洋常春藤甲羟戊酸焦磷酸脱羧酶基因 HhMVD 的克隆与表达 分析

甲羟戊酸焦磷酸脱羧酶（Mevalonate diphosphate decarboxylase，MVD）是三萜皂苷生物合成途径中的关键酶， 为研究其在洋常春藤中的基因功能，通过 RT-PCR 和 RACE 技术从洋常春藤叶片中克隆获得 HhMVD 基因的 cDNA 全 长序列，并通过 RT-qPCR 技术分析其表达规律。结果表明：RACE 克隆获得的 HhMVD 基因 cDNA 序列全长 1 799 bp， 包含一个完整开放阅读框（ORF）1 263 bp、5′非编码区（5′UTR）192 bp、3′非编码区（3′UTR）344 bp；该基因编码 420 个氨基酸，分子质量 46.6 ku，理论等电点为 6.57，不含跨膜区，属于非分泌型蛋白；HhMVD 蛋白具有 GHMP 激 酶 N 端结构域，属于甲羟戊酸激酶系列，与刺五加、人参、三七等同科植物亲缘关系较近。RT-qPCR 结果表明，洋常 春藤中 HhMVD 基因的时空表达相对稳定，但与常春藤皂苷含量差异变化之间存在一定的负相关性。洋常春藤 HhMVD 基因的成功克隆及表达分析研究，为深入探讨其基因功能、阐明其在常春藤皂苷生物合成途径中的作用提供理论依据。

Cloning and Expression Analysis of Mevalonate Diphosphate Decarboxylase Gene in Hedera helix L.

Mevalonate diphosphate decarboxylase is a key enzyme in the biosynthesis pathway of triterpenoid saponins. In order to analyze its gene function, the full-length cDNA sequence was cloned successfully by RT-PCR and RACE methods in the leaves of H. helix, the expression trends were illustrated through RT-qPCR analysis. Results indicated that the cDNA sequence of HhMVD was 1 799 bp which contained a ORF from 193 bp to 1 455 bp and a 5′UTR with 192 bp and a 3′UTR with 344 bp. It encoded a 420-amino-acid protein with a molecular weight of 46.6 ku and an isoelectric point (pI) of 6.57. The HhMVD protein belonged to non-secreted protein and didn’t have a transmembrane region. The HhMVD protein had the characteristic GHMP kinase N-terminal domain and transmembrane region of plant MVD protein and closer relationship with Eleutherococcus senticosus, Panax ginseng, Panax notoginseng et al. The RT-qPCR results indicated that it had negative correlation between the relative expressing level of HhMVD gene and contents of saponins in H. helix leaves. The cloning and expression analysis results of HhMVD would provid a theoretical and technical basis for elucidating the role of HhMVD in the saponins biosynthetic pathway and metabolic regulation.