药用真菌猪苓菌核细胞色素P450基因的克

发布时间：2018-11-23 06:40

【摘要】：目的克隆药用真菌猪苓细胞色素P450基因并进行生物信息学分析。方法利用RT-PCR技术取得基因c DNA全长;利用在线工具预测蛋白的理化性质、结构域等分子特征;用MEGA 6.0分别对氨基酸进行多序列比对和进化关系分析。结果本实验从中国野生猪苓菌核(Polyporus umbellatus)中克隆得到11个P450超家族基因,该11个基因c DNA包含的完整开放阅读框在1 326~1 635 bp之间,编码蛋白含442~545个氨基酸之间,相对分子质量在(48.9~61.5)×103之间,理论等电点在6.3~8.9之间。序列分析发现其具有保守的P450结构域。氨基酸序列多重比对及系统发育树结果显示这11个细胞色素P450基因都隶属于担子菌类。实时荧光定量RT-PCR分析表明,这9个基因在不同的菌核部分都有表达,除基因comp18720、comp26906、comp32003及comp33717在被蜜环菌侵染部位的表达量要远远低于未被蜜环侵染的猪苓菌核,其他7个基因都是表达显著上调。结论药用真菌猪苓11种P450酶基因的分子特征为进一步研究其在猪苓菌核防御蜜环菌侵染的作用奠定理论基础。
[Abstract]:Objective to clone the cytochrome P450 gene of medicinal fungus Polyporus umbellata and analyze it by bioinformatics. Methods the full length of c DNA gene was obtained by RT-PCR technique, the physicochemical properties and domain characteristics of the protein were predicted by on-line tools, and the amino acid sequence alignment and evolutionary relationship were analyzed by MEGA 6.0, respectively. Results in this study, 11 P450 superfamily genes were cloned from the sclerotia sclerotiorum (Polyporus umbellatus) of Chinese umbellus umbellata. The complete open reading frame contained in c DNA was between 1 326 and 1 635 bp, and the encoding protein contained 442n545 amino acids. The relative molecular weight is (48.9 ~ 61.5) 脳 10 ~ 3 and the theoretical isoelectric point is between 6.3 ~ 8.9. Sequence analysis showed that it had a conserved P450 domain. The results of multiple amino acid sequence alignment and phylogenetic tree showed that the 11 cytochrome P450 genes belonged to basidiomycetes. Real-time fluorescence quantitative RT-PCR analysis showed that all the 9 genes were expressed in different sclerotia, except for comp18720,comp26906,comp32003 and comp33717, which were significantly lower than those in the sclerotia of Polyporus umbellata infected by Armillaria mellea. The other seven genes were significantly up-regulated. Conclusion the molecular characteristics of 11 P450 enzyme genes of medicinal fungus Polyporus umbellata lay a theoretical foundation for further study on the role of P450 enzyme gene in the prevention of Armillaria mellea infection by Polyporus umbellata sclerotia.
【作者单位】：中国医学科学院北京协和医学院药用植物研究所中草药物质基础与资源利用教育部重点实验室;江苏理工学院电气信息工程学院生物信息与医药工程研究所;
【基金】：国家重点基础研究发展计划资助(2014CB13830X) 国家自然科学基金资助项目(31201666) 山西省煤基重点科技攻关项目资助项目(FT2014-03) 河北山区特色中药材种质资源评价与仿野生栽培技术研究与示范资助项目(16232503D)
【分类号】：S567.3

【参考文献】