Construction Of Bacillus Thuringiensis Resolution Vector And Its Property

The dissertation mainly concerns the construction of resolution vector based on Bacillus thuringiensis transposon Tn4430. The research results are summarized as following:1. Feasibility of the resolution vector.A new resolution vector based on TnpI-mediated site-specific recombination system of B. thuringiensis transposon Tn4430 was developed. The crylAc10 or other target DNA, and the gene ori1030, from a plasmid of wide type B. thitringiensis subsp. kurstaki strain HD73, were inserted into two copy sets of res sites. The res sites have same direction. When -the recombinant plasmid was introduced into crystal negative B. thuringiensis host BMB171, antibiotic resistance genes and other non-5, thuringiensis DNA can be selectively eliminated after the selection by antibiotic resistance marker. This strategy can resolve the gene safety problem for genetic engineered strains and should facilitate regulatory approval for its development as a commercial biopesticide.2. Effect of integratase TnpI on resolution reaction of resolution vectorThe integratase TnpI has great effect on the speed and time of resolution reaction. When the resolution vector based on transposon Tn4430, it can not contain integratase gene (tnpI) on the vector, otherwise it can not get the E. coli transformant because the whole plasmid resolved very quickly when it was introduced into the host. When tnpI gene was deleted the 3'-stop coden, it still has function in E. coli. But it can't mediate all the resolution vectors resolved. On the other hand, when it introduced into B. thuringiensis strains and selected the transfonnants by antibiotic resistance, it can not resolve spontaneously. By this means, modifying the tnpI gene can get the different vectors with different resolution speed.3. Construction of two vectors containing different plasmid original repliconThis work constructed four resolution shuttle vectors, pBMB1205, pBMB1205R, pBMB1206 and pBMB1206R. There are multiple clone sites between two copies of res sites. For vector pBMB1205 and pBMB1205R, the plasmid original replicon ori44 was come from B. thuringiensis subsp. kurstaki strain HD263. The plasmid original replicon ori1030 of vector pBMB1206 and pBMB1206R was cloned from B. thuringiensis subsp.kurstaki strain HD73. These two types plasmid original replicons were very stable and they can exist in the same cell and have similarity in the resolution reaction and genetic stability, so they can resolve the plasmid incompatibity problem. They also have the same effect on the expression of gene crylAclO. crylC and cry3A. The replication director of plasmid has no effect on the expression of gene cry1Ac10 and cry1C.4. Effect of resolution vector on the expression of pesticidal crystal protein genesThis work successfully introduced pesticidal crystal protein genes into crystal negative strain BMB171 by using resolution shuttle vectors. After resolution, the expression of cry1Ac and cry 1C genes have no obvious change, but the expression of cry3A genes has great increase in the same condition.5. Effect of cry3A promoter on expressionBy using resolution vector, the cry1Ac and cry1C genes with cry3A promoter or it itself promoter were introduced into B. thuringiensis crystal negative strains. The expression of these strains is quit similar, however, the expression of cry 1 Ac and crylC with different promoter in same cell has no complete inhibition.