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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。
A plasmid pBMB175 of about 15.2 kb in size was cloned from the B. subtilis B. subtilis YBT_1765. The restriction map of the plasmid was constructed and its minimal replication region was located on a 1151 bp fragment by functional verification. A 4152 bp nucleotide sequence containing this copy region was analyzed and contained three coding sequences (ORF1, OFR2 and ORF3). Amino acid sequence homology comparison showed that ORF1 (767AA) had 20% -30% similarity with UvrD-gyrase, recombinase RecD and RecB families; ORF2 (149AA) did not find homology with any known sequence ; ORF3 (83AA) has 34% similarity with an unknown function protein (ORF7) in pGI3. It is deduced that ORF2 may encode a new type of replication protein by deletion and sequence comparison analysis. Therefore, the replication type of pBMB175 may belong to a new type of replication family. The recombinant plasmids constructed by using the minimal replication region can stably inherit for more than 40 generations without selection pressure of antibiotics, and has the potential of constructing a stable genetic plasmid vector.