中度嗜盐菌Halomonas sp.BYS-1耐盐相关DNA片段的克隆

Cloning of a Salt-tolerance Related DNA Fragment of a Moderate Halophilic Bacterium Halomonas sp.BYS-1

通过固体亚硝基胍诱变获得了Halom onassp.BYS-1的盐敏感突变株BYS-1M.以pBBR1MCS-2为载体在E.coliDH5α中构建了BYS-1的基因文库.以文库菌E.coliDH5α(pBBR-X)为供体菌、E.coliWD803(pRK2013)为辅助菌、BYS-1M为受体菌进行三亲接合,筛选到了恢复耐盐性能的接合子HR-23,提取接合子HR-23的重组质粒pHR23,酶切确定其插入的耐盐相关DNA片段大小分为5.4 kb.该片段为Halom onassp.BYS-1耐盐相关基因的克隆奠定了基础.

A salt-sensitive mutant BYS-1M of strain Halomonas sp. BYS-1 was obtained by nitriguanidine mutagenesis. The gene library of BYS-1 was constructed in E. coli OHSα with pBBR1MCS-2 as the vector by the method of shotgun cloning. Triparental conjucation was conducted with BYS-1M as recipient, the library strain E. coil DHSα （pBBR-X） as donors and E. coil WD803 （pRK2013） as helper, In this way, the gene library of BYS-1 was transferred into BYS-1M to look for the complementation of the mutant, One hybrid named HR-23, which had recovered the same salt-tolerance characteristics as the original strain BYS-1 was obtained, The recombinant plasmid pHR-23 was extracted from the HR-23 and subjected to restriction analysis; the results showed that the length of inserted salt-tolerance related DNA fragment in pHR-23 was 5.4 kb. This fragment laid the foundation for the cloning of salt-related gene of Halomonas sp, BYS-1. Fig 5, Tab 1, Ref 14