利用Tn5-1063转座诱变法分离苜蓿中华根瘤菌042BM noeB基因的研究

Study on Isolation of noeB of Sinorhizobium meliloti 042BM by Tn5-1063 Mutagenesis

采用三亲本杂交方法将带有Tn5 10 6 3(含luxAB)的质粒pRL10 6 3a导入苜蓿中华根瘤菌 (Sinorhizobiummeliloti) 0 4 2BM ,进行转座子插入诱变 ,在含有氯霉素、卡那霉素的TY平板上筛选接合子。通过结瘤试验 ,从 10 0 0个突变株中 ,筛选到 3个结瘤突变株 0 4 2BMR5、0 4 2BMR11和 0 4 2BRM2 9。它们都表现出发光酶活性 ,表明转座子正向插入到基因组中的某个启动子下游。Southern杂交结果证实 ,转座子均为单一位点插入。对 0 4 2BMR5突变株基因组进行反向PCR ,扩增位于Tn5 10 6 3两端的侧翼序列。测序结果表明 ,转座子插入到苜蓿中华根瘤菌的共生质粒pSymAnoeB基因内。根据基因组中noeB上游和下游序列扩增出 0 4 2BMnoeB ,其与苜蓿中华根瘤菌 10 2 1noeB的同源性为 98% ,而与NoeB蛋白的氨基酸序列相似性为 95 %。疏水性分析发现 ,NoeB是一个跨膜蛋白 ,在N末端有 4个跨膜区 ,其中包含 3个初级螺旋和 1个次级螺旋。

The Tn5-1063 containing promoterless luxAB genes was used to mutagenize S. meliloti 042BM and 1000 insertion mutants were subsequently screened for nodulation mutants. Three mutants involved in nodulation were obtained, named as 042BMR5, 042BMR11 and 042BM29, respectively .All of them showed some changes of ability in symbiosis. Further, their luciferase activities were determined in TY media. It is indicated that Tn5-1063 was inserted directly into loci downstream of promoters in 042BM genome. DNA sequences flanking Tn5-1063 of 042BMR5 was amplified using inverse PCR, and it was found that Tn5-1063 was inserted into noeB gene. The noeB of 042BM is identical to that of S. meliloti 1021 at 98% level , and similarity of amino acid sequences of their NoeB was 95%. Hydrophobicity analysis of the NoeB showed that it is a transmembrane protein and includes four transmembrane regions at N-terminal, consisting of three primary helixs and one secondary helix.