丛枝菌根真菌砷酸盐还原酶基因RiarsC的克隆和功能分析
Cloning and Characterization of Arsenate Reductase Gene RiarsC from Arbuscular Mycorrhizal Fungi
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摘要: 丛枝菌根真菌(AMF)在增强植物砷(As)抗性方面发挥着重要作用。已有相关研究表明，接种AMF能提高植物体内三价砷As(III)的比例，AMF可能参与了将五价砷As(V)还原为As(III)的过程从而提高了菌根植物的As抗性，但目前尚缺乏直接分子证据。本文从异形根孢囊霉(Rhizophagus irregularis)菌丝中克隆得到了一个砷酸盐还原酶基因RiarsC并进行序列分析。将该基因转入arsC缺陷型大肠杆菌(Escherichia coli)菌株WC3110 (ΔarsC)中，通过As(V)抗性生长曲线和As形态测定，分析了该基因的功能。结果显示，RiarsC属于谷氧还蛋白-谷胱甘肽依赖的砷酸盐还原酶家族；RiarsC基因的表达显著提高了As敏感型E. coli菌株对As(V)的抗性，当培养基中As(V)浓度为100 μmol·L-1时表现更加明显。As形态分析表明，表达RiarsC的E. coli菌株能够将培养基中71.03%的As(V)还原为As(III)；与表达空载体的菌株相比，还原效率提高了61.98%。本研究证明了AMF的砷解毒还原能力，为进一步开展AMF的砷代谢机制研究提供了一定的分子生物学基础。Abstract: Arbuscular mycorrhizal fungi (AMF) play a vital role in improving plant arsenic (As) tolerance. Studies have shown that AMF colonization could increase As(III) proportion in plants, suggesting that AMF might involve in reduction of As(V) into As(III). However, the molecular evidence of arsenate reduction in AMF is still lacking. Here, an arsenate reductase gene (RiarsC) from AM fungus Rhizophagus irregularis was cloned and characterized. RiarsC was transferred to Escherichia coli strain WC3110 (ΔarsC), whose arsenate reductase gene was knocked out and was sensitive to As(V). The function of RiarsC was verified through growth curve comparison and As speciation analysis. The results showed that RiarsC belonged to the Grx/GSH linked ArsC family. Heterologous expression of RiarsC enhanced As(V) tolerance of WC3110, especially under the As(V) concentration of 100 μmol·L-1 in LB medium. E. coli expressing RiarsC reduced 71.03% of As(V) into As(III) in LB medium. The As(V) reduction rate of E. coli bearing RiarsC increased by 61.98% compared to the strain expressing empty vector. The present study proved the ability of AMF to alleviate As toxicity by As(V) reduction, and advanced the research into the mechanism of As detoxification in AMF.
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