A recombinant insecticidal gene for protecting agricultural crop

Abstract

The present invention relates to a recombinant insecticidal gene for protecting agricultural crops and a method for protection from insect pests by modifying Chimeric δ-endotoxin protein Cry1EC to provide mCry1EC gene having SEQ ID NO.2 for a broader insecticidal host range and expressing the peptide in crop plants. The modification includes mutations at positions 475,476, and 483 in Domain III, wherein the amino acid Leucine, Glycine, and Lysine are replaced with Valine, Tryptophan, and Threonine respectively. The transgenic crop plant causes insect mortality upon feeding and renders protection from the plurality of insect pests.

Description

[0001] PT / 2025 / 16752

[0002] A RECOMBINANT INSECTICIDAL GENE FOR PROTECTING AGRICULTURAL CROP

[0003] FIELD OF INVENTION

[0004] The present invention relates to a recombinant insecticidal gene for protecting agricultural crops. The present invention also relates to a method for protecting crops from lepidopteran insect pests by engineering Chimeric b-endotoxin proteinCryl EC for a broader insecticidal host range and expressing the said engineered protein in crop plants. The transgenic crop plant causes larval mortality upon feeding and renders protection from the plurality of lepidopteran insect pests.

[0005] BACKGROUND OF THE INVENTION

[0006] Loss in crop yield due to the attack of various insect pests and associated diseases is a worldwide problem for sustainable agriculture. Insect pests are primarily of two types: chewing (insects of the order Lepidoptera and Coleoptera) and sucking type (insects of the order Hem iptera). Both edible and non-edible crops are highly vulnerable to these insect pests. Several agriculturally important crops, such as cotton, corn, soybean, etc., have been genetically engineered to produce insecticidal toxins from Bacillus thuringiensis (Bt) to safeguard against the major damaging lepidopteran insect pests. Approximately 68% of the cultivated cotton crop worldwide is now genetically modified (GM), containing Bt genes, namely CrylAc, Cry2Ab, Cryl Fa, Vip3A, etc (Zaidi et al. 2021 ). All these potent molecules exhibit lower LC50 values and increased toxicity against the target pests and have been utilized in the development of gene-pyramided next-generation genetically modified crop plants. The lepidopteran pests targeted by Bt cotton include the Helicoverpa armigera (cotton bollworm), Heliothis spp, Pectinophora gossypiella (Pink bollworm), and Spodoptera spp., which are major pests in many leading cotton, com, and soybean-producing countries. GM crops expressing insecticidal protein also require lower pesticides, which kill both destructive and economically useful crop insect pests globally (Qaimef al. 2009). Bt cotton technology has been embraced by millions of farmers worldwide, including numerous smallholders in developing nations. PT / 2025 / 16752

[0007] Although BT crops have remained effective for decades against most targeted pest populations, however, the rapid evolution of pest resistance to Bt crops has been observed. The 26 listed cases of practical resistance in a population of 11 pest species collectively reduce the effectiveness of nine Bt toxins in seven countries (Tabashnikef al. 2023). As a result, P.gossypiella (Pink bollworm) and S.frugiperda (Fall armyworm) became a significant problem in many major cotton, com, and soybean-producing countries, causing losses of millions of dollars every year. Additionally, managing S.Htura (Leaf armyworm) has become increasingly challenging due to its resistance to numerous insecticides, impacting various edible crops (Ahmad et al. 2007). Among all pest species, the pink bollworm resistant to existing Bt technology, is particularly difficult to control with insecticides due to its feeding inside the cotton boll, where pesticides do not reach. To overcome the challenges, there is an urgent need to develop a sustainable solution to control the menace of insect pests.

[0008] Several control strategies have been developed and implemented to protect the crop against lepidopteran insect pests. Pesticides are the most preferred strategy due to their quick action and rapid control of the insect population. Availability in the local market and ease of use have made pesticides the first choice for farmers (Aktaref al., 2009; Smith et al., 2019; Sarkar et al., 2021 ).

[0009] The sterile insect technique (SIT) in which mass-reared insects are irradiated with Gamma or X-rays. Treated insects remain sexually competitive but cannot produce offspring. SIT has been used to control major insect pests, including the pink bollworm in the USA, a global pest of cotton (Morrison et al. 2012).

[0010] The Integrated Pest Management (IPM) approach for control of target insect pests has been implemented for several years in many countries. The following are some key components of an IPM approach for managing the problem of emerging lepidopteran insect pests. a) Crop rotation with non-host plants like corn or sorghum to disrupt the life cycle of target insect pests, especially monophagous insect pests like pink bollworm. b) Pheromone trap strategy for capturing / diverting / confusing male moths by releasing female-specific sesquiterpene pheromone. PT / 2025 / 16752 c) Uses of biological control, such as parasitic wasps, which are natural enemies of pink bollworm, and predators like ladybirds. d) Chemical control of lepidopteran worms through rotational use of different chemical categories to reduce the risk of resistance development.

[0011] Genetically modified crops (Bt cotton) expressing cry proteins of Bacillus thuringiensis in combination with the refugia approach have been cultivated worldwide for the last two decades to protect the crop from target insect pests. For example, Bt cotton expressing CrylAc and Cry2Ab protects cotton bollworm, pink bollworm, and leaf armyworm, etc.

[0012] A variety of plant extracts, such as saponins, tannins, alkaloids, and di and triterpenoids, have been used in the past few years to control the major crop insect pests. These metabolites cause various impacts on insect populations, including inhibitory effects on many insects, acting as repellent, antifeedant, ovicidal, insecticidal, cellular toxicity inducer, mortality inducer, reproductive suppressor, fertility and fecundity reducer, and growth inhibitor (Carvalhoef al. 2013).

[0013] Excessive use of pesticides and substandard methods of application raise multifaceted problems, like killing of predators, development of pesticide resistance in insect pests, and environmental hazards (Aggarwal et al., 2006; Horowitz et al., 2011 ; Raj mohan, 2020).

[0014] The sterile insect technique (SIT) is an effective and environment-friendly approach, but it has several limitations, such as high cost, the requirement of infrastructure and expertise, the requirement of continuous release of sterile insects, and ineffective for high pest populations, etc.

