药用真菌猪苓菌核细胞色素P450基因的克隆、序列分析及表达差异的鉴定  

Cloning,Sequence Analysis and Expression Analysis of Eleven Cytochrome P450 Encoding Genes in Polyporus umbellatus

作　　者：刘蒙蒙[1,2] 邢咏梅[1] 郭顺星[1] 

机构地区：[1]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,北京100193 [2]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,江苏常州213001

出　　处：《中国药学杂志》2017年第11期918-923,共6页Chinese Pharmaceutical Journal

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)

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。OBJECTIVE To clone the cytochrome P450 genes from a medicinal fungus Polyporus umbellatus and carry out bioinfor- matic analysis. METHODS The full length cDNAs of the genes were obtained by RT-PCR. Some bioinformatic tools were used to characterize the physiochemical properties of the 11 deduced protein. The analyses of multiple alignment and phylogenetic trees were performed with MEGA 6.0 software. RESULTS Eleven P450 genes were cloned using RT-PCR method from the Polyporus umbellatus sclerotium. The full open reading frame cDNA sequence of these eleven genes was between 1 326 and 1 635 bp, the putative encoding proteins were between 442 and 545 amino acids, the molecular weight was between 48. 9 x 103 and 61.5 x 103 , and the theoretical pI was between 6. 3 and 8.9. Protein sequence analysis found that there was conserved P450 domain in the 11 genes. Phylogenetic tree analysis indicated that all these 11 P450 genes belonged to basidiomycetes. Quantitative real-time PCR showed that these 11 genes were expressed in both the in- fected partand non-infectedpart. Meanwhile, the expressions of seven genes were significantly up-regulated in the infected part except comp18720, comp26906, comp32003 or comp33717. CONCLUSION Molecular characterization of the llCytochrome P450 genes will be useful for further functional determination of the genes involved in the defense progress of Polyporus umbellatus.

关 键 词：猪苓 细胞色素P450 基因克隆 特异性表达 

分 类 号：R284[医药卫生—中药学]