F0RM2
THE PATENTS ACT, 1970
(39 of 1970)
&
The Patents Rules, 2003
COMPLETE SPECIFICATION
(See section 10; rule 13)
1. Title of the invention. - SYNTHETIC DNA MOLECULES ENCODING
DELTA ENDOTOXIN PROTEINS IMPARTING RESISTANCE AGAINST INSECT PESTS IN PLANTS"
2. Applicants)
(a) NAME : KRISHIDHAN RESEARCH FOUNDATION
PRIVATE LIMITED
(b) NATIONALITY: An Indian Company.
(c) ADDRESS: Krishidhan Bhavan, Plot No. 3-D6, Additional MIDC,
Jalna - 431 213, Maharashfra, India
3. PREAMBLE TO THE DESCRIPTION
The following specification particularly describes the invention and the manner in which it is to be performed:

FIELD OF THE INVENTION
The present invention relates to the field of plant genetic engineering, for the purpose of developing transgenic plants improved for insect resistance. Provided are novel, isolated, synthetic nucleotide sequences encoding modified CrylAc pesticidal protein(s), engineered for efficient expression in plants. The invention further provides a method for developing such nucleotide sequence(s) and its use in developing insect resistant transgenic plants. More specifically, the invention provides synthetic DNA molecules encoding three domain CrylAc delta endotoxin protein toxin, truncated for C-terminal half but having 56 amino acids extra for in-vivo protein stability, for developing insect resistant transgenic plants. The said synthetic crylAc gene, comprised of DNA sequence of SEQ ID No. 1 is modified for high transcription, increased transcript stability, removal of polyadenylation signals and incorporation of intron in the mid of the coding sequence for selective and efficient expression in higher plants. Additionally, a variant of SEQ ID No. 1, represented in SEQ ID No. 2, comprised of removal of first 56 amino acids and substitution of two amino acids, is presented for modified crylAc gene truncated at both N-terminal and C-terminal to compare the efficacy of these two CrylAc proteins against random mating population of target insects. Both the synthetic nucleic acid sequences of the present invention encoding modified crylAc protein (s) also possess significant advantages of optimized plant codons for fidel expression and encodes protein with high in-vivo stability (because of retention of extra 56 amino acids at C-terminal) in the plant cell milieu. The encoded proteins possess four tryptic sites at C-terminal allowing rapid and fidel conversion of protoxin to active toxin which resultantry leads to efficient action of the toxin protein in the insect mid gut.
BACKGROUND OF THE INVENTION:
Bacillus thuringiensis (Bt) is an endospore forming, Gram-positive, aerobic soil bacterium characterized by the ability to produce crystalline protein inclusions within the cytoplasm of sporulating cell. The proteins within these crystalline inclusions are known as d-endotoxins including crystal proteins (Cry proteins) and cytolysins (Cyt). No other protein as toxic to target pests as d-endotoxins, yet as specific as these and therefore safe to non- target organisms as well as plants is known.
The Cry proteins exhibit insecticidal activity against lepidopteran, dipteran and coleopteran insect larvae (reviewed in Hofte and Whiteley, 1989, Microbiol. Rev. 53: 242-255). Most of the cry genes

reside on plasmids (Gonzalez et al.,1981, Plasmid 5: 351-365), often as a part of composite structure that includes mobile genetic elements (Lereclus et al., 1983, Mol. Gert.Genet. 191: 307-313). The Bt - Cry proteins constitute a family of related proteins, for which over 612 genes have been described and have recently been reclassified into 70 major classes (Crickmore, updated 2012, website : http : // www.biols.susx.ac.uk/Home / NeilCrickmore / Bt / index.html), primarily on the basis of amino acid sequence homology. The different toxins have different specificities for different orders of insects although the susceptibility of different species within a susceptible order varies enormously (Slaney et al.,1992, Insect, Biochem, Mol.Biol. 22: 9-18). The CrylA class of proteins are a group of Bt toxins that kill important insects of Lepidoptera. Out of these Cry 1 Ac is the protein which show highest efficacy against Heliothines, particularly Heliothis virescens, Helicoverpa armigera etc.
Chemical pesticides like synthetic pyrethroids have been effective in controlling insect pests, but there is growing fear that development of resistance by the insects may soon make pyrethroids also ineffective. Bt- based spray formulations have also been used in past to control and manage insect pests. However, an attractive alternative is the expression of proteins like Bt toxins with target insect specific activity in the crop plant itself. Genetic engineering can pave way to produce such transgenic plants by assessing a much wider gene pool for novel insect resistance characters not present in any of the plant species or their close relatives.
Transgenic plants expressing native cry genes in different crop species have been developed as early as 1987 (tobacco-Barton etal., 1987, Cell 32: 1033-1043; Vaecketal.,1987, Nature 327: 33-37 and Adang et aL,1987, Molecular strategies for crop protection: UCLA symposia on molecular and cellular biology, New Series, Alan R Liss, New York. 46: 345-353; tomato- Fischhoff et al.,1987, Bio/Tech. 5: 807-813 ). The level of expression of native cry gene from nucleus was very poor in these plants, although they have been shown to exhibit mortality of Manduca sexta larvae. However, many of the serious lepidopteran pests needed greater amount of plant expressed Bt-toxin for effective control. Native cry genes also have poor coding capacity in plants (Murray et al., 1991, Plant Mol. Biol. 16:1035-1050).
Subsequent group of workers have engineered and improved the d-endotoxin genes for achieving high level expression in plants. A number of strategies have been used to increase the expression of Bt genes in plants including use of plant preferred codon usage (Fujimoto et al., 1993, Bio/Tech. 11:1151-1155; Perlak et al, 1990, Bio / Tech. 8: 939 -943 etc), removal of polyadenylation signals from the coding sequence (Kumar PA, 2010, Indian patent IN 242768) as well as expression of toxin genes in chloroplasts (McBride et al., 1995, Bio/Tech. 15: 362-365) etc.

However, none of the strategy has been found to be absolutely effective in increasing the expression of gene and efforts are still going on in the domain of insecticidal proteins to achieve nucleotide sequences which have high expression efficiency in plants so as to ensure high toxicity against insects.
Gene fragments from some Bt strains, encoding insecticidal proteins, have been identified and integrated into plant genomes in order to provide the insect-resistant plants. In order to achieve optimal expression of an insecticidal protein in plant cells, it was found necessary to engineer each Bt gene fragment in a specific manner so that it encodes a part of a Bt protoxin that retains substantial toxicity against its target insects (known through European patent application ("EPA") 86/300,291.1 and 88/402,115.5; U.S. patent application Ser. No. 821,582).
U.S. Pat. No. 6,500,617 provides methods of obtaining pest resistance genes that are improved over naturally occurring genes for use in conferring upon plants resistance to pests. The methods involve the use of DNA shuffling of pest resistance genes to produce libraries of recombinant pest resistance genes, which are then screened to identify those that exhibit the improved property or properties of interest.
In the case of Cry 1 family of delta endotoxins, use of genes encoding C-terminal half truncated protein in the transgenics development program is known in the prior art. With relation to Cry 1 Ac delta endotoxin, wherever, a truncated protein has been used, protein of length upto 615 amino acids have been used (613 aa long protein, Adang et al.1996; 615 aa long protein, Perlak et al., 1990,608 aa long protein, Kaur S& Kumari GK-NCBIADT 61968.1). In most of these prior art, however, trypsin site around 603 aa of native protein was considered for processing of toxin to active state.
Moreover, introns have been used in plant genetic engineering prior art to increase the expression of a prokaryotic gene in eukaryotes like plants. A good example is use of gusA reporter gene with intron to transform rice. (Hiei et al., 1994, Plant J. 6: 271-282). US Patent no. 6114608 describes the use of maize alcohol dehydrogenase intron in the start of a cryl Ab gene to produce transgenic maize improved for insect resistance. Introns have mostly been used in the first domain or start of the gene to increase the expression of a particular gene in the transgenics.
Thus, there exists need of nucleotide sequences encoding insecticidal protein which exhibit higher expression efficiency, along with achieving other advantages like high insecticidal toxicity. These features are: selection of a stable truncated amino acid length of amino acids from native Cryl Ac

protein ensuring more number of tryptic sites at C-terminal for better processing and efficacy in terms of action on target insects and a synthetic gene to encode this truncated protein, the synthetic gene encompassing all known features of gene designing like high GC content, removal of polyadenylation signals etc along with purposeful interventions like codon optimization in way where only most preferred codons have been used achieving a CAI value suitable for both monocotyledonous and dicotyledonous group of plants, inclusion of intron in the middle of the coding sequence and a unique nucleotide composition..
QBJECTQFTHE INVENTION
A basic object of the present invention is to overcome the disadvantages/drawbacks of the known
art.
Another basic object of the invention is to provide compositions of DNA molecules encoding
Cry 1 Ac delta endotoxin proteins and associated methods to create such type of DNA molecules and
use of these DNA molecules in plants to confer pesticidal activity to plants, plant cells, tissues and
seeds.
Yet another object of the present invention is to provide synthetic DNA sequence or variant thereof,
represented by SEQ ID No.l and SEQ ID No.2 encoding Cryl Ac delta endotoxins, modified for
exhibiting selective and higher expression level in plants.
Yet another object of the present invention is to provide synthetic DNA sequence or variant thereof
encoding Cryl Ac delta endotoxin with high in-vivo stability.
Yet another object of the present invention is to provide synthetic nucleotide sequence or variant
thereof which has higher GC content.
Yet another object of the present invention is to provide synthetic nucleotide sequence or variant
thereof with higher Codon Adaptation Index (CAI) value for monocot as well as dicot plants
Yet anotfier object of the present invention is to provide synthetic DNA sequence or variant thereof
comprising intron in the middle of the coding region.
Yet another object of the present invention is to provide synthetic nucleotide sequence or variant
thereof encoding truncated Cryl Ac delta endotoxin comprising large number of tryptic sites at C-
terminalto ensure rapid and fidel conversion of protoxin to active toxin core and resultantly causes
better processing of the toxin protein in the insect mid gut.
Yet another object of the invention is to provide method for designing and producing synthetic
nucleotide sequence or variant thereof encoding Cryl Ac delta endotoxin having higher expression
level in plants.
Yet another object of the present invention is to demonstrate expression of 1he synthetic genes
represented by SEQ ID No. 1 and SEQ ID No. 2 in transgenic plants, wherein said transgenic

plants get improved for insecticidal activity comprising synthetic nucleotide sequence or variant thereof represented by SEQ ID No.l or SEQ ID No. 2.
Yet another object of the invention is to show the comparative efficiency of the two truncated Cryl Ac toxin proteins (One having N-terminal unchanged and other having 56 amino acids deleted from N-terminal, both having 56 amino acids extra at C-terminal, encoded by genes represented in SEQ ID No. 1 and SEQ ID No.2 respectively) against Helicoverpa armigera and other stem, shoot and fruit borers' insects larvae.
SUMMARY OF THE INVENTION
The invention provides synthetic DNA molecules that encode different point truncated three domain delta endotoxin CrylAc proteins and using the same for genetic engineering of crop plants to develop insect resistant transgenic plants. More specifically, isolated, synthetic DNA molecules sequences as set forth in SEQ ID No. 1 and SEQ ID No.2 are provided that encode different point truncated CrylAc proteins. Preferably, nucleotide sequences that are complementary to the SEQ ID Nos. 1 and / or 2 of this invention, or any sequence that hybridize to the nucleic acid sequence of invention are also encompassed. Methods are disclosed to create and / or design such DNA molecules as mentioned in invention to produce a pesticidal three domain CrylAc protein, their transfer to appropriate vectors and ultimately to plant cells and tissues, and for using those pesticidal proteins for controlling or killing Lepidopetran pests comprising of stem, shoot and fruit borers and more specifically Helicoverpa armigera, Leucinodes orbonalis or Chilo partellus. The isolated, synthetic gene(s) encoding different point truncation CrylAc proteins were designed in-silico to incorporate features like plant preferred codon usage, intron in the coding sequence, a comparatively higher GC content, removal of polyadenylation signals for eukaryotic expression and extra 56 amino acids at the C-terminal end. The extra 56 amino acids at C-terminal of these proteins provide an efficient in-vivo stability (at the time of its expression and folding in plants) and higher number of tryptic sites, allowing for fidel conversion of protoxin to toxin core in the insect mid gut which resuhantly leads to efficacy of the plant expressed toxin protein against target insects.
These and other advantages of the present invention will become readily apparent from the following detailed description.
BRIEF DESCRIPTION OF THE DRAWINGS:
Figure 1: A. domain architecture of native cryl Ac protein,