[0015] IPM approaches also have some limitations, including high labour and time demand for regular monitoring, and delayed pest control due to the lower effectiveness of biological and cultural methods. IPM can also be less effective in managing severe pest outbreaks in crops (like pink bollworm emergence in Bt cotton). (Bajwaef al. 2002, Koul et al. 2008) PT / 2025 / 16752

[0016] It is well studied and documented that many lepidopteran insect pests, viz., pink bollworm and fall armyworm, have currently evolved to tolerate the Bt toxin, diminishing the productivity of Bt crops (Tabashnikef al. 2008, Selvarani et al. 2024).

[0017] Plant extracts often provide short-term protection and degrade quickly under various environmental conditions that require frequent re-application (Ismanef al. 2006).

[0018] Cotton crops are facing a menace from Pink Bollworm (P.gossypiella). A few Bt proteins, including those deployed in GM cotton, were effective against P. gossypiella. However, the insect has developed resistance by modifying its receptors. At present, an insecticidal protein that can kill its larvae at less than 100 ppm is not known. Thus, GM cotton resistant to P. gossypiellais not available anywhere in the world. S. frugiperda (Fall armyworm) is a deadly insect pest of Maize all over the world, while a sporadic pest of cotton. A GM technology for defense against this insect is also not available to the farming community. These pests are controlled by synthetic pesticides, yet they cause yield loss of over a billion US dollars.

[0019] Chimeric b-endotoxin protein Cryl EC disclosed in Indian patent no: 243632 is efficacious against S.Htura only. Cryl EC protein (Indian patent no: 243632) is found deleterious to cotton, causing abnormal growth and development and delaying its flowering. The mCryl EC protein provided in the present invention is toxic to larvae of three field crop insect pests S. litura, S. frugiperda, and P. gossypiella, in a range of 22-310 ng / g of diet. mCryl EC exhibits a broad range of insecticidal activity and is highly efficacious.

[0020] OBJECTIVES OF THE INVENTION

[0021] The main objective of the present invention is to provide a recombinant insecticidal gene for plant protection .

[0022] Another objective of the present invention is to provide a recombinant mCryl EC gene for its broad-spectrum insecticidal activity.

[0023] Another objective of the present invention is to provide the recombinant gene for the expression of mCryl EC. PT / 2025 / 16752

[0024] Another objective of the present invention is to select and modify specific amino acid residues in region 464 to 493 of the Cry1 EC protein.

[0025] Yet another objective of the present invention is to identify candidate regions in domain III for enhancement of the host range and toxicity of Cry1 EC.

[0026] Yet another objective of the present invention is to mutate the Leucine, Glycine, and Lysine at positions 475,476, and 483 with other amino acids to prepare mCryl EC.

[0027] Yet another objective of the present invention is to express Cry1 EC variant proteins in E. coli and evaluate their insecticidal activity against P. gossypiella, S. litura, and S. frugiperda.

[0028] Yet another objective of the present invention is to construct a plant expression cassette in a binary vector, harbouring mcrylEC gene under the regulation of a woundinducible plant promoter for the expression of mCryl EC protein in plants.

[0029] Another objective is to evaluate the toxicity of the mCryl EC protein expressed in stable transgenic cotton against S. / / a( Cotton leaf armyworm), S. frugiperda(Fa\\ armyworm), and P. gossypiella(Pin bollworm).

[0030] Still another objective of the present invention is to introduce the mCryl EC expression cassette into the cotton genome through Agrobacterium-mediated transformation and develop stable transgenic cotton lines.

[0031] Still another objective of the present invention is to obtain insect pest-resistant GM cotton lines without any phenotypic anomalies by expressing mCryl EC protein.

[0032] SUMMARY OF THE INVENTION

[0033] Accordingly, the present invention relates to a recombinant insecticidal gene for protecting agricultural crops having SEQ ID NO.2. More particularly the present invention provides a modified chimeric 5-endotoxin protein mCryl EC of SEQ ID NO: 1 , with broad-spectrum insecticidal properties than the original Cry1 EC protein (Indian patent no: 243632) and a gene of SEQ ID NO: 2, encoding the said modified chimeric 5-endotoxin protein (mCryl EC), and also a method of protecting crop plants from three major lepidopteran insect pests by expressing mCryl EC protein of SEQ ID NO: 1. PT / 2025 / 16752

[0034] Since Domain III of Cry toxins play an important role in host specificity same of Cry1 EC protein is selected for modification. Amino acids Leucine, Glycine, and Lysine at positions 475,476, and 483 in Domain III are identified and replaced with Valine, Tryptophan, and Threonine, respectively by site-directed mutagenesis. The mCryl EC expression cassette is introduced in the cotton genome through Agrobacterium- mediated genetic transformation. Stable transgenic lines expressing mCryl EC protein cause >90% larval mortality to three lepidopteran insect pests, Spodoptera litura (Leaf armyworm), Spodoptera frugiperda (Fall armyworm), and Pectinophora gossypiella (Pink bollworm) and show nearly complete protection from them. The mCryl EC protein expressed is also efficacious to Bollgard Il-resistant larvae of P. gossypiella, thus the GM cotton lines expressing mCryl EC are highly efficacious to resistant strains of P. gossypiella also.

[0035] In an embodiment, the present invention provides a recombinant gene (mCryl EC) useful as insecticidal agent having SEQ ID NO. 2.

[0036] In another embodiment of the present invention, the recombinant gene corresponding peptide sequence is having SEQ ID NO. 1 .

[0037] In yet another embodiment of the present invention, the peptide sequence (SEQ ID NO.2) having mutations at positions 475,476, and 483, wherein the amino acid Leucine, Glycine, and Lysine are replaced with Valine, Tryptophan, and Threonine respectively.

[0038] In another embodiment the present invention provides an expression cassette (PPCDI- mcryl EC-T nos) having SEQ ID NO: 3 containing the recombinant gene (SEQ ID NO.1 ).

[0039] In yet another embodiment of the present invention, the expression cassette consisting of: a) mcrylEC gene having seq id no.2 b) RbPCDI promoter c) NosT as transcriptional terminator PT / 2025 / 16752

[0040] In yet another embodiment of the present invention, wherein the target insect is selected from the group consisting of Pectinophora gossypiella, Spodoptera litura, and Spodoptera frugiperda.

[0041] Still another embodiment of the present invention, wherein the insecticidal toxicity is in the range of 22-310 ng / g.

[0042] Yet another embodiment of the present invention mCryl EC gene having seq id no.2 is toxic to broad range of insect pests, S. litura, S. frugiperda and P. gossypiella, with the LC50 in the range of 91 - 310, 22-107, and 34-141 ng / g of artificial diet, respectively.

[0043] BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure. 1 : Homology analysis of Cry1 EC protein with other Cry1 class of proteins.

