| The dissertation mainly concerns the construction of resolution vector with green fluorescent protein. The research results are summarized as following.1. Construction of a resolution vector with a small plasmid origional replionA small plasmid original replion about 2062bp come from B. thuringiensis subsp. kurstaki strain YBT-1520 and the res site of Tn4430 were used to construct a resolution vector. The resolution vector based on Tnpl-mediated site-specific recombination system of B. thuringiensis transposon Tn4430. The gene of or/2062 and other target DNA were inserted into two copy sets of res sites. The res sites have the same direction. With the help of the integrase( Tnpl) the antibiotic resistance genes and other non-5, thuringiensis DNA can be eliminated. When the engineered strain, which had the recombinant plasmid without resistance gene cultured for 40 generations the very recombinant plasmid could still be detected.2. Construction of green fluorescent protein resolution vector with various promoters and different origional repliconsIn this study, three kinds of specific promoter of B.thuringiensis: crySA promoter, Btl-BtH promoter and 5. cereus promoter were chosen to drive the expression of gfp. Three kinds of plasmid original replion of B.thuringiensis subsp. kustaki: ori44, oriW30 and ori2062 were chosen to construct the GFP resolution vector. There were 9 kinds of GFP resolution vector. All of the recombinant plasmids were introduced into crystal negative B. thuringiensis 8MB 171.The fluorescence of the 9 kinds engineered strain can be detected by the fluorescent microscope.3. Construction of fusion genes with insecticidal protein gene and gfpThe fusion genes was constructed by PCR. The pesticidal crystal protein gene crylAc10 and gfp were chosen to construct the fusion genes. The gfp gene was fused to the C-terminate of crylAclO. The two genes were in an open reading frame. The fragment of pro3A+crylAc10 without terminated structure was inserted into plasmid pBMB1205 when it was digested by BamHI and SphI. The recombinant plasmid pBMB1205Ac was obtained. Recombinant plasmid of pUC19G was constructed when PCR product of gfp was digested by Sphl and inserted into pUC19. The gfp fragment with Sphl and BglII enzyme sites from pUC19G was inserted into pBMB1205Ac. The very plasmid pBMB0474 was constructed. When the recombinant plasmid pBMB0474 was transferred into crystal negative Bt strain BMB171 throughelectroporation, the expression of the fusion genes about 150kDa-160kDa can be detected by SDS-PAGE.4. Microcalorinetric study on B. thuringiensisBy using an LKB-2277 Bioacitivity Monitor, the thermogenic curves of different B thuringiensis strains YBT-833 and YBT-833-2-I, have been determined. The metabolism heat output revealed the heat output was correlated to the yield of the protein, the higher yield protein, the less heat output. A microcalorimetric technique based on the bacterial heat-output was explored to evaluate the effect of various promoters and different plasmid original replicons on the expression of GFP. In this study the heat output also suggested there was obvious relation between the gene copy number and heat output. The heat output rate of different strains with various copy number is BMB304GFP) BMB315GFP. The relation of the heat output rate with original replion is or/1030) or/44) or/2062. The heat output indicated that different promoter had various impact on the expression of GFP. The drive impact was promoter Btl-Btll } promoter B. cereus ) promoter cry3A.5. Wild-type B.thuringiensis tabled with green fluorescent proteinPlasmid pBMB0472 and pBMB0474 were introduced into wild type B.thuringiensis subsp. tenebrionis YBT-1765 by electropotation. The transformams YBT1765-72B and YBT1765-74. were obtained. SDS-PAGE result suggested GFP about 27kDa was expressed in YBT1765-72. But the fusion protein could not be detected in YBT 1765-74. Fluorescent microscope observation results indicated there was fluorescence in YBT1765-72B. but there was no flu...
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