B. Sequence architecture and important features of modified crylAc gene-MV of the
present invention,
C. Fragment "X" for modified crylAc gene-V2a cloning, sequence represented in SEQ
ID No. 3.
Figure 2: A. Plasmid map of plant transformation vector pKRI-57 IN, B. Plasmid map of plant transformation vector pKRI-572N. Figure 3: PCR of modified cry I Ac-MV gene from transgenic tobacco
Figure 4: Insect bioassay of transgenic tobacco (with modified cryl Ac-MV gene) with Helicoverpa armigera larva.
Figure 5: PCR of modified cryl Ac-V2a from transgenic tobacco
Figure 6: Insect bioassay of transgenic tobacco (with modified cry lAc-V2a gene) with Helicoverpa armigera larvae.
Figure 7: Insect bioassay with Helicoverpa armigera larvae on artificial diet mixed with plant expressed modified Cryl Ac-MV protein.
Figure 8: Dose response of Helicoverpa armigera surviving larvae on artificial diet mixed with purified modified Cryl Ac-MV protein (A) and artificial diet mixed with purified modified Cryl Ac-V2a protein (B).
Figure 9: Leaf painting of purified proteins based bioassay experiment with Helicoverpa armigera 1st instar larvae.
DETAILED DESCRIPTION OF THE INVENTION:
The invention disclosed provides compositions and methods for conferring protection against damage by insect pests to plants. Compositions comprise of synthetic DNA molecules encoding three domain CrylAc delta endotoxin proteins and methods encompass methods to create such type of DNA molecules and methods to confer pesticidal activity to plants, plant cells, tissues and seeds comprising such synthetic DNA molecules encoding CrylAc protein as mentioned in this invention. It also includes vectors comprising these DNA molecules and host cells comprising the vectors. Accordingly, isolated synthetic DNA molecules, sequences as mentioned in SEQ ID No. 1 or SEQ ID No. 2 are provided that encode different point truncated three domain cryl Ac proteins and which may be used to develop insect resistant transgenic plants expressing crylAc delta endotoxin protein for controlling or killing Lepidopetran pests comprising of stem, shoot and fruit borers, more specifically from species of Helicoverpa armigera, Leucinodes orbonalis or Chilo partellus.
Definitions
The invention is to be further understood in accordance with the following definitions. All technical

and scientific terms used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which invention pertains, unless stated otherwise. It is also to be
understood that the terminology used herein is for the purpose of describing particular embodiments
only, and is not intended to be limiting.
As referred herein the term "polypeptide sequence" is intended for an amino acid sequence, which
is a polymer of amino acids or a character string representing an amino acid polymer, depending on
context; used interchangeably with "protein" or "polypeptide".
Similarly by the term "isolated" polypeptide or nucleic acid is meant it is partially or completely
separated from components with which it is normally associated (other proteins, genes, nucleic
acids, molecules, cells or biochemical reagents etc).
"Nucleic acid molecule" is intended to include DNA molecules (e.g. cDNA, genomic DNA,
recombinant DNA or synthetic DNA) and RNA molecules (e.g. mRNA) and analogs of the DNA or
RNA generated using nucleotide analogs. The nucleic acid molecule can be single-stranded or
double-stranded. A nucleic acid or a polypeptide is "recombinant", when it is artificial or
engineered, or derived from artificial or engineered protein or nucleic acid.
Nucleotide sequences encoding the Cry 1 Ac delta endotoxin proteins of the present invention
include the sequences set forth in SEQ ID no. 1, SEQ ID No. 1 (i), SEQ ID No. 2 and SEQ ID No. 2
(i) and complementary sequence thereof. By "complementary" is intended to two nucleotide
sequences which comprise antiparallel nucleotide sequences capable of pairing with one another
upon formation of hydrogen bonds between the complementary base residues in the antiparallel
nucleotide sequences.
"Antiparallel" refers herein to two nucleotide sequences paired through hydrogen bonds between
complementary base residues with phosphodiester bonds running in the 5-3' direction in one
nucleotide sequence and in the 3-5' direction in the other nucleotide sequence.
"GC" content is the percentage of Guanosine and Cytosine nucleotides in the DNA.
By the term "codon adapatation index" or "CAP' is meant a value ranging from 0 to 1 which
represents fitness of nucleotide sequence open reading frame codons for expression in a particular
organism expression system and calculated on the basis of codon frequencies of highly expressed
genes in that particular organism, thereby representing high CAI means better expression in that
system and vice versa.
For the purpose of this invention, "Vector" refers to a nucleic acid construct designed for transfer
between different host cells containing the said construct under control of regulatory elements
required for expression in a particular host system.
As referred herein, native CrylAc protein stands for insecticidal delta endotoxin protein of CrylA
subfamily and specifically naturally existing CrylAc protein present in natural isolates of Bacillus

thurtngiensis strains.
Similarly a "truncated" delta endototoxin protein refers to C-terminal half removed naturally
existing Cryl Ac protein protoxin, but retaining the toxin core of the protein.
As mentioned herein, "three domain" truncated delta endotoxin signifies N-terminal half portion of
native Cryl Ac protoxin containing all the three domains required for toxin core action in insects.
As referred herein, "modified" Cryl Ac protein stands for a truncated three domain CrylAc protein
designed from native CrylAc protein and incorporating some olher amino acids changes (like 56
amino acids extra at C-terminal and some mutations). Similarly "modified cryl Ac" gene (s) of the
present invention refers to DNA sequence (s) encoding modified CrylAc protein of the invention. It
also refers to all the changes and engineering done at nucleic acid sequence level to enhance its
expression in plants.
Bt proteins and encoding genes from Bacillus thuringiensis are named and classified on the basis of
amino acid sequence homology of the domains. Thus standard nomenclature of naturally existing Bt
Cry protein from Bt strain HD-73, selected for interventions and modifications in this invention is
CrylAc. As different versions of the CrylAc protein have been invented in this invention, for the
sake of clarity they have been suffixed with version codes. Thus for the purpose of this invention,
modified crylAc-MV gene (MV stands for main version) contains an intron and codes for a 659
amino acids long protein. Similarly, modified crylAc-V2a (V2a stands for Version 2a) also contains
an intron but codes for a 603 amino acids long protein.
As referred herein, terms "MV" or "V2a" are confined to invention description only and these have
been post-scripted to term "cryl Ac" to differentiate between two subtypes of the invention for die
sake of clarity and distinction. Adding MV (stands for main version) and V2a (Version 2a) are not
having any relationship with standard nomenclature.
The invention will now be elaborated in greater detail of specifications.
Selection of different point truncated three domain CrylAc toxin protein sequence
Many of the delta endotoxin encoding cry genes have been cloned and characterized for their respective efficacy against insect pests. CrylAc is a well characterized protein for its showing highest efficacy against Heliothines. One CrylAc molecule, classified so in Crickmore classification of Bt proteins (NCBI entry: M 11068) is chosen for designing the said gene of the present invention. Domain I of this protein spans from aa 36 to 254, domain II spans from aa 259 to aa 461 and domain III spans from 464 to aa 603. Synthetic genes encoding truncated CrylAc have been used in past to develop transgenic plants improved for resistance to insects. Often a CrylAc protein of 603-615 aa length (to take into account a trypsin site around 603 aa in the 1178 aa long

native Cry 1 Ac polypeptide sequence) is chosen to develop a truncated Cry 1 Ac protein and its encoding crylAc gene. In the present invention, 659 aa length protein is chosen from native Cryl Ac protein (exemplified by NCBI: Ml 1068) to design nucleotide sequences encoding a protein functional in plant cell milieu. Selecting C-terminal additional 56 aa extra has two advantages: (1) In terms of stability in plant cell milieu: Online analysis of secondary structure of native CrylAc protein reveals that amino acids from 604 to 659 constitute a helix-loop-helix-loop portion. Thus a truncated protein if selected upto aa 659, it will provide better in-vivo stability to the protein at the time of expression and folding in plant cells. (2) Online analysis of native CrylAc protein for the presence of tryptic sites reveal that it has three additional tryptic sites in the zone of aa 604 to 659. Thus retaining extra 56 amino acids also retains extra trypsin sites for more efficient processing and efficacy in insect mid-gut or stated differently, it will lead to rapid and fidel conversion of protoxin to active toxin core. Thus isolated synthetic DNA sequence as mentioned in SEQ ID No. 1 encodes a CrylAc protein of 659 amino acids length (referred hereafter as modified CrylAc- Main Version) and which mostly resembles corresponding portion from native CrylAc protein (M11068), except for a few amino acids mutations (424 'Q' of the native protein was changed to 424 'N' in the present invention, 628 'L' of the native protein was changed to 628 T in the present invention; 634 'Q' of the native protein was changed to 634 'G' in the present invention; 648 'Q' of the native protein was changed to 648 'G' in the present invention and 649 V of the native protein was changed to 649 Y' in the present invention ). The change from 424 'Q' to 424 "N1 was incorporated to introduce an intron splice site in the corresponding coding sequence. Rest of the mutations were introduced to create diversity in the selected protein, all of them falling in a zone which is not essential for toxin protein action. The identities search based on polypeptide sequences shows that modified CrylAc-MV resembles to the extent of only 55.45% (when compared with full length native CrylAc Sequence, Ml 1068) and to the extent of 99.24% (when compared with 659 aa coverage of native CrylAc Sequence Ml 1068).
When native Bt protoxin is ingested by insects, proteolytic cleavage by insect mid gut protease occurs not only at C-terminal, but also at N-terminal (approx. 25-36 amino acids) to release the active toxin core. According to pore formation model, this active toxin core then interacts with primary receptors before binding to cadherin. The binding to cadherin induces cleavage of some more amino acids from N-terminal end and eventual ologomerization of the toxin core, which now binds to Aminopeptidase receptors and is inserted into insect mid gut brush-border membrane microdomains inducing pore formation that finalry causes insect death (Bravo et al., 2004, Biochim. Biophys. Acta 1667, 38-46). A variant of modified Cryl Ac-MV protein was chosen to seethe effect of deletion of N-terminal 56 amino acids and to compare its efficacy with that of modified CrylAc

against insects. In this variant ( referred hereafter as modified Cryl Ac- Version 2a ), amino acids 1-56 of modified Cry] Ac-MV have been deleted and two amino acids substituted ( 57V and 58 'L ' of the modified Cryl Ac-MV is substituted by 1 'M' and 2 'A') giving rise to a 603 amino acids long protein. For rest of the sequence, modified CrylAc-V2a is exactly similar to modified Cryl Ac-MV. This variant chosen protein, modified CrylAc- V2a also harbors five mutations mentioned described earlier (424 'Q' to 368 N*, 628 'L' to 572 T, 634 'Q' to 578 'G', 648 'Q' to 592 'G' and 649 'Vto593'Y').
The designs and specifications of genes encoding different point truncation three domain CrylAc proteins for efficient expression in plants
Development of Modified and synthetic cryl Ac-MV gene :
The modified crylAc -MV gene was designed in-silico to encompass all features of gene designing known in prior art like high GC content, removal of poryadenylation signals etc as well as purposeful interventions like codon optimization in a way where only most preferred codons have been used achieving a CAI value suitable for both monocotyledonous and dicotyledonous group of plants, inclusion of intron in the middle of the coding sequence and a unique nucleotide composition.
The chosen 659 aa long sequence modified CrylAc protein was used for designing a nucleic acid sequence of SEQ ID No. 1, referred hereafter as modified crylAc- MV gene, in such a way that it has 42.81 % GC content and 57.19% AT content and has nucleotide composition of A:30.01%,C : 21.58%, G : 21.23% & T : 27.18 % for intron less gene, while the native crylAc gene encoding full length protein from Bacillus thuringiensis strain HD-73 (Adang et al.,1985, Gene 36(3)-289-300, incorporated here as reference in its entirety) had 39.13% GC content and 60.87% AT content and had nucleotide composition of A : 32.29%, C : 16.91%, G : 22.22% & T : 28.58%.. In engineering this modified crylAc of the present invention for enhanced expression in plants, the DNA sequence was designed in such a way to achieve relatively higher GC content so as to attain an AT content in nucleotide composition substantially as that found in plants and to eliminate sequences that cause improper polyadenylation, transcript destabilization and transcript termination. The nucleotide composition for intron containing crylAc gene represented by SEQ ID No.l comprises A : 29.31 %, C : 20.80 %, G : 20.66 %, T : 29.22 % . A comparison of DNA composition and other features of native crylAc gene (corresponding to Ml 1068 protein) with those of modified cryl Ac-MV gene of present invention is presented in Table 1.

