利用TAIL-PCR克隆耐盐基因及其分析

Screening of Salt-tolerance rhizobium Strains and Cloning and Analysis of Salt-tolerance Gene

通过大量筛选得到一株高耐盐的豇豆根瘤菌WME7,最高可耐15g/L NaCl,利用Tn5-sacB转座子对该菌株进行随机插入突变,从突变子中筛选获得30个共生缺陷型突变株。利用TAIL-PCR(thermal asymmetric interlaced PCR)方法克隆了突变株Tn5-sacB侧翼序列,通过BLAST发现有1个突变株的插入失活基因与鼠伤寒沙门氏菌抗银的结合蛋白SilE有94%同源性,表明有关Na+离子的抗性基因可能与Ag+离子的抗性基因有某种关系。该基因在其它菌中也能抗其它金属离子(铜、锌、钴、铬)。

A high salt-tolerance rhizobium strain was screened,which can grown on 15 g/L NaCl YMA medium at most.Using Tn5-sacB insertion mutagenesis,30 salt-tolerance-defective mutants were screened by salt-tolerance experiments.The DNA sequences of the contiguous region from the Tn5 insertion site were determined by thermal asymmetric interlaced PCR（TAIL-PCR）.A new gene was cloned and BLAST analysis of the gene showed a sequence similarity（94%）to Salmonella typhimurium silver binding protein SilE gene,indicating there are certain relationship between gene of resistance to Ag＋ and Na＋.BLAST indicating this gene can resist copper,cadmium,zinc and cobalt et al.