人参皂苷合成生物学关键元件HMGR基因克隆与表达分析

Cloning and expression analysis of a key device of HMGR gene involved in ginsenoside biosynthesis of Panax ginseng via synthetic biology approach

3-羟基-3-甲基戊二酸单酰辅酶A还原酶(3-hydroxy-3-methylglutaryl coenzyme-A reductase,HMGR)是甲羟戊酸途径中的第一个限速酶,直接催化人参皂苷的生物合成,是人参皂苷合成生物学研究中的重要元件之一。本研究克隆了人参(Panax ginseng)中一个新HMGR(命名为PgHMGR2)的编码区序列,并对其编码的蛋白进行生物信息学预测及基因表达分析。PgHMGR2与GenBank中的另一条人参HMGR的序列同源性仅为71.6%,因此,PgHMGR2是人参中HMGR基因家族的一个新成员。PgHMGR2基因全长1 770 bp,编码589个氨基酸残基。生物信息学预测PgHMGR2编码蛋白不含信号肽,包含两个跨膜区,具有HMGR催化作用的活性中心结构域。PgHMGR2编码蛋白与西洋参(Panax quinquefolius)的HMGR(GenBank登录号为ACV65036)具有99%的序列相似性。实时荧光定量PCR检测到PgHMGR2在人参花中表达量最高,其次为叶和根,茎中表达量最低。本研究是国内外首次获得的人参PgHMGR2基因的全长序列,为进一步鉴定PgHMGR2的功能及开展人参皂苷的合成生物学研究奠定基础。

3-Hydroxy-3-methylglutaryl coenzyme-A reductase （HMGR）, the first enzyme of mavalonic acid pathway, is one of the key devices involved in ginsenoside biosynthesis based on synthetic biology approach. The open reading frame of a novel HMGR gene from Panax ginseng （PgHMGR2） was cloned and analyzed in this study. PgHMGR2-encoding protein showed 71.6% sequence similarity to a P. ginseng HMGR in GenBank. The full-length cDNA sequence of PgHMGR2 containing 1 770 bp, which encodes 589 amino acids, was cloned by RT-PCR strategy from P. ginseng. The bioinformatic analysis showed that PgHMGR2-encoding protein contained two transmembrane regions and the HMG_CoA_reductase domain, without signal peptide. The protein sequence of PgHMGR2 had the highest sequence similarity （99%） with Panax quinquefolius HMGR （GenBank accession No. ACV65036）. The expression level of PgHMGR2 was the highest in flower based on a real-time PCR analysis, followed by leaf and root, and the lowest was in stem. The result will provide a foundation for exploring the molecular function of PgHMGR2 involved in ginsenoside biosynthesis based on synthetic biology approach in P. ginseng plants.