重瓣铁线莲鲨烯合酶SQS基因的克隆及表达分析  

Cloning and Expression of Squalene Synthase Gene from Clematis florida Thunb.var. plena D. Don

作　　者：何舒澜 杨谨 黄娴 陈观水[2] HE Shulan;YANG Jin;HUANG Xian;CHEN Guanshui(Department of Pharmacy,Fujian Health College,Fuzhou,Fujian 350101,China;College of Life Science,Fujian Agriculture and Forestry University,Fuzhou,Fujian 350002,China)

机构地区：[1]福建卫生职业技术学院,福建福州350101 [2]福建农林大学生命科学学院,福建福州350002

出　　处：《福建农业学报》2022年第11期1430-1437,共8页Fujian Journal of Agricultural Sciences

基　　金：福建省中青年教师教育科研项目职业院校专项(JZ180548);福建省大学生创新创业项目(S202110389044)。

摘　　要：【目的】鲨烯合酶是三萜类物质生物合成途径中的限速酶之一。克隆重瓣铁线莲的鲨烯合酶基因(Clematis florida squalene synthase,CfSQS),为利用基因工程技术提高重瓣铁线莲的三萜类次生代谢物的含量、提升重瓣铁线莲的药用价值奠定理论基础。【方法】根据重瓣铁线莲转录组数据并参考其他植物SQS基因设计引物,提取重瓣铁线莲叶片总RNA为模板,利用RT-PCR法扩增并克隆获得CfSQS基因全长cDNA序列,并对其进行生物信息学分析和时空表达分析。【结果】CfSQS ORF序列长度为1 227 bp,编码408个氨基酸,预测编码蛋白相对分子量为46.69 kDa,为不稳定、无信号肽的跨膜疏水蛋白,二级结构以α-螺旋(69.61%)和不规则卷曲(22.06%)为主;CfSQS含有鲨烯合酶家族高度保守的氨基酸残基,包括含有4个高度保守且典型的17~23个氨基酸长度的结构域(Ⅰ~Ⅳ)和1个高度差异的疏水区(Ⅴ)。系统进化分析表明,CfSQS与黑种草(Nigella sativa L.)等毛茛科植物SQS聚在一类,说明其亲缘关系最近。时空表达分析表明,CfSQS在重瓣铁线莲的根、茎和叶器官中都有表达。【结论】首次克隆了重瓣铁线莲三萜类皂苷上游合成的关键酶基因CfSQS的全长编码序列,并对其进行了生物信息学预测和组织表达分析。研究结果可为进一步研究重瓣铁线莲三萜类皂苷生物合成途径提供参考。【Objective】 The squalene synthase(SQS), the first key rate-limited enzyme in the triterpenoids biosynthetic pathway, from Clematis florida Thunb. var. plena D. Don was cloned, and its expression analyzed in preparation for an investigation to increase the medicinal value of the plant by means of genetic engineering. 【Method】 Primers were designed based on the transcriptome data on the SQS genes in C. florida and in other plants. Total RNA was extracted from C. florida leaves, and full-length cDNA obtained by RT-PCR. Bioinformatics and spatiotemporal expression of the gene were analyzed.【Result】 The ORF of the SQS, designated as CfSQS, was 1 227 bp encoded 408 amino acids. It was an unstable hydrophobic protein comprised 69.61% of α helix and 22.06% of random coil. The deduced amino acid sequence predicted the presence of4 highly conserved domains with 17–23 amino acids residues(I–IV) and one variable hydrophobic region(V). The predicted 3D structure of CfSQS by various programs was similar to those of SQSs from other species. It had a close phylogenetic relationship and in the same clade with the SQS of Nigella sativa. A spatiotemporal expression analysis indicated the gene to be expressed in all tissues. 【Conclusion】 The full-length cDNA sequence and bioinformatics of the squalene synthase gene CfSQS of C. florida were determined for the first time. The information helped to further clarify the synthesis of the saponin compounds and regulatory mechanism that would pave the way for future studies on the biosynthetic pathways of triterpene saponins in C. florida.

关 键 词：重瓣铁线莲 三萜皂苷 鲨烯合酶 生物信息学分析 

分 类 号：S567[农业科学—中草药栽培]