic nucleotide sequence(s) of the invention encoding the Cry2Ai toxin is expressed in a higher organism, eg, a plant, by virtue of the codon-optimized nucleotide sequence of the invention. In this case, transgenic plants expressing effective amounts of the toxin protect themselves from insect pests. When an insect begins to feed on such a transgenic plant, it also consumes the expressed toxin. This will keep the insect from further eating 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 into the plant genome.

[00153] Embodiments also include 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 directs expression in the plant cell.

[00154] While the embodiments are independent of a particular biological mechanism for increasing plant pest resistance, expression of the nucleotide sequences of the embodiments in the plant may result in the production of the pesticidal proteins of the embodiments and increased plant pest resistance. The plants of the embodiments can be used in agriculture to control insect pests. Particular embodiments relate to transformed crops, such as, for example, rice and tomato plants, used in methods for controlling plant pests, such as, for example, various lepidopteran insects, including Cnaphalocrocis medinalis - rice leafworm and Scirpophaga incertulas - rice stem borer.

[00155] An "experimental plant or plant cell" is an entity that has undergone a genetic change, such as a transformation with a gene of interest, or is a plant or plant cell that is derived from a plant or cell that has been altered in this manner and has such a change. The "control" or "control plant" or "control plant cell" provides a starting point for determining changes in the phenotype of the experimental plant or plant cell. A control plant or plant cell may include, for example: (a) a wild-type plant or cell, that is, with the same genotype as the source material for the genetic change that results in the experimental plant or cell; (b) a plant or plant cell with the same genotype as the parent material but which has been transformed with a "null" construct (i.e., a construct that has no known effect on the trait of interest, such as a construct containing a marker gene); (c) a plant or plant cell which is a non-transformed segregant among the progeny of the experimental plant or plant cell; (d) a plant or plant cell that is genetically identical to the experimental plant or plant cell, but which has not been subjected to conditions or stimuli that would induce expression of the gene of interest; or (e) the experimental plant itself, or the plant cell, under conditions in which the gene of interest is not expressed.

[00156] The transfer (or introgression) of cry2Ai trait-determining nucleotides into inbred plants such as cotton, rice, eggplant (brinjal), tomato, and legumes can be achieved by recurrent breeding, for example, by backcrossing. In this case, the desired recurrent parent is first bred to an inbred donor (non-recurrent parent) carrying the nucleotide sequence disclosed herein for cry2Ai detectable traits. The offspring from this cross are then backcrossed to the recurrent parent, followed by selection of the resulting offspring for the presence of the desired trait(s) passed on from the non-recurrent parent. After three, preferably four, more preferably five or more generations from backcrossing with the recurrent parent, selecting for the desired trait(s), the offspring will be heterozygous for the loci that control the carried trait(s), but will be similar to the recurrent parent for most or almost all other genes.

[00157] Embodiments also refer to the propagating material of the transformed plant of the embodiments, including, but not limited to, seeds, tubers, corms, bulbs, leaves, and root and shoot cuttings.

[00158] 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 methods, including, but not limited to, monocots and dicots. Examples of plants of interest include, but are not limited to, cereals, cereals, vegetables, oilseeds, fruit, ornamentals, lawns, and other plants. Например, рис ( Oryza sativa , Oryza spp.), кукурузу ( Zea mays ), рожь ( Secale cereale ), сорго ( Sorghum bicolor, Sorghum vulgare ), просо (например, просо американское ( Pennisetum glaucum ), просо обыкновенное ( Panicum miliaceum ), просо итальянское ( Setaria italica ), просо пальчатое ( Eleusine coracana )), пшеницу ( Triticum aestivum, Triticum sp.), овес ( Avena sativa ), ячмень ( Hordeum vulgare L), сахарный тростник ( Saccharum spp .), хлопчатник ( Gossypium hirsutum, Gossypium barbadense , Gossypium sp .), томаты ( Lycopersicon esculentum ), бринджал/баклажан ( Solanum melongena ), картофель ( Solanum tuberosum ), сахарную свеклу ( Beta vulgaris ), сладкий картофель ( Ipomoea batatus ), маниоку ( Manihot esculenta ), салат-латук ( Lactuca sativa ), капусту ( Brassica oleracea var. capitata ), цветную капусту ( Brassica oleracea var. botrytis ), брокколи ( Brassica oleracea var. indica ), виды Brassica (например, B. napus, B. rapa, B. juncea ), в частности, те виды Brassica, которые полезны в качестве источника масла семян, сою ( Glycine max ), подсолнечник ( Helianthus annuus ), сафлор ( Carthamus tinctorius ), арахис (Arachis hypogaea), голубиный горох (Cajanus cajan), нут ( Cicer arietinum ), зеленую фасоль ( Phaseolus vulgaris ), лимскую фасоль ( Phaseolus limensis ), горох ( Pisum sativum ), чину ( Lathyrus spp .), огурец ( Cucumis sativus ), дыню ( Cucumis cantalupensis ) и мускусную дыню ( Cucumis melo ), люцерну ( Medicago sativa ), табак ( Nicotiana tabacum ) и арабидопсис ( Arabidopsis thaliana ).

[00159] Plants of interest include cereal plants producing seeds of interest, oil plants, and leguminous plants. The seeds of interest include seeds of cereals such as corn, wheat, barley, rice, sorghum, rye, millet and so on. Oil plants include cotton, soybean, safflower, sunflower, cabbage, corn, alfalfa, palm, coconut, flax, castor bean, olive and so on. Leguminous plants include beans and peas. Beans include guar, carob, fenugreek, soybeans, horticultural beans, cowpeas, mung beans, lima beans, green beans, lentils, chickpeas, and so on.

[00160] In specific embodiments, at least one codon-optimized synthetic nucleotide sequence(s) of the invention can be packaged with any combination of polynucleotide sequences of interest to generate plants with the desired phenotype. For example, the codon-optimized synthetic nucleotide sequence can be packaged with any other polynucleotide encoding a polypeptide having pesticidal and/or insecticidal activity, such as other toxic Bt proteins, and the like. The resulting combinations may also include multiple copies of any of the polynucleotides of interest. Nucleotide sequences of embodiments can also be packaged with any other gene or combination of genes to produce plants with various combinations of traits desired, including, but not limited to, traits desirable for animal feed, such as genes for high oil content, balanced amino acid content, resistance to abiotic stress, and so on.

[00161] The nucleotide sequences of the invention can also be packaged with traits desirable for disease or herbicide resistance, genes associated with avirulence and disease resistance, acetolactate synthase (ALS) inhibitors, glutamine synthetase inhibitors such as phosphinothricin or basta (e.g., the bar genome), glyphosate resistance, traits desirable for processing, or products processing such as high oil content, modified oils, modified starch (e.g., ADPG pyrophosphorylase (AGPase), starch synthases (SS), starch branching enzymes (SBE) and starch branching enzymes (SDBE)). It is also possible to combine polynucleotides of the embodiments with polynucleotides providing agronomic traits such as male sterility, stem strength, flowering time, or traits useful for transformation technology such as cell cycle regulation or gene targeting.

[00162] These stacked combinations can be created by any method, including, but not limited to, crossing plants by conventional methods, genetic transformation, or any other method known in the art. If the traits are packaged by plant genetic transformation, the polynucleotide sequences of interest can be combined at any time and in any order. For example, a transgenic plant having one or more desired traits can be used as a target for introducing additional traits through subsequent transformation. Features can be entered simultaneously in a co-transformation protocol with polynucleotides of interest provided by any combination of transforming cassettes. For example, if two sequences are introduced, the two sequences may be contained in separate transforming cassettes (trans) or contained in the same transforming cassette (cis). Expression of the sequences may be driven by the same promoter or by different promoters. In some cases, it may be desirable to introduce a transforming cassette that will repress expression of the polynucleotide of interest. It can be combined with any combination of other suppression or overexpression cassettes to produce the desired combination of traits in the plant. In addition, it is recognized that polynucleotide sequences can be packaged at any desired location in the genome using a site-specific recombination system.

Analysis of transgenic plants

[00163] Polymerase chain reaction (PCR)

[00164] The present invention relates to a method for PCR amplification of a fragment of the codon-optimized nucleotide sequence shown in SEQ ID NO: 2, disclosed herein and encoding a Cry2Ai protein having the amino acid sequence shown in SEQ ID NO: 1, including DNA amplification by PCR in the presence of a set of primers shown in SEQ ID NO: 8-9. Similarly, a fragment of the codon-optimized nucleotide sequence shown in SEQ ID NO: 3-8, disclosed herein, can be amplified using primers specific for the specified nucleotide sequence. A person skilled in the art can design a set of primers to amplify said nucleotides. Protocols and conditions for PCR amplification of a DNA fragment from a DNA template are described elsewhere in this document or are known in the art.

[00165] Oligonucleotide primers can be designed for use in PCR reactions to amplify appropriate DNA sequences from the codon-optimized DNA sequences of the invention. Methods for constructing PCR primers and PCR cloning are generally known in the art and are described in Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2nd ed., Cold Spring Harbor Laboratory Press, Plainview, NY), hereinafter "Sambrook". Known PCR methods 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.

[00166] In one embodiment, the present invention relates to a set of primers suitable for PCR amplification of a fragment of the codon-optimized synthetic nucleotide sequence(s) of the present invention encoding a pesticidal polypeptide, and methods of using the primer set in PCR amplification of DNA. Primer sets include forward and reverse primers that have been designed to anneal with the nucleotide sequences of the present invention.

[00167] The primer sets for amplifying the nucleotide sequence shown in SEQ ID NO: 2 of the invention include the forward and reverse primers shown in SEQ ID NO: 8 and SEQ ID NO: 9.

[00168] Southern hybridization

[00169] In hybridization methods, all or part of a known nucleotide sequence is used as a probe that selectively hybridizes to other relevant nucleotide sequences present in a population of cloned genomic DNA fragments or from a selected organism. Hybridization probes can be PCR-amplified DNA fragments of the codon-optimized DNA sequence(s) of the present invention, or a linearized plasmid containing the nucleotide sequence(s) or other oligonucleotides that are capable of hybridizing to the corresponding sequences of the synthetic polynucleotide disclosed herein and can be labeled with a detectable group such as ³² P or any other detectable marker. Thus, for example, hybridization probes can be made by labeling synthetic oligonucleotides based on the sequences of the embodiments. Methods for making probes for hybridization are generally known in the art and are described in Sambrook.

[00170] For example, the entire sequence disclosed herein, or one or more fragments thereof, can be used as a probe capable of specifically hybridizing to appropriate sequences. To achieve specific hybridization under various conditions, such probes include sequences that are unique to those of the embodiments and are typically at least about 10 or 20 nucleotides in length. Such probes can be used to amplify the corresponding cry2Ai nucleotide sequence(s) from the nucleotide sequences by PCR.

[00171] Hybridization of such sequences can be carried out under stringent conditions. As used herein, "stringent conditions" or "stringent hybridization conditions" means conditions under which a probe will hybridize to its target sequence to a significantly greater extent than to other sequences (e.g., at least 2-fold, 5-fold, or 10-fold background). Strict conditions depend on the sequences and will be different under different circumstances. By controlling the stringency of the hybridization and/or washing conditions, it is possible to detect target sequences that are 100% complementary to the probe (homologous probing). Alternatively, the stringency of the conditions can be adjusted to allow for some sequence mismatch, whereby a lower degree of similarity is found (heterologous probing).

[00172] Generally, stringent conditions are those where the salt concentration is less than about 1.5 M Na ions, typically about 0.01-1.0 M Na ions (or other salts) at pH 7.0-8.3, and the temperature is at least about 30°C for short probes (e.g., 10-50 nucleotides) and at least about 60°C for long probes (e.g., over 50 nucleotides). Stringent conditions can also be achieved by adding destabilizers such as formamide. Exemplary low stringency conditions include hybridization with a buffer solution containing 30% - 35% formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulfate), at 37°C and washing in 1x - 2x SSC (20x SSC = 3.0 M NaCl/0.3 M trisodium citrate) at 50°C - 55°C. Illustrative conditions of moderate stringency include hybridization in buffer containing 40% - 45% formamide, 1.0 M NaCl, 1% SDS, at 37°C and washing in 0.5x - 1x SSC at 55-60°C. Exemplary high stringency conditions include hybridization in buffer containing 50% formamide, 1 M NaCl, 1% SDS at 37°C and a final wash in 0.1x SSC at 60°C - 65°C for at least about 20 minutes. Optionally, wash buffers may contain from about 0.1% to about 1% SDS. The duration of hybridization is typically less than about 24 hours, typically from about 4 to about 12 hours.

[00173] Specificity generally depends on washes after hybridization, with ionic strength and temperature of the last wash solution being critical factors. In the case of DNA-DNA hybrids, Tm (melting point) can be approximated from the Meinkoth and Wahl (1984) Anal equation. Biochem. 138:267-284: Tm=81.5°C +16.6 (log M)+0.41 (% GC)-0.61 (% form)-500/L; where M is the molarity of monovalent cations, % GC is the percentage of guanosine and cytosine nucleotides in DNA, "% form" is the percentage of formamide in the hybridization solution, and L is the length of the hybrid in base pairs. ^Tm is the temperature (at a given ionic strength and pH) at which 50% of a complementary target sequence hybridizes to a perfectly matched probe. Washes are typically performed until at least equilibrium is reached and a low background level of hybridization is reached, for example, for 2 hours, 1 hour, or 30 minutes. T ^m is reduced by about 1°C for each 1% mismatch; thus, T ^m hybridization and/or washing conditions can be adjusted for hybridization with sequences having the desired identity. For example, if sequences with 90% identity are needed, T ^m can be reduced by 10°C. Typically, stringent conditions are chosen such that the temperature is about 5° C. lower than the T ^m for a particular sequence and its complement at a particular ionic strength and pH. However, for very stringent conditions, hybridization and/or washing at 1, 2, 3, or 4° C. lower than T ^m can be used; for moderately stringent conditions, hybridization and/or washing at 6°C, 7°C, 8°C, 9°C or 10°C lower than T ^m can be used; for non-stringent conditions, hybridization and/or washing at 11°C, 12°C, 13°C, 14°C, 15°C or 20°C lower than T ^m can be used.

[00174] Using the equation, the hybridization and wash compositions, and the desired T ^m , those skilled in the art will appreciate that variations in hybridization conditions and/or wash solutions are inherently described. If the desired degree of mismatch results in a T ^m of less than 45° C. (aqueous solution) or 32° C. (formamide solution), the SSC concentration may be increased so that a higher temperature can be used. An extensive guide to nucleic acid hybridization can be found in Tijssen (1993) Laboratory Techniques in Biochemistry and Molecular Biology--Hybridization with Nucleic Acid Probes, Part I, Chapter 2 (Elsevier, New York); and Ausubel et al., eds. (1995) Current Protocols in Molecular Biology, Chapter 2 (Greene Publishing and Wiley-Interscience, New York). See also Sambrook et al .

[00175] The present invention encompasses a nucleotide sequence that is complementary to the nucleotide sequence shown in SEQ ID NO: 2, or a nucleotide sequence that specifically hybridizes to at least 10 nucleotides of the nucleotide sequence shown in SEQ ID NO: 2 at nucleotide positions 251 to 402 and/or 1456 to 1628, or its complementary sequence. Those skilled in the art are capable of making probes based on the nucleotide sequences disclosed herein for nucleic acid hybridization.

Bioanalysis

[00176] A variety of bioassay methods are known to those skilled in the art. Common methods include adding the experimental compound or organism to a food source in a closed container. Pesticidal activity can be assessed based on, but not limited to, changes in mortality, weight loss, attraction, repulsion, and other behavioral and physical changes following ingestion and exposure over an appropriate period of time. The bioassays described herein can be used with any larval or adult feeding insect pest.

insecticidal composition

[00177] Biological pesticide is one of the most promising alternatives to conventional chemical pesticides, it causes less or no harm to the environment and biota. Bacillus thuringiensis (known as Bt) is an insecticidal Gram-positive spore-forming bacterium that produces crystalline proteins called delta-endotoxins (δ-endotoxins) during its stationary phase or retardation of its growth. Bt was originally discovered in a diseased silkworm ( Bombyx mori ) by the scientist Shigetane Ishiwatari in 1902. But it was formally characterized by Ernst Berliner while studying diseased flour moth caterpillars ( Ephestia kuhniella ) in 1915. The first record of its use for insect control was made in Hungary at the end of 1920, and in Yugoslavia in the early 1930s it was used to combat the European corn grain grinder. Bt, the leading biorational pesticide, was initially characterized as an insect pathogen, and its insecticidal activity was attributed predominantly, or entirely, to parasporal crystals. It is active against more than 150 types of insect pests. The toxicity of Bt culture lies in its ability to produce a crystalline protein, this observation has led to the development of Bt-based bioinsecticides to control certain insect species from the orders Lepidoptera, Diptera and Coleoptera.

[00178] The compositions of the embodiments find use in protecting plants, seeds, and plant products in a variety of ways. For example, the compositions may be used in a method comprising placing an effective amount of the pesticidal composition into the habitat of the pest by a method selected from the group consisting of spraying, dusting, spreading, or seed coating.

