Wang Li, Guo Suxia, Huang Junyan, Yu Ziniu, Sun Ming
College of Life Science and Technology, State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan 430070, China.
Wei Sheng Wu Xue Bao. 2008 Jan;48(1):15-20.
The largest detected plasmid pBMB165 from Bacillus thuringiensis subsp. tenebrionis strain YBT-1765 (H(8ab)) 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.
对来自苏云金芽孢杆菌暗黑变种菌株YBT - 1765（H(8ab)）中检测到的最大质粒pBMB165进行了克隆，并分析了其物理图谱。为了进行克隆，分别用其质粒DNA和基因组DNA构建了两个BAC文库。质粒DNA BAC文库是通过用BamHI对质粒DNA进行部分酶切，然后克隆到pBeloBAC11载体上获得的，而基因组DNA BAC文库则是用HindIII进行部分酶切构建的。采用染色体步移策略，首先根据先前鉴定的3.6kb DNA片段（pBMB165 - F4A）上编码复制蛋白Rep165的序列设计引物，对质粒BAC文库进行筛选。最终获得了5个覆盖大部分质粒pBMB165的克隆。在筛选基因组DNA BAC文库时，分离出了8个覆盖整个质粒pBMB165的克隆。通过对这13个BAC克隆的酶切分析，构建了质粒pBMB165的物理图谱和线性连锁图谱，并计算出pBMB165的大小为82kb。基于BAC插入末端的DNA序列以及重组质粒pBMB165A2上先前发表的20kb片段，发现该大质粒上存在冗余的转座元件。本研究为从苏云金芽孢杆菌中克隆大质粒、通过构建BAC文库绘制物理图谱以及解决从苏云金芽孢杆菌中克隆大质粒的问题提供了一种新方法。
