苏云金芽胞杆菌拟步行甲亚种YBT-1765中稳定质粒pBMB175的克隆和功能分析

从苏云金芽胞杆菌拟步行甲亚种YBT_1765中克隆得到一个大小约15.2kb的质粒pBMB175,构建了该质粒的限制性图谱,通过功能验证,将其最小的复制区定位在一个1151bp的片段上。分析了包含有这个复制区的一个大小为4152bp的核苷酸序列,该片段包含有3个编码框(ORF1、OFR2和ORF3)。氨基酸序列同源性比较发现,ORF1(767AA)与UvrD_旋促酶、重组酶RecD和RecB家族具有20%～30%的相似性;ORF2(149AA)没有发现与任何已知序列具有同源性;ORF3(83AA)与pGI3中一个未知功能的蛋白(ORF7)具有34%的相似性。通过缺失及序列比较分析推测ORF2可能编码一种新的复制蛋白。因此pBMB175的复制类型可能属于一类新的复制家族。利用最小复制区构建的重组质粒在无抗生素选择压力下可稳定遗传40多代,具备构建稳定遗传质粒载体的潜力。

Cloning and functional analysis of the stable plasmid pBMB175 in Bacillus thuringiensis subsp. tenebrionis strains YBT-1765

A 15.2 kb plasmid pBMB175 from Bacillus thuringiensis subsp. tenebrionis strains YBT-1765 was cloned and the restriction map was constructed. The mini-replicating region of pBMB175 was located in a 1151 bp fragment by functional analysis. The sequence of a 4152 bp fragment which contained the mini-replicating region was analyzed and results showed that the fragment had three potential open reading frames (ORF1, ORF2 and ORF3). Sequence comparison and homology search revealed that ORF1 (767AA) has 20% approximately 30% similarity to UvrD-helicase, RecD and RecB family proteins; no homology was found between ORF2 (149AA) and other known proteins; ORF3 shared 34% identification to a potential protein (ORF7) in pGI3. Deletion and sequence analysis presumed that the protein encoded by ORF2 maybe a new replication protein. Above all, pBMB175 likely belongs to a new plasmid family with a new replicon. The recombinant plasmid harboring the mini-replicating region is very stable, even after growth for more than 40 generations without selection, so it might be used as a cloning and expression vector.