[0045] Figure 2: Electropherogram showing the site-directed mutagenesis in crylEC gene for strategic amino acid substitution wherein (A) represents electropherogram of crylEC variants for single amino acid substitution at positions 476, 483 and 475, respectively, (B)represents amino acid substitutions at positions 476 and 483 and (C) represents variants for three amino acid substitutions at positions 475, 476, and 483.

[0046] Figure.3: Expression of Cry1 EC and mCryl EC proteins, purification, and their toxicity to P. gossypiella (A). Cryl EC and mCryl EC proteins expressed in E. coll after IPTG induction. Lane 1 represents the molecular weight marker, and lanes 2-3, 4-5, and 6- 7 represent protein expression from the empty, crylEC and mcrylEC harbouring vector, respectively, before and after IPTG induction. (B) Immunoblot analysis of the same gel showing expression of Cryl EC and mCryl EC after IPTG induction. Hise- tagged proteins were detected with Anti-Penta His antibody. (C)A representative gel image showing purification of Cry1 EC variants, partially purified mCryl EC is marked with an arrow. (D-E) Comparative toxicity of Cryl EC and mCryl EC protein against P. gossypiella at 500 ng / g and 50 ng / g of artificial diet, respectively, showing efficacy of mCryl EC against P. gossypiella. Cry1 EC did not cause any insect mortality and led to weight loss only at 500 ng / g.

[0047] Figure 4: mCryl EC expression cassette for production of mCryl EC in plants and map of the binary vector harbouring mCryl EC expression cassette for plant PT / 2025 / 16752 transformation.

[0048] Figure 5: Expression of mCryl EC protein in selected transgenic cotton line and toxicity of transgenic cotton line to larvae of S. litura, P. gossypiella, and S. frugiperda (A) Expression of mCryl EC in fresh leaf tissue of three independent transgenic lines (519, 508, and 523), expression in squares and flower bracts, and expression in boll bracts, epicarp, and immature seeds. (B) Average percent mortality of neonatal larvae of S. litura across 13 lines. Damage in non-transgenic cotton leaf and protection of transgenic cotton leaf establishes toxicity of mCryl EC to S. litura. (C) Protection of boll of transgenic cotton from P. gossypiella. 5 neonatal larvae were release on each boll of 12-15 day and covered with red kite paper. In non-transgenic cotton, larvae entered in bolls by making small entry hole and came out by making a big exit hole and damaged the boll completely. Larvae of P. gossypiella could not enter in cotton bolls of transgenic cotton (GM) and died due to feed of mCryl EC expressed in epicarp of cotton bolls. (D) Mortality of larvae of S. frugiperda after feed on transgenic cotton expressing mCryl EC protein.

[0049] LIST OF ABBREVIATIONS USED

[0050] IPTG-lsopropyl-[3-D-thiogalactopyranoside

[0051] P. gossypiella, - Pectinophora gossypiella

[0052] S. litura, -Spodoptera litura

[0053] S. frugiperda. - Spodoptera frugiperda

[0054] Bt - Bacillus thuringiensis

[0055] Cry1 Ac - Crystal protein 1 Ac

[0056] Cry2Ab - Crystal protein 2Ab

[0057] IPM - Integrated pest management mCryl EC- Modified Crystal protein 1 EC

[0058] Cry1 EC- Crystal protein 1 EC

[0059] E. coll- Escherichia coll PT / 2025 / 16752

[0060] GM- Genetically modified

[0061] DETAILED DESCRIPTION OF THE INVENTION

[0062] The present invention relates to a recombinant insecticidal gene for plant protection having SEQ ID NO.2. The present invention relates to modification of Cryl EC for its broad-spectrum insecticidal activity. For this, amino acids Leucine, Glycine, and Lysine at positions 475,476, and 483 are replaced with Valine, Tryptophan, and Threonine, respectively. After these mutations, the protein is named mCryl EC.

[0063] The present invention also provides a construct of plant expression cassette in a binary vector, harbouring mcrylEC gene under the regulation of a wound inducible plant promoter for the expression of mCryl EC protein in plants.

[0064] The present invention relates to the strategic modification of previously developed chimeric 5-endotoxin protein Cryl EC of 641 amino acid residues (IN 243632) by substituting its three crucial amino acid residues. This modification broadened its insecticidal host range enabling more effective protection of the crop plants against three target insect pests, Spodoptera litura (Leaf armyworm), Spodoptera frugiperda(Fa\\ armyworm), and Pectinophora gossypiella (Pink bollworm).

[0065] The present invention describes the substitution of three amino acid residues Leucine, Glycine, and Lysine with Valine, Tryptophan, and Threonine at positions 475, 476, and 483, respectively. It is achieved by site-directed mutagenesis of crylEC gene. This generated a gene of 1926 base pairs of SEQ ID NO: 2 to encode a modified chimeric 5 endotoxin protein Cry1 EC (mCryl EC) of 641 amino acid residues of SEQ ID NO: 1 .

[0066] The Cryl EC protein was originally toxic to S. litura, with the LCsoin the range of 133- 850 ng / g of artificial diet. The protein did not show significant toxicity to two other important insect pests S. frugiperda and P.gossypiella. The present invention describes the improvement of Cryl EC by making its variants through site-directed mutagenesis, evaluating their insecticidal activity against three target pests, and selecting the most efficacious protein.

[0067] For this purpose, the amino acid sequence from 464 to 493 of Cry1 EC was selected and compared for homology with 14 proteins of the Cry1 class of BtS-endotoxins (Fig. 1 ). Based on the homology analysis, a few amino acid residues are selected for protein PT / 2025 / 16752 engineering through site-directed mutagenesis. Cryl EC and its modified Cryl EC variants are expressed in E. coli strain BL21 -Codon Plus (DE3)-RIL(Agilent Technologies, USA) using pET28a(+) expression vector (Novagene / Merck Millipore, USA), which included 6 Histidine residues at the C-terminus. Proteins are partially purified on a Ni-NTA column (Qiagen, Germany) and quantified with anti-Penta His antibody (Sigma-Aldrich, USA). The variant proteins are mixed in insect-specific artificial diets individually and qualitatively tested against neonatal larvae of the three target insect pests (collected from field and reared in laboratory). Substitution of three amino acid residues at positions 475 (Leucine), 476 (Glycine), and 483 (Lysine) with Valine, Tryptophan, and Threonine, respectively, made the protein highly toxic to P. gossypiella, S. frugiperda, and S. litura. The mutant variant protein is named mCryl EC.

