茶树甲羟戊酸激酶基因CsMVK克隆及表达特性分析

摘要

[目的]克隆茶树甲羟戊酸激酶基因(CsMVK),分析其生物学信息及在不同组织及乌龙茶做青过程中的表达特性,为研究茶叶加工过程中香气形成机制提供参考.[方法]RT-PCR扩增CsMVK基因,应用生物信息学软件对其编码蛋白(CsMVK)的氨基酸序列进行预测分析,通过实时荧光定量PCR(qPCR)分析CsMVK在茶树不同组织及乌龙茶做青过程中的表达特性.[结果]克隆获得的CsMVK基因(GenBank登录号MF668187)全长1455 bp,开放阅读框(ORF)长度为1164 bp,编码387个氨基酸,蛋白质分子量41.18 kD,理论等电点(pI)5.88.CsMVK蛋白具有GHMP_kinases_N domain和GHMP_kinases_C domain 2个功能结构域,定位于细胞质中,不存在跨膜结构和信号肽.系统发育进化树分析结果显示,CsMVK与三七、常春藤的MVK聚为一类,表明茶树与三七、常春藤的亲缘关系最近.qPCR检测结果显示,CsMVK基因在果实中的表达量显著高于其他组织(P<0.05,下同),在不同组织中的表达量排序为:果实>根>叶>茎>花;在乌龙茶做青过程中,从鲜叶到晒青叶,CsMVK基因的表达量上升,晒青到一摇过程中表达量下降,经过3次摇青,其表达量持续升高并在杀青前达最大值,与其他做青阶段差异显著.[结论]CsMVK基因表达与茶叶香气品质形成有关.%[Objective]Mevalonate kinase gene(CsMVK)from tea plant was cloned,and its biological information and expression in different tissues and Oolong tea during fine manipulation procedure were analyzed,to provide theoreti-cal basis for exploring the formation machanism of aroma during tea processing.[Method]Gene CsMVK was amplified by RT-PCR,and the amino acid sequence of its encoded protein(CsMVK)was predicted by bioinformatics software. Ex-pressions of gene CsMVK in different tissues of tea plant and Oolong tea during fine manipulation procedure were ana-lyzed by real-time fluorescence quantitative PCR(qPCR).[Result]The full-length cDNA of cloned gene CsMVK(Gen-Bank accession number MF668187)was 1455 bp in length,containing a 1164 bp open reading frame(ORF)encoding 387 amino acids. The protein molecular was 41.18 kD and theoretical isoelectric point(pI)5.88. Protein CsMV contained two functional domains,GHMP_kinases_N domain and GHMP_kinases_C domain functional domain. It was primarily located in cytoplasm,with no transmembrane structure and signal peptide. The phylogenetic tree analysis showed that CsMVK and MVK of Panax notoginseng and Hedera helix belonged to the same category,which indicated that tea plant, P. notoginseng and H. helix had the closest genetic relationship. According to qPCR detection,expression of gene CsMVK in fruit was significantly higher than those in other tissues(P<0.05,the same below). The order of gene CsMVK expression levels was fruit>root>leaf>stem>flower. During fine manipulation procedure of Oolong tea,expression of gene CsMVK up-regulated from leaves to solar withering and down-regulated from solar withering to first tumblings. Through three tum-blings,the expression of gene CsMVK reached its peak before fixation and showed significant difference with other stages.[Conclusion]Expression of gene CsMVK might be related to aroma formation in tea processing.