Study On The Activity And Expression Of A Recombinant Bacillus Thuringiensis Cry1Ac Gene With Tobacco Chitinase-Encoding TchiA21 Gene

Bacillus thuringiensis (Bt) is one of the most widely usedinsecticidal microbe, because of its ability to produceinsecticidal crystal proteins (ICPs) during sporulation, andits structure and function mechanism is a hot topic study recent.However there were some drawbacks for all traditional Btproducts as biopesticides, such as narrow host range, lowtoxicity target pest, resistance from insect, therefore strainimprovement is needed by biotechnology. In order to improve theinsecticidal activity and construction some new effectiverecombinant strains, in this work, the cry1Ac gene and tchi21gene was recombinanted and constructed into XBU001 to enhancethe toxicity of crystal proteins.First, this paper was performed on the Bt 4.0718 (CCTCCNo.M200016) strain which were stored by our lab. The cry1Ac geneand the terminator gene was amplified from the plasmid of Bt4.0718 strain using primer Ac-F/Ac-R and ter-F/ter-R; and theChitinase gene was amplified from the plasmid pBG1112 usingprimer Chi-F/Chi-R, the two PCR fragment were digested byNcoâ… , and then the two PCR fragment were linked by T4 DNA ligase,the chi-ter fusing gene was amplified from the two PCR linkedfragment. The plasmid pUAc19 which contain the cry1Ac geneandthe chi-ter fusing gene were digested by Bglâ…¡/Smaâ… , purifiedand linked to construct the plasmid pUAccB19, and then theplasmid pUAccB19 and the shuttle plasmid PHT315 were digestedby Salâ… /Smaâ… to perform the recombinant plasmid pHUAccB5 andtransformant into Acrystalliferous Bacillus thuringiensis.The expression of Bt transformant XBU-HUAccB5 analysis by SDS-PAGE and western-blotting, in SDS-PAGE, the XBU-HUAccB5produced 130-kDa cry1Ac protein and 30-kDa chitinase protein,the result also was proved by the western-blotting. Theobservation of Atomic Force Microscopy demonstrated that therecombinant crystals appeared bipyramidal crystals. During thechitinase active analysis, the HTX-42 and Bt acrystalliferousstrain XBU001 chitinase active were kept at 1.5U/ml all the time,the XBU-HUAccB5 chitinase active were much higher than theother two strains, the XBU-HUAccB5 chitinase active was goingup until it reached a maxim of 7.5U/ml at 72h in the NYSM medium,was 5 times higher than the XBU001. The insecticidal activityof transformant was evaluated against Helicoverpa armigeraHubner and compared with those of the contrast strains afterautolysis. The toxicity Activity against Helicoverpa armigeraHubner was tested at two different times. The HTX42 LC₅₀(117.7780μg/ml) was 11. 30 times higher than the XBU-HUAccB5LC₅₀ (10.4240μg/ml) at 48h, and the HTX42 LC₅₀ (89.8691μg/ml)was 18.76 times higher than the XBU-HUAccB5 LC₅₀ (4.7904μg/ml)at 72h. The XBU-HUAccB5 LC₅₀ went down sharply than the HTX42LC₅₀ from 48h to 72h. The results show that chitinase has beenfound to increase the efficacy and potency of B. thuringiensisCry toxins in insect control.This work constructed the fusion of cry1Ae gene and tchi21gene successfully which made a good ground for constructing thefusion genes of Bt cry gene and other foreign toxin genes.