Table 1: Comparison of Features between native crylAc gene and modified cry 1Ac gene-MVof present invention

S.No. Parameters Native crylAc Mod ified cryl Ac-MV
NCBI: M11068 CDS
1. GC content 39.13% 42.81%
mRNA degradation signal 17 0
2 (ATTTA)
Poh/adenylation signal
3a.AATAAA 0 0
3b.AATAAT 4 0
3c.AATTAA 5 0
3 3d. AACCAA 4 0
RNA polymerase II termination 0 0
4 signal { CAN (7-9) AGTNNA }
The synthetic nucleotide sequence and variant thereof for heterologous expression of a prokaryotic gene are specifically designed with distinct codon bias to a particular expression system in which the gene is to be expressed. This is done to ensure enough stalling of t-RNA and fidel expression and folding of the protein. Codon Adaptation Index (CAT) is a value based on analysis of frequency of codons to a particular amino acid in highly expressed genes of a particular organism and it tells us the capacity of a particular coding sequence or gene for expression in a particular organism. Thus, higher the index, higher is the expression. The codon adaptation index (CAI) of native crylAc gene (exemplified by NCBI Ml 1068) for dicot plants (e.g. cotton) is 0.755 while for monocots (e.g. maize) is 0.590 whereas for E. coli K12 is 0.315. This represents the poor expression capability of the native gene in higher plants.
The modified crylAc- MV gene of the present invention represented by coding sequence of SEQ ID No. 1 is designed in such way so as to incorporate codons preferred by higher plants. The CAI of modified crylAc gene of this invention (represented by SEQ ID No. 1) for dicot (say, cotton) is 0.937 and for monocots (say, maize) is 0.736 & for E. coli is 0.337 (calculated through OPTIMIZER, a web server program- Puigb P., Guzmn E.Romeu A. and Garcia-Vallv S. 2007 OPTIMIZER: A web server for optimizing the codon usage of DNA sequences. Nucleic Acids

Research, 35: W126-W131. And Puigb P., Romeu A. and Garcia-Vallve S. 2008. HEG-DB: a database of predicted highly expressed genes in prokaryotic complete genomes under translational selection. Nucleic Acids Research. 36: D524-7). A comparison of Codon Adaptation Index (CAT) of the modified crylAc gene-MV of present invention and native cry 1 Ac gene is given below (Table 2):
Table 2: The comparison of codon adaptation Index of the modified crylAc gene-MV of present invention and native crylAc gene

S.No. Codon Adaptation Index Native cry 1 Ac
NCBI:
M11068 Modified cry1Ac-MV CDS
1 Monocot (Maize) 0.590 0.736
2 Dicot (cotton) 0.755 0.937
3 Prokaryote (E.coli K12) 0.315 0.337
The difference in CAI of the native gene for dicot and monocot and that of gene of the present invention was found to be substantially increased by 24 % over native gene CAI for dicots and 24.74% for monocots. Thus, probability of expressing modified crylAc-MV gene of the present invention is found to be much better than the native gene in higher plants. . However, this fact is also to be seen in the light of the fact that native gene has low GC content and some polyadenylation signals as shown in Table 1. Thus, the comparative analysis of native gene with the modified gene shows that the native gene seems to be inappropriate for high expression in plants while modified crylAc gene (s) of the present invention has been designed for the purpose of better expression in plants.
The frequency and number of each codons for the said modified crylAc gene is represented in Table 3 (calculated through countcodon program, http:Vwww.kazusa.or. jp/codon/c ountco don .html).

fields [triplet] [frequency: per thousand] ([number])

UUU 0.0 ( 0) UCU 0.0( 0) UAU 0.0( 0) UGU 0.0( 0)
UUC 56.0( 37) UCC 0.0( 0)UAC46.9( 31) UGC 3.0( 2)
UUA 0.0( 0) UCA 0.0( 0) UAA 1.5( 1) UGA 1.5( 1)
UUG 0.0( 0) UCG 0.0( 0) UAG 0.0( 0) UGG 15.1( 10)
CUU81.7( 54) CCU 0.0( 0) CAU 15.1( 10) CGU 0.0( 0)
CUC 0.0( 0) CCC 0.0( 0) CAC 0.0( 0) CGC 0.0( 0)
CUA 0.0( 0) CCA49.9( 33) CAA34.8( 23) CGA 0.0( 0)
CUG 0.0( 0) CCG 0.0( 0) CAG 6.1( 4) CGG 0.0( 0)
AUU 69.6( 46) ACU 62.0( 41) AAU 0.0( 0) AGU 0.0{ 0)
AUC 4.5( 3) ACC 0.0( 0)AAC81.7( 54)AGC96.8( 64)
AUA 0.0( 0) AC A 0.0{ 0) AAA 0.0( 0) AGA60.5( 40)
AUG 12.1( 8) ACG 0.0( 0) AAG 6.1( 4) AGG 6.1( 4)
GUU68.1{ 45) GCU59.0( 39) GAU42.4( 28) GGU 1.5( 1)
GUC 0.0( 0) GCC 0.0( 0) GAC 0.0( 0) GGC 0.0( 0)
GUA 0.0( 0) GCA 0.0( 0) GAA 0.0( 0) GGA71.1( 47)
GUG 0.0( 0) GCG 0.0( 0) GAG46.9{ 31) GGG 0.0{ 0)
Warning: [start codon unexisl [][stop codon unexist]
Translation exception table: Standard
While optimizing the codons of a particular gene, mostly available codons for a particular amino acid are guided in random frequency across the sequence.
In a preferred embodiment of the present invention, at most of the position highly preferred codons have been used for a particular amino acid. For example, Glycine is present at 48 positions in the modified Cryl Ac of the present invention, but GGA as a codon for Glycine has been used at 47 positions (97.91%) and GGT has been used at one position (2.08%) only.
Incorporation of intron to the coding sequence:
The nucleic acid sequence of the modified cryl Ac gene-MV was further modified to incorporate

one intron in the coding sequence to serve following two advantages:
a) enhancement in expression of the gene (s) in the plant cells and;
b) prevention of horizontal transfer to prokaryotes, if any, when transgenic plants comprising of this cry 1 Ac gene (s) as mentioned in the invention get released to open field.
Introns, wherever added to any prokaryotic sequence destined for high expression in eukaryotes, (a recombinant DNA procedure much known to those who are skilled in the art), are added before start of coding sequence, or so far as possible around the N-terminal part of the coding sequence. This depends on the availability of intron splice site in the coding sequence. If splice site for a particular intron is not available in the coding sequence, mostly an untranslated region is added at the N-terminus and intron is incorporated at the junction of untranslated region and start of actual coding sequence. After examining DNA sequence of the modified crylAc-MV gene of the present invention it was found that only a single intron splice site was incidentally present in the portion of the gene coding for domain III of the protein. Domain III, however, being involved in receptor binding was very crucial for the very function of the protein and was not a logical choice for incorporation of the intron. Other option was to have it around N-terminus, but no splice site was present in the N-terminal portion of the coding sequence of the modified crylAc-MV gene of this invention. N-terminal intron or intron with untranslated region at N-terminus was also not considered because of design of modified crylAc-V2a gene (described below) which was for deletion of first 56 amino acids from the modified crylAc-MV. Thus if N-terminal intron would have been selected, after deletion of first 56 aa coding sequence from modified crylAc-MV gene, intron portion would have also been deleted. The logical option thus was to have the intron in the middle or around mid of the coding sequence, the splice site for which was not present in the coding sequence of the modified cryl Ac-MV gene. To incorporate intron and to create a proper splice site, 424 'Q' of native CrylAc protein was changed to 424 'N' in the modified Cryl Ac protein of the present invention and accordingly, castor bean catalase intron of 190 bp was integrated in the gene sequence of modified cryl Ac gene represented by SEQ ID No. 1 of the present invention. The changed amino acid with similar biochemical property will not have larger implication on the very function of CrylAc protein, but incorporating an intron will have better implication for expression in plants. Castor bean catalase intron is a preferred intron in the ambit of this invention. However, any intron which fulfills the criteria of having splice site at defined position in the nucleic acid sequence, where it has been mentioned in this invention is also encompassed.
The in-silico designed nucleotide sequence of modified crylAc-MV gene was analyzed for restriction enzyme sites and unwanted sites were removed without changing the amino acid. Also,

some nucleotide sequences were added at N-terminal (before ATG or start site) and at C-terminal (after stop codon) to have restriction sites with the help of which gene can be cloned to plant transformation vectors or preferably, binary vectors, much known in the art. Figure IB shows the sequence architecture and important features of modified cry 1 Ac gene-MV of the present invention. SEQ ID No. 1 represents the nucleic acid sequence of the modified crylAc-MV gene which also shows the sequence and position of intron, whereas SEQ ID No. 1 (i) shows sequence of cDNA of modified cryl Ac-MV gene and its translation.
Development of Modified and synthetic crv1Ac-V2a gene:
Another aspect of the invention provides a variant of modified cryl Ac-MV gene, represented as modified cryl Ac-V2a gene, which is to encode a Cry 1 Ac protein truncated at both N-terminal and C-terminal (mentioned as CrylAc-V2a protein in previous section on selection of truncated Cryl Ac proteins). This variant of cry 1 Ac-MV, produces N-terminal 56 amino acids deleted protein. This form will have other wise the same sequence as in 659 aa long modified Cryl Ac-MV protein of the present invention, except for N-terminal 56 amino acids deleted and two amino acids substituted -57'V and 58 'L ' of the modified Cryl Ac-MV is substituted by 1 'M and 2 'A' of modified CrylAc-V2a, thus giving rise to a 603 aa long protein.The chosen 603 aa long sequence of this modified Cryl Ac-V2a protein is used for designing a modified cry lAc nucleic acid sequence of SEQ ID No. 2, referred hereafter as modified cry 1 Ac- V2a.
The nucleotide sequence has been designed in such a way that it has 42.84 % GC content and 57.16 % AT content and has nucleotide composition of A : 30.07%, C : 21.56%, G : 21.28% & T : 27.09 % for intron less gene. The nucleotide composition for intron containing cryl Ac gene represented by SEQ ID No. 2 comprises A: 29.38 %, C: 20.65 %, G : 20.65 % and T : 29.33 %. In the most preferred embodiment, CAI of modified crylAc gene- V2a of this invention (represented by SEQ ID No. 2) for dicot (say, cotton) is 0.937 and for monocots (say, maize) is 0.735. The nucleotide sequence of SEQ ID No. 2 of the present invention has also been designed to incorporate the castor bean catalase intron in the domain II sequence at the same position as in and for SEQ ID No. 1 and it has all the other modifications and amino acid mutations done for SEQ ID No. 1. The SEQ ID No. 2 has sequence similar to SEQ ID No. 1 except that it will not have N-terminal 56 amino acids of modified cryl Ac gene-MV and it has been designed to illustrate the comparative efficacy of SEQ ID No. 1 and SEQ ID No. 2 encoded plant expressed proteins. After the nucleotide sequence of modified crylAc-V2a of the present invention has been designed in-silico (as represented by SEQ ID No. 2), it was observed that it has BseYl site in common with

