Cloning And Physical Map Analysis Of Large Plasmid PBMB165 In Bacillus Thuringiensis

The bacteria is Bacillus thuringiensis in this study, the largest detected plasmid pBMB165 of strain YBT-1765 were cloned and physical map analysed. Enhancin gene of BMB 171 had been cloned and sequenced.In the first part of this paper, B. thuringiensis subsp, tenebrionis YBT-1765(H_8ab) was isolated from a warehouse. In this study, the largest detected plasmid pBMB 165 of strain YBT-1765 were cloned and physical map analysed. It is an effective, quick and simple way to clone large plasmid by constructing the plasmid BAC library and genomic DNA BAC library of YBT-1765. These libraries were obtained by the BamHI partly digested plasmid and the Hindâ…¢partly digested genomic DNA of YBT-1765 into cloning pBeloBAC11 vector respectively. With the use of chromosome walking strategy, the plasmid BAC library of YBT-1765 was screened by the primers designed according the sequence coding Rep165 on 3.6kb DNA fragment (pBMB165-F4A)containing plasmid pBMB165 replicon, 5 clones covering the uncompleted whole plasmid pBMB 165 were screened. The genomic DNA BAC library of YBT-1765 was also screened by the primers designed according the terminal sequence of 5 positive clones in the same way, 8 clones covering different regions of plasmid pBMB 165 were isolated. Analyzing the digestion of these 13 recombinant plasmids with various combinations of restriction enzymes, the physical map and the linear contig linkage map of plasmid pBMB165 were constructed and the size of pBMB165 was calcuLated to be 82Kb. The sequencing resuLts of one recombinant plasmid pBMB165A2 supported the probability of the transposable element's appearance in plasmid pBMB165 according to the bioinformatics analysis. There are redundant transposable elements in the plasmid of B. thuringiensis on fixed quantity. So, construction of the plasmid BAC library and genomic DNA BAC library of Bacillus thuringiensis and the drawing of physical map have successfuLly resolved the problem of cloning the large plasmid from Bacillus thuringiensis.In the second part, to rule out a possible implication of Bacillus Enhancin in insect viruLence, Enhancin protein gene had been cloned and sequenced. A DNA fragment carrying the Enhancin protein gene was amplified by PCR using BMB 171 that is crystal minus mutant chromosomal DNA as template and primers based on the genome sequence of B. cereus ATCC14579. The PCR fragment was sequenced on both strands. The sequence revealed an ORF of 2229 bp potentially encoding a polypeptide of 743 amino acid residues with a calculated molecular mass of 85.4 kDa. Comparisons of Enhancin proteins from various origins (Bacillus, Yersinia and BacuLovirus) were made in order to determine a relationship between conserved domains, function and phylogeny. The gene is predicted to encode a polypeptide showing 23-25% identity with Enhancins from several BacuLoviruses and 31% with that of Yersinia pestis. With the use of homologous recombination, the Enhancin protein gene's knocked out in Bacillus thuringiensis BMB171, constructed the mutant BMB1084. Genetic complementary assay was also performed by expression the crylAc10 gene cloned in a modified E. Coli-Bacillus thuringiensis shuttle vector pHT304 in the strain BMB171 and BMB1084 respectively. Three days after inoculation, the LD₅₀ for BMB1087 and BMB1088 were 0.46 and 2.73 mL/mL respectively. The result indicated that the Enhancin protein gene of Bacillus thuringiensis has a very significant Enhancement on the toxicity of Bacillus thuringiensis to Helicoverpa armigera.