[00179] Before a plant propagation material (fruit, tuber, bulb, rhizome, grains, seeds), but especially seeds, is sold as a commercial product, it is usually treated with a protective coating including herbicides, insecticides, fungicides, bactericides, nematicides, molluscicides, or mixtures of some of these drugs, if desired, together with other carriers, surfactants or application aids commonly used in formulation areas to provide protection against damage caused by bacterial, fungal or animal pests. For seed treatment, a protective coating may be applied to seeds either by impregnating tubers or grains with a liquid preparation or by coating them with a combined wet or dry preparation. In addition, in special cases, other methods of application to plants can be used, for example, treatment aimed at buds or fruits.

[00180] The seed of the plant of the embodiments, containing the nucleotide sequence encoding the pesticidal protein of the embodiments, can be treated with a protective coating comprising a seed treatment compound such as, for example, captan, carboxin, thiram, metalaxyl, pirimiphos-methyl, and others commonly used for seed treatment. In one embodiment, a seed protection coating comprising the pesticidal composition of the embodiments is used alone or in combination with one of the seed protection coatings commonly used in seed treatment. The compositions of the embodiments may be in a form suitable for direct use, or as a base composition concentrate that must be diluted with an appropriate amount of water or other diluent prior to use. The concentration of the pesticide will vary depending on the nature of the particular formulation, in particular whether it is a concentrate or can be used directly. The composition contains 1-98% solid or liquid inert carrier, and 0-50% or 0.1-50% surfactant. These compositions will be applied at the stated commercial product rate, eg, about 0.01-5.0 pounds per acre dry and about 0.01-10 pints per acre liquid.

[00181] The codon-optimized synthetic nucleotide sequence disclosed herein can also be used to produce a transformed Bacillus thuringiensis capable of producing the Cry2Ai protein. The transformed Bacillus thuringiensis can then be used to make an insecticidal and/or pesticide composition useful for agricultural activities such as pest control.

[00182] В вариантах осуществления трансформированный микроорганизм (который включает целые организмы, клетки, споры), такой как Bacillus thuringiensis , трансформированный кодон-оптимизированными синтетическими нуклеотидными последовательностями, раскрытыми в настоящем документе, пестицидным белком(ами), пестицидным компонентом(ами), поражающим вредителей компонентом(ами), мутантом(ами), живыми или мертвыми клетками и клеточными компонентами, включая смеси живых и мертвых клеток и клеточных компонентов, и включая разрушенные клетки и клеточные компоненты, или выделенный пестицидный белок, может быть сформулирован с приемлемым носителем в пестицидную композицию(и), то есть, например, суспензию, раствор, эмульсию, порошок, диспергируемую гранулу или драже, смачиваемый порошок и эмульгируемый концентрат, аэрозоль или спрей, пропитанную гранулу, адъювант, пасту для нанесения покрытия, коллоид, а также инкупсулирован, например, в полимерные вещества. Such formulated compositions can be obtained by conventional methods such as dehydration, lyophilization, homogenization, extraction, filtration, centrifugation, sedimentation or concentration of cell culture containing the polypeptide.

[00183] Such compositions as described above can be prepared by adding a surfactant, an inert carrier, a preservative, a wetting agent, a nutritional stimulant, an attractant, an encapsulating agent, a binding agent, an emulsifier, a coloring agent, a UV protectant, a buffer, a flow agent, or fertilizers, micronutrient donors, or other drugs that affect plant growth. One or more agrochemicals, including, but not limited to, herbicides, insecticides, fungicides, bactericides, nematicides, molluscicides, acaricides, plant growth regulators, yield enhancers, and fertilizers, may be combined with carriers, surfactants, or adjuvants commonly used in the formulation art, or other components to facilitate product handling and application to specific target pests. Suitable carriers and adjuvants may be solid or liquid, and are substances commonly used in formulation technology, such as natural or regenerated minerals, solvents, dispersants, wetting agents, tackifiers, binders, or fertilizers. The active ingredients of the embodiments are usually used in the form of compositions and they can be applied to the area occupied by agricultural plants, plants or seeds to be treated. For example, compositions of embodiments can be applied to grain in preparation for storage or during storage in a granary or silo, and so on. The compositions of the embodiments can be used simultaneously or sequentially with other compounds. Methods for using the active ingredient of the embodiments or the agrochemical composition of the embodiments that contains at least one of the pesticidal proteins produced by the bacterial strains of the embodiments include, but are not limited to, foliar application, seed coating, and soil application. The number of applications and application rate depend on the intensity of the infestation of the respective pest.

[00184] In a further embodiment, the compositions, as well as the transformed microorganisms and the pesticidal protein of the embodiments, may be treated prior to formulation to prolong pesticidal activity when applied to the habitat of the target pest, provided that the pretreatment does not adversely affect the pesticidal activity. Such treatment can be carried out by chemical and/or physical means, provided that it does not adversely affect the properties of the composition(s). Examples of chemicals include, but are not limited to, halogenating agents; aldehydes such as formaldehyde and glutaraldehyde; anti-infective drugs such as zefiran chloride; alcohols such as isopropanol and ethanol.

[00185] In other embodiments, it may be useful to treat the Cry toxin polypeptides with a protease, such as trypsin, to activate the protein prior to introducing the pesticidal protein composition of the embodiments into the habitat of the target pest. Methods for activating a protoxin by a serine protease are well known in the art.

[00186] The compositions (including the transformed micro-organisms and the pesticidal protein of the embodiments) can be applied to the habitat of the insect pest by, for example, spraying, spraying, pollinating, spreading, coating or pouring, applying to, or onto, soil, applying to irrigation water, seed treatment, or general application or spraying at the time the pests begin to appear or before the pests appear as a protective measure. For example, the pesticidal protein and/or the transformed microorganisms of the embodiments can be mixed with the grain to protect the grain during storage. As a rule, it is important to provide good protection against pests in the early stages of plant growth, since this is the time when the plant can be most severely damaged. The compositions of the embodiments may contain another insecticide if deemed necessary. In one embodiment, the composition is applied directly to the soil at the time of planting in a granular form of a composition of the carrier and dead cells of the Bacillus strain or transformed microorganism according to the embodiments. Another embodiment is a granular form of a composition containing an agrochemical such as, for example, a herbicide, an insecticide, a fertilizer, an inert carrier, and dead cells of the Bacillus strain or transformed microorganism of the embodiments.

[00187] Those skilled in the art understand that not all compounds are equally effective against all pests. The embodiment compounds exhibit activity against insect pests, which may include pests of economically important agricultural, forestry, greenhouse, nursery, ornamental, food and fiber, human and animal health, domestic and commercial, household, stored products. Insect pests include insects from the order Lepidoptera. Insect larvae of the order "Lepidoptera" include, but are not limited to, "marching worms", caterpillars of winter cutworms, moths, and cutworm larvae in the family Noctuidae Spodoptera frugiperda JE Smith (grass cutworm); S. exigua Hubner (small cutworm); S. litura Fabricius (Asian cotton bollworm, cluster caterpillars); Mamestra configurata Walker (large walking worm); M. brassicae Linnaeus (Cabbage cutworm); Agrotis ipsilon Hufnagel (upsilon cutworm); A. orthogonia Morrison (rectangular cutworm); A. subterranea Fabricius (cotton bollworm); Alabama argillacea Hubner (American cotton bollworm); Trichoplusia ni Hubner (Cabbage cutworm); Pseudoplusia includens Walker (soy worm); Anticarsia gemmatalis Hubner (velvet bean scoop); Hypena scabra Fabricius (clover cutworm); Heliothis virescens Fabricius (tobacco leaflet); Pseudaletia unipuncta Haworth (meadow cutworm); Athetis mindara Barnes and Mcdunnough (rough cutworm); Euxoa messoria Harris (black-sided cutworm); Earias insulana Boisduval (Egyptian cotton bollworm); E. vittella Fabricius (spotted cutworm); Helicoverpa armigera Hubner (Bristle-legged mop cutworm); H. zea Boddie (corn cutworm or cotton cutworm); Melanchra picta Harris (scoopworm); Egira (Xylomyges) curialis Grote (citrus cutworm); grinders, case moths, spider worms, cap moths and parsleys in the family Pyralidae Ostrinia nubilalis Hubner (corn stem moth); Amyelois transitella Walker (moth); Anagasta kuehniella Zeller (mill moth); Cadra cautella Walker (dried fruit moth); Chilo suppressalis Walker (rice stem grinder); C. partellus (swarf armyworm); Corcyra cephalonica Stainton (rice moth); Crambus caliginosellus Clemens (moth); C. teterrellus Zincken (moth); Cnaphalocrocis medinalis Guenee (rice leaflet); Desmia funeralis Hubner (vine leaflet); Diaphania hyalinata Linnaeus (melon moth); D. nitidalis Stoll (moth); Diatraea grandiosella Dyar (southwestern corn moth), D. saccharalis Fabricius (cane grinder); Eoreuma loftini Dyar (rice moth); Ephestia elutella Hubner (tobacco moth (chocolate)); Galleria mellonella Linnaeus (large wax moth); Herpetogramma licarsisalis Walker (meadow moth); Homoeosoma electellum Hulst (sunflower moth); Elasmopalpus lignosellus Zeller (grain grinder); Achroia grisella Fabricius (small wax moth); Loxostege sticticalis Linnaeus (meadow moth); Orthaga thyrisalis Walker (tea tree moth); Maruca testulalis Geyer (Acacia moth); Plodia interpunctella Hubner (Indian flour moth); Scirpophaga incertulas Walker (yellow stem grinder); Udea rubigalis Guenee (rusty brown moth); as well as leafworms, budworms, seed worms and fruitworms in the family Tortricidae Acleris gloverana Walsingham (western black-headed leafworm); A. variana Fernald (eastern black-headed leafworm); Archips argyrospila Walker (fruit tree leaf roller); A. rosana Linnaeus (cut leafroller) and other species of Archips, Adoxophyes orana Fischer von Rosslerstamm (netted leafroller); Cochylis hospes Walsingham (sunflower broom); Cydia latiferreana Walsingham (codling moth); C. pomonella Linnaeus (apple codling moth); Platynota flavedana Clemens (variegated golden leaflet); P. stultana Walsingham (omnivorous leafroller); Lobesia botrana Denis & Schiffermuller (cluster leaf roller); Spilonota ocellana Denis & Schiffermuller (bud leaf roller); Endopiza viteana Clemens (vine leaflet); Eupoecilia ambiguella Hubner (bunch leaf roller); Bonagota salubricola Meyrick (apple leaflet); Grapholita molesta Busck (eastern peach leafroller); Suleima helianthana Riley (sunflower moth); Argyrotaenia spp.; Choristoneura spp.

[00188] Selected other pests from the order Lepidoptera include, but are not limited to, Alsophila pometaria Harris (moth moth); Anarsia lineatella Zeller (fruit stripe moth); Anisota senatoria J. E. Smith (orange-striped oak moth); Antheraea pernyi Guerin-Meneville (Chinese oak silkworm); Bombyx mori Linnaeus (mulberry silkworm); Bucculatrix thurberiella Busck (cotton cutworm; Collas eurytheme Boisduval (alfalfa cutworm); Datana integerrima Grote & Robinson (nut cutworm); Dendrolimus sibiricus Tschetwerikov (Siberian silkworm), Ennomos subsignaria Hubner (elm moth); Erannis tiliaria Harris (linden moth); Euproctis chtysorrhoe a Linnaeus (goldtail), Harrisina americana Guerin-Meneville (grape moth), Hemileuca oliviae Cockrell (grazing armyworm), Hyphantria cunea Drury (American white butterfly), Keiferia lycopersicella Walsingham (tomato pinworm), Lambdina fiscellaria fiscellaria Hulst (eastern hemlock moth), L. fi scellaria lugubrosa Hulst (western hemlock moth), Leucoma salicis Linnaeus (willow silkworm), Lymantria dispar Linnaeus (gypsy moth), Manduca quinquemaculata Haworth (spotted hawk hawk, tomato hawk moth), M. sexta Haworth (tomato hawk hawk, tobacco hawk moth), Operophtera brumata Linnaeus (moth moth) winter); Paleacrita vemata Peck (spring moth); Papilio cresphontes Cramer (giant sailboat, sailboat); Phryganidia californica Packard (California oak moth); Phyllocnistis citrella Stainton (citrus leaf miner); Phyllonotycter blancardella Fabricius (fruit miner moth); Pieris brassicae Linnaeus (cabbage whitefish); P. rapae Linnaeus (turnip whitefish); P. napi Linnaeus (rutabaga whitefish); Platyptilia carduidactyla Riley (Artichoke Fingerwing); Plutella xylostella Linnaeus (cabbage moth); Pectinophora gossypiella Saunders (pink bollworm); Pontia protodice Boisduval & Leconte (southern cabbage); Sabulodes aegrotata Guenee (omnivorous moth); Schizura concinna J.E. Smith (corydalis); Sitotroga cerealella Olivier (grain moth); Thaumetopoea pityocampa Schiffermuller (pine mottled silkworm); Tineola bisselliella Hummel (furniture moth); Tuta absolute Meyrick (tomato leaf miner); Yponomeuta padella Linnaeus (ermine moth); Heliothis subflexa Guenee; Malacosoma spp. and Orgyia spp.

[00189] The pesticidal activity of the compositions of the embodiments can be tested on insect pests in the early stages of development, for example, as larvae or other immature forms. Insects can be grown in total darkness at a temperature of about 20° C. to about 30° C. and a relative humidity of about 30% to about 70%. Methods for rearing insect larvae and performing bioassays are well known to those skilled in the art.

[00190] The method of controlling insects, in particular lepidopteran insects, of the present invention may include applying (e.g., spraying) to the area or plant requiring protection, to the site (area) where protection is required, the insecticidal/pesticide composition disclosed herein, containing host cells transformed with codon-optimized cry2Ai nucleotide sequences of the invention. The area where protection is required may include, for example, an area where insect pests or plants grow, or an area where plants will be grown.

[00191] The present invention relates to a method for controlling lepidoptera insect pests of eggplant, comprising applying the insecticidal/pesticide composition disclosed herein to an area or plant requiring protection, planting eggplant plants transformed with the cry2Ai nucleotide sequence(s) of the invention, or spraying a composition containing the Cry2Ai protein of the invention. The invention also relates to the use of the composition of the invention against lepidoptera insect pests of eggplant, while minimizing damage to eggplant plants.

[00192] Настоящее изобретение также относится к способу борьбы с чешуекрылыми насекомыми-вредителями риса, в частности, чешуекрылыми стеблевыми рисовыми точильщиками, рисовыми листовертками, рисовыми толстоголовками, рисовыми совками, рисовыми походными червями, или куколками рисовых насекомых-вредителей, предпочтительно, с насекомым, выбранным из группы, состоящей из: Chilo suppressalis, Chilo partellus, Scirpophaga incertulas, Sesamia inferens, Cnaphalocrocis medinalis, Marasmia patnalis, Marasmia exigua, Marasmia ruralis, Scirpophaga innotata , включающему нанесение инсектицидной/пестицидной композиции, раскрытой в настоящем документе, на площадь или растение, требующие защиты, посадку растений риса, трансформированных нуклеотидной последовательностью(ми) cry2Ai по изобретению, или распыление композиции, содержащей белок Cry2Ai по изобретению. The invention also relates to the use of the composition disclosed herein against lepidopteran pests of rice while minimizing damage to rice plants.

[00193] The present invention also relates to a method for controlling lepidoptera insect pests of tomato, in particular the lepidopteran insect Helicoverpa armigera , comprising applying the insecticidal/pesticide composition disclosed herein to an area or plant requiring protection, planting tomato plants transformed with the cry2Ai nucleotide sequence(s) of the invention, or spraying a composition containing the Cry2Ai protein on invention. The invention also relates to the use of the composition disclosed herein against lepidoptera insect pests of tomato, while minimizing damage to tomato plants.

[00194] The present invention also relates to a method for controlling Lepidoptera insect pests of cotton, comprising applying the insecticidal/pesticide composition disclosed herein to an area or plant requiring protection, planting cotton plants transformed with cry2Ai nucleotide sequence(s) of the invention, or spraying a composition containing the Cry2Ai protein of the invention. The invention also relates to the use of the composition disclosed herein against Lepidoptera pests of cotton, while minimizing damage to cotton plants.

[00195] To obtain the Cry2Ai toxin protein (SEQ ID NO: 1), recombinant host cells expressing the Cry2Ai protein can be grown in a conventional manner on a suitable culture medium, and then lysed by conventional methods such as degradation with enzymes or detergents, or the like. The toxin can then be isolated and purified by standard methods such as chromatography, extraction, electrophoresis, or the like.

[00196] Accordingly, the present invention relates to compositions and methods for controlling insect pests, in particular plant pests. More specifically, the invention relates to codon-optimized polynucleotides encoding biologically active insecticidal polypeptides against insect pests, such as, but not limited to, Lepidoptera insect pests, for example, Helicoverpa armigera - cotton bollworm and corn bollworm, Cnaphalocrocis medinalis - rice leafworm and Scirpophaga incertulas - rice stem borer, and also Pectinophora gossypiella .

[00197] One of the embodiments of the present invention relates to a codon-optimized synthetic nucleotide sequence encoding a protein having the amino acid sequence shown in SEQ ID NO: 1, where the specified nucleotide sequence is

a. the sequence shown in SEQ ID NO: 2, or its complementary nucleotide sequence, or

b. a nucleotide sequence that specifically hybridizes to at least 10 nucleotides of the nucleotide sequence shown in SEQ ID NO: 2 at nucleotide positions 251 to 402 and/or 1456 to 1628, or its complementary sequence.