the sequence of modified cryl Ac gene-MV represented by SEQ ID No. 1.
After the nucleotide sequence of modified crylAc-V2a of the present invention has been designed in-silico (as represented by SEQ ID No. 2), it was observed that it has BseY1 site in common with the sequence of modified cryl Ac gene-MV represented by SEQ ID No. 1. In another useful aspect of the invention, a DNA fragment was designed having sequence of SEQ ID No. 2 only upto Bse Y1 site ( Thus representing N-terminal 56 amino acid deleted and two amino acid substituted N-terminal portion of SEQ ID No. 2 gene only), and spanning upto 210 nucleotide length. For this DNA fragment, represented as Fragment "X" for V2a cloning in SEQ ID no. 3, some nucleotide sequences were added at N-terminal (before ATG or start site) and at C-terminal (after BseYl site) to have restriction sites, with the help of which modified cryl Ac gene-V2a can be cloned to cloning vector or plant transformation vectors or preferably, binary vectors, much known in the art by replacing corresponding portion from modified crylAc gene-MV with Fragment "X", thus achieving right frame translatable modified crylAc gene-V2a . Figure 1C shows the sequence architecture and important features of Fragment "X" of the present invention. SEQ ID No. 2 represents the nucleic acid sequence of the modified crylAc-V2a gene which also shows the sequence and position of intron, whereas SEQ ID No. 2 (i) shows sequence of cDNA of modified cryl Ac-V2a gene and its translation.
The synthesis and cloning of modified crylAc qene(s) to plant transformation vectors:
(A) Modified cryl Ac-MV gene
The in-silico designed nucleic acid sequence of modified crylAc-MVgene of the present invention was chemically synthesized through custom DNA service and cloned in custom service provider cloning vector between appropriate sites. The sequence of mis was confirmed through procedures established in the molecular biology.
The modified cryl Ac-MV gene pertaining double stranded DNA molecule was cloned to binary vector (preferably pBI121, a derivative of it or any other binary vector) and mobilized to Agrobacterium tumefaciens strains (preferably LBA 4404) containing disarmed Ti plasmid via electroporation. However, any other Agrobacterium tumefaciens strain containing disarmed Ti plasmid may also be utilized. The plasmid map of the recombinant binary vector, hereafter referred to as pKRI-571N) is shown in Figure 2A. Binary vector pBI121 has been preferentially used (by replacing CaMV35S promoter and gusA gene with CaMV35S duplicated promoter and modified cryl Ac-MV nucleic acid sequence) to constitute a functional plant expression cassette for modified crylAc-MV gene of this invention. However, cloning of such a modified crylAc-MV gene as

described in this invention to any plant transformation vector, binary vector, marker free vector with any promoter capable of expressing cry1 Ac-MV gene and with or without any kind of selection marker are also encompassed.
(B) Modified cryl Ac-V2a gene
For the cloning of modified cryl Ac gene- V2a to plant expression cassette, Fragment "X" of Ihe SEQ ID No. 3 was chemically synthesized along with N-terminal appropriate restriction site and C-terminal BseY1 site. The modified cryl Ac-V2a gene was cloned by replacing corresponding portion from modified crylAc-MV gene preferably to binary vector pBI121 and mobilized to Agrobacterium tumefaciens strains (preferably LBA 4404) containing disarmed Ti plasmid via electroporation. However, any other Agrobacterium tumefaciens strain containing disarmed Ti plasmid may also be utilized. The plasmid map of the recombinant binary vector (hereafter referred to as pKRI-572N) is shown in Figure 2B. The recombinant binary vector pKRI-572N has been constructed (by replacing CaMV35Spromoter and gusA gene of pBI121 with CaMV35S duplicated promoter and modified crylAc-V2a nucleic acid sequence) to constitute a functional plant expression cassette for modified crylAc-V2a gene of this invention. However, cloning of such a modified cryl Ac-V2a gene as described in this invention to any plant transformation vector, binary vector, marker free vector with any promoter capable of expressing cryl Ac-V2a gene and with or without any kind of selection marker are also encompassed.
The expression of modified crvlAc gene (s) in plants and determining the efficacy of plants comprising these gene (s):
The modified nucleic acid sequences represented as SEQ ID No.l and 2 were chemically synthesized and then cloned to plant transformations vectors and introduced to Agrobacterium tumefaciens by the methods well known in molecular biology. The synthetic cryl Ac genes of SEQ ID Nos. 1 & 2 were transformed via Agrobacterium mediated transformation to tobacco. The transgenic tobacco lines regenerated were grown further, analyzed for presence and expression of respective transgene. These transgenic lines were progressed further for checking the expression of respective transgene in next generation also. The bioefficacy of transgenic plants were checked by challenging plant leaves to Helicoverpa and other insect larvae, which showed that these plants were resistant to target insects. The bioefficacy of plant expressed modified Cryl Ac proteins were also checked by mixing total soluble proteins from some of the best expressing transgenic plants to artificial diet and subjecting Helicoverpa armigera larvae to this, which also showed that a

functional and efficacious Cry 1 Ac protein is present in the total soluble proteins from transgenic plant comprising of modified cry 1 Ac-MV gene. After analysis of expression data and bioassay data of different transgenic plant lines with random mating laboratory population of Helicoverpa armigera, plant expressed modified CrylAc-MV protein was found better than modified CrylAc-V2a protein.
To validate the function of intron in elevating the level of expression and to analyze whether intron containing gene is appropriately expressed in plant cell milieu and a functional CrylAc toxin protein is produced, in another useful aspect of the invention, modified cryl Ac-MV gene (as represented in SEQ ID No. 1 ) was further modified for the removal of intron. Thus intron from cryl Ac-MV gene was removed by standard techniques of molecular biology and an intron less modified crylAc-MV gene, referred hereafter as modified crylAc-V3 is cloned in plant transformation vector, giving rise to pKRI-573N. The recombinant vector pKRI-573N was mobilized to Agrobacterium tumefaciens strain LBA 4404 and transformed to tobacco. The regenerated transgenic tobacco plants were analyzed for the presence and expression of modified crylAc-V3 gene. The expression of these plants was compared to expression of modified cryl Ac-MV of this invention on the basis of ELISA. This comparative analysis showed that intron significantly enhances the expression of modified cryl Ac gene (s) of the present invention. Embryogenic calli of cotton were transformed with modified cryl Ac-MV, modified crylAc-V2a and modified crylAc-V3 genes via Agrobacterium mediated transformation. After 3 cycles of selection on selection media, these calli were analyzed for the expression of respective transgene. The data obtained revealed that these genes express efficiently in the cotton cells also.
The comparative efficacy of the chosen 659 aa long modified Cryl Ac-MV and modified Cryl Ac-V2a purified proteins against target insects:
The polypeptide sequences of chosen 659 aa long modified CrylAc-MV protein and 603 aa long modified CrylAc-V2a protein were used to design respective genes for expression in prokaryotes. The codons of these genes were optimized as per E.coli K12 codon table cloned to expression vector along with His-tag. The respective proteins were expressed, purified and His-tag was removed to finally give rise to purified protein.
The modified CrylAc-MV protein, expressed in and purified from E.coli as well as modified CrylAc-V2a protein, expressed in and purified from E.coii, in varying concentrations were used in bioassay experiments to test the comparative efficacy against Helicoverpa armigera larvae. In artificial diet mixed protein feeding experiments, modified CrylAc-MV clearly showed a dose response curve whereas the same was not found in case of modified CrylAc-V2a protein. A leaf

painting experiment with cotton leaves was done in order to see the effect of the two proteins on Heltcoverpa armigera. This also has showed that both modified CrylAc-MV and modified CrylAc-V2a were effective against HeJicoverpa armigera insect larvae. These demonstrations reveal that modified crylAc-MV gene of this invention and encoded protein will serve better insecticidal candidate molecule in transgenics development program. The purified CrylAc-MV protein was also found to be highly effective against larvae of Chilo partellus.
The present invention will be explained further with reference to non-limiting embodiments of the invention.
In an embodiment of the present invention, two truncated cryl Ac toxins of different lengths- 659 aa
and 603 aa (One having N-terminal unchanged and other having 56 amino acids deleted from N-
terminal but both having 56 amino acids extra at C-terminal) were chosen to develop synthetic
DNA molecules encoding proteins for higher expression in plants to impart the transformed plants
with insect resistance activity.
In another embodiment of the present invention, the synthetic nucleotide sequence having
nucleotide sequence of SEQ ID No. 1 or a sequence having at least 90% sequence identity to the
nucleotide sequence of SEQ ID No. 1 is used for producing insect resistant transgenic plants.
In another embodiment of the present invention, the modified synthetic nucleotide sequence having
nucleotide sequence of SEQ ID No. 2 or a sequence having at least 90% sequence identity to the
nucleotide sequence of SEQ ID No. 2 is used for producing insect resistant transgenic plants.
In another embodiment of the present invention, the synthetic DNA sequence or its variant have
been designed for high expression in eukaryotic systems like plants.
In another embodiment of the present invention, the synthetic DNA sequence or its variant are
optimized with highly expressed codons in a way that the said synthetic gene or its variants express
to a high level in both monocotand dicot group of plants.
In yet another embodiment of the present invention, the nucleotide sequence or its variant does not
contain any polyadenylation signal or transcript splicing site.
In another embodiment of the present invention, the synthetic DNA sequence or its variant possess
intron in the mid of the coding sequence and further possess intron splicing site for the removal of
the said intron, post transcription.
In yet another embodiment of the invention, insecticidal protein encoding synthetic DNA
sequence(s) have been provided which possess higher GC content than the native gene and unique
nucleotide compositions.
In another embodiment of the present invention, the synthetic DNA sequence or its variant encodes

protein that differs from native CrylAc in being shorter by 519 amino acids. In yet another embodiment of the invention, synthetic nucleotide sequence or variant thereof have been provided which encode truncated CrylAc delta endotoxin proteins comprising more number of tryptic sites at C-terminal to ensure rapid and fidel conversion of protoxin to toxin core.
The invention is further explained with the help of following examples. These examples are provided to better elucidate the practice of the present invention and should not be interpreted in any way to limit the scope of the present invention. Those skilled in the art will recognize that various modifications, additions, substitutions, truncations, etc., can be made to the methods and genes described herein while not departing from the spirit and scope of the present invention.
1. DNA constructions expressing modified Cry toxins which contain plants preferred codons and intron in the sequence:
To the in-silico designed and optimized 2173 bp long sequence of modified crylAc gene of the present invention, some sites were added at N-terminal end (preferably Ncol) and some sites were added at the C-terminal end, after the stop codons (preferably Sad). The resulting nucleic acid sequence was chemically synthesized via techniques much known in the art through a contract DNA synthesis firm. The synthesized DNA was cloned in a routine cloning vector (proprietary of contract firm, referred hereafter as synthesis vector 1) and it was sequenced and confirmed. The synthesis vector 1 (SV1) was digested with two restriction enzymes - Ncol and Sad. The digested products were electrophoresed on 0.8% agarose gel, to separate out NcoI-SacI gene containing fragment from vector backbone. The Ncol- Sad modified crylAc- MV gene containing fragment was excised from gel and eluted utilizing gel elution kit. In a separate digestion reaction, Krishidhan proprietary cloning vector (referred hereafter as pKRI-920N and which harbors a CaMV 35S enhanced promoter between Sbf 1 and Ncol sites, MCS between Ncol and Sad sites and nos terminator between Sad and EcoRI sites) was digested with Ncol and Sad and digestion products were electrophoresed on 0.8% gel. The Ncol -Sad vector backbone was excised and eluted utilizing gel elution kit. The Ncol- SacI modified crylAc- MV gene containing fragment was ligated with Ncol -SacI vector backbone of pKRI- 920N to get the vector pKRI-92lN. The CaMV 35S enhanced promoter-modified crylAc gene-MV segment between Sbf I and SacI sites were excised frompKRI-921Nand ligated to vector pBI121 digested at corresponding enzymes. The ligation mix was transformed to E.coli competent cells and positive clones were identified for the recombinant binary vector harboring modified crylAc as represented in SEQ ID No. 1 via restriction digestion analysis. One of the positive clone was electroporated to Agrobacterium tumefaciens strain (preferably LBA 4404) containing disarmed Ti plasmid. The plasmid map of the recombinant

