ABSTRACT
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.
Subject(s)
Escherichia coli/genetics , Genes, Bacterial , Sinorhizobium/genetics , Sodium Chloride/pharmacology , Cloning, MolecularABSTRACT
A 23 kb DNA fragment related to salt tolerance was obtained from the gene library of S. fredii strain RT19. In this study, BamH I was selected to digest 23 kb DNA fragment into different length of DNA fragments. The resulting fragments were ligated with plasmid pML122, then the recombinant plasmids were transformed to competent cells of E. coli S17-1 on selective medium and three transformants TR were obtained. Two-parental mating experiments were carried out with these transformants as donor and salt sensitive S. fredii strain RC3-3 as recipient, and the transconjugant BR2 was selected on FY plates containing gentamycin and 0.4 mol/L NaCl. Thus, a 4.4 kb DNA fragment related to salt tolerance was obtained. Based on its physical map, six restriction fragments were subcloned into plasmid pUC18 for DNA sequencing. Subsequently, sequencing and analysis of 4.4 kb DNA fragment showed that fixO, fixN genes and three ORFs were obtained.
