苜蓿中华根瘤菌与耐盐有关的DNA片段的克隆

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\%Eco\%RⅠ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\%Eco\%RⅠ将其切成线状，然后用T\-4DNA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4kb与耐盐有关的片段。

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

Total DNA partially digested by EcoR I was prepared for S. meliloti 042B, in which 15-25 kb DNA fragments were collected. Vector pLAFR I was purified and digested by EcoR I, and then the various DNA fragments of 042B were ligated with pLAFR I by T4DNA ligase. Gene library of S. meliloti 042B was constructed with pLAFR I 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 0.5 mol/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 0.5 mol/L NaCl. Then the transconjugants were grown on FY plates containing tetracycline (20 micrograms/mL) and 0.5 mol/L NaCl. A 7 kb inserted DNA fragment related to salt tolerance was obtained. In subcloning experiment, a 4 kb DNA fragment related to salt tolerance was obtained.