[00198] Another embodiment relates to a codon-optimized synthetic nucleotide sequence of the present invention, wherein the nucleotide sequence that specifically hybridizes to the nucleotide sequence shown in SEQ ID NO: 2 is selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7.

[00199] Another embodiment relates to a recombinant DNA containing a codon-optimized synthetic nucleotide sequence of the present invention, where the nucleotide sequence is operably linked to a heterologous regulatory element. Another embodiment relates to the recombinant DNA disclosed herein, wherein said codon-optimized synthetic nucleotide sequence optionally contains a selectable marker gene, a reporter gene, or a combination thereof. Another embodiment relates to the recombinant DNA disclosed herein, wherein said codon-optimized synthetic nucleotide sequence optionally contains a DNA sequence encoding a guide or transit peptide for direction to a vacuole, mitochondria, chloroplast, plastid, or for secretion.

[00200] Another embodiment relates to a DNA construct for expressing an insecticidal protein of interest, comprising a 5'-untranslated sequence encoding a sequence encoding a Cry2Ai insecticidal protein comprising the amino acid sequence of SEQ ID NO: 1, or an insecticidal fragment thereof, and a 3'-untranslated region, said 5'-untranslated sequence containing a promoter functional in a plant cell, specified The coding sequence is a codon-optimized synthetic nucleotide sequence disclosed herein, and said 3'-untranslated sequence contains a transcription termination sequence and a polyadenylation signal.

[00201] Another embodiment relates to a plasmid vector containing the recombinant DNA or DNA construct of the present invention.

[00202] Another embodiment relates to a host cell containing a codon-optimized synthetic nucleotide sequence of the present invention. The host cell of the present invention includes a plant cell, a bacterial cell, a virus, a fungal cell and a yeast cell. In another embodiment, the described host cell is a plant cell, Agrobacterium or E. coli.

[00203] Another embodiment of the present invention relates to a method for imparting insect resistance to a plant, comprising

a. introducing into a plant cell a codon-optimized synthetic nucleotide sequence disclosed herein, wherein the nucleotide sequence is operably linked to (i) a plant cell-functional promoter and (ii) a terminator;

b. obtaining a transformed plant cell from a plant cell of step (a), wherein said transformed plant cell contains said codon-optimized synthetic nucleotide sequence disclosed herein; And

c. obtaining a transgenic plant from said transformed plant cell of step (b), wherein said transgenic plant contains said codon-optimized synthetic nucleotide sequence disclosed herein.

[00204] Another embodiment relates to a transgenic plant obtained by the method of conferring insect resistance to a plant disclosed herein. Another embodiment of the present invention relates to a transgenic plant containing a codon-optimized synthetic nucleotide sequence disclosed herein. Another embodiment of the present invention relates to a transgenic plant comprising a codon-optimized synthetic nucleotide sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7. Another embodiment of the present invention relates to a transgenic plant disclosed herein, which is selected from the group consisting of rice, wheat, Corn, Sorghum, Oats, Millet, Legumes, Cotton, Tomato, Eggplant, Cabbage, Cauliflower, Broccoli, Brassica spp., Beans, Peas, Pigeonpeas, Potatoes, Peppers, Pumpkins, Lettuce, Sweet Potatoes, Canola, Soybeans, Alfalfa, Peanuts, Sunflowers, Safflower, Tobacco, Sugarcane, Mango yoki, coffee, pineapple, citrus, cocoa, tea, banana and melon.

[00205] Another embodiment of the present invention relates to tissues, seeds or progeny derived from a transgenic plant of the present invention, where said seeds or progeny contain a codon-optimized synthetic nucleotide sequence disclosed herein. Another embodiment of the present invention relates to a biological sample derived from the tissues or seeds or progeny disclosed herein, wherein said sample contains a detectable amount of said codon-optimized synthetic nucleotide sequence of the present invention. Another embodiment of the present invention relates to a commercial product derived from a transgenic plant disclosed herein, wherein said product contains a detectable amount of said codon-optimized synthetic nucleotide sequence disclosed herein.

[00206] Another embodiment of the present invention relates to a composition comprising Bacillus thuringiensis containing a codon-optimized synthetic nucleotide sequence of the present invention encoding a Cry2Ai protein having the amino acid sequence shown in SEQ ID NO: 1. The composition disclosed herein may optionally contain an additional insecticidal agent that is toxic to the same insect pest, but has the mechanism of its insecticide action, different from the mechanism of action of the specified insecticidal protein. The insecticidal agent of the composition of the invention is selected from the group consisting of Bacillus toxin, Xenorhabdus toxin, Photorhabdus toxin, and a dsRNA that specifically represses one or more important genes in said insect pest.

[00207] Another embodiment of the present invention relates to a method for controlling and providing insect resistance to crop plants, comprising contacting said crop plant with an insecticidal effective amount of the composition described above.

[00208] Another embodiment of the present invention relates to the use of a codon-optimized synthetic nucleotide sequence, DNA construct or plasmid of the invention for the production of insect-resistant transgenic plants. Another embodiment of the present invention relates to the use of the codon-optimized synthetic nucleotide sequence disclosed herein for the production of an insecticidal composition that contains cells of Bacillus thuringiensis containing said nucleotide sequences.

[00209] Another embodiment of the present invention relates to transgenic plants expressing at least one codon-optimized synthetic nucleotide sequence disclosed herein. Another embodiment relates to a transgenic plant obtained by the method disclosed herein. The transgenic plant disclosed herein is selected from the group consisting of rice, wheat, corn, sorghum, oats, millet, legumes, cotton, tomato, eggplant, cabbage, cauliflower, broccoli, Brassica spp., beans, peas, pigeon peas, potatoes, peppers, cucurbits, lettuce, sweet potatoes, canola, soybeans, alfalfa, peanut, sunflower, safflower, tobacco, sugarcane, cassava, coffee, pineapple, citrus, cocoa, tea, banana and melon. Some embodiments of the invention relate to tissues, seeds or progeny obtained from the transgenic plant(s) according to the invention, where these seeds or progeny contain a codon-optimized synthetic nucleotide sequence described herein. Some embodiments of the invention relate to biological samples obtained from tissues or seeds or progeny, where the sample contains a detectable amount of the specified codon-optimized synthetic nucleotide sequence. One embodiment relates to a commercial product derived from a transgenic plant of the invention, wherein the product contains a detectable amount of a codon-optimized synthetic nucleotide sequence.

[00210] One embodiment relates to a composition comprising Bacillus thuringiensis comprising at least one codon-optimized synthetic nucleotide sequence of the invention, wherein the nucleotide sequence encodes a Cry2Ai protein having the amino acid sequence shown in SEQ ID NO: 1. The composition optionally contains an additional insecticidal agent that is toxic to the same pest but has a mechanism for its insecticidal action, different from the mechanism of action of said insecticidal protein. The insecticidal agent is selected from the group consisting of Bacillus toxin, Xenorhabdus toxin, Photorhabdus toxin, and a dsRNA that specifically represses one or more important genes in said insect pest.

[00211] Another embodiment relates to a method of controlling and providing insect resistance to crop plants, comprising contacting said crop plant with an insecticidal effective amount of the composition described herein.

[00212] Another embodiment relates to the use of a codon-optimized synthetic nucleotide sequence, DNA construct, or plasmid of the invention for the production of insect-resistant transgenic plants. Another embodiment relates to the use of the codon-optimized synthetic nucleotide sequence disclosed herein for the production of an insecticidal composition that contains Bacillus thuringiensis cells containing said nucleotide sequences.

[00213] These and/or other embodiments of the present invention are reflected in the claims, which form part of the description of the invention.

[00214] Various modifications and other embodiments of the present invention may be proposed by a person skilled in the art to which the present invention pertains, taking into account the ideas set forth in the description and the accompanying drawings. Thus, it is to be understood that the present invention is not limited to the specific embodiments disclosed herein, and that modifications and other embodiments are to be included within the scope of the appended claims.

[00215] The following examples illustrate the invention and are not intended to limit the invention or the patent protection sought.

EXAMPLES

[00216] The following examples are set forth to provide those skilled in the art with a full disclosure and description of how to practice and apply the present invention, and should not limit the scope of what the authors consider their invention, nor should they be taken as if the experiments below represent all, or the only, experiments performed. Efforts have been made to ensure the accuracy of the figures used (eg quantities, temperatures, etc.), but some experimental errors and deviations should be taken into account.

General Methods

[00217] Manipulation of DNA was performed using methods standard in the art. These methods can often be modified and/or replaced without significantly changing the result. Except where otherwise indicated, most of these procedures are described in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, second edition, 1989.

Example 1: Development of a codon-optimized sequence encoding the CRY2Ai protein (SEQ ID NO: 1)

[00218] A DNA sequence characterized by monocot plant codon preference was designed and synthesized to express the Cry2Ai protein having the amino acid sequence shown in SEQ ID NO: 1 in transgenic plants. A codon usage table for a monocot plant was generated based on coding sequences derived from SEQ ID NO: 1 of the Cry2Ai protein (NCBI GenBank ACV97158.1). The DNA sequence was optimized using the Monte Carlo algorithm (Villalobos A, Ness JE, Gustafsson C, Minshull J and Govindarajan S (2006) Gene Designer: a synthetic biology tool for constructing artificial DNA segments; BMC Bioinformatics 67:285-293) as the main criterion for codon selection. While optimizing the DNA sequence, the probabilities from the codon usage table were considered. Rare and rarer codons were replaced by the most common codons. A weighted average plant codon set was calculated after excluding any excess codon used in less than 10% of the total codon count for a given amino acid. The weighted average representation for each codon was calculated using the formula:

[00219] Weighted % value of CI=1/(%C1 + %C2 + %C3+etc) x %C1×100, where CI is the codon in question, and %C2, %C3, and so on are the averages of % usage of the remaining synonymous codons.

[00220] To derive a codon-optimized monocot DNA sequence encoding the Cry2Ai protein of SEQ ID NO: 1, codon substitution was performed in the DNA sequence encoding the Cry2Ai protein such that the resulting DNA sequence had an overall codon composition optimized according to the table of preferred codon usage in monocots. Additional sequence refinement was performed to remove undesired restriction enzyme recognition sites, potential intron splicing sites in the plant, a long string of A/T or C/G residues, and other motifs that may interfere with RNA stability, transcription, or translation of the coding region in plant cells. The gene was optimized in-silico using gene design software with a cutoff value of 6.0 kcal/mol to form a stable mRNA secondary structure. Other changes were made to include the desired restriction enzyme recognition sites and to remove long internal open reading frames (frames other than +1). Restriction enzyme recognition sites Xba I, Nco I, and BamH I were included before the start ATG codon, and a restriction enzyme recognition site SmaI was included before the stop codon to allow the optimized gene to be cloned into a prokaryotic vector for fusion of C-terminal protein tags. Restriction enzyme recognition sites EcoR I, Hind III, and Sac I were included after the stop codon. The TGA stop codon was used in the optimized gene.

[00221] All of these changes were made within the constraints of maintaining the approximate composition of the codons preferably used in monocot plants. The complete codon-optimized monocotyledonous plant coding sequence for the Cry2Ai protein (SEQ ID NO: 1) is shown in SEQ ID NO: 2-7. In-silico translation of the codon-optimized monocot DNA sequence showed 100% identity with the natural Cry2Ai protein (SEQ ID NO: 1). Codon-optimized monocot DNA sequences (SEQ ID NOs: 2-7) were designated 201M1, 201M2, 201M3, 201M4, 201M5 and 201M6. Synthesis of DNA fragments 201M1-M6 (SEQ ID NO: 2-7) was performed by a commercial service provider (Genscript Inc., USA). The 201M1 DNA fragment was cloned into the pUC57 vector in a manner known in the art and the construct was named pUC57-201M1. Similarly, 201M2 DNA, 201M3 DNA, 201M4 DNA, 201M5 DNA and 201M6 DNA were cloned into the pUC57 vector and the constructs were named pUC57-201M2, pUC57-201M3, pUC57-201M4, pUC57-201M5 and pUC57-201M6, respectively.

Example 2: Construction of an expression vector

[00222] The pUC57-201M1 vector of Example 1 was digested with BamH I and Hind III to obtain the 201M1 DNA fragment (SEQ ID NO: 2) (reaction volume 20 μl, plasmid DNA 8.0 μl, 10X buffer 2.0 μl, BamH I restriction enzyme 0.5 μl, restriction enzyme Hind III - 0.5 µl, distilled water - 9.0 µl). All reagents were mixed to form a reaction mixture and incubated at 37° C. for 30 min, then the reaction mixture was analyzed by 2% agarose gel electrophoresis, and the 201M1 DNA fragment was excised from the agarose gel with a clean, sharp scalpel under UV light. The DNA fragment was eluted from the gel using a gel elution kit. The section of the gel containing the DNA fragment was transferred to a 2 ml Eppendorf tube and buffer DE-A was added in 3X the volume of the sample. The gel was resuspended in DE-A buffer by vortexing and the contents of the tube were heated at 75° C. until the gel was completely dissolved, after which 0.5X DE-A buffer was added and mixed with DE-B. An Eppendorf tube was prepared by placing the column in it, and the binding mixture was transferred to the column. The Eppendorf tube with the column was centrifuged for a short time. The column was placed in a new Eppendorf tube and 500 µl of WI wash buffer (Qiagen Kit) was added, followed by centrifugation. The supernatant was discarded and 700 μl of W 2 wash buffer was added along the column walls to wash out residual buffer, followed by centrifugation. This step was repeated with a 700 µl aliquot of buffer W 2. The column was transferred to a new Eppendorf tube and centrifuged at 6000 rpm for 1 min to remove residual buffer. The column was again placed in a new Eppendorf tube and 40 μl of elution buffer was added to the center of the membrane. The column with the elution buffer was left for 1 minute at room temperature and the tubes were centrifuged at 12,000 rpm for 1 minute. The eluted 201M1 DNA fragment was stored at -20°C until use.

[00223] The isolated and purified 201M1 DNA fragment thus obtained was ligated into the linearized pET32a vector. Ligation was performed using T4 DNA ligase enzyme (reaction volume 30 µl, 10X ligation buffer 3.0 µl, vector DNA 5.0 µl, insert DNA 15.0 µl, T4 DNA ligase enzyme 1.0 µl, distilled water 6.0 µl). The reagents were thoroughly mixed, and the resulting reaction mixture was incubated at 16°C for 2 hours. E. coli BL21 (DE3) competent cells were then transformed with a ligation mixture containing the pET32a vector carrying 201M1 DNA by adding 10 μl of the ligation mixture to 100 μl of competent BL21 DE3 cells. The cell mixture thus obtained was placed on ice for 30 min, incubated in a water bath at 42°C for 60 sec for heat shock, and again placed on ice for 5-10 min. Then, 1 ml of LB broth was added to the mixture and further incubated at 37°C for 1 hour in an incubator shaker at 200 rpm. The cell mixture was applied to LB agar with 50 μg/ml carbenicillin and incubated at 37°C overnight. Positive clones were identified by restriction digestion analysis. The expression vector thus obtained was designated pET32a-201M1.

[00224] Similarly, expression vectors were constructed carrying other codon-optimized for monocot DNA sequences of SEQ ID NO: 3-7, which were designated pET32a-201M2, pET32a-201M3, pET32a-201M4, pET32a-201M5 and pET32a-201M6.

Example 3: Expression of the CRY2Ai protein encoded by 201M1 (SEQ ID NO: 2)

[00225] 201M1 DNA (SEQ ID NO: 2) cloned in pET32a, designated pET32a-201M1, was expressed in E. coli BL 21 D3. Expression was induced with 1 mM IPTG at an OD nm cell density of about 0.5-1.0. After induction, E. coli cells were incubated in a shaker for 24-40 hours at 16° C. for protein production. Protein Cry2Ai (SEQ ID NO: 1) was expressed in the cell in a soluble form (not secreted). The culture was then transferred to a centrifuge tube and centrifuged at 10,000 rpm for 5 min. The supernatant was discarded, 10 ml of cells were digested with lysozyme and incubated for 1 hour at room temperature. Samples were centrifuged at 10,000 rpm for 5 minutes and the supernatant discarded. The pellet was suspended in sterile distilled water and centrifuged at 10,000 rpm for 10 min. This step was repeated twice and the precipitate was stored at -20°C. The pellet containing proteins from the induced cells of the recombinant strain was analyzed in 10% SDS-PAGE.

[00226] Similarly, the DNA sequences shown in SEQ ID NO: 3-7 were cloned into pET32a and expressed in E. coli BL 21 D3.

Example 4 Construction of a Plant Transformation Vector Containing 201M1 DNA (SEQ ID NO: 2) Encoding CRY2Ai Protein (SEQ ID NO: 1)

[00227] Vector pUC57-201M1 with 201M1 DNA (SEQ ID NO: 2) was digested with restriction enzymes to release the 201M1 DNA fragment (reaction medium volume 20 µl, plasmid DNA 8.0 µl, 10X buffer 2.0 µl, EcoR V restriction enzyme 0.5 µl, distilled water - 9.5 µl). All reagents were mixed and the mixture was incubated at 37°C for 30 min. The product obtained after restriction digestion was analyzed by gel electrophoresis and further purified.

