苏云金芽胞杆菌大质粒pBMB165的克隆与分析

以pBeloBAC11为载体,成功构建了苏云金芽胞杆菌YBT-1765的基因组人工染色体(BAC)文库和质粒BAC文库.根据已克隆的包含复制子ori165在内的3.6kb片段中编码复制蛋白Rep165的核苷酸序列设计探针,通过染色体步移方式,对质粒文库和基因组文库进行筛选,得到13个覆盖YBT-1765菌株中质粒pBMB165不同区域的克隆子.通过Hind Ⅲ和BamH Ⅰ酶切分析,建立了质粒pBMB165的物理图谱和线状重叠连锁图,并测算出该质粒的大小为82kb.根据部分核苷酸序列初步统计了pBMB165上转座因子的存在机率.YBT-1765菌株基因组文库的构建和物理图谱的绘制为克隆苏云金芽胞杆菌大质粒提供了一套可行的方案,成功解决了大质粒难克隆的问题.

Cloning and physical map construction of a large plasmid pBMB165 in Bacillus thuringiensis

The largest detected plasmid pBMB165 from Bacillus thuringiensis subsp. tenebrionis strain YBT-1765 (H8ab) was cloned and its physical map was analyzed. For the cloning, two BAC libraries were constructed with their plasmid DNA and genomic DNA, respectively. The plasmid DNA BAC library was obtained by partially digesting plasmid DNA with BamHI and then cloning to pBeloBAC11 vector, whereas the genomic DNA BAC library was done with HindIII partial digestion. With the chromosome walking strategy, the plasmid BAC library was initially screened by the primers designed according the sequence coding replication protein Rep165 on a previously identified 3.6kb DNA fragment (pBMB165-F4A). Finally, 5 clones covering the most of plasmid pBMB165 were obtained. When screening the genomic DNA BAC library, 8 clones covering whole plasmid pBMB165 were isolated. By restriction analysis of these 13 BAC clones, the physical map and the linear linkage map of plasmid pBMB165 were constructed and the size of pBMB165 was calculated to be 82kb. Based on the DNA sequence of the BAC insertion ends and a previously published 20kb fragment on recombinant plasmid pBMB165A2, there were redundant transposable elements appeared on this large plasmid. This study provided a novel way to clone large plasmid from B. thuringiensis, to draw the physical map by construction of BAC library, and to dissolve the problem in cloning large plasmid from B. thuringiensis.