binary vector, hereafter referred to as pKRJ-571N is shown in Figure 2A. The recombinant binary vector pKRI-571N thus harbors modified crylAc gene-MV of the present invention under regulation of CaMV 35S enhanced promoter and nos terminator. At all steps of molecular cloning, standard molecular biology practices or techniques as detailed in Molecular cloning ( Sambrook and Maniatis) were followed ( incorporated herein by reference).
For making the DNA construction that can code for modified crylAc gene-V2a, a DNA fragment "X" was designed. To the in-silico designed 210 bp long DNA sequence of fragment "X" of SEQ ID no. 3 of the present invention, some sites were added atN-terminal end (preferably Ncol) and some sites were added at the C- terminal end, after the site BseY1. The resulting nucleic acid sequence was chemically synthesized via techniques much known in the art through a contract DNA synthesis firm. The synthesized DNA was cloned in a routine cloning vector (proprietary of contract firm, referred hereafter as synthesis vector 2) and it was sequenced and confirmed. The synthesis vector 2 (SV2) was digested with Ncol and BseYl and ligated to vecor backbone of pKRI-92IN digested with corresponding enzymes to get the vector pKRI-922N, thus bringing the right frame sequence of modified crylAc gene-V2a under the control of CaMV35S enhanced promoter and nos terminator. The CaMV 35S enhanced promoter-modified crylAc gene-V2a segment between Sbf I and Sad sites were excised from pKRI-922N and ligated to vector pBI121 digested at corresponding enzymes. The ligation mix was transformed to E.coli competent cells and positive clones were identified for the recombinant binary vector harboring modified crylAc as represented in SEQ ID No. 2 via restriction digestion analysis. One of the positive clone was electroporated to Agrobacterium tumefaciens strain (preferably LBA 4404) containing disarmed Ti plasmid. The plasmid map of the recombinant binary vector, hereafter referred to as pKRI-572N is shown in Figure 2B. At all steps of molecular cloning, standard molecular biology practices or techniques as detailed in Molecular cloning ( Sambrook and Maniatis) were followed ( incorporated herein by reference).
2. Development of insect resistant transgenic plants: Expression of modified, synthetic crylAc gene (s) in plants and evaluating the efficacy of plant expressed proteins against insects:
A. Transformation and expression of modified crylAc-MVgene to model species tobacco:
Nicotiana tabacum var Petit Havana SR-1 leaf explants (from axenically grown seedlings), approx. 1 cm2 pieces, were transformed by Agrobacterium tumefaciens strain LBA 4404 containing recombinant binary vector pKRI-571N (harboring binary vector containing modified crylAc gene-

MV of SEQ ID No. 1 of the present invention under the regulation of CaMV 35S enhanced promoter) However, this gene may be transformed to tobacco by any other method of transformation like direct DNA delivery as well. Moreover, for transformation, any other vector, with or without selection marker and any other Agrobacterium strain may also be used for transformation of modified crylAc-MV gene to plants. Similarly, regulatory elements from any other group comprising of constitutive or inducible or tissue promoters and terminators from a group comprising of ocs terminator, nos terminator or CaMV35S polyA may also be used to express the modified crylAc-MV gene of the present invention to plants. The transformed explants were regenerated on selection medium containing MS basal salts, RT vitamins, 100 mg/L myo-inositoL 3% w/v Sucrose, 0.075 mg/L NAA, 1.25 mg/L BAP, 0.8% w/v agar, 300mg/L Kanamycin and 300 mg/L Cefotaxime. Twenty transgenic plants were regenerated and acclimatized in pots containing potting mix in transgenic containment. All these putative transgenic plants were found to be positive for the presence of modified-cryl Ac MV gene of the invention as revealed by PCR analysis. The putative transgenic plants were further analyzed for expression of modified cryl Ac of the present invention by commercially available anti-CrylAc monoclonal antibody coated ELISA plates (from Amar Immunodiagnostics, Hyderabad, India). Four plants were found to have very good expression level and ELISA OD value. The ELISA data of these plants along with untransformed control plants are tabulated in Table 4.
Table 4: Expression of modified crylAc-MVgene in transgenic tobacco as detected by
ELISA

S.No. Event/ transgenic plant line no. ELISAOD value
1 Transgenic plant no. A-3 1.743
2 Transgenic plant no. A-12 1.873
3 Transgenic plant no. A-16 1.620
4 Transgenic plant no. A-18 1.739
5 Untransformed, Control plant no. 1 0.017
6 Untransformed Control plant no. 2 0.011
B. Efficacy of transgenic tobacco expressed modified CrylAc-MV protein against different
insects:
Four of the PCR confirmed transgenic plant, transformed with modified crylAc-MV gene with
intron of the present invention (pKRI-571N), with high expression value as revealed by ELISA and

normal, untransformed tobacco plant(s) were selected for insect toxicity experiment with target and alternative insect pests larval stages. Fully expanded leaves from 2nd to 4th node from top of 30-45 days old plants were used in bioassay experiments. Leaf from each plant line was challenged with 5-6 insect larvae and the experiment was repeated. Leaves were placed in cylindrical boxes and insect larvae were released on it. The mouths of boxes were covered with wet muslin cloth to maintain sufficient humidity. When necessary, larvae on transgenic plants were re-fed with leaves of equivalent size and physiological age from within the same clonal line and those on control plants, given leaves from the same genetic plant type. The 100 % mortality of Helicoverpa armigera larvae of different stages and weight reduction in alternate pests within 72 hrs of challenge, showed that modified crylAc-MV gene with intron of the present invention is adequately expressed in plants and it revealed the utility of this modified gene in providing complete protection of crop plants or any plant against insects. The results are summarized in Table 5.
Table 5: Efficacy of modified cryl Ac-MV transgenic tobacco expressed toxin protein against target {Helicoverpa armigera and Leucinodes orbonalis) and alternate pest (Spodoptera litura).

s. Plant No. Bioassay with Bioassay with Bioassay with
No. Helicoverpa armigera Spodoptera litura Leucinodes orbonalis
% mortality * % mortality * % mortality *
neonata 1 1ST i nstar neonata 1 1st i nstar neonatal 1sti nstar
larvae larvae larvae larvae larvae larvae
Transg. plant A-3 67, weight 50, weight NT
1 100 100 reduction reduction 100
Transg. plant A-12 17, weight 0, weight NT
2 100 100 reduction reduction 100
Transg. plant A-16 67, weight 33, weight NT
3 100 100 reduction reduction 100
Transg. plant A-18 33, weight 0, weight NT
4 100 100 reduction reduction 100

Untransf. Cont. C- NT
5 1 0 0 0 0 0
Untransf. Cont. C- NT
6 2 0 0 0 0 0
* As calculated after 72 hours of challenging plant leaves with insects larvae. NT= Not tested.
C. Stability and inheritance of transgenic tobacco harboring modified crvlAc-MVqene: PCR, ELISAand bioefficacy of T1 generation:
The transgenic tobacco plant lines (A-3, A-12, A-16 and A-18) with modified crylAc-MV gene of the present invention were grown further in transgenic containment. The flowers were selfed and seeds were collected. The seeds from individual lines were germinated in petri plates containing Hoagland solution plus 300 mg/L kanamycin. Kanamycin positive seedlings remained green and some of them were grown further for molecular analysis of these plants for presence and expression of modified cryl Ac-MV transgene.
PCR of genomic DNA of kanamycin positive Tl plants of transgenic plant line nos. A-3, A-12, A-16 and A-18 with Forward Primer (as mentioned in SEQ ID no. 4) and Reverse Primer (as mentioned in SEQ ID no. 5) revealed expected amplicon size of 854 bp whereas no amplification was observed in gDNA of untransformed tobacco plant(s). Figure 3 shows an image of the representative samples of this PCR.
The Tl plants from each selected TO plant were also tested for expression of modified cryl Ac-MV gene via ELISA utilizing commercially available (from Amar Imunodiagnostics, Hyderabad) anti-Cry 1 Ac monoclonal antibody coated ELISA plate kit. The results obtained are tabulated in Table 6.
Table 6: Expression of modified cryl Ac-MV gene in transgenic tobacco (Tl generation) as
detected by ELISA

S.No. Plant No. ELISAOD
value S.No. Plant No. ELISA OD value
1 Untransf. Contr, 1 0.002 23 Tl plant no. A-l 2/10 1.348
2 Untransf. Contr, 2 0.009 From TO plant no. A-16
3 Untransf. Contr, 3 0.009 24 Tl plant no. A-l 6/1 1.731
4 Untransf. Contr, 4 From TO plant no. A-3 0.004 25 26 Tl plant no. A-l6/2 Tl plant no. A-l 6/3 1.341 1.410

5
Tl plant no. A-3/1 1.168 27 Tl plant no. A-l 6/4 1.711
6 Tl plant no. A-3/2 1.607 28 Tl plant no. A-l 6/5 1.883
7 Tl plant no. A-3/3 1.163 29 Tl plant no. A-l 6/6 1.717
8 Tl plant no. A-3/4 1.533 30 Tl plant no. A-l 6/7 2.092
9 Tl plant no. A-3/5 1.323 31 Tl plant no. A-l 6/8 1.565
10 11 Tl plant no. A-3/6 Tl plant no. A-3/7 1.313 1.396 32 33 Tl plant no. A-l6/9 Tl plant no. A-l6/10 1.119 1.665
12 Tl plant no. A-3/8 1.069 From TO plant no. A-18
13 Tl plant no. A-3/9 1.758 34 Tl plant no. A-l 8/1 1.448
From TO plant no. A-12 35 Tl plant no. A-l 8/2 1.858
14 Tl plant no. A-l2/1 1.331 36 Tl plant no. A-l 8/3 1.631
15 Tl plant no. A-l2/2 0.887 37 Tl plant no. A-l 8/4 1.705
16 Tl plant no. A-l2/3 1276 38 Tl plant no. A-l 8/5 1.681
17 Tl plant no. A-l 2/4 0.831 39 Tl plant no. A-l 8/6 2.025
18 Tl plant no. A-l2/5 1.167 40 Tl plant no. A-l 8/7 1.520
19 Tl plant no. A-l2/6 1.694 41 Tl plant no. A-l8/8 1.361
20 Tl plant no. A-l2/7 1.290 42 Tl plant no. A-l 8/9 1.662
21 Tl plant no. A-12/8 1.570 43 Tl plant no. A-l 8/10 1.806
22 Tl plant no. A-l2/9 1.473
Selected, PCR and ELISA confirmed plants of Tl plants were also checked for bioefficacy in terms of its action on Helicoverpa armigera insects larvae. After 72 hrs of challenging these Tl plant leaves with Helicoverpa armigera 1st instar larvae, 100% mortality of insects larvae were observed whereas all the larvae on control untransformed leaf survived well. Figure 4 shows a picture of representative sample from the bioassay experiment.
The data obtained clearly showed that modified crylAc-MV gene of the present invention got stably integrated in the genome of tobacco TO plants and was inherited to Tl generation, where it

was expressing well and the expressed protein caused mortality of larvae of target insects larvae. No other difference in morphology and physiology of these plants were noticed compared to control, untransformed tobacco plants.
D. Transformation of modified cry1Ac-V2a gene to model species tobacco, molecular analysis and bioefficacy of transgenic plants:
Nicotiana tabacum var Petit Havana SR-1 leaf explants (from axenically grown seedlings), approx. 1 cm2 pieces, were transformed by Agrobacterium tumefaciens strain LBA 4404 or any other competent Agrobacterium strain harboring binary vector containing modified cry 1 Ac gene-V2a of SEQ ID No. 2 of the present invention under the regulation of CaMV 35S enhanced promoter and nos terminator or other regulatory elements for constitutive or inducible expression ( in a preferred embodiment with pKRI-572N), and transformed explants were regenerated on selection medium containing MS basal salts, RT vitamins, 100 mg/L myo-inositol, 3% w/v Sucrose, 0.075 mg/L NAA, 1.25 mg/L BAP, 0.8% w/v agar, 300mg/L Kanamycin and 300 mg/L Cefotaxime. Fifteen transgenic plants were regenerated and acclimatized in pots containing potting mix in transgenic containment. The putative transgenic plants were analyzed for expression of modified cryl Ac-V2a of the present invention by commercially available anti-CrylAc monoclonal antibody coated ELISA plates. Three plants were found to have good expression level and ELISA OD value. The ELISA data of these plants along with untransformed control plants are tabulated in Table 7.
Table 7: Expression of modified cryl Ac-V2a gene in transgenic tobacco as detected by
ELISA