[00228] The vector, Ti plasmid pGreen0029, was prepared by restriction digestion with EcoR V (reaction medium 20 µl, plasmid DNA 8.0 µl, 10X buffer 2.0 µl, EcoR V restriction enzyme 0.5 µl, distilled water 9.5 µl). All reagents were mixed and the mixture was incubated at 37°C for 30 min. The product obtained after restriction digestion was analyzed by gel electrophoresis.

[00229] The purified 201M1 DNA fragment (SEQ ID NO: 2) was ligated into the linearized vector pGreen0029. Ligation was performed using T4 DNA ligase enzyme (reaction volume 30 µl, 10X ligation buffer 3.0 µl, vector DNA 5.0 µl, insert DNA 15.0 µl, T4 DNA ligase enzyme 1.0 µl, distilled water 6.0 µl). The reagents were thoroughly mixed, and the resulting reaction mixture was incubated at 16°C for 2 hours. E. coli BL21 (DE3) competent cells were then transformed with a ligation mixture containing the pGreen0029 vector carrying 201M1 DNA (SEQ ID NO: 2) by adding 10 μl of the ligation mixture to 100 μl of competent BL21 DE3 cells. The cell mixture thus obtained was placed on ice for 30 min, incubated in a water bath at 42°C for 60 sec for heat shock, and again placed on ice for 5-10 min. Then, 1 ml of LB broth was added to the mixture and further incubated at 37°C for 1 hour in an incubator shaker at 200 rpm. The cell suspension (100 μl) was evenly distributed on LB agar medium containing 50 μg/ml carbenicillin. Cups were incubated at 37°C overnight. Positive clones were identified by restriction digestion analysis. The recombinant vector thus obtained was designated pGreen0029 CaMV35S-201M1 .

[00230] Thus, the recombinant plasmid pGreen0029-CaMV35S-201M1 contains the plant optimized DNA sequence 201M1 (SEQ ID NO: 2) under the transcriptional control of the 35SCaMV promoter. In addition, pGreen0029-CaMV35S-201M1 contains the nptII gene, a plant selectable marker gene, under the transcriptional control of the NOS promoter. The physical layout of the pGreen0029-CaMV35S-201M1 T-region components is as follows:

[00231] RB>35SCaMV: 201M1 CDS: CaMV polyA>NOS promoter: nptII CDS: NOS poly A>LB

[00232] Similarly, the DNA sequences shown in SEQ ID NO: 3-7 were cloned into the Ti plasmid pGreen0029, and the recombinant vectors thus obtained were designated pGreen0029-CaMV35S-201M2, pGreen0029-CaMV35S-201M3, pGreen0029-CaMV35S-201M4, p Green0029-CaMV35S-201M5 and pGreen0029-CaMV35S-201M6. The physical arrangement of pGreen0029-CaMV35S components with the indicated DNA sequences (SEQ ID NOs: 3-7) in the T region is as follows:

[00233] RB>35SCaMV: 201M2 CDS: CaMV polyA>NOS promoter: nptII CDS: NOS Poly A>LB

[00234] RB>35SCaMV: 201M3 CDS: CaMV polyA>NOS promoter: nptII CDS: NOS Poly A>LB

[00235] RB>35SCaMV: 201M4 CDS: CaMV polyA>NOS promoter: nptII CDS: NOS Poly A>LB

[00236] RB>35SCaMV: 201M5 CDS: CaMV polyA>NOS promoter: nptII CDS: NOS Poly A>LB

[00237] RB>35SCaMV: 201M6 CDS: CaMV polyA>NOS promoter: nptII CDS: NOS Poly A>LB

[00238] Then, the DNA sequences shown in SEQ ID NO: 2-7 were also cloned into the Ti plasmid pGreen0179, and the recombinant vectors thus obtained were designated pGreen0179-CaMV35S-201M1, pGreen0179-CaMV35S-201M2, pGreen0179-CaMV35S-201M3, pG reen0179-CaMV35S-201M4, pGreen0179-CaMV35S-201M5 and pGreen0179-CaMV35S-201M6. Thus, the recombinant plasmid pGreen0179-CaMV35S-201M1 contains the plant-optimized 201M1 DNA sequence (SEQ ID NO: 2) under the transcriptional control of the 35SCaMV promoter. In addition, pGreen0029-CaMV35S-201M1 contains the hptII gene, a plant selectable marker gene, under the transcriptional control of the CaMV35S promoter. The physical arrangement of the components of pGreen0179-CaMV35S with the DNA sequence in the T region is as follows:

[00239] RB>35SCaMV promoter: 201M1 : CaMV polyA>35SCaMV promoter: hptII : CaMV PolyA>LB

[00240] RB>35SCaMV promoter: 201M2 : CaMV polyA>35SCaMV promoter: hptII : CaMV PolyA>LB

[00241] RB>35SCaMV promoter: 201M3 : CaMV polyA>35SCaMV promoter: hptII : CaMV PolyA>LB

[00242] RB>35SCaMV promoter: 201M4 : CaMV polyA>35SCaMV promoter: hptII : CaMV PolyA>LB

[00243] RB>35SCaMV promoter: 201M5 : CaMV polyA>35SCaMV promoter: hptII : CaMV PolyA>LB

[00244] RB>35SCaMV promoter: 201M6 : CaMV polyA>35SCaMV promoter: hptII : CaMV PolyA>LB

[00245] Agrobacterium tumefaciens transformation recombinant vector pGreen0179-CaMV35S-201M1

[00246] Agrobacterium tumefaciens strain LBA440 was transformed with recombinant plasmid pGreen0179-CaMV35S-201M1 carrying the DNA sequence shown in SEQ ID NO: 2. 200 ng of plasmid pGreen0179-CaMV35S-201M1 DNA was added to a 100 μl aliquot of competent cells A tumefaciens strain LBA440. The mixture was incubated on ice for 30 min and transferred to liquid nitrogen for 20 min, followed by thawing at room temperature. Agrobacterium cells were then transferred into 1 ml of LB broth and incubated at 28°C for 24 hours in a water bath shaker at 200 rpm. The cell suspension was evenly distributed on LB agar medium containing 50 μg/ml rifampicin, 30 μg/ml kanamycin and 5 μg/ml tetracycline. Cups were incubated at 28°C overnight. Transformed Agrobacterium cells were analyzed by plasmid extraction and restriction digestion, positive colonies of A. tumefaciens were selected and stored for later use.

Example 5 (A) Indirect Agrobacterium transformation of rice with construct pGreen0179-CaMV35S-201M1

[00247] mediatedAgrobacterium rice transformation was performed using the pGreen0179-CaMV35S-201M1 recombinant vector containing T-DNA as shown in FIG. 1. Immature rice embryos were used as explants for transformation (Hiei, Y. and T. Komari, 2008,Agrobacterium-mediated transformation of rice using immature embryos or calli induced from mature seed. Nat. Protocols., 3: 824-834) conducted at the Department of Plant Biotechnology, Center for Molecular Biology and Plant Biotechnology, Tamil Nadu Agricultural University, Coimbatore, India. The details of the rice transformation experiments are described below.

[00248] Those skilled in the art of rice transformation understand that other methods of rice transformation and selection of transformed plants are available using other plant-expressed selectable marker genes.

plant material

[00249] The local elite rice cultivar ASD16 from the Department of Rice Studies, Center for Plant Breeding and Genetics, Tamil Nadu Agricultural University (TNAU), Coimbatore, India was used in the transformation experiments. Plant material was accessed by TNAU.

Preparation of immature rice germ explants for transformation

[00250] Immature rice seeds were harvested 12-14 days after pollination at the Rice Breeding Station, TNAU, for transformation experiments. Immature seeds were dehusked and treated with 70% ethanol for one minute, followed by treatment with 1.5% sodium hypochlorite solution containing a drop of Tween-20, and then washed with sterile water. Immature seed embryos were excised aseptically using sterile forceps under a microscope (ZEISS, Germany or Leica, Switzerland) and cultured on a 0.8% (w/v) agar plate. For transformation, immature embryos ranging in size from 1.3 mm to 1.8 mm were used.

Pretreatment of immature embryos

[00251] Immature embryos were incubated in a water bath at 43°C for 30 minutes, immediately cooled in a water bath for 1 minute and centrifuged at 1100 rpm for 10 minutes at 25°C. The pretreated embryos were used as explants in subsequent transformation experiments.

Pretreatment and co-cultivation of explants with Agrobacterium tumefaciens

[00252] Three days prior to co-culture, A. tumefaciens LBA4404 carrying plasmid pGreen0179-CaMV35S-201M1 described in Example 4 was inoculated into 5 ml of YEP broth containing rifampicin (20 mg/l), kanamycin (100 mg/l), streptomycin (50 mg/l) and tetracycline (5 mg/l), and incubated at 28°C in a shaker at 200 rpm for 2-3 days. During transformation of immature embryos, a complete loop of A. tumefaciens culture was suspended in 1.0 ml of AA infection medium (Table 1; pH 5.2) at a density of 1×10 ⁹ colony forming units (CFU) per ml with an OD of 1.0 at 660 nm.

The composition of the YEP medium :

Yeast extract: 10.0 g

Peptone: 10.0 g

NaCl: 5.0 g

Distilled water: up to 1000.0 ml

Agar: 18.0 g

pH: 6.8

[00253] YEP broth has the same composition but without agar.

[00254] The pre-treated immature embryos were then transferred to NB-As medium (Table 2) shield up. Five μl of the A. tumefaciens culture suspension described above was applied to each of the immature embryos and incubated at 25°C for 15. The embryos were then transferred to a Petri dish with NB-As medium, shield up, and incubated in the dark at 25°C for 7 days.

Callus induction and selection

[00255] After 7 days of co-culture, callus formed from immature embryos. The callus thus obtained was transferred to CCMC medium (Table 3) and incubated at 31° C. for 5 days under continuous illumination (5000 lux). After 5 days, the callus was cut into 4 parts (depending on the size of the callus), transferred to CCMC medium and incubated at 31°C for 10 days under continuous illumination (5000 lux). Next, the calli were transferred to the CCMCH50 selection medium containing hygromycin (Table 4), incubated at 31°C for 10 days under continuous illumination (5000 lux) with periodic selection on the CCMCH50 medium.

[00256] After two rounds of selection on CCMCH50 medium, hygromycin-resistant calli were transferred to NBPRCH40 pre-regeneration medium (Table 5) and incubated at 31°C for 7 days under continuous illumination (5000 lux).

[00257] Yellowish-white, well-proliferated calli were transferred to RNMH30 regeneration medium (Table 6) and incubated at 31° C. for 14 days under continuous light (5000 lux) to produce shoots. The shoots thus obtained were transferred to 1/2 MS rooting medium (Murashige, T; Skoog, F, 1962, "A Revised Medium for Rapid Growth and Bio Assays with Tobacco Tissue Cultures", Physiologia Plantarum. 15 (3): 473-497) with 30 mg/l hygromycin B and incubated at 31°C for 14 days with continuous lighting (5000 lux) for further development. Then, well-grown regenerated rice seedlings were transferred to culture flasks containing hygromycin B 30 mg/l rooting medium, well-rooted rice plants were transferred to soil and kept in a greenhouse. Thus, eight T ₀ putatively transgenic rice plants (FIG. 2) were generated from hygromycin-resistant calli with varying numbers of regenerated plants per event.

Example 5 (B) Molecular Analysis of Putatively Transgenic Rice Plants Transformed with 201M1 DNA Sequence (SEQ ID NO: 2)

(i) Isolation of genomic DNA

[00258] Total genomic DNA was extracted from leaf tissues of putatively transgenic rice plants of Example 5 (A) and control non-transgenic ASD16 cultivar plants. Leaves were harvested from putatively transgenic rice plants and non-transgenic rice plants, homogenized with 300 µl of extraction buffer (1M Tris-HCl, pH 7.5, 1M NaCl, 200 mM EDTA, and 10 percent SDS) using a QIAGEN TissueLyser II (Retsch) and centrifuged at 12,000 rpm for 10 minutes. The supernatant was transferred into a sterile microcentrifuge tube. A mixture of chloroform-isoamyl alcohol (24:1) was added to the supernatant and centrifuged at 12,000 rpm for 10 minutes. The aqueous layer was transferred to a microcentrifuge tube. There was added an equal volume of ice-cold isopropanol and kept at -20°C for 20 minutes. The supernatant was then discarded and 300 µl of ethanol was added to the pellet. After centrifugation at 10,000 rpm for 5 minutes, the supernatant was discarded, the DNA pellet was air-dried for 15 minutes and then dissolved in 40 μl of 0.1X TE buffer (Tris pH 8.0: 10.0 mM and EDTA pH 8.0: 1.0 mM). The quality and quantity of genomic DNA was assessed using a Nanodrop 1000 ^® spectrophotometer (Thermo Scientific, USA) by measuring OD at 260 nm. DNA integrity was also assessed by electrophoresis in a 0.8% agarose gel.

PCR analysis

[00259] Polymerase chain reaction (PCR) was performed on genomic DNA (100 ng) of putatively transgenic rice plants and non-transgenic control rice plants to analyze synthetic cry2Ai-201M1 DNA using primers with the sequences shown in SEQ ID NO: 8 and SEQ ID NO: 9, and the hptII gene using primers with the sequences shown in SEQ ID NO : 12 and SEQ ID NO: 13.

[00260] PCR Conditions

94°C for 5 min: 1 cycle

94°C for 45 sec

58°-62°C for 45 sec 30 cycles

72°C for 45 sec

72°C for 10 min: 1 cycle

[00261] All eight putatively transgenic rice plants were positive for cry2Ai-201M1 DNA having the nucleotide sequence shown in SEQ ID NO: 2 and the hptII gene.

(ii) Biochemical analysis of presumably transgenic rice lines (T0) by ELISA

[00262] A sandwich ELISA was performed using the EnviroLogix Quantiplate kit (EnviroLogix Inc., USA) according to the manufacturer's instructions to quantify the Cry2Ai protein (SEQ ID NO: 1) in putatively transgenic PCR positive rice plants. The positive and negative controls provided in the kit were used as a reference. For this experiment, the second leaf from the main offshoot of a putatively transgenic rice plant was used. Approximately 30 mg of leaf tissue was homogenized in 500 μl of extraction buffer, centrifuged at 6000 rpm at 4° C. for 7 minutes, and the supernatant was used for analysis. The supernatant (100 µl) was applied to the wells of the tablet, previously coated with an antibody against the Cry2Ai protein. The plate was covered with parafilm and incubated at room temperature (24±2°C) for 15 minutes. Enzyme conjugate (100 μl) was added to each well. After one hour, the wells were thoroughly washed with 1X wash buffer. Substrate (100 μl) was added to each well and incubated for 30 minutes. The reaction was stopped by adding a stopping solution (0.1 N hydrochloric acid). The optical density (OD) of the plate was determined at 450 nm using the negative control as a blank. Each sample was analyzed in duplicate and each well was considered a duplicate.

[00263] A graph was plotted based on the absorbance values of the calibration solutions at different concentrations (provided in the kit). The concentration of the Cry2Ai protein (SEQ ID NO: 1) was determined by correlating its OD value with the corresponding concentration level on the graph. The concentration was calculated as described below.

[00264] The amount of Cry2Ai protein (SEQ ID NO: 1) present in the samples was expressed as micrograms per gram of fresh leaf tissue. All eight PCR-positive transgenic rice plants (T0) screened using the Cry2Ai quantification ELISA kit were positive for expression of the Cry2Ai protein (SEQ ID NO: 1) in young transgenic rice leaf tissue of the transgenic rice plants. The expression of the Cry2Ai protein (SEQ ID NO: 1) in these transgenic rice plants was found to range from 0.083 to 0.766 μg/g fresh leaf tissue during the vegetative stage (Table A).

Table A: Expression of Cry2Ai protein (SEQ ID NO: 1) in T0 transgenic rice plants

Sample No. rice plants Cry2Ai protein concentration at the vegetative stage
(µg/g fresh leaf tissue)* 1 Control plant - non-transgenic rice plant - ASD 16 0.0 2 OS-1 0.145 3 OS-2 0.107 4 OS-3 0.083 5 OS-4 0.143 6 OS-5 0.095 7 OS-6 0.135 8 OS-7 0.118 9 OS-8 0.766

*Average of two replicas

(iii) Southern hybridization

[00265] Seven ELISA positive rice plants (OS-1 to OS-7) were selected for Southern hybridization. Genomic DNA (5 μg each) of T ₀ ELISA positive transgenic rice plants was digested with Nco I or Xba I or Bam HI restriction enzymes and Southern blotted using a 1077 bp cry2Ai [α- ^32P ]-dCTP-labeled probe.

[00266] Genomic DNA of PCR and ELISA positive transgenic rice plants and non-transgenic rice plants were digested with restriction enzyme Nco I at 37° C. for 16 hours. The pGreen 0179-CaMV35S-201M1 plasmid DNA was used as a positive control. Digested genomic and plasmid DNA samples were electrophoresed on a 0.8% agarose gel at 20 V overnight in 1X TAE buffer, visualized after staining with ethidium bromide in a UV transilluminator, and documented in a gel documentation system (SYNGENE).

