[费氏中华根瘤菌RT19耐盐相关基因的克隆及其在大肠杆菌中的表达]
[Cloning of gene related to salt tolerance from Sinorhizobium fredii RT19 and its expression in Escherichia coli].
作者信息
Ge S C, Liu Y N, Yang S S
机构信息
Department of Microbiology, College of Biological Sciences, China Agricultural University, Beijing 100094, China.
出版信息
Yi Chuan Xue Bao. 2001;28(6):575-82.
A 4.4 kb DNA fragment related to salt tolerance containing three open reading frames was isolated from the gene library of S. fredii strain RT19. By subcloning and functional analysis, only ORF2 related to salt tolerance was obtained. The ORF2 was ligated to expression vectors pThioHisA, B and C, respectively, and recombinant expression vectors pGA, pGB and pGC containing 1.5 kb DNA fragment related to salt tolerance were constructed. These recombinant expression vectors were transformed into E. coli DH5 alpha. Inducing by IPTG and analyzing with SDS-PAGE, it was found that the fusion protein encoded by pGC was expressed, and its molecular weight was equal to the sum of thioredoxin encoded by trxA and ORF2 putative protein molecular weight. The Western blot demonstrated that the target gene was successfully expressed in E. coli.
从费氏中华根瘤菌菌株RT19的基因文库中分离出一个与耐盐性相关的4.4 kb DNA片段，该片段包含三个开放阅读框。通过亚克隆和功能分析，仅获得了与耐盐性相关的开放阅读框2（ORF2）。将ORF2分别连接到表达载体pThioHisA、B和C上，构建了包含与耐盐性相关的1.5 kb DNA片段的重组表达载体pGA、pGB和pGC。将这些重组表达载体转化到大肠杆菌DH5α中。经异丙基-β-D-硫代半乳糖苷（IPTG）诱导并用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）分析，发现pGC编码的融合蛋白得以表达，其分子量等于硫氧还蛋白（由trxA编码）与ORF2推定蛋白分子量之和。蛋白质免疫印迹法（Western blot）表明目标基因在大肠杆菌中成功表达。
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