Cloning Of Insecticidal Gene From Bacillus Thuringiensis And Construction Of Pseudomonas Insecticidal Engineered Bacteria

Because of the disadvantages of Bacillus thuringiensis in the application and the resistance of the insect pest, people today turn to transfer Bt toxin protein gene into some salutary microorganism. In order to construct antimicrobial and insecticidal engineered bacteria, the novel vip3A and crylBa gene were cloned and transferred into Pseudomonas sp.Using the PCR-RFLP technique, the genotypes of Bt strains separated by this lab were identified. The results showed the WZ-7 strain contained crylAb and vip3A, sfwl2 contained crylBa.According to the gene sequences of vip3A and crylBa, two pairs of primer were designed. vip3A gene and part crylBa gene were amplified from the two B. thuringiensis WZ-7 and sfwl2 strains respectively and then cloned into T-vector. The obtained vip3A-WZ7 gene and cry 1Ba-sfv12 gene were sequenced respectively. The results indicated the homology between Vip3A-WZ7 and Vip3A (a) was 99%;the homology between Cry1Ba-sfw12 and Cry1Ba was 99%.By inserting vip3A gene into the vector pAG408, which carried Tn5 transposon, medium plasmids containing vip3A gene were gotten and transformed into E. coli S17-1. Using the Tn5 transposon, vip3A were recombined to the genome of Pseudomonas sp. in company with by conjugation. The Pshvip9 strain was gotten by hand-light and bioassay. PCR analysis demonstrated that vip3A gene had been inserted into the pseudomonas randomly. The lab bioassay indicated that the insecticidal activities (LC₅₀) of engineered strain were 1.02×10⁸ cells/mL and 6.95×10⁷ cells/mL against 2-day instar larvae of Helicoverpa armigera and Spodostera exigua. The engineered strain still remained strong antifungal activity against Bipolaris sorokinianum;Fusarium oxysporum, Bt and E.coli. The test of growth curve to the engineered strain showed that its growth can get to stable time in 18h. It was tested that the colonized ability of the engineered bacteria on wheat roots in sterile and natural soil. The results indicated the engineered bacteria can reach 3 4 ×10⁶CFU/g in 20 days. The results above showed that the antifungal activity and colonization of PHB158 have not been influenced after vip3A were transferred into.Cultivating the engineered strain utilizing several different culture mediums, the result showed the BPY medium was the best suitable to the strain.