灰葡萄孢BcKMO在病菌生长、发育和致病过程中的功能

摘要

[Objective]The objective of this study is to investigate the function ofBcKMO gene in growth, development and pathogenicity ofBotrytis cinerea, to further clarify the molecular mechanism of theBcKMO gene in development and pathogenicity of the fungus and provide a theoretical basis for the gray mould control.[Method] Bioinformatics methods were used to analyze theBcKMO gene and its encoding product. TheBcKMO gene coding region was amplified and cloned into pBARKS1-eGFP, and pBARKS1-BcKMO-eGFP was constructed. The protoplasts of mutant BCG183 were prepared and transformed with pBARKS1-BcKMO-eGFP by PEG-mediated method. PCR, Southern blotting and real-time PCR were used to identify the transformant. Phenotype including colony morphology, mycelium morphology, growth rate, conidial yield, and so on, and pathogenicity assays of wild-type strain BC22 (WT), mutant BCG183 and complemutant BCG183/BcKMO were performed to verify the function of the BcKMO gene in growth, development and pathogenicity.[Result]TheBcKMO gene encodes kynurenine 3-monooxygenase (KMO) having monooxygenase FAD conserved domain and four Aromatic-ring hydroxylase-like motifs. The BcKMO protein displayed high homology to rossmann-fold NAD(P)(+)-binding protein (gi156049701) ofSclerotinia sclerotiorum and FAD-dependent oxidoreductase (gi512192943) ofOphiostoma piceae. The expression vector of theBcKMO gene, pBARKS1-BcKMO-eGFP, was successfully constructed and transformed into the protoplasts of mutant BCG183. Results of PCR, Southern blotting and Real-time PCR showed that theBcKMO gene complementing mutant (BCG183/BcKMO) was successfully obtained. Phenotype assay showed that the mutant BCG183 growed slowly, did not produce conidia and sclerotia, and hyphae slender, but the phenotypes of the BCG183/BcKMO were all similar to that of WT. Compared with WT and the BCG183/BcKMO, the pathogenicity of the mutant BCG183 on kidney bean leaves and cucumber leaves were dramatically increased.[Conclusion]TheBcKMO gene is a negative regulation in growth and development, and a positive regulation in pathogenicity ofB. cinerea.%明确灰葡萄孢BcKMO在病菌生长、发育和致病过程中的功能，为阐明该基因调控灰葡萄孢生长、发育和致病的分子机理奠定基础，并为灰霉病的防治提供理论依据。利用生物信息学方法，对BcKMO及其编码产物进行分析。扩增 BcKMO 的编码区，与 pBARKS1-eGFP 载体连接，构建基因的表达载体pBARKS1-BcKMO-eGFP；利用PEG介导的原生质体转化方法，转化BcKMO的T-DNA插入突变体BCG183的原生质体；利用PCR、Southern blotting和real-time PCR技术，对所获得转化子进行鉴定；对野生型菌株BC22、突变体BCG183和回复突变体 BCG183/BcKMO的表型（菌落形态、菌丝形态、生长速度、产孢量等）和致病力进行分析。BcKMO编码犬尿氨酸单氧酶（kynurenine 3-monooxygenase，KMO），含有单氧酶FAD的保守结构域和4个类似芳香环羟化酶（Aromatic-ring hydroxylase-like）的基序，与核盘菌（Sclerotinia sclerotiorum）的Rossmann卷曲NAD(P)(+)-结合蛋白（Rossmann-fold NAD(P)(+)-binding proteins，gi156049701）和从线嘴壳（Ophiostoma piceae）的FAD依赖性氧化还原酶（FAD-dependent oxidoreductase，gi512192943）同源性较高。成功构建了BcKMO的表达载体 pBARKS1-BcKMO-eGFP，利用 PEG 介导的转化方法，转化突变体 BCG183的原生质体，获得了草胺膦抗性的转化子；利用PCR、Southern blotting和real-time PCR技术鉴定转化子，获得了BcKMO的回复突变体BCG183/BcKMO。对突变体BCG183和回复突变体BCG183/BcKMO的表型进行了分析，发现BCG183突变体的生长速率减慢，不产生分生孢子和菌核，菌丝纤细；回复突变体和野生型菌株的生长速率、菌丝形态基本一致，均产生分生孢子和菌核。致病力分析发现，BCG183突变体在芸豆叶片和黄瓜叶片上的致病力均较野生型和回复突变体明显增强。灰葡萄孢BcKMO正调控病菌的生长、发育，负调控病菌的致病力。