Study On The Cloning And Identifcation Of Bacilluds Thuringiensis Cry2Ac Gene

Bacillus thuringiensis(Bt) is a spore-forming gram-positive bacterium. During sporulation,the Insecticidal Crystal Proteins (Cry proteions) are produced. Those proteins shows towic to the larvae of Lepidopetera, Diptera and Coleoptera.The Cry Proteins from Bt has developed as one of the most successful biological insectidides.In this study, 24 isolates of Bt were obtained from nearly 300 soil samples from four provinces in the north of China. The bioassay in lab showed LL-6 and FY-2 are high toxicity against three lepidopetera pests,including Helicoverpa armigera, Spodoptera exigua, Mythimna separata (Walker). Especially, LC₅₀ of FY-2 to 2 pests(Helicoverpa armigera, , Spodoptera exigua) respectively was 3.3×10⁶cells/mL, 1.8×10⁶cells/mL.FY-2 strain cry-type genes were identified by PCR-RFLP method. The results showed it contained cry1A, ,crylg, cry2Ac,and all genes of FY-2 were located on plasmids.A pair of specific primer was designed for amplification of cry2Ac gene.The amplified product of cry2Ac gene was directly inserted into pET-21b vector and transformed into E. coli strain DH5α . The linkage sequences of cry2Ac gene from Bt strain FY-2 were cloned and sequenced.The results of sequencing and structural analysis showed that the gene is 1872 bp encoded a 69.7kDa protein with 633 amino acids. The isoelectric point of Cry2Ac-FY is pH 9.00. This gene was registered in GenBank ,The accession number is DQ020578.Recombinant plasmid, pFY2A, with full length cry2Ac-FY gene was constructed and transformed into E.coli BL21. Result of SDS- PAGE analysis indicated that the cry2Ac-FY gene was expressed in the receptor.The result of Bioassay show that the expressed protein lias very strong lose weight to Helicoverpa armigera larvae.