[00267] Restriction-cleaved and electrophoresis-separated genomic DNA was denatured by immersing the gels in two volumes of denaturing solution for 30 minutes with gentle agitation. The gels were neutralized by immersing them in two volumes of neutralizing solution for 30 minutes with gentle agitation. The gel was washed quickly in sterile deionized water and the DNA was transferred to a positively charged nylon membrane (Sigma) by upward capillary transfer in 20X SSC buffer for 16 h following a standard protocol. After complete transfer of the genomic DNA, the nylon membrane was quickly washed in 2X SSC buffer and air dried for 5 minutes. DNA was crosslinked by exposing the membrane to crosslinking UV radiation (UV ^{Stratalinker®} 1800 Stratagene, CA, USA) at 1100 μJ for 1 minute. The cross-linked membrane was sealed in plastic bags and stored at 4° C. until used in Southern hybridization.

Denaturing solution :

NaCl: 1 M

NaOH: 0.5 N

Neutralizing solution :

NaCl: 1.5M

Tris: 0.5M

20X SSC :

NaCl: 3.0M

Sodium citrate: 0.3M

pH: 7.0 with conc. HCl

20x SSC solution can be diluted as follows:

Final concentration of SSC 20x SSC solution Distilled or deionized water 3x SSCs 30 ml 170 ml 2x SSCs 20 ml 180 ml 1x SSC 10 ml 190 ml 0.5x SSC 5 ml 195 ml

Getting a Probe

[00268] Approximately 100 ng of a 1077 bp cry2Ai-201M1 DNA fragment amplified from a binary vector and purified using a mini gel extraction kit (Bio Basic Inc., Canada) radiolabeled with [α- ^32P ]-dCTP was used as a labeling probe. Four µl of template DNA (DNA cry2Ai-201M1 ) was mixed with 10 µl of random primer (DecaLabel DNA Labeling Kit, Thermo Fisher Scientific Inc. USA) in a microcentrifuge tube. The volume was adjusted to 40 μl with sterile distilled water, denatured by heating for 5 minutes in a boiling water bath and cooled on ice. 5 µl of a labeling mixture containing dATP, dGTP, dTTP, 5 µl of [α- ^32P ]-dCTP (50 µCi) and 1 µl of the Klenow fragment of DNA polymerase I was added to the denatured DNA and incubated at 37°C for 10 minutes in a water bath. The reaction was stopped by adding 1 μl of 0.5 M EDTA, incubated in a boiling water bath for 4 minutes and transferred to ice for 4-5 minutes.

[00269] Prehybridization and Probe Hybridization

[00270] The cross-linked DNA membrane described above was carefully placed in a hybridization vial containing 30 ml hybridization buffer. The vial was tightly closed and placed in a 65° C. oven for 45 minutes - 1 hour for pretreatment prior to hybridization.

[00271] The hybridization buffer was decanted from the vial and replaced with a 30 ml hybridization solution (maintained at 65°C) containing the denatured labeled [α- ^32P ]-dCTP DNA probe. The vial with the hybridization solution was tightly closed and placed in an oven at 65°C for 16 hours.

Solution for hybridization :

Na ₂ HPO ₄ , pH 7.2: 0.5 M

SDS: 7% (w/v)

EDTA, pH 7.2: 1 mM

[00272] The hybridization buffer was decanted from the vial. Wash solution I was added and the vial was placed in an oven on a slowly rotating platform at 65° C. for 10 minutes. After 10 minutes wash solution I was replaced with 30 ml wash solution II and incubated at 65° C. for 5-10 min with gentle agitation. Wash solution II was then discarded and the amount of radioactivity on the membrane was determined using a Gregor-Muller counter. Depending on the results of the determination, 30 ml of washing solution III was added to the vial and incubated at 65°C for 30 seconds - 1 minute with gentle stirring. Wash solution III was then removed, the membrane was dried on Whatman No. 1 filter paper for 5-10 minutes at room temperature, and X-ray film irradiation (Kodak XAR) was performed in a dark room with a signal-enhancing screen (Hyper cassette ^® from Amersham, USA) for 2 days at -80°C.

[00273] After 2 days of exposure, the x-ray film was removed in a dark room and immersed in a developer solution for 1 minute, followed by immersion in water for 1 minute. Finally, the x-ray film was immersed in the fixative solution for 2 minutes, then washed in water for 1-2 minutes and dried in air.

Washing solution

Wash Solution I: 3X SSC+0.1% SDS

Wash solution II: 0.5X SSC + 0.1% SDS

Wash solution III: 0.1X SSC + 0.1% SDS

Developer solution :

[00274] 13.2 g of the contents of package A were dissolved in 800 ml of distilled water, after complete dissolution, 89 g of the component from package B was added and dissolved with slow stirring. After complete dissolution, the volume was adjusted to 1000 ml and stored in a dark glass bottle.

fixative solution :

[00275] 268 g of the fixative was dissolved in 800 ml of distilled water with slow stirring, after complete dissolution, the volume was adjusted to 1000 ml, filtered through filter paper and stored in a dark glass bottle.

[00276] In the case of all seven ELISA-positive rice transgenic events, hybridization signals of different sizes were detected, indicating integration of the transgene into the rice genome. In the transgenic rice plant OS-6, the integration of the cry2Ai gene into one locus took place. A hybridization signal was also observed in the positive control, with no hybridization signal in the case of the non-transgenic control rice plant (ASD16).

(iv) Obtaining the next generation of cry2Ai transgenic rice (T2) line OS 6

[00277] Thirty T1 seeds of transgenic rice plant OS 6 were sown in trays. Twenty-three seeds germinated and the 15-day-old seedlings were potted and placed in the greenhouse. Twenty out of 23 T1 progeny of the transgenic rice plant OS 6 were found to express the cry2Ai gene by quantitative ELISA analysis. The concentration of Cry2A protein (SEQ ID NO: 1) expressed in OS 6 transgenic rice T1 plants ranged from 0.192-0.588 μg/g at 55 DAP and 0.082-0.387 μg/g fresh leaf tissue at 84 DAP. Southern hybridization analysis of six T1 plants of OS 6 transgenic rice using the Nco I enzyme showed integration of cry2AiM1 (SEQ ID NO: 2) at one locus similar to that of the T0 transgenic rice plant.

[00278] Fifty (50) seeds of transgenic T1 rice plants, namely OS 6-8 and OS 6-20, were sown to produce T2 transgenic rice plants. A total of 83 T2 plants, 46 from OS 6-8 and 37 from OS 6-20, were screened for the presence of cry2AiM1 DNA (SEQ ID NO: 2) by polymerase chain reaction (PCR). PCR results showed the presence of this DNA in all 83 transgenic T2 rice plants. The results of quantitative ELISA analysis of representative ten T2 plants showed a concentration of Cry2Ai protein (SEQ ID NO: 2) from 0.433 to 0.857 μg/g of fresh leaf tissue 77 days after planting (DPP) (Table B).

Table B: Cry2Ai protein expression and insecticidal activity in T2 rice plants

Sample No. T2 plant Cry2Ai protein concentration, µg/g
(77 DPA, 77 day old plants) Mortality, in percent, of rice marching worm larvae (86 DPP) 1 OS 6-8-13 0.433 100 2 OS 6-8-25 0.625 100 3 OS 6-8-28 0.746 100 4 OS 6-8-31 0.665 90 5 OS 6-8-35 0.471 100 6 OS 6-20-3 0.741 100 7 OS 6-20-7 0.594 100 8 OS 6-20-8 0.649 100 9 OS 6-20-9 0.857 100 10 OS 6-20-10 0.539 95 eleven Control plant - non-transgenic rice plant - ASD 16 No expression 0

Example 5 (C) Insect Bioassay

Bioassay with separated stem fragments for activity against rice stem borer

[00279] Egg mass of rice stem borer ( Scirpophaga incertulas) was collected directly from the rice field (Paddy Breeding Station, TNAU) and incubated in the laboratory at 25±1°C and 60 percent relative humidity to hatch from the eggs. Alternatively, adult rice borer butterflies were harvested from the field and released in insect cages containing 30-day-old rice seedlings of vulnerable TN1 variety to lay eggs. The eggs laid on the leaves were then harvested from the cells and incubated in the laboratory by keeping the leaves with the eggs in a microtube at 25±1°C and 60 percent relative humidity to hatch from the eggs. The larvae hatched from the egg mass were used for bioassay with insects.

[00280] The stem of ELISA-positive transgenic rice plants was cut into six pieces (approximately 5 cm long), each of the pseudo-stem fragments was placed on wet filter paper in a separate plastic petri dish, and five newly hatched larvae of the rice stem borer were placed on each pseudo-stem (Chakraborty et al ., 2016). Pseudo-stems of non-transgenic ASD16 rice were used as controls. Petri dishes were covered with a thin film to prevent larvae from escaping from the dishes and maintained at 25±1° C. and 60 percent relative humidity. After 6 days, the pseudo-stem fragments were carefully opened using a thin blade and larval mortality in the transgenic as well as control plants was assessed. Bioassays with detached stem fragments and using newly hatched larvae of the rice borer on T2 rice plants demonstrated larval mortality in the range of 80-85 percent. In control plants, the larvae did not die. In six days, the surviving larvae in the control destroyed most of the internal tissues of the stem and drilled holes in the stem fragments, leaving a large amount of insect excrement (Fig. 5).

[00281] Bioassay activity against rice marching worms

The egg mass of paddy marching worms ( Spodoptera mauritia) was collected directly from the paddy field (Paddy Breeding Station, TNAU) and incubated in the laboratory at 25±1°C and 60 percent relative humidity to hatch from the eggs. The larvae hatched from the egg mass were used for bioassay with insects.

A bioassay for activity against rice marching worms was performed using leaf fragments of ELISA positive transgenic rice plants and corresponding control plants.

Bioassay with detached leaf fragments and using newly hatched rice marching worm larvae demonstrated larval mortality in T2 rice plants. In the control plants, the larvae did not die and most of the leaf tissue was destroyed by the surviving larvae within six days (Fig. 6).

Bioassay using newly hatched marching worm ( Spodoptera mauritia) larvae demonstrated 100 percent mortality in eight of ten T2 plants tested, and the remaining two plants had 90 and 95 percent mortality (Table B).

Example 6(A): Mediated Agrobacterium tomato transformation

[00282] Agrobacterium- mediated tomato transformation was performed using the pGreen0029-CaMV35S-201M1 recombinant vector containing the T-DNA construct shown in FIG. 3. Tomato cotyledon explants were used as transformation explants.

[00283] Agrobacterium- mediated tomato transformation was performed at the Department of Plant Biotechnology, Center for Molecular Biology and Plant Biotechnology, Tamil Nadu Agricultural University, Coimbatore, India. Details of the tomato transformation experiments are described below.

[00284] Those skilled in the art of tomato transformation understand that other methods of tomato transformation and selection of transformed plants are available using other plant-expressed selectable marker genes.

plant source

[00285] PKM1 cultivar tomato seeds were obtained from College and Research Institute of Horticulture, Coimbatore, India by Tamil Nadu Agricultural University (TNAU), PN Pudur, Coimbatore, Tamil Nadu-641003, India. Access to tomato seeds was obtained from the Department of Plant Biotechnology, Center for Molecular Biology and Plant Biotechnology, TNAU.

In vitro seed germination and pre-cultivation

[00286] PKM1 cultivar tomato seeds were washed 3 times with sterile distilled water, treated with 70% ethanol for 5 minutes and 4% sodium hypochlorite solution containing Tween 20 for 5-7 minutes. Then the seeds were washed with sterile distilled water and dried in air on sterile paper. Sterilized seeds were germinated for 8-10 days on half-strength MS medium (as described above) in the dark, followed by a 16-hour photoperiod cycle using cold white fluorescent lamps (intensity 110-130 nanomoles/m ² /s) and 8 hours of darkness at 25°C in a growth chamber.

[00287] Cotyledon explants from in vitro grown 8-10 day old seedlings were excised and cultured on pre-culture medium, which is a modified MS medium containing zeatin (1.0 mg/l), for two days before co-culture.

Modified medium composition MS/l

MS Modified Medium Powder (PT025-Himedia, Mumbai): 4.2 g

Sucrose: 30.0 g

Agar: 8.0 g

Distilled water: 1000 ml

pH: 5.8

Co-cultivation, selection and regeneration of plants

[00288] Two days later, explants from the pre-culture medium were infected with suspension cultureA. tumefaciensLBA4404 carrying plasmid pGreen0029-CaMV35S-201M1, within 10-12 minutes. Excess suspension culture was removed by blotting with sterile filter paper, and the explants were transferred to co-cultivation medium, which was a modified medium MS + zeatin (1.0 mg/l) + acetosyringone (100 µM). After 48 hours of co-culturing in the dark at 26°C, the explants were washed with sterile distilled water and an aqueous solution containing 250 mg/l cefotaxime, and then 2 times with sterile distilled water. The co-cultured explants were dried on sterile filter paper and cultured on a selection medium consisting of MS modified medium + zeatin (1.0 mg/l) + kanamycin (50 mg/l) + cefotaxime (250 mg/l). The explants were subcultured on the same medium three times with an interval of 15-20 days. The resulting well developed shoots were transferred to a root development medium containing half strength 1.0 MS+IBA (1 mg/l) + kanamycin (30 mg/l) (FIG. 4).

[00289] After abundant root development (15-20 days), the seedlings were carefully removed from the culture bottles and hardened in small paper cups with a hardening mixture (red earth:chernozem:vermicompost in the ratio 1:1:1), kept at 25±2°C for 7-8 days, and then transferred to the greenhouse.

Example 6(B): Molecular and Biochemical Analyzes of Putatively Transgenic Tomato Plants

[00290] Putatively transgenic tomato plants were screened for the presence of the cry2Ai 201M1 DNA fragment (SEQ ID NO: 2) by PCR analysis, and protein expression was analyzed by ELISA.

Tomato genomic DNA isolation and PCR analysis

[00291] Genomic DNA was extracted from the leaves of putatively transgenic tomato plants and non-transformed tomato plants (as a negative control). For DNA extraction, the method described above was used.

[00292] PCR was performed as described above with isolated genomic DNA to confirm the presence of the cry2Ai (201M1) DNA fragment using primers with the sequences shown in SEQ ID NO: 8 and SEQ ID NO: 9. Twenty-three of the 29 putatively transgenic tomato plants (T0) were confirmed to have the cry2Ai (201M1) DNA fragment.

[00293] PCR analysis was performed using primers with the sequences shown in SEQ ID NO: 10 and SEQ ID NO: 11 to amplify the nptII gene (kanamycin resistance selectable marker gene) to check for the presence of the nptII gene in putative tomato transformants (T0). Analysis showed amplification of the nptII gene.

Screening of T0 transgenic tomato plants in quantitative ELISA and Southern analysis

[00294] Seventeen of the twenty-three PCR positive tomato plants were positive in the ELISA assay (using the Envirologix Cry2A kit) and the Cry2A protein concentration ranged from 0.006-0.159 µg/g fresh leaf tissue during the vegetative stage.

[00295] Southern hybridization was performed on five ELISA-positive tomato events using Xba I-digested DNA. The results of Southern hybridization demonstrated integration of the transgene at one to two loci in T0 plants, and in the SL-34 event, integration of the cry2Ai gene occurred at one locus.

Obtaining the next generation of cry2Ai transgenic object SL-34 tomato

[00296] All 18 seeds of one fruit from the object SL-34 tomato were placed for germination on MS medium at half strength. Twelve day old seedlings were transferred for hardening. Of the 16 seedlings, 12 T1 offspring were placed in the greenhouse and subjected to PCR screening. Eleven of the twelve T1 offspring were PCR positive and all 11 offspring were found to express the cry2Ai gene by ELISA. The concentration of the Cry2Ai protein expressed in eleven T1 plants ranged from 0.320 to 0.695 μg/g fresh leaf tissue at 82 DAP (Table C). Southern hybridization of five T1 plants showed integration of the cry2Ai gene into one locus, similar to that in the T0 transgenic plant.

Table C. Cry2Ai protein expression and insecticidal activity in T1 progeny of tomato SL-34 event

Sample No. T1 tomato plant PCR Cry2Ai protein concentration (mcg/g) at 82 DPP Mortality of Helicoverpa armigera (%) at 83 DPP 1 SL-34-1 Negative 0.000 0 2 SL-34-2 Positive 0.442±0.001 100 3 SL-34-3 Positive 0.474±0.000 100 4 SL-34-4 Positive 0.695±0.004 100 5 SL-34-6 Positive 0.649±0.008 100 6 SL-34-7 Positive 0.382±0.008 100 7 SL-34-8 Positive 0.358±0.008 100 8 SL-34-9 Positive 0.370±0.012 100 9 SL-34-10 Positive 0.591±0.000 100 10 SL-34-11 Positive 0.566±0.008 100 eleven SL-34-12 Positive 0.320±0.004 100 12 SL-34-13 Positive 0.524±0.000 100 13 Control plant - non-transgenic tomato plant Negative 0.000 0

[00297] Of all 19 seeds of one T1 fruit of plant SL-34-11, 14 T2 progeny were grown in the greenhouse. All fourteen T2 progeny were found to express the cry2Ai gene, and the amount of protein was determined by ELISA. The concentration of the Cry2Ai protein expressed in T2 transgenic plants ranged from 0.540-0.783 µg/g at 80 DAP and 0.287-0.547 µg/g fresh leaf tissue at 104 DAP.

