苜蓿中华根瘤菌与耐盐有关的DNA片段的克隆  

CLONING OF DNA FRAGMENT RELATED TO SALT TOLERANCE IN \%SINORHIZOBIUM MELILOTI\% 042B\+*

作　　者：陈雪松[1] 张海瑜[1] 高为民[1] 朱坤 阚凤玲[1] 杨苏声[1] 

机构地区：[1]中国农业大学生物学院微生物学系,北京100094

出　　处：《微生物学报》1999年第6期489-494,共6页Acta Microbiologica Sinica

基　　金：高等学校博士学科点专项科研基金;欧盟研基金

摘　　要：以耐盐的苜蓿中华根瘤菌（Ｓｉｎｏｒｈｉｚｏｂｉｕｍ ｍｅｌｉｌｏｔｉ）０４２Ｂ为材料，制备其总ＤＮＡ，经过限制性内切酶ＥｃｏＲⅠ的部分酶解，利用电洗脱方法回收１５ ～２５ｋｂ 大小的ＤＮＡ 片段。以碱法制备载体质粒ｐＬＡＦＲⅠ，用ＥｃｏＲⅠ将其切成线状，然后用Ｔ４ＤＮＡ 连接酶将回收片段与线状载体连接，利用包装蛋白进行包装后，感染大肠杆菌（ Ｅｓｃｈｅｒｉｃｈｉａ ｃｏｌｉ）Ｓ１７１，构建了０４２Ｂ的基因文库。以固体亚硝基胍作为诱变剂处理出发菌株，在０５ｍｏｌ／ＬＮａＣｌ 的条件下，从２０００ 个菌落中筛选得到１２ 株０４２Ｂ的盐敏感突变株，以其中稳定的盐敏突变株ＧＺ１７ 为受体菌，利用两亲本杂交将含有０４２Ｂ的ＤＮＡ 片段的ｐＬＡＦＲⅠ重组质粒转移到ＧＺ１７ 中，在含有四环素和０５ｍｏｌ／ＬＮａＣｌ 的基本培养基上筛选出能够耐盐的阳性克隆，获得了与耐盐有关的７ｋｂ 长的ＤＮＡ片段。对该片段进行亚克隆，最终获得了４ｋｂTotal DNA partially digested by Eco RⅠ was prepared for \%S.meliloti\% 042B, in which 15～25kb DNA fragments were collected. Vector pLAFRⅠ was purified and digested by Eco RⅠ, and then the various DNA fragments of 042B were ligated with pLAFRⅠ by T\-4DNA ligase. Gene library of \%S.meliloti\% 042B was constructed with pLAFRⅠ using \%E.coli\% S17\|1 as recipient. The number of bacterial recombinants obtained was about 8 000 and 95% of them contained foreign DNA fragments. Using NTG, 042B was mutated on FY plates and 12 sensitive strains were screened at 0 5mol/L NaCl from 2 000 colonies. One of them was named GZ17 and selected as a recipient strain. By biparental mating the foreign DNA fragments were introduced from gene library of strain 042B into recipient strain GZ17 which is sensitive to 0 5mol/L NaCl. Then the transconjugants were grown on FY plates containing tetracycline (20μg/mL) and 0 5mol/L NaCl. A 7kb inserted DNA fragment related to salt tolerance was obtained. In subcloning experiment, a 4kb DNA fragment related to salt tolerance was obtained.

关 键 词：苜蓿中华根瘤菌 耐盐 基因文库 亚克隆 

分 类 号：S154.381[农业科学—土壤学] Q789[农业科学—农业基础科学]