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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。
Objective To clone the medicinal fungus Phyllostachys P450 gene and analyze its bioinformatics. Methods RT-PCR was used to obtain the full-length cDNA of c DNA. The on-line tools were used to predict the physical and chemical properties of the proteins. The molecular characteristics of the proteins were analyzed by MEGA 6.0. The amino acid sequences were analyzed by MEGA 6.0. Results Eleven P450 superfamily genes were cloned from Chinese Polyporus umbellatus. The complete open reading frame (ORF) of the 11 cDNAs contained between 1 326 and 1 635 bp, encoding 442 ~ 545 amino acids, the relative molecular mass between (48.9 ~ 61.5) × 103, the theoretical isoelectric point between 6.3 ~ 8.9. Sequence analysis revealed that it has a conserved P450 domain. Amino acid sequence multiple alignment and phylogenetic tree results show that these 11 cytochrome P450 genes belong to basidiomycetes. Real-time quantitative RT-PCR analysis showed that these nine genes were expressed in different sclerotia, and the expression levels of comp18720, comp26906, comp32003 and comp33717 were much lower than those of the uninfected Meloid ring infected Ling bacteria nucleus, the other seven genes are significantly up-regulated expression. Conclusion The molecular characteristics of eleven P450 genes of medicinal fungi Polyporus umbellatus lay a theoretical foundation for further study of its role in the defense against Mycorrhizal fungi in Polyporus umbellatus.