[00298] Of all 33 seeds of one T2 fruit of plant SL-34-11-1, 26 plants were grown in the greenhouse. Of all 42 seeds from another fruit of the same plant (SL-34-11-1), 37 seedlings were grown in the greenhouse. A total of 63 T3 offspring were grown from all 75 seeds of two fruits of the same T2 plant. All 63 T3 plants were positive by PCR screening and quantitative ELISA. This suggested the homozygous nature of the T2 plant SL-34-11-1. Cry2Ai protein concentration was assessed by quantitative ELISA in 37 of the T3 transgenic plants and ranged from 0.329-0.881 µg/g fresh leaf tissue at 42 DAP. The concentration of Cry2A protein in immature and ripe fruits of four T3 offspring during 119-128 DPT was approximately 0.118-0.330 µg/g fresh fruit tissue.

Example 7 Insect Bioassay

Insect bioassay of activity of cry2Ai transgenic tomato plants against H. armigera

[00299] Tomato transformants that were confirmed to have the Cry2Ai protein by ELISA were bioanalyzed to assess their resistance to H. armigera . A leaf disc bioassay was performed to determine the degree of insect resistance of cry2A transgenic tomato plants in the laboratory. The second leaf from the top of transgenic plants and control (non-transformed) tomato plants was cut off at the reproductive stage and used for bioassay with leaf disks. The leaf disc was placed in a sterile Petri dish with wet filter paper. One newly hatched H. armigera larva was placed on each piece of leaf placed on filter paper using a fine camel hair brush. After placing the insect, the cups were wrapped in Klinfilm and placed in a dark place. Each treatment option was performed in two replicates, with ten cups per replica. The experiment was maintained at 26-28°C and 60 percent relative humidity. Larval mortality and leaf damage were recorded 48 hours later at 24 hour intervals for six days.

[00300] Bioassay results showed 100% mortality of H. armigera in five of 12 T0 ELISA positive plants. Insect analysis using newly hatched H. armigera larvae on T1, T2 and T3 leaf disks of cry2Ai transgenic tomato event SL-34 progeny also demonstrated 100% mortality for all transgenic plants tested (Figure 7). On leaf disks of control plants, the larvae did not die.

[00301] Although the foregoing invention has been described in some detail with the help of illustrations and examples for clarity of understanding, it is obvious that certain changes and modifications can be applied in practice within the scope of the present invention.

Table 1. AA infection media (Hiei and Komari, 2008) Chemical reagents final conc./liter Stock/1000 ml Quantity/liter 20X basic AA salts KCl 29.50 g 59.0 g 50 ml CaCl ₂ -2H ₂ O 0.15 g 3.00 g MgSO ₄ -7H ₂ O 0.50 g 10.0 g NaH ₂ PO ₄ -H ₂ O 0.15 g 3.00 g 100X Fe EDTA FeSO ₄ -7H ₂ O 27.80 mg 2.78 g 10 ml EDTA, disodium salt 37.30 mg 3.73 g 10X trace element salts B5 MnSO ₄ -4H ₂ O 13.20 mg 1.320 g 10 ml ZnSO ₄ -7H ₂ O 2.00 mg 0.200 g CuSO ₄ -5H ₂ O 25.00 mg 2.500 g Na ₂ MoO ₄ -2H ₂ O 0.25 mg 0.025 g CoCl ₂ -6H ₂ O 25.00 mg 2.500 g _{H3BO3 _} 3.00 mg 0.300 g KI 0.75 mg 0.075 g 100X vitamins B5 Myo-inositol 100.0 mg 10,000 g 10 ml Thiamine hydrochloride 10.0 mg 1,000 g Pyridoxine hydrochloride 1.0 mg 0.100 g A nicotinic acid 1.0 mg 0.100 g 10X AA amino acids (pH-5.8) Gln 0.876 g 8.760 g 100 ml asp 0.266 g 2.660 g Arg 0.174 g 1.740 g gly 7,500 mg 0.075 g 100 mM Acetosyringone 0.392 mg 0.392 g 1 ml sucrose 20.0 g - - Glucose 10.0 g - - Casmino acids, vitamin analysis 0.5 g - -

pH to 5.2 and sterilize by filtration through a 0.22 mm cellulose acetate filter

Table 2. NB-As environment (Hiei and Komari, 2008) Chemical reagents final conc./liter Stock/1000 ml Quantity/liter 10X base salts N6 KNO ₃ 2.830 g 28.30 g 100 ml CaCl ₂ -2H ₂ O 0.166 g 1.66 g MgSO ₄ -7H ₂ O 0.185 g 1.85 g _{KH2PO4 _} 0.400 g 4.00 g 100X Fe EDTA FeSO ₄ -7H ₂ O 27.80 mg 2.78 g 10 ml EDTA, disodium salt 37.30 mg 3.73 g 100X trace element salts B5 MnSO ₄ -4H ₂ O 13.20 mg 1.320 g 10 ml ZnSO ₄ -7H ₂ O 2.00 mg 0.200 g CuSO ₄ -5H ₂ O 25.00 mg 2.500 g Na ₂ MoO ₄ -2H ₂ O 0.25 mg 0.025 g CoCl ₂ -6H ₂ O 25.00 mg 2.500 g _{H3BO3 _} 3.00 mg 0.300 g KI 0.75 mg 0.075 g 100X vitamins B5 Myo-inositol 100.0 mg 10,000 g 10 ml Thiamine hydrochloride 10.0 mg 1,000 g Pyridoxine hydrochloride 1.0 mg 0.100 g A nicotinic acid 1.0 mg 0.100 g 2,4-D 2,000 mg 0.100 g 20 ml NAA 1,000 mg 0.100 g 10 ml 6BA 1,000 mg 0.100 g 10 ml sucrose 20,000 g - - Glucose 10,000 g - - Proline 0.500 g - - Casmino acids, vitamin analysis 0.500 g - - Agarose type I 8,000 g - - 100 mM Acetosyringone 0.392 mg 0.392 g 1 ml

[00302] Separately prepare 10X N6 basic salt solution, 100X Fe EDTA solution, 100X B5 trace element salt solution, 100X vitamin solution, 100 mg L ^-1 2,4-D solution, 100 mg L ^-1 NAA solution, 100 mg L ^-1 6BA solution, and mix the required amount or the above stock solution with sucrose, glucose, proline and casamino acids for vitamin analysis. Add the required amount of agarose after adjusting the pH to 5.2 and sterilize. After sterilization, the required amount of acetosyringone is added from the stock solution under sterile conditions.

Table 3. CCMC environment Chemical reagents final conc./liter Stock/1000 ml Quantity/liter 10X base salts CC KNO ₃ 1.212 g 12.12 g 100 ml _{NH4NO3 _} 0.640 g 6.40 g CaCl ₂ -2H ₂ O 0.588 g 5.88 g MgSO ₄ -7H ₂ O 0.247 g 2.47 g _{KH2PO4 _} 0.136 g 1.36 g 100X Fe EDTA FeSO ₄ -7H ₂ O 27.80 mg 2.78 g 10 ml EDTA, disodium salt 37.30 mg 3.73 g 100X micronutrient salts CC MnSO ₄ -4H ₂ O 11.150 mg 1.115 g 10 ml ZnSO ₄ -7H ₂ O 5.760 mg 0.576 g CuSO ₄ -5H ₂ O 0.025 mg 2,500 mg Na ₂ MoO ₄ -2H ₂ O 0.240 mcg 0.024 mg CoSO ₄ -7H ₂ O 0.028 mg 2,800 mg _{H3BO3 _} 3,100 mg 0.310 g KI 0.830 mg 0.083 g 100X vitamins CC Myo-inositol 90.00 mg 9,000 g 10 ml Thiamine hydrochloride 8.50 mg 0.850 g Pyridoxine hydrochloride 1.00 mg 0.100 g A nicotinic acid 6.00 mg 0.600 g Glycine 2.00 mg 0.200 g 2,4-D 2,000 mg 0.100 g 20 ml NAA 1.00 mg 0.100 g 10 ml 6BA 0.200 mg 0.100 g 2 ml Maltose 20,000 g - - Mannitol 36,000 g - - Proline 0.500 g - - Casmino acids, vitamin analysis 0.500 g - - Gelrite 5,000 g - - Cefotaxime 0.250 g 250 g 1.0 ml

[00303] Prepare separately 10X CC basic salt solution, 100X Fe EDTA solution, 100X CC trace element salt solution, 100X CC vitamin solution, 100 mg l ^-1 2,4-D solution, 100 mg l ^-1 NAA solution, 100 mg l ^-1 6BA solution, 250 g l ^-1 cefotaxime solution, and mix the required amount or the above stock solution with maltose, mannitol, proline and casamino acids for vitamin analysis. Add the required amount of gelrite after adjusting the pH to 5.8 and sterilize. After sterilization, the required amount of cefotaxime is added from the stock solution under sterile conditions.

Table 4. CCMCH50 environment (Hiei and Komari, 2008) Chemical reagents final conc./liter Stock/1000 ml Quantity/liter 10X base salts CC KNO ₃ 1.212 g 12.12 g 100 ml _{NH4NO3 _} 0.640 g 6.40 g CaCl ₂ -2H ₂ O 0.588 g 5.88 g MgSO ₄ -7H ₂ O 0.247 g 2.47 g _{KH2PO4 _} 0.136 g 1.36 g 100X Fe EDTA FeSO ₄ -7H ₂ O 27.80 mg 2.78 g 10 ml EDTA, disodium salt 37.30 mg 3.73 g 100X micronutrient salts CC MnSO ₄ -4H ₂ O 11.150 mg 1.115 g 10 ml ZnSO ₄ -7H ₂ O 5.760 mg 0.576 g CuSO ₄ -5H ₂ O 0.025 mg 2,500 mg Na ₂ MoO ₄ -2H ₂ O 0.240 mcg 0.024 mg CoSO ₄ -7H ₂ O 0.028 mg 2,800 mg _{H3BO3 _} 3,100 mg 0.310 g KI 0.830 mg 0.083 g 100X vitamins Myo-inositol 90.00 mg 9,000 g 10 ml Thiamine hydrochloride 8.50 mg 0.850 g Pyridoxine hydrochloride 1.00 mg 0.100 g A nicotinic acid 6.00 mg 0.600 g Glycine 2.00 mg 0.200 g 2,4-D 2,000 mg 0.100 g 20 ml NAA 1.00 mg 0.100 g 10 ml 6BA 0.200 mg 0.100 g 2 ml Maltose 20.00 g - - Mannitol 36,000 g - - Proline 0.500 g - - Casmino acids, vitamin analysis 0.500 g - - Agarose 8,000 g - - Cefotaxime 0.250 g 250 g 1.0 ml Hygromycin B 0.050 g 50 g 1.0 ml

[00304] Prepare separately 10X CC basic salt solution, 100X Fe EDTA solution, 100X CC trace element salt solution, 100X CC vitamin solution, 100 mg l ^-1 2,4-D solution, 100 mg l ^-1 NAA solution, 100 mg l ^-1 6BA solution, 50 g l ^-1 hygromycin B solution, and mix the required amount or the above stock solution with maltose, mannitol, proline and casamino acids for vitamin analysis. Add the required amount of agarose after adjusting the pH to 5.8 and sterilize. After sterilization, the required amount of 50 g l ^-1 solution of hygromycin B is added from the stock solution.

Table 5. NBPRCH40 medium (Hiei and Komari, 2008) Chemical reagents final conc./liter Stock/1000 ml Quantity/liter 10X base salts N6 KNO ₃ 2.830 g 28.30 g 100 ml CaCl ₂ -2H ₂ O 0.166 g 1.66 g MgSO ₄ -7H ₂ O 0.185 g 1.85 g _{KH2PO4 _} 0.400 g 4.00 g 100X Fe EDTA FeSO ₄ -7H ₂ O 27.80 mg 2.78 g 10 ml EDTA, disodium salt 37.30 mg 3.73 g 100X trace element salts B5 MnSO ₄ -4H ₂ O 13.20 mg 1.320 g 10 ml ZnSO ₄ -7H ₂ O 2.00 mg 0.200 g CuSO ₄ -5H ₂ O 25.00 mg 2.500 g Na ₂ MoO ₄ -2H ₂ O 0.25 mg 0.025 g CoCl ₂ -6H ₂ O 25.00 mg 2.500 g _{H3BO3 _} 3.00 mg 0.300 g KI 0.75 mg 0.075 g 100X vitamins B5 Myo-inositol 100.0 mg 10,000 g 10 ml Thiamine hydrochloride 10.0 mg 1,000 g Pyridoxine hydrochloride 1.0 mg 0.100 g A nicotinic acid 1.0 mg 0.100 g 2,4-D 2,000 mg 0.100 g 20 ml NAA 1,000 mg 0.100 g 10 ml 6BA 1,000 mg 0.100 g 10 ml Maltose 30,000 g - - Proline 0.500 g - - Casmino acids, vitamin analysis 0.500 g - - Agarose type I 8,000 g - - Glutamine 0.300 g 30.00 g 10 ml Cefotaxime 0.250 g 250.0 g 1.0 ml Hygromycin B 0.040 g 50.00 g 0.8 ml

[00305] Prepare separately 10X N6 basic salt solution, 100X Fe EDTA solution, 100X B5 trace element salt solution, 100X vitamin solution, 100 mg l ^-1 2,4-D solution, 100 mg l ^{-1 NAA solution, 100 mg l -1 6BA} solution, 30 g l ^-1 glutamine solution, 250 g l ^-1 solution cefotaxime, 50 g l ^-1 solution of hygromycin B, and mix the required amount or the above stock solution with maltose, proline and casamino acids for vitamin analysis. Add the required amount of agarose after adjusting the pH to 5.8 and sterilize. After sterilization, the required amount of glutamine, cefotaxime and hygromycin B is added from the stock solution under sterile conditions.

Table 6. RNMH30 medium (Hiei and Komari, 2008) Chemical reagents final conc./liter Stock/1000 ml Quantity/liter 10X base salts N6 KNO ₃ 2.830 g 28.30 g 100 ml CaCl ₂ -2H ₂ O 0.166 g 1.66 g MgSO ₄ -7H ₂ O 0.185 g 1.85 g _{KH2PO4 _} 0.400 g 4.00 g 100X Fe EDTA FeSO ₄ -7H ₂ O 27.80 mg 2.78 g 10 ml EDTA, disodium salt 37.30 mg 3.73 g 100X trace element salts B5 MnSO ₄ -4H ₂ O 13.20 mg 1.320 g 10 ml ZnSO ₄ -7H ₂ O 2.00 mg 0.200 g CuSO ₄ -5H ₂ O 25.00 mg 2.500 g Na ₂ MoO ₄ -2H ₂ O 0.25 mg 0.025 g CoCl ₂ -6H ₂ O 25.00 mg 2.500 g _{H3BO3 _} 3.00 mg 0.300 g KI 0.75 mg 0.075 g 100X vitamins B5 Myo-inositol 100.0 mg 10,000 g 10 ml Thiamine hydrochloride 10.0 mg 1,000 g Pyridoxine hydrochloride 1.0 mg 0.100 g A nicotinic acid 1.0 mg 0.100 g NAA 1,000 mg 0.100 g 10 ml 6BA 1,000 mg 0.100 g 10 ml Maltose 30,000 g - - Proline 0.500 g - - Casmino acids, vitamin analysis 0.500 g - - Agarose type I 7.500 g - - Glutamine 0.300 g 30.00 g 10 ml Cefotaxime 0.250 g 250 g 1.0 ml Hygromycin B 0.030 g 50 g 0.6 ml

[00306] Prepare separately 10X N6 basic salt solution, 100X Fe EDTA solution, 100X B5 trace element salt solution, 100X vitamin solution, 100 mg l ^-1 NAA solution, 100 mg l ^-1 6BA solution, 30 g l ^-1 glutamine solution, 250 g l ^-1 cefotaxime solution, 50 g l ^-1 ghee solution gromycin B, and mix the required amount or the above stock solution with maltose, proline and casamino acids for vitamin analysis. Add the required amount of agarose after adjusting the pH to 5.8 and sterilize. After sterilization, the required amount of glutamine, cefotaxime and hygromycin B is added from the stock solution under sterile conditions.