S.No. Event/ transgenic plant line no. ELISA OD value
1 Transgenic plant no. B-19 0.650
2 Transgenic plant no. B-34 0.467
3 Transgenic plant no. B-60 0.311
5 Untransformed, Control plant no. 1 0.002
6 Untransformed Control plant no. 2 0.006
PCR of genomic DNA of some selected putatively transformed TO plants with Forward Primer (as mentioned in SEQ ID no. 6) and Reverse Primer (as mentioned in SEQ ID no. 7) revealed expected amplicon size of 1023 bp whereas no amplification was observed in gDNA of untransformed

tobacco plants). Figure 5 shows an image of the. representative samples of this PCR.
TO Transgenic plant line no. B-19 with modified crylAc-V2a gene with best expression was progressed ftirther, flowers selfed and seeds collected. Tl seeds of B-19 were germinated on Haogland solution containing 300 mg/L kanamycin and kanamycin positive seedlings were progressed furlher to grow up in plants. 25 Tl plants of plant no. B-19 were screened via ELISA for the expression of modified crylAc-V2a gene and some selected plants were checked for efficacy against Helicoverpa armigera 1st instar larvae. TO plants of B-34 and B-60 were also included in the experiment as also some untransformed control tobacco plants. Fully expanded leaves from 2nd to 4 node from top of these plants were used in bioassay experiments. Leaf from each plant line was challenged with 4 insect larvae and the experiment was repeated. Leaves were placed in cylindrical boxes and insect larvae were released on it. The mouths of boxes were covered with wet muslin cloth to maintain sufficient humidity. When necessary, larvae on transgenic plants were re-fed with leaves of equivalent size and physiological age from within the same clonal line and those on control plants, given leaves from the same genetic plant type. 100 % larval mortality was observed post 72 hours of challenge in better expressing plants whereas low expressing plants gave mortality in the range of 42 to 87.5 %. A pictorial representation of a sample from this experiment has been given in Figure 6 and results are summarized in Table 8.
Table 8: Expression of modified crylAc-V2a in transgenic tobacco and efficacy of transgenic lines in insect bioassay with Helicoverpa armigera 1st instar larvae

S.No. Plant No. ELISAOD value % mortality * in bioassay
1 T0plantno.B-19 0.650 100
2 Tl plant no. B-19/1 0.407 71.42
3 Tl plant no. B-19/9 0.350 75
4 Tl plant no. B-19/12 0.853 100
5 Tl plant no. B-19/14 0.451 75
6 Tl plant no. B-19/16 0.366 75
7 Tlplantno.B-19/17 0.437 62.5
8 Tl plant no. B-19/18 0.846 100

9 Tl plant no. B-19/20 0.569 57.14
10 TO plant no. B-34 0.467 100
11 TO plant no. B-60 0.311 87.5
* As calculated after 72 hours of challenging plant leaves with insects larvae.
The data obtained clearly showed that modified crylAc-V2a gene of the present invention got stably integrated in the genome of tobacco TO plants and was inherited to Tl generation, where it was expressing well and the expressed protein caused mortality of larvae of target insects larvae. No other difference in morphology and physiology of these plants were noticed compared to control, untransformed tobacco plants. However, in general, it was noticed that modified crylAc-V2a was not better than crylAc-MV in terms of its efficacy against insects. Whereas 100 % mortality was observed in insect bioassay experiments with crylAc-MV gene transformed plants with good expression, 100 % mortality was not always noticed in all the plant lines transformed with cryl Ac-V2a.
E. Checking the effect of plant expressed modified CrylAc-MV protein in artificial diet against Helicoverpa armigera larvae:
The leaves of some selected Tl transgenic plant lines (transformed with modified crylAc-MV) were used to extract total soluble protein. Approx. 200 mg of plant leaf tissue from these transgenic plants and control, untransformed plants were crushed in liquid nitrogen and homogenized in 1 ml of a protein extraction buffer containing lx PBS pH 7.4, 0.5 mM EDTA pH 8.0, 0.5 mM DTT and 100 uMPMSF. The homogenate was incubated on ice for 30 minutes and then centrifuged at 10000 rpm for 10 minutes at 4°C to a clear supernatant containing total soluble protein. The protein obtained from each sample was estimated with BIO-RAD's Bradford dye reagent kit. Routine artificial diet was prepared for Helicoverpa armigera and to the 8 ml of molten artificial diet, 2 ml of extracted protein from a particular line was added, mixed and immediately kept for solidification at 4°C in a refrigerator. After 20 minutes, artificial diet containing total soluble protein from these plants was transferred to plastic vials to which insect larvae were released. A buffer control was also included in the experiment to observe the effect of protein extraction buffer, if any on insect growth. The vials were covered with wet muslin cloth to retain moisture. These vials were observed on daily basis and weight of larvae in each treatment was recorded after 7 days. The results obtained are summarized in Table 9. A pictorial representation of larvae on artificial diet control and artificial diet mixed with total soluble protein of transgenic plant line with modified cryl Ac-MV gene is provided in Figure 7.

Table 9: Effect of plant expressed modified Cry1 Ac-MV protein on insects in artificial diet.

S.No. Plant No. Diet plus protein/buffer Avg. larval wt (mg)*
1 Control untransformed plant, CI 8 ml + 2 ml 20.12
2 Protein Extraction buffer 8 ml+ 2 ml 27.34
3 Tl transgenic plant no. A-12/6 8 ml + 2 ml 14.6
4 Tl transgenic plant no. A-l 6/7 8ml+2ml 12.53
5 Tl transgenic plant no. A-l 8/6 8ml+2ml 14.94
* Starting avg. larval wt. = 0.5 mg.
A significant degree of weight loss was observed in larvae fed on artificial diet with transgenic plant protein. The experiment further proved that modified crylAc-MV gene of this invention was expressing well in the transgenic tobacco and was able to form a functional three domain Cryl Ac toxin.
3. Use of DNA constructions to develop an intron less version of modified cryl Ac gene (modified crv1 Ac-V3): The effect of intron on expression of crylAc qene(s) in plants:
The DNA sequence of modified crylAc-MV of this invention from 887bp to 1776 bp (having intron in its middle and having Hindi 11 site at the start and Bgll I site at last) was chosen to design in-silico a sequence without intron. The sequence from HindllI site to last of first exon and from start of 2nd exon to Bgll I site was joined (thus removing intron) giving rise to a 660 bp long fragment, referred hereafter as Fragment "Y"(sequence not shown). To the start and to the last of this sequence, some more nucleotides were added to allow restriction enzymes to properly acquire the site at the time of its restriction digestion reaction. The Fragment "Y" along with prefixing and suffixing nucleotide pairs was chemically synthesized via techniques much known in the art through a contract DNA synthesis firm. The synthesized DNA was cloned in a routine cloning vector (proprietary of contract firm, referred hereafter as synthesis vector 3) and it was sequenced and confirmed. The synthesis vector 3 (SV3) was digested with HindiII and Bglll. The cloning vector pKRI-921N harboring modified crylAc-MV gene was digested in separate reactions with Ncol-Hindllf (Fragment from N-terminal to H ind 11 () and with N col -Bgl 11 (to isolate vector backbone). In a triple ligation reaction, HindiII-BgllI fragment from vector SV3 and Ncol-Hindlll fragment from pKRI-92IN were ligated to Ncol-Bglll vector backbone of pKRI-921N to give rise to cloning vector

pKRI-923N harboring modified crylAc-V3 gene, which is an intron less version of the gene modified crylAc-MV. This right frame translatable gene modified crylAc-V3 gene having no intron was now under the regulation of CaMV 35s duplicated promoter and nos terminator. The CaMV 35S enhanced promoter-modified crylAc gene-V3 segment between Sbf I and SacI sites were excised from pKRI-923N and ligated to vector pBI121 digested at corresponding enzymes. The ligation mix was transformed to E.coli competent cells and positive clones were identified for the recombinant binary vector (referred herafter as pKRI-573N) harboring modified cryl Ac-V3 gene as represented in CDS of SEQ ID No. 1 (i) via restriction digestion analysis. One of the positive clone was electroporated to Agrobacterium tumefaciens strain (preferably LBA 4404) containing disarmed Ti plasmid. At all steps of molecular cloning, standard molecular biology practices or techniques as detailed in Molecular cloning ( Sambrook and Maniatis) were followed ( incorporated herein by reference!).
Nicotiana tabacum var Petit Havana SR-1 leaf fcxplants (from axenically grown seedlings), approx. 1 cm2 pieces, were transformed by Agrobacterium tumefaciens strain LBA 4404 or any other competent Agrobacterium strain harboring binary vector containing modified cry 1 Ac gene-V3 of SEQ ID No. 1 (i) of the present invention under the regulation of CaMV 35S enhanced promoter and nos terminator or other regulatory elements for constitutive or inducible expression ( in a preferred embodiment with pKRI-573N), and transformed explants were regenerated on selection medium containing MS basal salts, RT vitamins, 100 mg/L myo-inositol, 3% w/v Sucrose, 0.075 mg/L NAA, 1.25 mg/L BAP, 0.8% w/v agar, 300mg/L Kanamycin and 300 mg/L Cefotaxime. Twenty transgenic plants were regenerated and acclimatized in pots containing potting mix in transgenic containment. The putative transgenic plants were analyzed for expression of modified crylAc-V3 of the present invention by commercially available anti-CrylAc monoclonal antibody coated ELISA plates. For comparison of expression of the modified crylAc- V3 (i.e. intron less version of the gene) with that of modified cryl Ac-MVgene in plants, some more TO transgenic tobacco plants were developed with modified cryl Ac-MV (containing intron) of the present invention. These TO transgenic plants with modified cryl Ac-MV gene were also analyzed for expression by commercially available anti-CrylAc monoclonal antibody coated ELISA plates. The results in the form of ELISA data obtained with 12 best expressers from both of these group of plants along with unrransformed controls are presented in Table 8,
Table 8: Expression of modified cryl Ac-MV Gene (containing intron) and modified cryl Ac-V3 gene (without intron) in transgenic tobacco as detected by ELISA

Transg< gene ;nic tobacco with modified cryl Ac-MV Transgenic tobacco with modified cryl Ac -V3
S.No. Plant No. ELISAOD value S.No. Plant No. ELISA OD value
1 TO plant no. A-22 1.254 1 TO plant no. C-l 0.668
2 TO plant no. A-26 1.859 2 TO plant no. C-2 0.631
3 TO plant no. A-28 1.543 3 TO plant no. C-4 0.845
4 TOplantno.A-31 1.973 4 TO plant no. C-5 0.712
5 TO plant no. A-32 1.426 5 TO plant no. C-6 0.643
6 TO plant no. A-34 1.578 6 TO plant no. C-9 0.594
7 8 T0plantno.A-40 TO plant no. A-43 1.395 1.246 7 8 TO plant no. C-l2 TO plant no. C-l 5 0.621 0.793
9 T0plantno.A-47 1.134 9 TO plant no. C-l 6 0.348
10 TO plant no. A-49 1.373 10 TO plant no. C-l 8 0.572
11 T0plantno.A-52 1.854 11 TO plant no. C-l 9 0.461
12 TO plant no. A-55 1.642 12 TO plant no. C-20 0.389
13 Untransformed control 0.007 13 Untransformed control 0.006
The results obtained clearly indicate 1hat intron enhances the expression level of a prokaryotic sequence designed for eukaryotic expression in plants, preferably modified crylAc gene (s) of the present invention. The DNA sequences and the codons of the two genes (modified cryl Ac-MV and modified crylAc-V3) were exactly similar, except that one contained intron and other was not having it. Stated differently, incorporation of intron in the sequence has significantly improved the expression level of genes in plants. Another advantage of incorporating intron is that it prevents proakryotes to take use of the gene containing introns, post horizontal transfer, if any, from the transgenic plants in the field as prokaryotes in general are known for their inability to process intron from the transcript.
4. Use of DNA constructions to transform cotton embryoqenic cells: expression of modified crylAc gene (s) of the invention in cotton:

Cotton (Gossypium hirsutum) cv Coker 312 embryogenic calli were transformed with pKRI-571N (harboring modified crylAc-MV gene), pKRI-572N (harboring modified crylAc-V2a gene) and pKRI-573N (harboring modified crylAc-V3 gene) in separate experiments via Agrobacterium mediated transformation. The transformed calli in each experiment were selected for three subculturing cycles on medium containing MS (1962) basal salts, GAM B5 (1968) vitamins, 100 mg/L myo-inositol, 3% w/v Glucose, 0.8% w/V agar, 50mg/L Kanamycin and 250 mg/L Augmentin, pH 5.8. The selected embryogenic calli from each experiment were tested for expression of corresponding modified crylAc gene (s) in cotton cells via ELISA utilizing commercially available (from Amar Imunodiagnostics, Hyderabad) anti-Cry 1 Ac monoclonal antibody coated ELISA plate kit. The results obtained are tabulated in Table 9.
Table 9: Expression of modified crylAc- gene (s) in transformed cotton embryogenic cells as detected by ELISA.

