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以耐盐的弗氏中华根瘤菌(Sinorhizobium fredii)RT19菌株为材料,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得到15～25kb大小的DNA片段.用QIAGEN-tip100试剂盒方法,提取并纯化质粒pLAFRI,用EcoRI将其切成线状.然后用T_4 DNA连接酶将回收片段与线性载体连接,并利用包装蛋白进行包装,感染大肠杆菌(Esherichia coli)SP17.1,构建了RT19的基因文库.以固体亚硝基胍作为诱变剂,在0.4mol·L_(-1)NaCI的条件下,从2500个菌落中筛选得到了5株RT19的盐敏感突变株.以其中稳定的突变株RC3-3作为受体菌,在辅助质粒pRK2013的协助下,将RT19的DNA片段转移到RC3-3中.在含有四环素和盐的基本培养基上筛选出能够耐盐的阳性克隆.提取其重组质粒,用EcoRI完全酶切,得到15～25kb的外源DNA片段.
The salt-tolerant Sinorhizobium fredii strain RT19 was used as the material to prepare the total DNA, which was partially digested with restriction endonuclease EcoRI to obtain a DNA fragment of 15-25 kb size by electroelution. Plasmid pLAFRI was extracted and purified by QIAGEN-tip100 kit and cut into linear sections with EcoRI.The recovered fragment was then ligated with a linear vector using T4 DNA ligase and packaged with packaging protein and infected with Escherichia coli ) SP17.1, a gene library of RT19 was constructed.Using solid nitrosoguanidine as a mutagen, 5 strains of RT19 were screened from 2,500 colonies under the condition of 0.4mol·L -1 NaCI Sensitive mutant.The stable mutant RC3-3 was used as the recipient bacteria, and the DNA fragment of RT19 was transferred to RC3-3 with the aid of the helper plasmid pRK2013.Screening was performed on minimal medium containing tetracycline and salt Salt-tolerant positive clones were obtained and their recombinant plasmids were extracted and completely digested with EcoRI to obtain foreign DNA fragments of 15-25 kb.