--->

SEQUENCE LIST

<110> DCM SRIRAM LIMITED

<120> CODON-OPTIMIZED SYNTHETIC NUCLEOTIDE

CRY2Ai PROTEIN ENCODING SEQUENCES AND THEIR APPLICATIONS

<130> PD033499IN-SC

<160> 13

<170>PatentIn version 3.5

<210> 1

<211> 633

<212> PROTEIN

<213> Artificial sequence

<220>

<223> SEQ ID NO: 1 is the amino acid sequence

Cry2Ai protein (NCBI GenBank: ACV97158.1)

<400> 1

Met Asn Asn Val Leu Asn Ser Gly Arg Asn Ile Thr Cys His Ala His

1 5 10 15

Asn Val Val Ala His Asp Pro Phe Ser Phe Glu His Lys Ser Leu Asn

20 25 30

Thr Ile Glu Lys Glu Trp Lys Glu Trp Lys Arg Thr Asp His Ser Leu

35 40 45

Tyr Val Ala Pro Ile Val Gly Thr Val Gly Ser Phe Leu Leu Lys Lys

50 55 60

Val Gly Ser Leu Val Gly Lys Arg Ile Leu Ser Glu Leu Gln Asn Leu

65 70 75 80

Ile Phe Pro Ser Gly Ser Ile Asp Leu Met Gln Glu Ile Leu Arg Ala

85 90 95

Thr Glu Gln Phe Ile Asn Gln Arg Leu Asn Ala Asp Thr Leu Gly Arg

100 105 110

Val Asn Ala Glu Leu Ala Gly Leu Gln Ala Asn Val Ala Glu Phe Asn

115 120 125

Arg Gln Val Asp Asn Phe Leu Asn Pro Asn Gln Asn Pro Val Pro Leu

130 135 140

Ala Ile Ile Asp Ser Val Asn Thr Leu Gln Gln Leu Phe Leu Ser Arg

145 150 155 160

Leu Pro Gln Phe Gln Ile Gln Gly Tyr Gln Leu Leu Leu Leu Pro Leu

165 170 175

Phe Ala Gln Ala Ala Asn Leu His Leu Ser Phe Ile Arg Asp Val Ile

180 185 190

Leu Asn Ala Asp Glu Trp Gly Ile Ser Ala Ala Thr Val Arg Thr Tyr

195 200 205

Arg Asp His Leu Arg Asn Phe Thr Arg Asp Tyr Ser Asn Tyr Cys Ile

210 215 220

Asn Thr Tyr Gln Thr Ala Phe Arg Gly Leu Asn Thr Arg Leu His Asp

225 230 235 240

Met Leu Glu Phe Arg Thr Tyr Met Phe Leu Asn Val Phe Glu Tyr Val

245 250 255

Ser Ile Trp Ser Leu Phe Lys Tyr Gln Ser Leu Met Val Ser Ser Gly

260 265 270

Ala Asn Leu Tyr Ala Ser Gly Ser Gly Pro Gln Gln Thr Gln Ser Phe

275 280 285

Thr Ala Gln Asn Trp Pro Phe Leu Tyr Ser Leu Phe Gln Val Asn Ser

290 295 300

Asn Tyr Ile Leu Ser Gly Ile Ser Gly Thr Arg Leu Ser Ile Thr Phe

305 310 315 320

Pro Asn Ile Gly Gly Leu Pro Gly Ser Thr Thr Thr His Ser Leu Asn

325 330 335

Ser Ala Arg Val Asn Tyr Ser Gly Gly Val Ser Ser Gly Leu Ile Gly

340 345 350

Ala Thr Asn Leu Asn His Asn Phe Asn Cys Ser Thr Val Leu Pro Pro

355 360 365

Leu Ser Thr Pro Phe Val Arg Ser Trp Leu Asp Ser Gly Thr Asp Arg

370 375 380

Glu Gly Val Ala Thr Ser Thr Asn Trp Gln Thr Glu Ser Phe Gln Thr

385 390 395 400

Thr Leu Ser Leu Arg Cys Gly Ala Phe Ser Ala Arg Gly Asn Ser Asn

405 410 415

Tyr Phe Pro Asp Tyr Phe Ile Arg Asn Ile Ser Gly Val Pro Leu Val

420 425 430

Ile Arg Asn Glu Asp Leu Thr Arg Pro Leu His Tyr Asn Gln Ile Arg

435 440 445

Asn Ile Glu Ser Pro Ser Gly Thr Pro Gly Gly Ala Arg Ala Tyr Leu

450 455 460

Val Ser Val His Asn Arg Lys Asn Asn Ile Tyr Ala Ala Asn Glu Tyr

465 470 475 480

Gly Thr Met Ile His Leu Ala Pro Glu Asp Tyr Thr Gly Phe Thr Ile

485 490 495

Ser Pro Ile His Ala Thr Gln Val Asn Asn Gln Thr Arg Thr Phe Ile

500 505 510

Ser Glu Lys Phe Gly Asn Gln Gly Asp Ser Leu Arg Phe Glu Gln Ser

515 520 525

Asn Thr Thr Ala Arg Tyr Thr Leu Arg Gly Asn Gly Asn Ser Tyr Asn

530 535 540

Leu Tyr Leu Arg Ala Ser Ser Ile Gly Asn Ser Thr Ile Arg Val Thr

545 550 555 560

Ile Asn Gly Arg Ala Tyr Thr Val Ser Asn Val Asn Thr Thr Thr Asn

565 570 575

Asn Asp Gly Val Asn Asp Asn Gly Ala Arg Phe Ser Asp Ile Asn Ile

580 585 590

Gly Asn Ile Val Ala Ser Asp Asn Thr Asn Val Thr Leu Asp Ile Asn

595 600 605

Val Thr Leu Asn Ser Gly Thr Pro Phe Asp Leu Met Asn Ile Met Phe

610 615 620

Val Pro Thr Asn Leu Pro Pro Leu Tyr

625 630

<210> 2

<211> 2041

<212> DNA

<213> Artificial sequence

<220>

<223> SEQ ID NO: 2 is a full size

codon-optimized synthetic sequence

cry2Ai DNA (201M1) encoding the Cry2Ai protein (SEQ ID NO: 1).

<400> 2

gcgcgccctg caggggccgg ccgtttaaac gcggccgcat ttaaaggtac ctactagacca 60

tggggatcca tgaataacgt cctgaatagt ggccgcaata tcacctgtca cgcccacaac 120

gttgtcgcac acgacccttt ctctttcgag cacaaatctc tgaacactat agagaaggag 180

tggaaagaat ggaaaagaac cgaccacagt ttgtatgtag cccctatcgt cgggactgtg 240

ggcagttttc tacttaagaa agtgggttcc ctggtcggca agcgtatttt atcagagttg 300

cagaacttga tttttccatc cggctccatc gatttgatgc aggagatcct tcgtgccacg 360

gaacagttca ttaatcagcg gctgaatgcg gacactctgg ggagggtcaa tgcggaactc 420

gccggactgc aagccaacgt agcggagttc aatagacagg tcgataactt cttgaaccct 480

aatcaaaacc cagtcccgct ggcaataatt gactccgtga ataccctgca acaactcttc 540

ctgtcccggt taccccagtt ccaaattcaa ggctaccagt tgctcttgct cccactcttc 600

gcacaagctg caaaccttca cttgtccttt atcagagatg tgatcctcaa cgccgatgag 660

tggggcatat cggctgctac ggttcggacc taccgggacc acctgcgcaa cttcacacgg 720

gactactcaa actattgtat caacacatat caaacagcgt ttcgcggatt gaacacacgc 780

ctgcacgaca tgctggagtt tcggacctac atgtttttga acgttttcga gtacgtgtcc 840

atatggtcct tgttcaaata tcaatcacta atggtctctt ctggcgctaa tctctatgcc 900

agcggttcag ggccccagca aacacaatcg tttacagccc aaaactggcc gttcctctac 960

agcctgttcc aggtgaatag caactacatt ctctccggca tttcaggtac acgactttcc 1020

ataactttcc caaacatagg cggcctgccc ggttcacga cgactcactc attaaacagc 1080

gcgagagtga actacagcgg cggtgttagt tctggtctca ttggggcaac caacctgaac 1140

cataatttca attgttcgac tgtcctacca cctttaagca ctcccttcgt ccgcagttgg 1200

1260

ttccaaacca ctctctccct ccgctgcgga gcgttttcag cacgaggcaa cagcaactat 1320

ttccctgact attttatccg taacattagc ggcgtcccgc tggtcataag aaatgaggac 1380

ttgactcgac ccctgcacta caatcagata cgcaacatcg aatccccttc gggaacacct 1440

gggggagcac gtgcttacct agtctcagta cacaaccgaa agaacaacat ctacgcggcc 1500

aatgagtacg ggaccatgat ccatctcgct cccgaggact acaccgggtt cacgatttct 1560

ccgatacatg ctacccaggt taataaccaa acccggactt tcatcagcga gaagttcggc 1620

aaccagggag attctctgag atttgagcaa agcaatacca cagctaggta caccctccgg 1680

ggaaatggca atagctataa cttgtatctg agggcgtctt caatcggcaa ttcaactatt 1740

agggtgacta ttaacggcag ggcatataca gtttctaatg tgaacacaac gactaacaac 1800

gatggcgtaa acgacaatgg ggcacgtttt tctgacatca atatagggaa catagtcgcc 1860

agtgataaca caaacgtgac actggatatt aacgtcaccc tcaactctgg tactccgttc 1920

gatctcatga acatcatgtt cgtacccaca aaccttcctc cgctttatta gggggctgaa 1980

ttcaagcttg agctctccgg agcgatcgcg agatcgcggc cggccgttta aacatttaaa 2040

t 2041

<210> 3

<211> 2041

<212> DNA

<213> Artificial sequence

<220>

<223> SEQ ID NO: 3 is a full size

codon-optimized synthetic sequence

cry2Ai DNA (201M2) encoding the Cry2Ai protein (SEQ ID NO: 1).

<400> 3

gcgcgccctg caggggccgg ccgtttaaac gcggccgcat ttaaaggtac ctactagacca 60

tggggatcca tgaataacgt gctgaacagc ggccgcaata tcacctgtca tgcccacaat 120

gttgtagccc acgacccttt ttccttcgag cacaaaagct tgaatacgat agagaaggaa 180

tggaaggagt ggaaaagaac cgaccatagt ttgtacgtcg caccaatcgt cgggactgtt 240

ggcagtttcc tacttaagaa agtgggttcc ctggtcggca agcgtatttt atcagagttg 300

cagaacttga tttttccatc cggctccatc gatttgatgc aggagatcct tcgtgccacg 360

gaacagttca ttaatcagcg gctgaatgcg gacactctgg ggagagtcaa tgcggagctt 420

gcaggactcc aagctaacgt cgcagagttc aacagacagg ttgataactt cctcaatccg 480

aatcagaacc cagtcccgct agccattata gactctgtca atactctcca acaactcttt 540

ttgtcccggt tacctcagtt ccaaattcaa ggctaccagc ttttgctgct accactcttt 600

gctcaagctg cgaacctcca cctgtcattc atcagagatg tcatcttgaa cgcggacgag 660

tggggcatat ccgctgcgac ggttcgcacc taccgggatc acctgcgtaa ttttacacga 720

gattacagca actattgcat caacacgtac cagacagcgt tccgaggatt gaacacaaga 780

ctacatgata tgctggagtt ccgaacctat atgtttttga acgtatttga gtatgtctcc 840

atttggtcat tgttcaaata tcaatcgtta atggtttctt ctggcgcgaa cctctatgcg 900

agcggcagtg gaccccagca aacacaatcc tttactgccc aaaattggcc gtttctctac 960

tcccttttcc aggtgaatag caactacata ctgtcggca

atcacctttc ctaacattgg agggctgccc gggtcaacaa cgacgcattc attgaacagt 1080

gcgagggtta attacagcgg cggagtaagt agcggactca tcggggcgac taacctcaac 1140

cacaacttta actgtagcac tgtgctacca cctttatcaa cacctttcgt gcggagttgg 1200

ctcgactcag gaacagatcg ggaaggcgtc gcgacgagca cgaattggca gactgagtcc 1260

ttccaaacaa ccctatctct ccggtgcggg gcgttttcag cgagaggcaa ctcgaactac 1320

tttcccgatt atttcatacg gaacatttct ggcgtccccc tggtcattag aaacgaagat 1380

ttgacgcgcc cactcacta caaccagata aggaacatag agtctccttc agggacacca 1440

ggaggcgctc gtgcgtacct agtctcagta cacaaccgaa agaacaacat ctacgcggcc 1500

aatgagtacg ggaccatgat ccatctcgct cccgaggact acaccgggtt cacgatttct 1560

ccgatacatg ctacccaggt taataaccaa acccggactt tcatcagcga gaagttcggc 1620

aaccagggcg actctctgag atttgagcaa agcaacacca ccgcccgata tacattacgg 1680

ggcaacggca acagttacaa cctctatctg cgtgcgtcct caataggcaa ctcaacgata 1740

agggtgacaa ttaacggtcg ggcgtataca gtgtcgaatg ttaataccac aacaaacaat 1800

gacggcgtca acgacaatgg tgcgcgtttt tctgatatta acattggcaa catcgtcgcc 1860

agtgacaaca ccaacgttac gttagacatc aacgtgacgc ttaactctgg cactcctttc 1920

gatcttatga acatcatgtt cgtgccgacc aatttgccgc cactctacta gggggctgaa 1980

ttcaagcttg agctctccgg agcgatcgcg agatcgcggc cggccgttta aacatttaaa 2040

t 2041

<210> 4

<211> 2041

<212> DNA

<213> Artificial sequence

<220>

<223> SEQ ID NO: 4 is a full size

codon-optimized synthetic sequence

cry2Ai DNA (201M3) encoding the Cry2Ai protein (SEQ ID NO: 1).

<400> 4

gcgcgccctg caggggccgg ccgtttaaac gcggccgcat ttaaaggtac ctactagacca 60

tggggatcca tgaataatgt gttgaacagt ggccgaaata taacttgcca cgcccacaac 120

gttgtcgcgc acgacccatt ttcgttcgag cacaagagtc tgaatacaat cgagaaggaa 180

tggaaagaat ggaaaagaac cgaccacagt ctatacgttg cacctattgt cgggactgtg 240

ggtagtttcc tacttaagaa agtgggttcc ctggtcggca agcgtatttt atcagagttg 300

cagaacttga tttttccatc cggctccatc gatttgatgc aggagatcct tcgtgccacg 360

gaacagttca ttaatcagcg gctgaatgcg gacactctgg ggcgggtcaa cgccgaacta 420

gcggggcttc aggctaacgt cgccgagttc aaccgacaag ttgataactt cctgaatccg 480

aaccagaacc cagtcccgct agctataatc gactccgtga acactttgca acaactattc 540

ctgtcccggt taccgcagtt ccaaattcaa ggctaccagc tactactgct accgctattc 600

gcccaagctg caaacctgca cttgagcttc atacgagatg tgatcttgaa tgccgacgag 660

tggggcatat ctgctgctac cgttcggacg tacagggatc acctcagaaa cttcacgcgg 720

gattattcta actactgcat caatacttac cagaccgcgt ttcgtggatt aaacacgaga 780

ctccacgata tgctagagtt tcggacctat atgtttttga acgttttcga atacgtgtcc 840

atctggtcat tgttcaaata ccaatcatta atggtgtctt ccggcgcgaa tctgtatgct 900

agcgggtcgg ggccccagca aacccaatcc tttacagccc aaaactggcc ttttttgtat 960

tccctattcc aagtgaatag caactacatc ctttccggca tttcagggac tcgcctctcc 1020

atcacgtttc caaacatagg aggcttgccc ggctctacca cgacccactc attgaactca 1080

1140

cataatttca actgctcaac tgtgctacca ccattaagca ctccttttgt cagaagttgg 1200

1260

tttcaaacta ccctgtctct gagatgtggt gcgttcagtg cccgtggcaa ttcaaactac 1320

tttcctgatt acttcattcg caatatctct ggcgtgcctc tggtcataag aaacgaggat 1380

ctcacacgac cactgcacta taaccagata cgcaacattg agtctccgag cggcactccg 1440

ggtggcgctc gtgcatacct agtctcagta cacaaccgaa agaacaacat ctacgcggcc 1500

aatgagtacg ggaccatgat ccatctcgct cccgaggact acaccgggtt cacgatttct 1560

ccgatacatg ctacccaggt taataaccaa acccggactt tcatcagcga gaagttcggc 1620

aaccagggcg acagcctacg atttgagcaa agcaacacca cggctcggta cacactccgg 1680

ggtaacggca attcttacaa cctctatctg cgtgcgtcgt caattggcaa ctcaacaatc 1740

agggtgacta tcaatggtcg ggcttatact gtctctaacg tcaatactac caccaataac 1800

gatggcgtga acgataacgg tgctcgtttc tctgacatca acatcggaaa tatcgtggct 1860

agtgacaaca ccaacgtcac tctcgatatt aacgtaacat tgaactctgg aacacctttc 1920

gatctgatga acattatgtt tgttcctact aacctaccgc cgctttacta gggggctgaa 1980

ttcaagcttg agctctccgg agcgatcgcg agatcgcggc cggccgttta aacatttaaa 2040

t 2041

<210> 5

<211> 2041

<212> DNA

<213> Artificial sequence

<220>

<223> SEQ ID NO: 5 is a full size

codon-optimized synthetic sequence

cry2Ai DNA (201M4) encoding the Cry2Ai protein (SEQ ID NO: 1).

