KRISHNARAJ, P. U.BHARTI, GUNJAN2019-01-282019-01-282015-06http://krishikosh.egranth.ac.in/handle/1/5810092687The present work involved screening of B. thuringiensis isolates for different coleopteran-specific cry gene/s, cloning and expression of variants of cry and vip. The presence of different coleopteran specific cry genes (cry1I, cry3, cry7,8, cry14, cry18, cry23, cry26, cry28, cry34, cry35, cry36, cry37 and cry55) was determined in 993 native B. thuringiensis isolates. Strains carrying cry26 and cry3 were the most abundant representing 6.16 and 6.03 per cent of the isolates respectively. Many of the isolates screened (~60 per cent) did not amplify for any cry gene. The crude proteins extracted from the native isolates along with positive control were subjected to SDS-PAGE. The protein bands corresponding to approximately 130kDa were observed in seven potent isolates. Six of the potent isolates showed the presence of a protein band size of 45-50 kDa. None of the 90 native isolates tested, amplified for vip1A. Of the 90 isolates, only five of the isolates (DBT 141, DBT142, DBT642, DBT637, DBT658) were found to be positive for vip3A. DBT 730 was found to be positive for vip2A. The variant cry1I gene from CFE20(3) based on ARFLP patterns was cloned and expressed in E. coli M15(pREP4). The nucleotide sequence showed 99 per cent homology with the reference sequence (JN226100.1). The cry34 gene from CFE43(1) was cloned and expressed in E. coli M15(pREP4). The nucleotide sequence showed 95 per cent homology with the cry34Ba(AY552555.1). The vip3A and vip2A variants were cloned from DBT642 and DBT730 respectively and expressed in E.coli M15(pREP4). The nucleotide sequence of vip3A and vip2A showed 99 per cent homology with the reference sequence (AF500478.2) and reference sequence (EU940372.1) respectively. A construct with both cry1I and chi gene has been developed using the Gibson assembly master mix.ennullThesis