[0068] Substitution of Glycine with Tryptophan at position 476 and Lysine with Threonine at position 483 changed the physicochemical properties of the protein and made it efficacious against three target insect pests. mCryl EC disclosed in the present invention is toxic to broad range of insect pests, S. litura, S. frugiperda and P. gossypiella, with the LCso in the range of 91 - 310, 22-107, and 34-141 ng / g of artificial diet, respectively.

[0069] Since Domain III of Cry toxins is crucial for host specificity, its peptide from position 464 to 493 selected for modification. The homology of the polypeptide is compared with the corresponding regions of 14 other proteins of the Cry1 class.

[0070] At position 476, significant variability observed, with the occurrence of Glycine in 4, Alanine in 3, Serine in 3, Threonine in 2, Phenylalanine in 1 , Tryptophan in 1 , and Glutamine in 1 of the 15 Cry proteins. Glycine in Cryl EC substituted with the above- mentioned amino acid by site-directed mutagenesis of thecrylEC gene.

[0071] Each variant cloned into pET28a (+) and introduced into the E.coli strain BL21 -Codon plus (DE3)-RIL for the production of recombinant proteins. Each variant protein is expressed and partially purified on Ni-NTA column. Their insecticidal activity is tested against the neonatal larvae of the three target insect pests. Substitution of Glycine with aromatic amino acids (Tryptophan or Phenylalanine) at position 476 enhanced the insecticidal activity and broadened the host range. PT / 2025 / 16752

[0072] Position 483 is dominated by positively charged amino acids, with Lysine in 7 proteins and Arginine in 6 proteins, whereas Serine and Threonine (uncharged polar amino acids) are found in one protein each. Substitution of Lysine at position 483 in Cry1 EC by site-directed mutagenesis with Serine or Threonine did not improve the insecticidal activity. However, when the substitutions with Tryptophan or Phenylalanine at 476 are combined with Serine or Threonine at 483 (double mutant), higher insecticidal activity obtained, with maximum activity in the case of Tryptophan at position 476 and Threonine at position 483.

[0073] Position 475 is dominated by Leucine, with its presence in 14 proteins. Only one protein has Valine at this position. Substitution of Leucine at position 475 of Cryl EC with Valine did not change the efficacy of the protein. When Leucine at position 475 was substituted with Valine, keeping Tryptophan at 476 and Threonine at 483, the toxicity of this variant protein (triple mutant) improved against three target insect pests as compared to the double mutant. This variant is named mCryl EC. mCrylEC gene, having codons for Valine, Tryptophan, and Threonine at positions 475, 476, and 483, cloned between a wound-inducible plant promoter and nos terminator to prepare mCryl EC expression cassette. The gene is introduced into the cotton genome through Agrobacterium-mediated genetic transformation. Stable transgenic lines expressing mCryl EC protein cause >90% larval mortality to neonatal larvae of P.gossypiella (Pink bollworm) and S.frugiperda (Fall armyworm) and > 70% mortality in S.Htura (Cotton Leaf armyworm), and show nearly complete protection of the transgenic cotton plants from three insects. The mCryl EC protein expressed in transgenic cotton is also efficacious against Boll gard Il-resistant larvae of P. gossypiella.

[0074] SEQ ID No 1

[0075] Amino acid sequence of mCryl EC (641 residues)

[0076] MAIVNNQNQCVPYNCLNNPENEILDIERSNSTVATNIALEISRLLASATPIGGILLGLFDAIWG SIGPSQWDLFLEQIELLIDQKIEEFARNQAISRLEGISSLYGIYTEAFREWEADPTNPALKEEM RTQFNDMNSILVTAIPLFSVQNYQVPFLSVYVQAANLHLSVLRDVSVFGQAWGFDIATINSR YNDLTRLIPIYTDYAVRWYNTGLDRLPRTGGLRNWARFNQFRRELTISVLDIISFFRNYDSRL YPIPTSSQLTREVYTDPVINITDYRVGPSFENIENSAIRSPHLMDFLNNLTIDTDLIRGVHYWA PT / 2025 / 16752

[0077] GHRVTSHFTGSSQVITTPQYGITANAEPRRTIAPSTFPGLNLFYRTLSNPFFRRSENITPTLGI

[0078] NVVQGVGFIQPNNADVLYRSRGTVDSLNELPIDGENSLVGYSHRLSHVTLTRSLYNTNITSL

[0079] PTFVWTHHSATNTNTINPDIITQIPLVKGFRVWGGTSVITGPGFTGGDILRRNTIGEFVSLQV

[0080] NINSPITQRYRLRFRYASSRDARVIVLTGAASTGVGGQVSVNMPLQKTMEIGENLTSRTFRY

[0081] TDFSNPFSFRANPDIIGISEQPLFGAGSISSGELYIDKIELILADATFKRRRWSVHKASRPLHL

[0082] HQQAGLAAD

[0083] SEQ ID No 2

[0084] Nucleotide sequence of mcry7EC(1926 bp)

