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3-羟基-3-甲基戊二酰辅酶A还原酶(3-hydroxy-3-methylglutaryl-CoA reductase,HMGR)是植物萜类代谢中甲羟戊酸途径的关键酶,本研究运用cDNA末端快速扩增(RACE)技术,首次从珍稀植物南京椴中克隆出HMGR的全长基因TmiHMGR,其长度为2160 bp,包含一个1758 bp的开放阅读框,其推导蛋白TmiHMGR编码585个氨基酸残基,相对分子量为62.9 kD,pI为6.11。将TmiHMGR与其他植物HMGR氨基酸序列构建进化树,结果显示Tmi-HMGR与苹果的HMGR聚为一枝。采用半定量RT-PCR分析TmiHMGR在根、茎和叶中的表达情况,结果表明该基因在茎中的表达量最高,根和叶中的表达量相对较弱。验证功能的颜色互补实验结果显示,TmiHMGR能够使代谢流明显朝类胡萝卜素合成的方向进行,说明TmiHMGR在萜类产物生物合成中是一个重要因子。
3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) is the key enzyme of mevalonate pathway in terpenoid metabolism. In this study, (RACE), the full-length gene TmiHMGR of HMGR was cloned from the rare plant Tilia amurensis for the first time. The length of the full length gene is 2160 bp, including a 1758 bp open reading frame. The deduced protein TmiHMGR encodes 585 amino acid residues, The molecular weight was 62.9 kD and the pI was 6.11. The phylogenetic tree was constructed using TmiHMGR and other plant HMGR amino acid sequences. The results showed that Tmi-HMGR and apple HMGR clustered together. The expression of TmiHMGR in roots, stems and leaves was analyzed by semi-quantitative RT-PCR. The results showed that the expression of TmiHMGR was the highest in stems and the expression of TmiHMGR was relatively weak in roots and leaves. The results of color complementation of validation function showed that TmiHMGR can make the metabolic flux obviously toward the direction of carotenoid synthesis, indicating that TmiHMGR is an important factor in the biosynthesis of terpenoid products.