摘要
转座因子在生物体内广泛存在,它在研究基因的重组机理以及生物染色体的进化方面有着重要意义。IS10是细菌中的一种转座因子,它既能单独作为插入序列,也能作为Tn10的一部分进行转座。利用含sacB基因的质粒pXT3sacB,获得了由转座因子IS10插入而导致sacB基因失活的突变体。通过对插入突变体质粒DNA的序列测定(GenBank登记号为AY580883.1),结果表明IS10两端分别包括22bp倒置重复区CTGAGAGATCCCCTCATAATTT和AAATCATT-AGGGGATTCATCAG,这与前人的报道一致;而IS10两端的插入靶位点序列为TGCTTGGTT,该9bp靶位点序列与前人报道的序列NGCTNAGCN不同。根据文献资料,本研究中的靶位点序列是首次报道。此外,通过Southernblot杂交分析,插入sacB基因中的IS10来源于宿主大肠杆菌DH5α染色体DNA,并且IS10在DH5α染色体中为两个拷贝。此外,本研究利用sacB基因捕获到转座因子IS10,该方法为研究其他插入序列提供了一个有益的体系。
A sacB mutant was obtained by transposon IS10 inactivation of a plasmid pXT3sacB carrying the sacB gene. Sequencing of this mutant plasmid DNA （GenBank accession No. AY580883.1） showed that the IS10 flanking the 22 bp inverted repeats were 5＇-CTGAGAGATCCCCTCATAATTT-3＇ and 5＇-AAATCATTAGGGGATTCATCAG-3＇, which were similar to those published in reports previously. However, the target sequence adjacent to IS10 was 5＇-TGCTTGGTT-3＇ instead of the previously reported 5＇-NGCTNAGCN-3＇. To our knowledge, this is the first report on the novel insertion site of IS10. In addition, Southern blot hybridization confirmed that the mobile IS10 originated from the chromosomal DNA of the host strain Escherichia coli DH5α and that there were two copies in the DH5α genome.
基金
This work was supported by the Natural Science Foundation of Zhejiang Province (No.Y304083), the Science Foundation of De-partment of Education at Zhejiang Province (No.20040069) and the Science Foundation for the talents of Hangzhou Normal Col-lege (No.87).