[0085] ATGGCTATCGTTAACAACCAGAACCAGTGCGTCCCTTACAATTGCCTCAACAACCCAGA

[0086] GAACGAGATCTTGGACATCGAAAGATCCAATTCTACCGTGGCCACCAACATTGCTCTTG

[0087] AGATTTCCAGATTGCTCGCTAGCGCAACTCCCATTGGTGGCATCCTCCTTGGATTGTTC

[0088] GACGCCATTTGGGGTTCCATCGGACCATCACAATGGGATCTCTTCCTTGAACAGATCGA

[0089] GTTGCTCATTGACCAGAAGATCGAAGAGTTTGCTAGGAACCAGGCAATTAGCCGTCTC

[0090] GAGGGGATCTCTTCCCTTTACGGAATCTATACAGAGGCCTTCAGAGAGTGGGAAGCTG

[0091] ACCCTACTAATCCAGCATTGAAGGAAGAGATGCGTACTCAATTCAACGATATGAACTCT

[0092] ATCTTGGTCACCGCCATTCCTCTCTTCTCAGTGCAGAACTACCAAGTGCCATTCCTCTC

[0093] CGTCTATGTTCAAGCTGCAAACTTGCACCTTTCTGTCCTTCGCGACGTGTCCGTCTTTG

[0094] GTCAAGCCTGGGGCTTCGATATCGCTACTATCAACTCCCGTTACAACGACCTCACAAGG

[0095] TTGATTCCTATCTACACTGACTACGCTGTTAGATGGTACAATACTGGGCTTGACAGACT

[0096] CCCACGTACCGGCGGATTGAGGAATTGGGCTCGCTTCAACCAGTTTAGGCGTGAGCTC

[0097] ACCATTAGCGTGTTGGACATCATTTCCTTCTTCAGAAACTACGACTCTAGACTTTATCCT

[0098] ATTCCAACTAGTTCTCAACTCACCAGGGAGGTCTACACCGATCCTGTGATCAACATTAC

[0099] CGACTATCGTGTGGGTCCCTCCTTCGAGAACATTGAAAACAGCGCTATCAGATCTCCAC

[0100] ACCTTATGGACTTCCTCAATAACTTGACTATCGATACAGACCTTATCAGAGGTGTTCACT

[0101] ACTGGGCTGGCCATAGGGTCACCTCTCACTTTACCGGTAGTTCCCAAGTGATCACAAC

[0102] CCCTCAATACGGAATTACTGCCAACGCAGAGCCAAGACGTACCATTGCTCCAAGTACCT

[0103] TTCCCGGGTTGAACCTCTTCTACCGCACATTGTCAAATCCATTCTTCAGGAGATCTGAG

[0104] AACATCACCCCTACCCTTGGGATCAACGTTGTCCAGGGAGTGGGTTTCATCCAGCCAA

[0105] ACAATGCTGATGTGCTCTACAGGTCTAGAGGCACAGTGGACTCCTTGAACGAACTTCCA

[0106] ATTGACGGTGAGAACTCACTCGTCGGATACAGTCACCGTCTTAGCCACGTTACTTTGAC

[0107] CAGGTCTCTCTATAACACTAATATCACTAGTTTGCCCACCTTCGTGTGGACTCACCACTC

[0108] AGCCACCAACACAAACACTATCAATCCCGATATCATTACACAAATCCCCCTTGTCAAGG

[0109] GCTTCCGCGTTTGGGGAGGGACCTCCGTCATTACTGGGCCCGGATTCACCGGTGGCG

[0110] ATATCCTCCGTAGAAACACCATTGGTGAGTTTGTGTCCCTCCAGGTTAACATTAACTCTC

[0111] CTATCACACAAAGGTACCGTCTTAGGTTCCGCTACGCTTCCTCTAGAGACGCAAGAGTC PT / 2025 / 16752

[0112] ATTGTGCTTACCGGTGCCGCTTCCACAGGAGTCGGTGGCCAAGTCAGCGTTAACATGC CATTGCAAAAGACTATGGAGATCGGAGAGAACCTCACTAGTAGAACCTTCAGGTATACC GACTTCTCTAACCCTTTCTCCTTCCGTGCTAACCCAGATATCATTGGCATCAGCGAACA ACCTCTCTTCGGCGCCGGCTCCATCAGCTCTGGTGAACTCTACATCGATAAGATCGAGT TGATCCTTGCTGACGCCACATTCAAGAGGAGACGATGGAGCGTGCACAAAGCCTCACG CCCTCTTCACCTCCACCAACAAGCTGGACTCGCTGCTGATTAA

[0113] Thus, another aspect of the present invention is the construction of mCryl EC expression cassette in a binary vector for high expression of mCryl EC protein in plants and obtain transgenic lines with control alike phenotype. For this, the mcrylEC gene is cloned downstream of a wound-inducible plant promoter; NosT used as the transcriptional terminator. It generated a mCryl EC expression cassette of 2723 base pairs of SEQ ID NO: 3, to express mCryl EC in plants.

[0114] SEQ ID NO: 3

[0115] Nucleotide sequence of mCryl EC expression cassette (2723 bp)

[0116] TAACCGCTAGGCAGTGAGCCGTTAGGTGAAAAACTTAGGCATGTTAAAGACTAGTCTTGT TTTCATTTTTGTTTTTTAATGAAGTTTCCAAGGTAGACAAAAGCTCTAAAGCCCAGGACAG CACAGGATACTATCTCTTTTAAGTCTTTTCCTAAGCAAATAGTAAGATACGAAAATGTTGGT GCATTTGGGCATAATACATAGAATTTTTGGGCATACGGGAAATACCCCTTTTTGAACACTG TATTCTCGTCATTCCGCTTATCGTTTTACTTTTACCATCTGAAAAAGAAGGGAGTTGTCAA AAAAGTTGAGCCATTTTTACCGCCCTGCTTGGCTAAGTTCACATGCTGTTGTTTCCTTGA TCTTATCCGATTGCTTTCCTCCAATAACAACGCCACACGTACCCCACAAACCCACAGAGA ATAAAACAAAATGCGTCCCACCTTGACCTTAAATAGAGCAACAATTCTTACACACTGATCC CATAAGCAGTTCCAAGTTTCAGGTAACAGAGAAGAAGAAGCTTTAAACCATGGCTATCGT TAACAACCAGAACCAGTGCGTCCCTTACAATTGCCTCAACAACCCAGAGAACGAGATCT TGGACATCGAAAGATCCAATTCTACCGTGGCCACCAACATTGCTCTTGAGATTTCCAGAT TGCTCGCTAGCGCAACTCCCATTGGTGGCATCCTCCTTGGATTGTTCGACGCCATTTGG GGTTCCATCGGACCATCACAATGGGATCTCTTCCTTGAACAGATCGAGTTGCTCATTGAC CAGAAGATCGAAGAGTTTGCTAGGAACCAGGCAATTAGCCGTCTCGAGGGGATCTCTTC CCTTTACGGAATCTATACAGAGGCCTTCAGAGAGTGGGAAGCTGACCCTACTAATCCAG CATTGAAGGAAGAGATGCGTACTCAATTCAACGATATGAACTCTATCTTGGTCACCGCCA TTCCTCTCTTCTCAGTGCAGAACTACCAAGTGCCATTCCTCTCCGTCTATGTTCAAGCTG CAAACTTGCACCTTTCTGTCCTTCGCGACGTGTCCGTCTTTGGTCAAGCCTGGGGCTTC GATATCGCTACTATCAACTCCCGTTACAACGACCTCACAAGGTTGATTCCTATCTACACTG ACTACGCTGTTAGATGGTACAATACTGGGCTTGACAGACTCCCACGTACCGGCGGATTG PT / 2025 / 16752

