葡萄乙醛脱氢酶VvALDH10A9基因的克隆、表达及酶活性分析

摘要

由葡萄座腔菌科Botryosphaeriaceae真菌引起的葡萄溃疡病Botryosphaeria dieback是葡萄上的主要枝干病害,严重影响葡萄产量和品质.鉴定和分析参与葡萄与葡萄溃疡病菌互作的基因,有助于揭示和阐明葡萄抗溃疡病的信号通路.本研究根据葡萄溃疡病菌侵染后的葡萄转录组数据信息,利用逆转录聚合酶链式反应技术(RTPCR)克隆了一个受葡萄溃疡病菌诱导上调表达的乙醛脱氢酶基因VvALDH10A9 (Vitis vini fera aldehyde dehydrogenase 10A9).系统进化树分析表明,该基因编码的蛋白与拟南芥AtALDH10A8亲缘关系最近.利用实时荧光定量PCR分析VvALDH 10A9的表达结果表明:VvALDH 10A9的表达具有组织特异性,在茎、叶和花中表达量最高,在根中表达量相对较低;葡萄溃疡病菌侵染后,VvALDH 10A9在葡萄抗病品种中表达变化不明显,而在葡萄感病品种中该基因显著上调表达.利用原核蛋白表达系统诱导合成的VvALDH 10A9蛋白,经纯化后可降解乙醛,表明VvALDH10A9蛋白具有乙醛脱氢酶活性.%Botryosphaeria dieback caused by pathogens of the fungal family Botryosphaeriaceae is currently one of the most important trunk diseases in the world's primary grape production areas.This disease had seriously affected the grape yield and quality.Investigating the genes that play roles in grape-Botryosphaeriaceae interactions helps us understand the defense mechanism in grapevine.In this study,depending on the transcription analysis,an aldehyde dehydrogenase gene VvALDH10A9 was cloned using reverse transcription polymerase chain reaction (RT-PCR) based on the transcriptome information.Phylogenetic analysis of VvALDH10A9 and other 37 characterized ALDH proteins revealed that VvALDH10A9 was more closely related to AtALDH10A8.Quantitative realtime PCR assays showed that VvALDH10A9 was preferentially expressed in leaf blades,stem and flower.Additionally,VvALDH10A9 was rapidly induced by Lasiodiplodia theobromae CSS-01s in susceptible cultivar but not in resistant cultivar.Further analysis indicated that purified VvALDH10A9 protein showed enzyme activities to acetaldehyde in vitro.