Embryogenic calli transformed Embryogenic calli transformed Embryogenic calli
with modified cry lAc-MV gene with modified cryl Ac-V2a gene transformed with modified
crylAc-V3 gene
S. Callus No. ELISAOD S. Callus No. ELISA S. Callus No. ELISA
No. value No. OD value No. OD value
1 Cott-EC- Al 1.356 1 Cott-EC- Bl 0.318 1 Cott-EC-CI 0.643
2 Cott-EC- A2 1.052 2 Cott-EC- B2 0.364 2 Cott-EC-C2 0.721
3 Cott-EC- A3 1.539 3 Cott-EC- B3 0.315 3 Cott-EC-C3 0.758
4 Cott-EC- A4 1.428 4 Cott-EC- B4 0.498 4 Cott-EC- C4 0.237
5 Cott-EC- A5 1.715 5 Cott-EC- B5 0.317 5 Cott-EC- C5 0.298
6 Cott-EC- A6 1.683 6 Cott-EC- B6 0.334 6 Cott-EC- C6 0.565
7 Cott-EC- A7 0.978 7 Cott-EC- B7 0.269 7 Cott-EC- C7 0.294
8 Cott-EC- A8 1.497 8 Cott-EC- B8 0.543 8 Cott-EC- C8 0.579
Cott-EC- UT (Untrans.Ctrl) Cott-EC- UT (Untrans.Ctrl) Cott-EC- UT (Untrans.Ctrl
9 0.005 9 0.006 9) 0.005
The results obtained showed that the modified crylAc gene (s) of the invention were expressing in

transformed cotton cells effectively.
5. Use of chosen 659 aa long Crv1 Ac-MV and 603 aa long Crv1 Ac-V2a polypeptide sequences to get the protein expressed in and purified from E.coli : Checking the efficacy of purified proteins in artificial diet mixing experiments on insects larvae'.
A. Expression and purification of protein(s) from E.coli:
A DNA sequence was designed for chosen 659aa long sequence of Cryl Ac-MV protein, utilizing the codons of E.coli K12 strain for high expression in bacteria. To the N-terminal of DNA sequence DNA sequences encoding Hex- His tag and a sequence encoding a protease site was added. This sequence was chemically synthesized and cloned in an appropriate expression vector (DNA sequence and vector details not shown). This was given to a contract research firm for expression and purification of the protein CrylAc-MV from bacteria viz. E.coli. The contract firm finally provided tag free and purified 659 aa long Cryl Ac-MV protein. Similarly a DNA sequence was also designed for chosen 603 aa long sequence of CrylAc-V2a protein and this was expressed and purified from E.coli by contract research organization. The concentrations of thus obtained protein(s) were estimated by BIO-RAD's Bradford dye reagent kit.
B. Artificial diet mixed with purified protein(s) based bioassay experiment with H, armiqera
neonatal larvae:
250 ml of artificial diet (containing Chickpea fluor 100 g/L, Wheat germ 30 g/L, Casein purified 40 g/L, Sucrose 25 g/L, Sorbic acid 2 g/L, Methyl paraben 2 g/L, Cholesterol 1.25 g/L, Choline chloride 125 g/L, Yeast extract 16 g/L, L-Ascorbic acid 5 g/L, Vitamin E capsules 2 nos./L, Vit-B-Complex capsules- 2nos/L, Formaldehyde 250ul/L and Agar 18 g/L of distilled water) for Helicoverpa armigera larvae was prepared. With the help of a syringe without needle,aliquots of 4 ml of boiled diet was dispensed in 16 petri-dishes and allowed to solidify at room temperature. To 4ml of the cooled, semi-solid diet, either purified protein in different concentration but in 1 ml volume (purified protein was diluted in carbonate buffer) was added and homogenized or 1ml of distilled water (treatment code 1 or treatment code 11 ) or 1 ml of carbonate buffer (treatment code 2 or treatment code 12) was added and homogenized. The treatments with water or carbonate buffer were used as controls. A small aliquot from each of different treatments artificial diet was transferred to bioassay vials and coded appropriately. To each vial 10-12 larvae were released and experiment was repeated. In any experiment, however, observation for mortality, if any, was taken on successive 24 hour basis and larval weight of survivors was recorded after each 72 hrs. Diet in each treatment was replaced with fresh diet from the same combination after 72 hrs of starting the

experiment. Experiment was run for 7 days and mortality after 7 days was recorded as percentage mortality and presented in Table 10.
Table 10: Bioassay of Purified, modified Cry1 Ac protein(s) on artificial diet with H. armigera neonatal larvae

Treatmen Description Avg. Larval Avg. Larval %
tCode Wt.*
of survivors
after 3 days
(mg) Wt.
of survivors
after 6 days
(mg) mortality**
Bioassay with purified modified Cry1Ac-MV prote n
Control 1
1 Artificial Diet with water 1.776 10.030 0.00
Control 2
2 Artificial Diet with Buffer 1.917 10.760 0.00
Artificial Diet mixed with purified protein @ lmicrogram protein per
3 ml of diet 0.902 2.590 33.33
Artificial Diet mixed with purified protein @ 2 microgram protein per
4 ml of diet 0.691 2.190 37.50
Artificial Diet mixed with purified protein @ 5 microgram protein per
5 ml of diet 0.500 1.530 62.50
Artificial Diet mixed with purified protein @ 10 microgram protein
6 per ml of diet 0.291 1.080 66.67
Artificial Diet mixed with purified protein @ 20 microgram protein
7 per ml of diet 0240 0.930 88.89
Artificial Diet mixed with purified
8 protein @ 50 microgram prote in 0240 0.670 88.89

per ml of diet
Bioassay with pu rifled modified Cry1Ac-V2a protein
Control 1
11 Artificial Diet with water 1.998 15.150 0.00
Control 2
12 Artificial Diet with Buffer 2.756 21.035 0.00
Artificial Diet mixed with purified protein @ 1 microgram protein per
13 ml of diet 1.988 5.795 25.00
Artificial Diet mixed with purified protein @ 2 microgram protein per
14 ml of diet 1.724 1.945 42.85
Artificial Diet mixed with purified protein @ 5 microgram protein per
15 ml of diet 1.570 39.570 66.67
Artificial Diet mixed with purified protein @ 10 microgram protein
16 per ml of diet 3.240 34.270 28.57
Artificial Diet mixed with purified protein @ 20 microgram protein
17 per ml of diet 2.843 13.320 70.00
Artificial Diet mixed with purified protein @ 50 microgram protein
18 per ml of diet 1.790 11.670 90.00
* Initial average larval weight was 0.3 mg, ** as calculated on 7 day of challenging the larvae with diet.
After closely examining the data, it was found that, whereas both the modified CrylAc-MV and modified CrylAc-V2a proteins caused significant mortality of insects, but only modified CrylAc-MV clearly showed a dose response in terms of average larval weight post 3 days and 6 days of challenging the larvae with the artificial diet mixed with purified protein. A graphical representation of the same for both of these proteins has been presented in Figure 8A and 8B.

C. Cotton leaf painting with purified protein(s) based bioassay experiment with H. armigera 1st instar larvae:
Small, square leaf pieces (approx. 2cm long/ 2 cm wide) were cut from a polyhouse grown untransformed plants of cotton (Gossypium hirsutum) and both the surfaces of the leaf pieces were painted with either purified protein(s) in different concentrations or with distilled water or with carbonate buffer. A range of protein concentrations were prepared in carbonate buffer, pH 9.6 (ranging from 1 microgram of protein per ml of carbonate buffer to 50 ug protein per ml of carbonate buffer). The painted cotton leaf pieces were kept at 4°C for 2hours so that protein or painted liquid could be adsorbed on the surface. Six painted leaf pieces per treatment were transferred to individual bioassay vial containing a wet filter paper so that leaf does not get dry during the experiment. To each bioassay vial containing one painted leaf piece, one 1st instar H. armigera was released. The experiment was run for 72 hours after which percent mortality per treatment was counted. The results obtained are presented in Table 11 and pictorial representation of a sample from this experiment has been given in Figure 9.
Table 11: Bioassay of cotton leaves painted with Purified modified Cry1 Ac protein(s) with H. armigera 1st instar larvae

Tr. Description % Tr. Description %
Cod e Mortality
* Cod e Mortality
*
Bioassay with purified modified ( cry1Ac- Bioassay with purified modified CrylAc-
MV protein V2a protein
Control 1 Control 1
1 Leaf pieces painted with water 0 11 Leaf pieces painted with water 0
Control 2
Leaf pieces painted with Control 2
Leaf pieces painted with
2 Buffer 0 12 Buffer 0
Leaf pieces painted with purified protein @ 1 Leaf pieces painted with purified protein @ 1
3 microgram protein per ml of 16.67 13 microgram protein per ml of 33.33

buffer buffer
Leaf pieces painted with purified protein @ 2 microgram protein per ml of Leaf pieces painted with purified protein @ 2 microgram protein per ml of
4 buffer 33.33 14 buffer 33.33
Leaf pieces painted with purified protein @ 5 microgram protein per ml of Leaf pieces painted with purified protein @ 5 microgram protein per ml of
5 buffer 66.67 15 buffer 50
Leaf pieces painted with purified protein @ 10 microgram protein per ml of Leaf pieces painted with purified prote in @ 10 microgram protein per ml of
6 buffer 66.67 16 buffer 33.33
Leaf pieces painted with purified protein @ 20 microgram protein per ml of Leaf pieces painted with purified protein @ 20 microgram protein per ml of
7 buffer 83.33 17 buffer 66.67
Leaf pieces painted with purified protein @ 50 microgram protein per ml of Leaf pieces painted with purified protein @ 50 microgram protein per ml of
8 buffer 100 18 buffer 100
* As calculated after 72 hrs of challenging the painted leaves with protein dilutions or control solutions.
In this case also, a good dose response for treatments was observed in case of modified Cryl Ac-MV protein but the same was not observed in modified Cry 1 Ac-V2a protein based treatments.
P. Artificial diet mixed with modified Crv1Ac-MV purified protein based bioassav experiment with Chilo partellus neonatal larvae:
Chilo partellus is a deadly lepidopteran pest of maize and other cereals in Indian subcontinent as well as other parts of world. To check the efficacy of chosen 659 aa long modified Cryl Ac-MV protein against the larvae of Chilo partellus, a bioassay experiment was done utilizing purified protein of Cryl Ac-MV protein and an artificial diet for maize stem borers. 250 ml of maize leaf

powder based artificial diet (containing Chickpea powder 100 g/L, Maize leaf powder 26 g/L, Yeast extract 10 g/L, Ascorbic acid 2.5 g/L, Sorbic acid 1 g/L, Methyl -p-hydroxy benzoate 2 g/L, Vit E capsules 200IU/L, Sucrose 5 g/L, Formaldehyde 1.5ml/L and Agar 12 g/L of distilled water) was prepared. With the help of a syringe without needle, aliquots of 4 ml of boiled diet was dispensed in 8 petridishes and allowed to solidify at room temperature. To 4ml of the cooled, semi-solid diet, either purified protein in different concentration but in 1 ml volume (purified protein was diluted in carbonate buffer) was added and homogenized or 1ml of distilled water (treatment code 1 or treatment code 11 ) or 1 ml of carbonate buffer (treatment code 2 or treatment code 12) was added and homogenized. The treatments with water or carbonate buffer were used as controls. A small aliquot from each of different treatments artificial diet was transferred to bioassay vials and coded appropriately. To each vial 8 larvae were released and experiment was repeated. In any experiment, however, observation for mortality, if any, was taken on successive 24 hour basis and larval weight of survivors was recorded after each 72 hrs. Diet in each treatment was replaced with fresh diet from the same combination after 72 hrs of starting the experiment. Experiment was run for 7 days and mortality after 7 days was recorded as percentage mortality and presented in Table 12.
Table 12: Bioassay of Purified, modified Cry1Ac-MV protein on artificial diet with Chilo partetlus neonatal larvae