[0117] AGGAATTGGGCTCGCTTCAACCAGTTTAGGCGTGAGCTCACCATTAGCGTGTTGGACAT CATTTCCTTCTTCAGAAACTACGACTCTAGACTTTATCCTATTCCAACTAGTTCTCAACTCA CCAGGGAGGTCTACACCGATCCTGTGATCAACATTACCGACTATCGTGTGGGTCCCTCC TTCGAGAACATTGAAAACAGCGCTATCAGATCTCCACACCTTATGGACTTCCTCAATAACT TGACTATCGATACAGACCTTATCAGAGGTGTTCACTACTGGGCTGGCCATAGGGTCACCT CTCACTTTACCGGTAGTTCCCAAGTGATCACAACCCCTCAATACGGAATTACTGCCAACG CAGAGCCAAGACGTACCATTGCTCCAAGTACCTTTCCCGGGTTGAACCTCTTCTACCGC ACATTGTCAAATCCATTCTTCAGGAGATCTGAGAACATCACCCCTACCCTTGGGATCAAC GTTGTCCAGGGAGTGGGTTTCATCCAGCCAAACAATGCTGATGTGCTCTACAGGTCTAG AGGCACAGTGGACTCCTTGAACGAACTTCCAATTGACGGTGAGAACTCACTCGTCGGAT ACAGTCACCGTCTTAGCCACGTTACTTTGACCAGGTCTCTCTATAACACTAATATCACTAG TTTGCCCACCTTCGTGTGGACTCACCACTCAGCCACCAACACAAACACTATCAATCCCG ATATCATTACACAAATCCCCCTTGTCAAGGGCTTCCGCGTTTGGGGAGGGACCTCCGTC ATTACTGGGCCCGGATTCACCGGTGGCGATATCCTCCGTAGAAACACCATTGGTGAGTT TGTGTCCCTCCAGGTTAACATTAACTCTCCTATCACACAAAGGTACCGTCTTAGGTTCCG CTACGCTTCCTCTAGAGACGCAAGAGTCATTGTGCTTACCGGTGCCGCTTCCACAGGAG TCGGTGGCCAAGTCAGCGTTAACATGCCATTGCAAAAGACTATGGAGATCGGAGAGAAC CTCACTAGTAGAACCTTCAGGTATACCGACTTCTCTAACCCTTTCTCCTTCCGTGCTAAC CCAGATATCATTGGCATCAGCGAACAACCTCTCTTCGGCGCCGGCTCCATCAGCTCTGG TGAACTCTACATCGATAAGATCGAGTTGATCCTTGCTGACGCCACATTCAAGAGGAGAC GATGGAGCGTGCACAAAGCCTCACGCCCTCTTCACCTCCACCAACAAGCTGGACTCGC TGCTGATTAATGAGAATTGGATCGTTCAAACATTTGGCAATAAAGTTTCTTAAGATTGAATC CTGTTGCCGGTCTTGCGATGATTATCATATAATTTCTGTTGAATTACGTTAAGCATGTAATA ATTAACATGTAATGCATGACGTTATTTATGAGATGGGTTTTTATGATTAGAGTCCCGCAATT ATACATTTAATACGCGATAGAAAACAAAATATAGCGCGCAAACTAGGATAAATTATCGCGC GCGGTGTCATCTATGTTACTAGATC

[0118] Another aspect of the present invention is the genetic engineering of cotton (Gossypium hirsutum) (procured from CSIR-National Botanical Research Institute, Rana Pratap Marg, Post Box No.436, Lucknow, Uttar Pradesh, India-226001 ) for the integration of mCryl EC expression cassette in its genome through Agrobacterium- mediated transformation and the development of 42 transgenic cotton lines. The presence of the mcrylEC gene is confirmed by PCR with mcryl EC-specific primers, and its expression is confirmed by qRT-PCR.

[0119] Another embodiment of the present invention involves advancing transgenic cotton lines for 3 generations, selecting them for the presence of mcryl EC, evaluating them PT / 2025 / 16752 on the basis of phenotypic characteristics, including plant phenotype (architecture), flowering time, and boll development, expression of mcrylEC through RT-PCR, and bioassay against the 3 target insects.

[0120] Another embodiment of the present invention is the three selected transgenic lines are advanced to T4 generation and evaluated for mcrylEC transcript level (through qRT- PCR), mCryl EC protein level (through ELISA), insect bioassay (toxicity of GM cotton plants to larvae o S. litura, S. frugiperda,and P.gossypiella), and phenotype of the plant.The site of integration of the mCryl EC expression cassette in the genome of the most promising line is also determined by genome walking.

[0121] Another important aspect of the present invention is a high-level expression of mCryl EC, holding about 0.1 % of total soluble plant protein in vegetative and various reproductive parts of three selected transgenic cotton lines. These lines exhibited sustainable expression of mCryl EC across the generations.

[0122] Another embodiment of the present invention is the mortality of the target insect larvae after feeding on selected transgenic cotton lines. They caused more than 70% mortality to 1stinstar larvae of S. litura, and more than 90% mortality to 1stinstar larvae S. frugiperdaand P.gossypiella, rendering nearly complete protection from the three insect pests at all the stages of transgenic cotton.

[0123] Another significant aspect of the present invention is to obtain transgenic cotton plants with no adverse effect of mCryl EC on the growth and life cycle of the transgenic cotton.

[0124] Examples

[0125] The following examples are given by way of illustration and therefore should not be construed to limit the scope of the present invention.

[0126] Example 1 : Process for preparing SEQ ID 1, 2, and 3

[0127] Cryl EC protein is toxic to S. litura but not efficacious to S. frugiperda and P. gossypiella. The main objective of the present invention is to make this protein efficacious against all three insect pests with high toxicity. For this purpose, the amino acid sequence from 464 to 493 of Cry1 EC is selected for its improvement. The said PT / 2025 / 16752 peptide of the Cry1 EC is compared for homology with 14 proteins of the Cry1 class of BtS-endotoxins (Figure 1 ). Based on the homology analysis, a few amino acids are selected for protein engineering through site-directed mutagenesis of crylEC gene (Indian patent no: 243632). A list of primers (procured from Eurofins Genomics, India) used for site-directed mutagenesis is provided in Table 1 .

[0128] Table1:List of primers used in site-directed mutagenesis to develop crylEC variants.

