盐单胞菌C6与耐盐有关DNA片段的克隆和序列分析  

Cloning and Sequencing of the DNA Fragment Related to Salt Tolerance of\% Halomonas\% sp. C6

作　　者：王海洪[1] 樊振川[1] 曾静[1] 杨苏声[1] 

机构地区：[1]中国农业大学生物学院微生物系,北京100094

出　　处：《农业生物技术学报》1999年第3期271-276,共6页Journal of Agricultural Biotechnology

基　　金：国家自然科学基金

摘　　要：提取盐单胞菌（ Ｈａｌｏｍｏｎａｓ）Ｃ６ 的总ＤＮＡ，用Ｓａｕ３ＡⅠ部分酶切，回收１５ ～３０ ｋｂ 的ＤＮＡ 片段，以ｐＬＡＦＲ３ 为载体在大肠杆菌（ Ｅｃｏｌｉ）ＤＨ５α中构建了该菌的基因文库。以Ｃ６ 的基因文库为供体，以费氏中华根瘤菌（Ｓｉｎｏｒｈｉｚｏｂｉｕｍ ｆｒｅｄｉｉ） 盐敏感菌株ＲＣ３３ｒ 为受体，在辅助质粒ｐＲＫ２０１３ 的协助下进行三亲本杂交，在选择培养基上筛选到接合子ＲＷＨ１５ 和ＲＷＨ１７ 。质粒检测表明，这两个接合子分别含有重组质粒ｐＷＨ１５ 和ｐＷＨ１７ 。通过三亲本杂交和功能互补的方法，对质粒ｐ ＷＨ１５ 进行两次亚克隆，最终得到３２ ｋｂ 的ＤＮＡ 片段。经检测，该片段能使ＲＣ３ － ３ ｒ 恢复耐盐性。通过ＤＮＡ 测序及开放阅读框架（ＯＲＦｓ） 分析，发现该片段上有４ 个开放阅读框架，其功能有待于进一步研究。Total DNA was partially digested by\% Sau\%3AⅠ from\% Halomonas\% sp. C6 and 15～30 kb DNA fragments were isolated. A gene library of C6 was constructed with pLAFR3,then transformed into E.\%coli\% DH\-\{5α\}. Triparental mating was conducted with recipient strain being RC3 3\+r,a salt sensitive mutant of\% Sinorhizobium fredii\% RT19,and donor being the gene library of C6. In this way,DNA fragments of C6 were transferred into RC3 3r to look for complementation of the mutant. Two hybrids were selected. A plasmid was extracted from one of the hybrids. From subsequent subcloning experiments,a 3 2 kb foreign DNA fragment was obtained which still retained the ability of complementing the salt sensitive mutant. DNA sequencing and ORFs analysis of this fragment showed that it consists of four ORFs. The function of ORFs needs to be studied further.

关 键 词：中度嗜盐菌 盐单胞菌 耐盐性 基因克隆 序列分析 

分 类 号：Q939.1[生物学—微生物学] S332.6[农业科学—作物遗传育种]