暗紫贝母鲨烯合酶基因的克隆及其组织表达分析

Cloning and Tissue Expression Analysis of Squalene Synthase Gene from Fritillaria unibracteata

【目的】:克隆名贵药材暗紫贝母(Fritillaria unibracteata)鲨烯合酶(Squalene synthase,SQS)基因的全长ORF序列(FuSQS),并对其进行生物信息学分析和不同组织表达水平分析。【方法】利用PCR方法,克隆得到FuSQS,利用生物信息学方法预测FuSQS蛋白的基本理化性质、保守结构域、二级及三级结构等蛋白特性,利用Clustal W和MEGA 7.0分别进行氨基酸序列对比和系统进化分析,通过Real-time PCR分析FuSQS在暗紫贝母不同组织的表达水平。【结果】FuSQS长度为1230 bp(GenBank登录号MW365404),编码1条长度为409个氨基酸残基的肽链,相对分子质量为46.84 kDa,等电点(pI)为6.33,含有法尼基焦磷酸结合口袋和Mg^(2+)结合位点等保守结构域。系统进化树表明FuSQS与油棕、宽瓣重楼、水稻、玉米等单子叶植物进化关系较近。Real-time PCR结果显示,FuSQS在不同组织中表达量有显著差异。【结论】FuSQS的克隆、生物信息学分析及其在鳞茎中的高表达特征,这为进一步研究FuSQS在暗紫贝母生物碱生物合成途径的调控机制提供科学依据。

【Objective】This study was carried out to clone ORF of Squalene synthase gene from Fritillaria unibracteata,a precious medicinal herb,followed by bioinformatics analysis and analysis of expression profile in different tissue,in order to lay a theoretical foundation for studying the regulation mechanism of SQS in the biosynthesis alkaloid pathway of Fritillaria unibracteata.【Method】RT-PCR was used to clone the Squalene synthase gene ORF cDNA,the characteristics of the basic physical and chemical properties,conserved domains,secondary and tertiary structure of the deduced FuSQS protein were determined with a series of bioinformatics tools,the analysis of multiple alignment and phylogenetic relationship were carried out by Clustal W and MEGA 7.0,and qPCR were employed to analyze the differences in the expression level of SQS gene in different tissues of F.unibracteata.【Result】A SQS ORF cDNA with a full length of 1230 bp,designed as FuSQS(GenBank accession number MW365404)was cloned from F.unibracteata.The gene encoded a 409 amino acids polypeptide with molecular weight of 46.84 kD and isoelectric point(pI)of 6.33.The deduced FuSQS protein contained conserved domains such as farnesyl pyrophosphate binding pocket and Mg^(2+)binding site.The phylogenetic tree showed that the protein encoded by FuSQS was closely related to Elaeisguineensis,Oryza sativa and Zea mays.The qPCR results indicated that the expression level of the FuSQS was different in various tissues.【Conclusion】The cloning,bioinformatic analysis and expression profile of FuSQS provided a scientific basis for further research on the regulation mechanism of FuSQS in the alkaloid biosynthesis of F.unibracteata.