[0129] Cryl EC and its modified variants are expressed in E. coli strain BL21 -Codon Plus (DE3)-RIL using pET28a(+) expression vector. These are partially purified on a Ni-NTA column. The protein quantified with anti-Penta His antibody. The variant proteins are mixed in an artificial diet individually at a concentration of 200 ng / gand qualitatively evaluated for their toxicity against neonatal larvae of the three target insect pests. The efficacy of the variants assessed on a scale of 1 to 10. PT / 2025 / 16752

[0130] As disclosed in Figure 1 , significant variability observed at the amino acid residue position 476, with the occurrence of Glycine in 4, Alanine in 3, Serine in 3, Threonine in 2, Phenylalanine in 1 , Tryptophan in 1 , and Glutamine in 1 of the 15 Cry1 class of proteins, including Cryl EC. Glycine in Cryl EC at position 476 was substituted with Alanine, Serine, Threonine, Phenylalanine, Tryptophan, and Glutamine. The substitution of Glycine with Tryptophan and Phenylalanine made the protein toxic to all three insects. The highest toxicity was recorded for S. / / a. These two were the most toxic proteins for this insect among all the variants developed in the present invention (Table 2).

[0131] Table 2: Toxicity of CrylEC variants with substitution of Glycine at position 476 c476Aetc., represent protein variants where Glycine substituted with Alanine, Serine, Threonine, Phenylalanine, Tryptophan, and Glutamine. Insecticidal activity (mortality and weight loss) was qualitatively monitored on the scale of 1 + to 10+. The substitution with Tryptophan and Phenylalanine increased the toxicity of the protein to all the insects to varying extents.

[0132] The amino acid at position 483 is frequently represented by positively charged polar amino acids (Lysine in 7 and Arginine in 6 Cry proteins). Threonine and Serine (neutral polar amino acids) are present in one protein each. Replacement of Lysine with Serine or Threonine reduced the toxicity of the protein significantly for S. litura. The substitution did not show significant improvement in the toxicity to S. frugiperdaand P. gossypiella(Tab\e 3).

[0133] Table 3: Toxicity of CrylEC variants with substitution of Lysine at position 483 PT / 2025 / 16752

[0134] K483T andK483s represent protein variants where Lysine was substituted with Threonine or Serine. The mutation did not increase the insecticidal activity in a major way. This reflects the importance of Lysine at position 483 for the toxicity of Cry1 EC protein against S. litura.

[0135] Double mutants prepared for amino acid positions 476 and 483 and tested for host range. Glycine at 476 substituted with Phenylalanine or Tryptophan, and Lysine at 483 is substituted with Serine or Threonine. The four variants of Cry1 EC evaluated for toxicity to the three insects (Table 4). The variant with Tryptophan at position 476 and Threonine at position 483 broadened the host range, making the protein toxic to P. gossypiella, S. frugiperda, and S. litura. However, the toxicity to S. liturawas reduced as compared to the original Cryl EC and single mutant (Tryptophan or Phenylalanine at position 476, Table 1 ). This result led to selecting Tryptophan at position 476 and Threonine at 483 as the most suitable residues for a broader host range of the protein.

[0136] Table 4: Toxicity of CrylEC variants with substitutions at position 476 and 483

[0137] Variants represent a double mutation. Glycine at 476 either substituted with Phenylalanine or Tryptophan, and Lysine at position 483 substituted with Threonine or Serine. The step generated 4 variants. G476WK483T is found most promising both in terms of host range and insecticidal activity. PT / 2025 / 16752

[0138] At position 475, Leucine is a frequently present amino acid among the Cry1 class of proteins, including Cry1 EC. It is replaced with Valine, which is a rare amino acid at this position. Such substitutions, made as standalone, did not significantly alter the host range or toxicity to the three insect pests. However, the 475 Valine taken along with Tryptophan 476 and Threonine 483 enhanced toxicity to the target pests, without affecting host range (Table 5).

[0139] Table 5: Toxicity of CrylEC variants with substitutions at position 475 and the triple mutant

[0140] Variants represent a single and triple mutation. L475V did not alter the insecticidal activity of the parent Cry1 EC protein. The triple mutant showed high toxicity to all three target insect pests.

[0141] This led to the development of Cryl EC three-amino acid variant (Valine 475, Tryptophan 476, and Threonine 483) as the best combination for the host range and insecticidal activity. This was named as mCryl EC disclosed in the present invention. mCryl EC was taken up for further improvement, and its Tryptophan at position 476 is substituted with aromatic amino acids (Phenylalanine or Tyrosine), or Alanine. The substitution with Phenylalanine or Tyrosine reduced the insecticidal activity of mCryl EC significantly against all three insects. The substitution with Alanine largely abolished the insecticidal activity. The present invention discloses the critical role of Tryptophan at position 476 in broadening the host range and larvicidal activity in combination with Threonine at position 483, and also with Serine at the same position. It also discloses that Tryptophan cannot be replaced with any other aromatic amino acids.

[0142] Table 6: Toxicity of other CrylEC triple mutant variants PT / 2025 / 16752

[0143] Variants represent triple mutant variant of Cryl EC. All the new substitutions (VFT, VYT, and VAT) were detrimental and led to a loss in insecticidal activity.

[0144] Finally, the triple mutant(i_475vG476wK483T) disclosed in the present invention is found as the most efficacious protein and effective against 3 lepidopteran insect pests. Cry1 EC proteins with Tyrosine or Tryptophan at 476 were the most efficacious proteins for S. litura.

[0145] The said three substitutions in Cryl EC generated mCryl EC as shown in SEQ ID No 1. To encode mCryl EC, the nucleotide sequence in Cryl EC is changed to obtain T1423G, G1425T, G1426T, T1428G, A1448C, and A1449T through site-directed mutagenesis. The gene is sequenced to ensure the presence of the desired mutations (SEQ ID No 2).

[0146] Example 2: Determination of LCso of mCrylEC against three target insect pests

[0147] The insecticidal activity of mCryl EC is quantitatively evaluated by estimating LCso of the protein. For this, mCryl EC protein of SEQ ID No. l and Cryl EC (IN 243632) produced in E. coli. The proteins are expressed at 20oCafter the induction with IPTG (Figure 3 A & B). The proteins are partially purified (Figure 3 C), estimated with an anti- penta-His antibody, and tested on three target insects- P.gossypiella, S. litura, and S.frugiperda. For this, proteins at six concentrations are mixed with artificial diets and fed to 1stinstar larvae of the three insects. 10 larvae released at each concentration of protein in triplicate. The whole experiment is repeated three times with one batch of protein prepared from E. coli. The experiment with Cryl EC served as a positive control. Protein extracted from E. coli harbouring pET-28a(+) without mcrylEC gene PT / 2025 / 16752 served as a negative control. The experiment repeated several times to estimate the range of LCso values. mCryl EC protein preparation showed significant insecticidal activity against the larvae of all three tested insects. Since Cry1 EC and mCryl EC proteins prepared from E. coli are not stable, there is a variability in their soluble expression (due to inclusion body formation). Thus, the range of LCso across the experiments was recorded. The LCso values of mCryl EC were in the range of 91 -310 ng / g for S.Htura, 22-107 ng / g for S.frugiperda, and 34-141 ng / g for P. gossypiella, when incorporated into the artificial diet (Table 7).

