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【目的】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。【方法】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。【结果】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。【结论】灰葡萄孢BcKMO正调控病菌的生长、发育,负调控病菌的致病力。
【Objective】 The objective of this study is to clarify the function of BcKMO in the growth, development and pathogenicity of Botryosphaeria bereria and to lay the foundation for clarifying the molecular mechanism of this gene regulating the growth, development and pathogenicity of Botrytis cinerea, in accordance with. 【Method】 Bioinformatics methods were used to analyze BcKMO and its coding products. The BcKMO coding region was amplified and ligated with the pBARKS1-eGFP vector to construct the gene expression vector pBARKS1-BcKMO-eGFP. The protoplast of the BCG183 mutant was transformed by PEG-mediated protoplast transformation. Transformants were identified by PCR, Southern blotting and real-time PCR. The phenotypes of the wild-type strain BC22, mutant BCG183 and the replicative BCG183 / BcKMO were determined (colony morphology, mycelial morphology, growth rate, Etc.) and pathogenicity were analyzed. 【Result】 BcKMO encoded kynurenine 3-monooxygenase (KMO), a conserved domain containing the oxidase FAD and four Aromatic-ring hydroxylase-like motifs (Rossmann-fold NAD (P) (+) - binding proteins, gi156049701) from Sclerotinia sclerotiorum and FAD from Ophiostoma piceae Dependent oxidoreductase (FAD-dependent oxidoreductase, gi512192943) higher homology. The BcKMO expression vector pBARKS1-BcKMO-eGFP was successfully constructed. The protoplasts of the mutant BCG183 were transformed by PEG-mediated transformation to obtain glufosinate-resistant transformants. PCR, Southern blotting and real-time PCR Technical identification of transformants, BcKMO accession mutant BCG183 / BcKMO was obtained. The phenotypes of the mutant BCG183 and the BCG183 / BcKMO mutant were analyzed and it was found that the growth rate of the BCG183 mutant was slowed down without producing conidia and sclerotia with mycelium slender; the growth of the recovery mutant and the wild-type strain Rate, mycelium basically the same shape, have produced conidia and sclerotia. Pathogenicity analysis showed that the pathogenicity of BCG183 mutants was significantly higher than that of wild type and restorer mutants on kidney bean and cucumber leaves. 【Conclusion】 Botrytis cinerea BcKMO is regulating the growth, development and negative regulation of pathogenic bacteria.