茯苓细胞色素P450基因PcCYP的克隆与序列分析  

Cloning,characterization and expression of a novel cytochrome P450 genes PcCYP in Poria cocos

作　　者：唐静 朱闻君 刘和平 苏振佳 刘金鑫 舒少华[1] TANG Jing;ZHU Wen-jun;LIU He-ping;SU Zhen-jia;LIU Jin-xin;SHU Shao-hua(College of Plant Science and Technology of Huazhong Agricultural University,Wuhan 430070,China;School of Biological and Pharmaceutical Engineering,Wuhan Polytechnic University,Wuhan 430023,China)

机构地区：[1]华中农业大学植物科学技术学院,湖北武汉430070 [2]武汉轻工大学生物与制药工程学院,湖北武汉430023

出　　处：《中草药》2023年第12期3962-3970,共9页Chinese Traditional and Herbal Drugs

基　　金：国家自然科学基金面上项目(81872948);名贵中药资源可持续利用能力建设项目(2060302-1907-05)。

摘　　要：目的筛选与茯苓Poriacocos生长发育相关的细胞色素P450基因序列,进一步丰富对茯苓生长发育理解。方法通过分子克隆到1个茯苓细胞色素P450基因Pc CYP,并利用在线工具分析其序列,预测蛋白的理化性质、结构域等分子特征;利用MEGA5.0分别对氨基酸进行多序列比对和进化关系分析,并采用q RT-PCR方法检测不同发酵培养时间6、8、10d的相对表达量。结果从茯苓中克隆到的Pc CYP存在可变性剪切,2个选择性剪切体PcCYP1和Pc CYP2CDS区长度分别为1590、1542bp,分别编码529、513个氨基酸。氨基酸序列分析发现其均具有保守的P450结构域,且具有信号肽和多个磷酸化修饰位点,属于分泌型的不稳定亲水蛋白。氨基酸序列多重比对及系统发育树结果显示PcCYP与担子菌Coprinopsis cinerea的CYP502距离最近,推测PcCYP和CYP502具有相似功能,即参与调控茯苓子实体的发育。定量PCR结果显示,选择性剪接变体Pc CYP1的表达量较Pc CYP2低,因此PcCYP2是Pc CYP基因的主要剪接体。结论Pc CYP与茯苓子实体的生长发育密切相关,为进一步研究Pc CYP在茯苓生长发育、遗传规律及育种等方面的生物学功能奠定基础。Objective A cytochrome P450 gene sequence related to the growth and development of Poria cocos was screened and reported to further enrich the understanding of the growth and development of P.cocos.Methods In this study,a cytochorome P450 gene PcCYP was cloned from P.cocos,and some bioinformatics tools were used to predict the physical and chemical properties of the protein,as well as the molecular characteristics of the domain;MEGA 5.0 was used to perform multi-sequence alignment and phylogenetic analysis of amino acids.The gene expression levels at 6d,8d,and 10d of fermentation were quantified by qRT-PCR.Results The results showed that two transcripts of PcCYP,PcCYP1 and PcCYP2 were founded from P.cocos for the first time.They were 1590 bp and 1542 bp in length and encoded 529 and 513 amino acids,respectively.Sequence analysis revealed that they both had a conserved P450 domain,a signal peptide and multiple phosphorylation sites,which secreted unstable hydrophobins;multiple alignments of amino acid sequences and phylogenetic tree results showed that the PcCYP was similar to CYP502 of the basidiomycete Coprinopsis cinerea;qPCR results showed that the expression of PcCYP1 is lower than that of PcCYP2,suggesting that PcCYP2 was the main transcript of PcCYP.Conclusion Hydrophobin PcCYP is closely related to the growth and development of the fruit body of P.cocos,which lays a foundation for further research on the biological functions of PcCYP in the growth and development,genetic rule and breeding of P.cocos.

关 键 词：茯苓 细胞色素P450 基因克隆 生物信息学分析 基因表达 生长发育 

分 类 号：R286.12[医药卫生—中药学]