<400> 5

gcgcgccctg caggggccgg ccgtttaaac gcggccgcat ttaaaggtac ctactagacca 60

tggggatcca tgaataatgt cttgaacagt ggccgaaata ttacgtgcca cgcccacaat 120

gttgtagcgc atgacccatt ctcgttcgag cacaaagct tgaacactat agagaaggaa 180

tggaaagagt ggaaaagaac cgaccacagt ctgtacgtcg cccccatcgt cgggactgtg 240

gggagtttcc tacttaagaa agtgggttcc ctggtcggca agcgtatttt atcagagttg 300

cagaacttga tttttccatc cggctccatc gatttgatgc aggagatcct tcgtgccacg 360

gaacagttca ttaatcagcg gctgaatgcg gacactctgg ggagggtgaa cgccgaactt 420

gccgggctgc aagcgaacgt cgcagagttt aacagacagg tggataattt cctgaaccct 480

aaccagaatc cagtcccgct tgccatcatt gactccgtta atacgctaca gcaacttttc 540

ctgtcccggt taccgcaatt ccaaattcaa ggctaccaac tcctgttgct cccgctgttt 600

gcacaggctg caaacctcca cctgtcattc attcgcgatg tgatcctgaa cgccgacgaa 660

tggggcatat ccgctgccac cgttcgtaca taccgggacc acctgcgtaa ctttactcgc 720

gattattcta actactgcat taacacatac cagacagcgt ttcggggctt gaatactaga 780

ctccacgata tgctggagtt ccgcacctat atgtttttga acgtattcga atatgtatcc 840

atttggtcct tgttcaaata tcaatcccta atggtatctt caggcgcgaa tctgtatgct 900

agcggtagtg ggccccagca aacccaatcc tttacagccc aaaactggcc gtttctgtac 960

agcctcttcc aggtgaatag caactacata ctgtccggca tttcaggaac gcggctcagt 1020

ataactttcc ccaatattgg cgggttgccc ggttctacca caacccattc attgaactca 1080

gctagagtga attacagcgg tggagtgtcg tcggggctca tcggggcgac taatcttaat 1140

cataacttta attgctcaac tgtgctacca cctttaagca cacctttcgt ccgcagttgg 1200

ctggattcgg gaacagatcg ggaaggagtc gccacatcca caaattggca aacagagtcg 1260

ttccagacca cactctcact gcggtgcgga gccttctcag cgagagggaa ctctaactac 1320

ttccctgatt actttattag gaacatatct ggcgtccccc tcgtcatcag aaacgaggac 1380

ttgacccgac cattacacta caaccagata cgtaatatag agtctccctc cggtacaccg 1440

ggcggtgcgc gtgcgtacct agtctcagta cacaaccgaa agaacaacat ctacgcggcc 1500

aatgagtacg ggaccatgat ccatctcgct cccgaggact acaccgggtt cacgatttct 1560

ccgatacatg ctacccaggt taataaccaa acccggactt tcatcagcga gaagttcggc 1620

aaccagggag acagcctcag attcgaacaa tccaacacta ccgctagata tacgcttcgc 1680

ggaaacggta atagctacaa cctctatttg agggcgtcct caataggcaa ttcaactatc 1740

cgggtgacta tcaatggaag ggcttatacg gtgtccaatg tcaatactac gaccaacaat 1800

gacggcgtca atgacaacgg cgcacgtttc tctgacatta acatcggcaa catcgtcgcg 1860

agtgacaaca caaacgtgac actcgacatc aacgtcacac ttaattctgg cacgccgttc 1920

gatctgatga acatcatgtt tgtccctact aacttacccc cactttacta gggggctgaa 1980

ttcaagcttg agctctccgg agcgatcgcg agatcgcggc cggccgttta aacatttaaa 2040

t 2041

<210> 6

<211> 2041

<212> DNA

<213> Artificial sequence

<220>

<223> SEQ ID NO: 6 is a full size

codon-optimized synthetic sequence

cry2Ai DNA (201M5) encoding the Cry2Ai protein (SEQ ID NO: 1).

<400> 6

gcgcgccctg caggggccgg ccgtttaaac gcggccgcat ttaaaggtac ctactagacca 60

tggggatcca tgaataacgt cctgaatagt ggccgcaata tcacctgtca cgcccacaac 120

gttgtcgcac acgacccttt ctctttcgag cacaaatctc tgaacactat agagaaggag 180

tggaaagaat ggaaaagaac cgaccacagt ttgtatgtag cccctatcgt cgggactgtg 240

ggcagttttc tacttaagaa agtgggttcc ctggtcggca agcgtatttt aagcgagctc 300

cagaacttga ttttcccatc cggctccatc gatttgatgc aggaaatact tcgtgcgact 360

gagcaattca ttaatcagcg gctgaatgcg gatactttgg gcagggtcaa tgcggaactc 420

gccggactgc aagccaacgt agcggagttc aatagacagg tcgataactt cttgaaccct 480

aatcaaaacc cagtcccgct ggcaataatt gactccgtga ataccctgca acaactcttc 540

ctgtcccggt taccccagtt ccaaattcaa ggctaccagt tgctcttgct cccactcttc 600

gcacaagctg caaaccttca cttgtccttt atcagagatg tgatcctcaa cgccgatgag 660

tggggcatat cggctgctac ggttcggacc taccgggacc acctgcgcaa cttcacacgg 720

gactactcaa actattgtat caacacatat caaacagcgt ttcgcggatt gaacacacgc 780

ctgcacgaca tgctggagtt tcggacctac atgtttttga acgttttcga gtacgtgtcc 840

atatggtcct tgttcaaata tcaatcacta atggtctctt ctggcgctaa tctctatgcc 900

agcggttcag ggccccagca aacacaatcg tttacagccc aaaactggcc gttcctctac 960

agcctgttcc aggtgaatag caactacatt ctctccggca tttcaggtac acgactttcc 1020

ataactttcc caaacatagg cggcctgccc ggttcacga cgactcactc attaaacagc 1080

gcgagagtga actacagcgg cggtgttagt tctggtctca ttggggcaac caacctgaac 1140

cataatttca attgttcgac tgtcctacca cctttaagca ctcccttcgt ccgcagttgg 1200

1260

ttccaaacca ctctctccct ccgctgcgga gcgttttcag cacgaggcaa cagcaactat 1320

ttccctgact attttatccg taacattagc ggcgtcccgc tggtcataag aaatgaggac 1380

ttgactcgac ccctgcacta caatcagata cgcaacatcg aatccccttc gggaacacct 1440

gggggagcac gtgcttacct agtctcagta cacaaccgaa agaacaacat ctacgcggcc 1500

aatgagtacg ggaccatgat ccatctcgct cccgaggact acaccgggtt cacgatttct 1560

ccgatacatg ctacccaggt taataaccaa acccggactt tcatcagcga gaagttcggc 1620

aaccagggag attctctgag atttgagcaa agcaatacca cagctaggta caccctccgg 1680

ggaaatggca atagctataa cttgtatctg agggcgtctt caatcggcaa ttcaactatt 1740

agggtgacta ttaacggcag ggcatataca gtttctaatg tgaacacaac gactaacaac 1800

gatggcgtaa acgacaatgg ggcacgtttt tctgacatca atatagggaa catagtcgcc 1860

agtgataaca caaacgtgac actggatatt aacgtcaccc tcaactctgg tactccgttc 1920

gatctcatga acatcatgtt cgtacccaca aaccttcctc cgctttatta gggggctgaa 1980

ttcaagcttg agctctccgg agcgatcgcg agatcgcggc cggccgttta aacatttaaa 2040

t 2041

<210> 7

<211> 2041

<212> DNA

<213> Artificial sequence

<220>

<223> SEQ ID NO: 7 is a full size

codon-optimized synthetic sequence

cry2Ai DNA (201M6) encoding the Cry2Ai protein (SEQ ID NO: 1).

<400> 7

gcgcgccctg caggggccgg ccgtttaaac gcggccgcat ttaaaggtac ctactagacca 60

tggggatcca tgaataacgt cctgaatagt ggccgcaata tcacctgtca cgcccacaac 120

gttgtcgcac acgacccttt ctctttcgag cacaaatctc tgaacactat agagaaggag 180

tggaaagaat ggaaaagaac cgaccacagt ttgtatgtag cccctatcgt cgggactgtg 240

ggcagttttc tacttaagaa agtgggttcc ctggtcggca agcgtatttt atcagagttg 300

cagaacttga tttttccatc cggctccatc gatttgatgc aggagatcct tcgtgccacg 360

gaacagttca ttaatcagcg gctgaatgcg gacactctgg ggagggtcaa tgcggaactc 420

gccggactgc aagccaacgt agcggagttc aatagacagg tcgataactt cttgaaccct 480

aatcaaaacc cagtcccgct ggcaataatt gactccgtga ataccctgca acaactcttc 540

ctgtcccggt taccccagtt ccaaattcaa ggctaccagt tgctcttgct cccactcttc 600

gcacaagctg caaaccttca cttgtccttt atcagagatg tgatcctcaa cgccgatgag 660

tggggcatat cggctgctac ggttcggacc taccgggacc acctgcgcaa cttcacacgg 720

gactactcaa actattgtat caacacatat caaacagcgt ttcgcggatt gaacacacgc 780

ctgcacgaca tgctggagtt tcggacctac atgtttttga acgttttcga gtacgtgtcc 840

atatggtcct tgttcaaata tcaatcacta atggtctctt ctggcgctaa tctctatgcc 900

agcggttcag ggccccagca aacacaatcg tttacagccc aaaactggcc gttcctctac 960

agcctgttcc aggtgaatag caactacatt ctctccggca tttcaggtac acgactttcc 1020

ataactttcc caaacatagg cggcctgccc ggttcacga cgactcactc attaaacagc 1080

gcgagagtga actacagcgg cggtgttagt tctggtctca ttggggcaac caacctgaac 1140

cataatttca attgttcgac tgtcctacca cctttaagca ctcccttcgt ccgcagttgg 1200

1260

ttccaaacca ctctctccct ccgctgcgga gcgttttcag cacgaggcaa cagcaactat 1320

ttccctgact attttatccg taacattagc ggcgtcccgc tggtcataag aaatgaggac 1380

ttgactcgac ccctgcacta caatcagata cgcaacatcg aatccccttc gggaacacct 1440

gggggagcac gtgcttacct agtgtctgtt cacaaccgaa agaacaacat ctacgcggcc 1500

aatgagtacg gcaccatgat ccatctcgct cccgaggact atacgggctt cacgatttct 1560

ccgatacatg ctacccaggt taacaatcag acccggacat ttattagcga gaagttcggc 1620

aatcaaggag attctctgag gttcgaacag agcaatacca cagcacggta caccctccgg 1680

ggaaatggca atagctataa cttgtatctg agggcgtctt caatcggcaa ttcaactatt 1740

agggtgacta ttaacggcag ggcatataca gtttctaatg tgaacacaac gactaacaac 1800

gatggcgtaa acgacaatgg ggcacgtttt tctgacatca atatagggaa catagtcgcc 1860

agtgataaca caaacgtgac actggatatt aacgtcaccc tcaactctgg tactccgttc 1920

gatctcatga acatcatgtt cgtacccaca aaccttcctc cgctttatta gggggctgaa 1980

ttcaagcttg agctctccgg agcgatcgcg agatcgcggc cggccgttta aacatttaaa 2040

t 2041

<210> 8

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> SEQ ID NO: 8 - Forward amplification primer

nucleotide sequence 201M1 (SEQ ID NO: 2)

<400> 8

aagaaagtgg gttccctggt 20

<210> 9

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> SEQ ID NO: 9 - Reverse primer for amplification

nucleotide sequence 201M1 (SEQ ID NO: 2)

<400> 9

tctctgtctg ccaattcgtg 20

<210> 10

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> SEQ ID NO: 10 - Forward primer for nptII gene amplification

<400> 10

ctgatgctct tcgtccagat 20

<210> 11

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> SEQ ID NO: 11 - Reverse primer for nptII gene amplification

<400> 11

agaggctatt cggctatgac t 21

<210> 12

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> SEQ ID NO: 12 Forward primer for amplification of the hptII gene

<400> 12

gctgttatgc ggccattggt c 21

<210> 13

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> SEQ ID NO: 13 Reverse primer for amplification of the hptII gene

<400> 13

gacgtctgtc gagaagtttg 20

<---

Claims (26)

1. A codon-optimized synthetic polynucleotide encoding a protein having the amino acid sequence shown in SEQ ID NO: 1, where the specified polynucleotide has the nucleotide sequence shown in SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 or SEQ ID NO: 7, or its complementary nucleotide sequence. 2. Recombinant DNA encoding the Cry2Ai insecticidal protein, containing the codon-optimized synthetic polynucleotide according to claim 1, wherein the polynucleotide is operably linked to a heterologous regulatory element. 3. The recombinant DNA of claim 2, wherein said codon-optimized synthetic polynucleotide optionally contains a selectable marker gene, a reporter gene, or a combination thereof. 4. The recombinant DNA of claim 2, wherein said codon-optimized synthetic polynucleotide optionally contains a DNA sequence encoding a guide or transit peptide for direction to a vacuole, mitochondria, chloroplast, plastid, or for secretion. 5. DNA construct for the expression of the Cry2Ai insecticidal protein, containing a 5'-untranslated sequence encoding a sequence encoding the Cry2Ai insecticidal protein containing the amino acid sequence of SEQ ID NO: 1, and a 3'-untranslated region, where the specified 5'-untranslated sequence contains a promoter functional in a plant cell, where the specified coding sequence is a codon-optimized a synthetic polynucleotide according to claim 1, and wherein said 3'-untranslated sequence contains a transcription termination sequence and a polyadenylation signal. 6. An expression plasmid vector containing recombinant DNA according to claim 2 or a DNA construct according to claim 5. 7. Host cell for the expression of the Cry2Ai insecticidal protein, containing the codon-optimized synthetic polynucleotide according to claim 1. 8. A host cell according to claim 7, which is a plant cell, bacterial cell, virus, fungal or yeast cell. 9. The host cell of claim 8, wherein said bacterial cell is an Agrobacterium or E. coli cell. 10. A method for producing a transgenic plant resistant to Scirpophaga incertulas , Spodoptera Mauritia and H. Armigera insects, comprising a) introducing into the plant cell a codon-optimized synthetic polynucleotide according to claim 1, wherein the polynucleotide is operably linked to (i) a promoter functional in the plant cell and (ii) a terminator; b) obtaining a transformed plant cell from a plant cell of step (a), wherein said transformed plant cell contains said codon-optimized synthetic polynucleotide according to claim 1; And c) obtaining a transgenic plant from said transformed plant cell of step (b), wherein said transgenic plant contains said codon-optimized synthetic polynucleotide according to claim 1. 11. Transgenic plant resistant to insects Scirpophaga incertulas , Spodoptera Mauritia and H. Armigera obtained by the method of claim 10, containing a codon-optimized synthetic polynucleotide according to claim 1. 12. Transgenic plant resistant to Scirpophaga incertulas , Spodoptera Mauritia and H. Armigera insects containing a codon-optimized synthetic polynucleotide according to claim 1. 13. A transgenic plant according to claim 11 or 12, selected from the group consisting of rice, wheat, corn, sorghum, oats, millet, legumes, cotton, tomato, eggplant, cabbage, cauliflower, broccoli, Brassica species, beans, peas, pigeon peas, potatoes, peppers, cucurbits, lettuce, sweet potatoes, canola, soy, alfalfa, peanut, sunflower, safflower, tobacco, sugarcane, cassava, coffee, pineapple, citrus, cocoa, tea, banana and melon. 14. Tissue resistant to insects Scirpophaga incertulas , Spodoptera Mauritia and H. Armigera obtained from the transgenic plant according to claim 11 or 12, wherein said tissue contains a codon-optimized synthetic polynucleotide according to claim 1. 15. Seed resistant to insects Scirpophaga incertulas , Spodoptera Mauritia and H. Armigera obtained from a transgenic plant according to claim 11 or 12, wherein said seed contains a codon-optimized synthetic polynucleotide according to claim 1. 16. A biological sample resistant to the insects of Scirpophaga incertulas , Spodoptera Mauritia and H. Armigera obtained from the tissue of claim 14 or the seed of claim 15, which contains a detectable amount of said codon-optimized synthetic polynucleotide of claim 1. 17. Commercial product resistant to Scirpophaga incertulas , Spodoptera Mauritia and H. Armigera insects, obtained from the transgenic plant according to claim 11 or 12, which contains a detectable amount of said codon-optimized synthetic polynucleotide according to claim 1. 18. An insecticidal composition against insects of Scirpophaga incertulas , Spodoptera Mauritia and H. Armigera , containing an effective amount of Bacillus thuringiensis containing a codon-optimized synthetic polynucleotide according to claim 1, encoding a Cry2Ai protein having the amino acid sequence shown in SEQ ID NO: 1. 19. The composition of claim 18, which optionally contains an additional insecticidal agent that is toxic to the same pest but has a different insecticidal mechanism of action than said insecticidal protein. 20. The composition of claim 19, wherein said insecticidal agent is selected from the group consisting of Bacillus toxin, Xenorhabdus toxin, Photorhabdus toxin, and a dsRNA that specifically represses one or more important genes in said insect pest. 21. A method for controlling insects of Scirpophaga incertulas, Spodoptera Mauritia and H. Armigera that infect agricultural plants and ensuring resistance of agricultural plants to insects of Scirpophaga incertulas, Spodoptera Mauritia and H. Armigera, including contacting the specified agricultural plant with an effective insecticidal amount of the composition according to claim 18. 22. Use of the codon-optimized synthetic polynucleotide of claim 1, the DNA construct of claim 5, or the plasmid vector of claim 6 for the production of insect-resistant transgenic plants that are resistant to the insects of Scirpophaga incertulas , Spodoptera Mauritia , and H. armigera . 23. The use of a codon-optimized synthetic polynucleotide according to claim 1 for the production of an insecticidal composition which contains Bacillus thuringiensis cells containing said polynucleotide and wherein the insecticidal composition is effective against Scirpophaga incertulas , Spodoptera Mauritia and H. Armigera insects.

Images