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
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摘要:
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。
Abstract: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.
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