摘要
目的克隆粉防己Stephania tetrandra苄基异喹啉类生物碱生物合成途径中的细胞色素P450基因StCYP80G,进行生物信息学分析、表达载体构建和组织特异性表达分析。方法设计特异性引物,从粉防己cDNA中克隆得到StCYP80G编码蛋白的基因序列;利用生物信息学在线工具预测StCYP80G蛋白的结构域、理化性质和跨膜区等分子特征;利用MEGA11.0对氨基酸进行多序列比对和系统进化关系分析;通过同源重组法构建含有StCYP80G基因的重组质粒;采用实时荧光定量PCR技术分析该基因在粉防己不同组织中的相对表达量。结果StCYP80G基因开放阅读框长度为1458 bp,编码485个氨基酸,蛋白质相对分子质量为54770,预测其具有CYP450的保守结构域,含有跨膜结构域,可能定位在内质网上。氨基酸序列比对与系统进化树分析显示,StCYP80G与莲NnCYP80G、智利桂Ls CYP80G的序列具有同源性且一致度较高,推测StCYP80G与这2条序列具有相似的功能。StCYP80G基因在粉防己叶中表达量显著高于根中。质粒序列分析结果表明,重组质粒pESC-Leu-StCYP80G构建成功。结论StCYP80G基因的克隆、生物信息学分析、重组质粒的构建、以及组织表达特异性的研究结果,为进一步探究其在苄基异喹啉类生物碱生物合成途径中的功能奠定了基础。
Objective To clone the cytochrome P450 gene,StCYP80G,involved in the benzylisoquinoline alkaloid biosynthetic pathway in Stephania tetrandra,and to characterize the gene by bioinformatics analysis,expression vector construction,and tissue-specific expression analysis.Methods Specific primers were designed to clone the gene sequence of StCYP80G encoding protein from cDNA of S.tetrandra.Online bioinformatics tools were utilized to predict the molecular characteristics of StCYP80G protein,including its structural domain,physicochemical properties,and transmembrane regions.Multi-sequence alignment and phylogenetic analysis of amino acids were performed using MEGA 11.0.A recombinant plasmid with StCYP80G gene was constructed using homologous recombination.The relative expression of the gene in different tissues was analyzed by real-time fluorescence quantitative PCR.Results The open reading frame of StCYP80G gene was 1458 bp in length,encoding 485 amino acids,and the relative molecular weight of protein was 54770.It was predicted that StCYP80G gene has a conserved CYP450 domain,including a transmembrane domain,and possibly localized in the endoplasmic reticulum.Amino acid sequence alignment and phylogenetic analysis revealed that StCYP80G had high homology and consistency with CYP80Gs from Nelumbo nucifera(NnCYP80G)and Laurelia sempervirens(LsCYP80G),suggesting StCYP80G had functional similarity with these two sequences.The expression of StCYP80G was significantly higher in leaves compared to roots.Sequence analysis of the plasmid confirmed the successful construction of the recombinant plasmid pESC-Leu-StCYP80G.Conclusion The successful cloning,bioinformatics analysis,recombinant plasmid construction,and tissue-specific expression analysis lay the groundwork for further functional research on StCYP80G in the benzylisoquinoline alkaloid biosynthesis.
作者
高雨
李姚婷
冯郁涵
康云
黄建明
翁伟宇
GAO Yu;LI Yaoting;FENG Yuhan;KANG Yun;HUANG Jianming;WENG Weiyu(Shanghai Key Laboratory of New Drug Design,School of Pharmacy,East China University of Science and Technology,Shanghai 200237,China;School of Pharmacy,Fudan University,Shanghai 201203,China)
出处
《中草药》
CAS
CSCD
北大核心
2024年第13期4508-4515,共8页
Chinese Traditional and Herbal Drugs