[0148] Table 7: LCso value of mCryl EC against neonatal larvae of three target insect pests.

[0149] The variability in the LCso was due to the following reasons-

[0150] 1 . A large proportion of recombinant proteins (both mCryl EC and Cry1 EC) are misfolded and form inclusion bodies. Thus, the solubilized protein shows batch- to-batch variability in the correct folding, leading to a variation in larvicidal activity.

[0151] 2. Partially purified protein is also not stable. Not at all the times, protein could be used immediately after preparation due to the unavailability of the required number of neonatal larvae of three insects.

[0152] 3. Variation in LC50 is also due to the insect fitness, as the field population is introduced in lab culture to overcome inbreeding depression.

[0153] Example 3: Insecticidal activity of mCrylEC protein expressed in transgenic cotton plants

[0154] PCR amplified DNA fragments of plant promoter RbPCDI (523 bp), mcrylEC gene (1926 bp coding 641 amino acid mCryl EC), and nos terminator (253 bp) assembled PT / 2025 / 16752 at BamHI and EcoRI digested pCAMBIA2300 binary vector (procured from Marker Gene Technology Inc., USA) through DNA homology-based assembly. The expression cassette (RbPCD1 - / 7?c / y7EC-Tnos) of 2723 bp is given as SEQ. ID. No 3. The binary vector is named pPK723. Expression cassette and binary vector map are provided in Figure 4. The binary vector is introduced into Agrobacterium tumefaciens LBA4404 (Takara Bio Inc., Japan). Coker 312 variety of cotton (procured from CSIR-National Botanical Research Institute, Rana Pratap Marg, Post Box No.436, Lucknow, Uttar Pradesh, India-226001 ) is transformed with Agrobacterium harbouring mCryl EC expression cassette. A total of 42 putative transgenic plants were developed through somatic embryogenesis.

[0155] These transgenic cotton plants are tested for the presence of mcrylEC gene with gene-specific PCR. All the PCR-positive plants are advanced to the T 1 generation and further evaluated for the presence of the gene by PCR and expression of the gene with qRT-PCR. T1 plants are challenged with neonatal larvae of P.gossypiella, S.Htura, and S.frugiperda. Non-GM cotton plant served as a control. The GM cotton lines caused 50-100 % mortality to the larvae of all three insect pests. The expression of mcrylEC and larval mortality are in good correlation. Based on gene expression and larval mortality, 8 cotton lines selected in the T2 generation, 5 lines in the T3 generation, and eventually 3 lines in the T4 generation. The three lines (Line 508, 519, and 523) evaluated for the expression of mcrylEC by qRT-PCR and ELISA. These lines express mCryl EC consistently at a high level (Figure 5 A).

[0156] Plants of the three selected lines in the T4 generation expressing mCryl EC of SEQ ID No. 1 cause 100% mortality to in-house reared neonatal larvae of P.gossypiella in 5 days and 3rdinstar larvae in 7-8 days. They also cause 100% mortality to field- collected (BGII resistant) larvae of P.gossypiella in 5-6 days and render nearly complete protection. Transgenic cotton lines cause more than 70 % mortality neonatal larvae of S itura in 3-4 days. The three lines cause 70-100% mortality in in-house reared 1stinstar larvae of S.frugiperda in 1 -2 days, 100% mortality to 2ndlarvae in 5-6 days, and 100% mortality to 3rdinstar larvae in 7-8 days.

[0157] ADVANTAGES OF THE INVENTION

[0158] 1. The mCryl EC geneof the present invention has a broad range of insecticidal activity and is toxic to larvae of three field crop insect pests S. litura, S. frugiperda, PT / 2025 / 16752 and P. gossypiellam a range of 10-100 ng / g of diet and can be deployed in a variety of crops for resistance to the three insect pests.

[0159] 2. The GM cotton lines developed with mCrylEC causes more than 90% larval mortality and render complete protection from all three insect pests. The invention also provides protection of a crop from three lepidopteran insect pests with a single protein / gene is a significant advantage.

[0160] 3. The transgenic cotton developed with mcryl ECgene is efficacious to all the developmental stages of field-collected BG Il-resistant larvae of P. gossypiella, and showing nearly complete protection is a major advantage.

[0161] 4. The transgenic cotton developed in the present invention can help restore the highest productivity of cotton in India (about 577 kg / hectare, recorded in 2013-14) which has declined to about 440 kg / hectare (2022-23) largely due to the eruption of P. gossypiella. For this mCryl EC GM cotton needs to be stacked with BGII cotton.

[0162] 5. The invention provides sustainable productivity of cotton and also spare the use of synthetic pesticides required for the control of P. gossypiella and S. litura on cotton.

[0163] 6. The mCryl EC gene is toxic to S. frugiperda, therefore, it can be utilized in developing GM maize and soybean for protectionfrom Fall armyworm.

Claims

PT / 2025 / 16752WE CLAIM:

1. A recombinant gene (mCryl EC) useful as insecticidal agent having SEQ ID NO.

22. The recombinant gene as claimed in claim 1 , wherein the corresponding peptide sequence is having SEQ ID NO.

13. The recombinant peptide as claimed in claim 2, having mutations at positions 475,476, and 483, wherein the amino acid Leucine, Glycine, and Lysine are replaced with Valine, Tryptophan, and Threonine respectively.

4. An expression cassette (PpcDi-mcry1EC-Tnos) having SEQ ID NO: 3 containing the recombinant gene as claimed in claim 1 ,5. The expression cassette as claimed in claim 4, wherein the cassette consisting of: a) mcrylEC gene as claimed in claim 1 b) RbPCDI promoter c) NosT as transcriptional terminator6. The recombinant peptide as claimed in claim 2, wherein the target insect is selected from the group consisting of Pectinophora gossypiella, Spodoptera litura, and Spodoptera frugiperda.

7. The recombinant peptide as claimed in claim 2, wherein the insecticidal toxicity is in the range of 22-310 ng / g.

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