花生根瘤菌Bradyrhizobium sp.MZ5 Ⅲ型分泌系统调节基因ttsI突变体的功能分析

Construction of regulatory gene ttsI mutant in type Ⅲ secretion system of Bradyrhizobium sp.MZ5 and its function analysis

【目的】探究慢生型花生根瘤菌Ⅲ型分泌系统在花生-根瘤菌互作的功能。【方法】本研究采用同源重组和三亲本接合转移的方法,构建Bradyrhizobium sp. MZ5的Ⅲ型分泌系统调节基因ttsI突变体;荧光定量PCR检测添加大豆苷元(Daidzein)和染料木黄酮(Genistein)诱导物后野生型和突变株转录水平上ttsI的表达量变化及其差异;蛭石结瘤实验分析ttsI基因突变对花生结瘤能力的影响。【结果】在转录水平上,大豆苷元和染料木黄酮对MZ5的Ⅲ型分泌系统调节基因ttsI的表达具有显著的抑制作用(P<0.05)。在MZ5△ttsI突变体中ttsI基因的表达量都明显下调,与野生型菌株的相比都达到极显著水平(P<0.001)。蛭石结瘤实验表明,与野生型菌株相比,MZ5△ttsI突变体在不同花生品种的结瘤数和地上部干重都显著性降低。根瘤石蜡切片表明,MZ5△ttsI突变体在根瘤内的含菌量少于野生型菌株。【结论】Bradyrhizobium sp. MZ5菌株中的Ⅲ型分泌系统在花生-根瘤菌互作中对结瘤有积极的促进作用。

[Objective]To explore the function of typeⅢsecretion system of bradyrhizbial strains in peanut-rhizobia interaction.[Methods]We constructed the ttsI mutant of typeⅢsecretion system in Bradyrhizobium sp.MZ5 by the way of homologous recombination and three parental conjugative transfer.The different expression of ttsI in MZ5 and MZ5ΔttsI were analyzed by real-time PCR by using the cDNA as templates after addition of Daidzein and Genistein.The vermiculite nodulation experiment was performed to comparative analysis of the nodulation ability of Bradyrhizobium sp.MZ5 and its ttsI mutant on Arachis hypogea(S523 and Y45).[Results]We found that daidzein and genistein had a significant inhibiting effect on the ttsI gene of the strain MZ5(P<0.05).In the vermiculite nodulation experiment,we found that there was a significant reduction in the number of nodules and the shoot dry weight on peanut S523 and Y45 for MZ5ΔttsI mutant(P<0.05)comparing with the wild-type strain MZ5.The semi-thin nodule section showed that the MZ5ΔttsI mutant contained fewer cells in the nodules than the wild-type strain.[Conclusion]The typeⅢsecretion system of Bradyrhizobium sp.MZ5 has a positive effect on peanut-rhizobia interaction.