豌豆根瘤菌RND外排转运基因tpaA的功能研究

Function of efflux RND transporter gene tpa A in Rhizobium leguminosarum

Rhizobium.leguminosarum 3841 tpaA基因编码RND家族内膜外排转运蛋白,RND外排泵的过量表达是革兰阴性菌细菌抗生素耐药性形成的重要原因之一.采用基因同源重组构建豌豆根瘤菌tpaA基因突变株RLtpaA,并研究了该基因突变对根瘤菌生长、抗生素耐药性以及共生固氮的影响.结果表明,tpaA基因突变会使根瘤菌在AMS基本培养基生长延迟,但不影响其在TY天然培养基中的正常生长.tpaA基因突变可能抑制了根瘤菌中的RND家族外排泵的过量表达,从而使其抗生素耐药性降低.接种了tpaA基因突变株的豌豆植株能形成红色有效根瘤,但其接种根瘤固氮酶活性降低.实时荧光定量RT-PCR分析表明tpaA基因在根瘤类菌体中的表达显著上调.

In Rhizobium leguminosarum 3841,tpaA gene encodes RND intima efflux transporter protein.Overexpression of RND family efflux pumps is one of the important reasons for the formation of antibiotic resistance in Gram-negative bacteria.R.leguminosarum tpaA mutant RLtpaA was constructed through homologous recombination,and the effects of tpaA gene mutation on growth,antibiotic resistance and symbiotic fixation were further studied.The results showed that the mutation of tpaA gene delayed the bacterial growth in minimal medium AMS,but did not affect the normal growth in natural medium TY.Antibiotic resistance experiments showed that tpaA gene mutation might inhibit the overexpression of RND family efflux pumps in R.leguminosarum and thus reduce its antibiotic resistance.Symbiotic experiments showed that pea plants inoculated with tpaA gene mutant strain formed effective red nodules,but could lead to a decline of the nitrogen fixation ability.Quantitative real-time RT-PCR result showed that tpaA gene expression could be induced under symbiotic condition.