Treatmen Description Avg. Larval Avg. Larval %
t Code Wt.*
of survivors
after 3 days
(mg) Wt.
of survivors
after 6 days
(mg) mortality**
lc Control 1
Artificial Diet with water 0.464 2.170 0
2c Control 2
Artificial Diet with Buffer 0.419 2.263 0
3c Artificial Diet mixed with purified protein @ lug protein per ml of
diet 0.390 1.976 12.5
4c Artificial Diet mixed with purified protein @ 2 ug protein per ml of
diet 0.360 1.930 25

5c Artificial Diet mixed with purified protein @ 5 ug protein per ml of
diet 0.315 1.670 37.5
6c Artificial Diet mixed with purified protein @ 10 ug protein per ml of
diet 0291 1.208 75
7c Artificial Diet mixed with purified protein @ 20 ug protein per ml of All died
diet 0.210 100
8c Artificial Diet mixed with purified protein @ 50 ug protein per ml of diet All died 100
* Initial average larval weight was 0.2 mg, ** as calculated on 7 day of challenging the larvae with diet.
The results obtained clearly provided for the fact that the chosen 659 aa long CrylAc-MV was highly effective against Chilo parte 11 us larvae and modified crylAc-MV gene can be deployed in transgenic plant development program of maize or sorghum or any other crop to which C. partellus is a pest, to render them improved for insect resistance.

WE CLAIM:
1. An isolated, synthetic DNA sequence or variant thereof coding for a truncated three domain delta
endotoxin Cry protein of Bacillus thuringiensis, said truncated delta endotoxin Cry protein is
selected from a group comprising of:
(a) a 659 amino acid long protein preferably from Cryl subfamily having three domain N-terminal portion and starting helix-loop-helix-loop portion of C-terminal half;
(b) a variant of part (a) further comprising removal of N-terminal 56 amino acids and substitution of 57th and 58* amino acids with Metl and Ala2 thus resulting into a 603 amino acid long protein; wherein said truncated three domain delta endotoxin comprises three or more number of tryptic sites at C-terminal end for rapid and fidel conversion of protoxin to toxin core in the insect gut.
2. The synthetic DNA sequence or variant thereof as claimed in Claim 1, wherein said DNA
sequence or variant is selected from a group comprising of:
a) Nucleotide sequence of SEQ ID No. 1 or a complement thereof;
b) Nucleotide sequence having at least 90% sequence identity to the nucleotide sequence of SEQ ID No.l or a complement thereof;
c) Nucleotide sequence comprising said variant of said synthetic DNA sequence comprising nucleotide sequence of SEQ ID No. 2 or a complement thereof;
d) Nucleotide sequence having at least 90% sequence identity to the nucleotide sequence of SEQ ID No. 2 or a complement thereof.
and wherein said DNA sequence or variant thereof is optimized for high expression in higher plants.
3. The synthetic DNA sequence or variant thereof as claimed in Claim 1 or 2, wherein delta endotoxin protein encoded by said nucleotide sequence is preferably from Cry 1A subfamily.
4. The synthetic DNA sequence or variant thereof as claimed in Claim 1 or 2, wherein Cry protein encoded by said nucleotide sequence comprises a basic helix-loop-helix-loop portion at the C-terminal for efficient in-vivo stability during expression and folding in plant cell milieu.
5. The synthetic DNA sequence or variant thereof as claimed in Claim 1 or 2, further comprising an intron in the coding sequence, preferably in the protein's domain II encoding sequence, so as to increase the expression of said DNA sequence or variant thereof to higher level in higher plants.
6. The synthetic DNA sequence or variant thereof as claimed in Claim 5, wherein said intron
comprises castor bean catalase intron or like.
7. The synthetic DNA sequence or variant tiiereof as claimed in Claim 1 or 2, wherein the
nucleotide composition of intron comprising synthetic DNA sequence of SEQ ID No.l comprises

A : 29.31 %, C : 20.80 %, G : 20.66 %, T : 29 22 % .
8. The synthetic DNA sequence or variant thereof as claimed in Claim 1 or 2, wherein the nucleotide composition of cDNA of synthetic DNA sequence of SEQ ID No.l comprises A: 30.01% , C: 21.58 %, G: 21.23 % andT : 27.18 %.
9. The synthetic DNA sequence or variant thereof as claimed in Claim 1 or 2, wherein the nucleotide composition of intron comprising synthetic DNA sequence of SEQ ID No.2 comprises A : 29.38 % , C : 20.65 %, G : 20.65 % and T : 29.33 %.

10. The synthetic DNA sequence or variant thereof as claimed in Claim 1 or 2, wherein the nucleotide composition of cDNA of synthetic DNA sequence of SEQ ID No.2 comprises A: 30.07 %, C: 21.56 %, G: 21.28 %,T : 27.09 %..
11. The synthetic DNA sequence or variant (hereof as claimed in Claim 1 or 2, comprises highly expressed plant preferred codons at more than 90% of the positions.
12. The synthetic DNA sequence or variant thereof as claimed in Claim 1 or 2, comprises 20 % to 30 % higher codon adaptive indices than the native gene for expression in higher plants.
13. The synthetic DNA sequence or variant thereof as claimed in Claim 1 or 2, wherein the coding sequences of said DNA sequence do not contain any eukaryotic polyadenylation signal or transcript termination signals.
14. The synthetic DNA sequence or variant thereof as claimed in Claim 1 or 2, comprises an intron splicing she allowing the removal of intron post transcription.
15. The synthetic DNA sequence or variant thereof as claimed in Claim 1 or 2, wherein said . nucleotide sequence of SEQ ID No.l and SEQ ID No.2 encode insecticidal protein (s)with additional 56 amino acids at C-terminal.
16. The variant of synthetic DNA sequence as claimed in Claim 1 or 2, wherein said nucleotide sequence of SEQ ID No.2 encodes insecticidal protein, said protein sequence comprises deletion of N-terminal 56 amino acids and substitution of 57 and 58 amino acids with Met 1 and Ala 2.
17.. The synthetic DNA sequence or variant thereof as claimed in Claim 1 or 2, wherein said
protein sequence encoded by SEQ ID No. 1 further comprises substitution with Asparagine at 424 th
position, Isoleucine at 628th position, Glycine at 634th and 648th position and tyrosine at 649th
position.
18.The synthetic DNA sequence or variant thereof as claimed in Claim 1 or 2, wherein said protein
sequence encoded by SEQ ID No. 2 further comprises substitution with Asparagine at 368th
position, Isoleucine at 572nd position, Glycine at 578 and 592 position and tyrosine at 593
position.
19.An in silico method for the preparation of truncated three domain delta endotoxin Cry protein of
Bacillus thuringiensis as claimed in claim 1, wherein said method comprises steps of:

a) Essential truncation at C-terminal half while addition of 56 amino acids extra at C-terminal which results into a 659 amino acid long protein as encoded by nucleotide sequence of SEQ ID No.l;
b) Further modification of the above protein of step (a) by the removal of 56 amino acids from N-terminal half and substitution of 57th and 58th amino acids with Metl and Ala2 thus resulting into a 603 amino acid long protein as encoded by nucleotide sequence of SEQ ID No. 2;
wherein said delta endotoxin comprises three or more number of tryptic sites at C-terminal end for
rapid and fidel conversion of protoxin to toxin core in the insect gut.
20.The in silico method as claimed in Claim 19, wherein said protein sequence encoded by SEQ ID
No. 1 further comprises substitution with Asparagine at 424 position, Isoleucine at 628 position,
Glycine at 634th and 648th position and tyrosine at 649th position.
21 .The in silico method as claimed in Claim 19, wherein said protein sequence encoded by SEQ ID
No. 2 further comprises substitution with Asparagine at 368* position, Isoleucine at 572nd
position, Glycine at 578th and 592nd position and tyrosine at 593rd position.
22.An in silico method for the preparation of synthetic DNA sequence or variant thereof encoding
truncated three domain delta endotoxin Cry protein of Bacillus thuringiensis as claimed in Claim
19, wherein said method comprises steps of:
a) Codon optimization of the coding sequence of the said DNA sequence or variant thereof in a way
that codons coding for a particular amino acid are not randomly distributed across the sequence and
at more than 90% of the positions for a particular amino acid only highly expressed codons are
used.
b) Codon optimization of the coding sequence of the said DNA sequence or variant thereof in a way that said optimized sequence has 20 % to 30% higher codon adaptation indices than the native gene.
c) incorporation of an intron in the coding sequence, preferably in the protein's domain II encoding sequence so as to increase the expression of said DNA sequence or variant thereof to higher level in higher plants.
d) a further step of incorporation of an intron splicing site allowing the removal of intron post transcription in the resulting nucleotide sequence from step (c);
wherein said DNA sequence or variant thereof is optimized for high expression in higher plants and wherein such higher plants come from a group comprising of monocotyledonous and dicotyledonous plants.
23 .A DNA construct for cloning and/or transforming prokaryotic or eukaryotic organisms, comprising the modified, synthetic DNA sequence or variant thereof as claimed in Claim 1 or 2. 24. A prokaryotic or eukaryotic host comprising said modified, synthetic DNA sequences as

claimed in Claim 1 or 2.
25. Aprokaryotic or eukaryotic host comprising said synthetic DNA sequence as claimed in Claim 1 or 2, wherein said host is resistant to insect pests selected from a group comprising of lepidopteran, dipteran and coleopteran pests or combination thereof.
26. The prokaryotic or eukaryotic host as claimed in claim 24 or 25 is selected from a group comprising of bacteria, algae and higher plants.
27. The higher plant host as claimed in Claim 26, wherein said plant is selected from a group comprising of tobacco, tomato, pigeonpea, cotton, okra, brinjal, cauliflower, rice, maize, sorghum, sugarcane or any other plant from monocotyledonous or dicotyledonous group of plants.
28. The prokaryotic or eukaryotic host as claimed in Claim 25, wherein said insect pest is selected from a group comprising of shoot borer, stem borer, fruit borers or other plant part eaters or combination thereof.
29. The prokaryotic or eukaryotic host as claimed in Claim 28, wherein said plant eater is selected from a group comprising of Helicoverpa armigera, Heliothis virescens, Leucinodes orbonalis, Pectinophora gossypiella, Manduca saxta, Chilo par tell us, Scirpophaga sp., Ostrinia nubilal is or like or any combination thereof.
30. The synlhetic DNA sequence or variant thereof as recited in Claim 2, wherein the said DNA sequence may be employed for pyramiding other insect resistance gene (s) encoding similar Bacillus thuringiensis Cry proteins, Cyt proteins, Vip proteins or other insecticidal proteins known to provide resistance to insect pests, to provide multi mechanistic host resistance to insect pests and to delay the potential insect resistance evolution.