Construction Of Bacillus Thuringiensis Engineered Strains And Study On Insecticidal Crystal Proteins

Bacillus thuringiensis is one of the most widely used insecticidal microbe. There were some drawbacks for all traditional Bt products as biopesticides, such as narrow host range, low toxicity to target pest, resistance from insect, therefore strain improvement is needed by biotechnology. A recombinant plasmid pSTK-3A containing cry3Aa7 gene encoding coleopteran-specific protein was constructed and introduced into the wild Bacillus thuringiensis strain G03 and UV17 with high toxicity to lepidopteran insect pests by electroporation, and the resulted genetically engineered strain was named G033A and UV173A respectively. Bioassay results in the lab showed that G033A were highly toxic not only to lepidopteran pests Spodoptera exigua (LC₅₀ 40.30 μg/ml) and Plutella xylostella (LC₅₀ 8.13 μg/ml) but also to Colopteran Pyrrhalta aenescens (LC₅₀ 3.10 mg/ml) and Leptinotarsa decernlineata(Say) (LC₅₀ 0.40 mg/ml), while UV173A against P. xylostella, P. aenescens and L. decernlineata with LC₅₀ 18.03 μg/ml, 1.47 mg/ml and 0.31 mg/ml respectively. Field experiments were conducted against S. exigua and L. decernlineata using G033A and UV173A. Biocontrol effect of G033A to S. exigua was 67.9% with the dosage 600 g/ha, while that of G033A and UV173A to L. decernlineata was 89.2% and 94.6% respectively at concentration 1g/L. The future of G033A and UV173A for application and exploration is very prosperous.The safty and risk of the engineered strain G033A and UV173A were assessed in field. The result showed that Bt spores could survive in soil for many months, but on the surface of leaves Bt has rarely detected more than a few days. The dispersing characterization of the two engineered strains indicated that they can disperse within 20 meter. And results of assessing non-target impacts showed that Bt has no significant impact against non-target organisms.To determine the minimal active fragment of Cry1Ba3 protein, nine different fragments of cry1Ba3 gene were amplified using PCR, and then were expressed in E. coli BL21 respectively. Bioassay results showed that the minimal active fragment was located at N-terminal of Cry1Ba3 between position 22 and 655, and this result is helpful for its use in transgenic plant.In order to recrystallize and dissolve the three dimensional structure of protein, Insecticidal crystal protein Cry1Ba3-3H and vegetative insecticidal protein Vip3A were purified using high potential liquid chromatography. The Crystal Screen and Crystal Screen 2 kits were screened for recrystalization of Cry1Ba3-3H and Vip3A by vapor diffusion method, and flat and octahedral crystals of Cry1Ba3-3H and Vip3A proteins were obtained respectively.To test our hypothesis that substitution of domain â…¢ of Bacillus thuringiensis delta-endotoxin (Cry) proteins might alter toxicity to insect pests, three chemeric proteins consisting of domain â… +â…¡ from Cry1Ca and domain â…¢ from Cry1Aa, Cry1Ab, Cry1Ac were obtained respectively. Bioassay showed that, compared with Cry1Ca, the toxicities of all chemeric toxins to Helicoverpa armigera increased to some extend, while decreased to S. exigua. These results showed that substitution of domain III may be a powerful tool to increase the activity of toxins for pests.