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羟甲基戊二酰辅酶A合酶(3-Hydroxy-3-methylglutaryl-coenzyme A synthase,HMGS)是甲羟戊酸途径(MVA)中的第一个催化酶。根据冬凌草转录组数据库中HMGS基因序列设计特异引物,采用RT-PCR技术克隆冬凌草IrHMGS基因cDNA全长,并对其序列进行生物信息学分析,通过荧光定量PCR的方法分析其组织表达特性。IrHMGS基因cDNA全长1 382bp,编码460个氨基酸,序列分析结果表明该基因编码的氨基酸序列含有羟甲基戊二酰辅酶A合成酶N末端、C末端等保守结构域。荧光定量PCR表明,IrHMGS在冬凌草组培苗叶和根中的表达量显著高于在组培苗花、茎和愈伤组织中的表达量。本研究为后续深入研究IrHMGS在冬凌草二萜类物质合成途径中的功能奠定了基础。
3-Hydroxy-3-methylglutaryl-coenzyme A synthase (HMGS) is the first catalytic enzyme in mevalonate pathway (MVA). According to the HMGS gene sequence of Rubia cordyceps transcriptome database, specific primers were designed, and the full-length cDNA of Rubescens IrrMGS was cloned by RT-PCR. The sequence of the gene was analyzed by bioinformatics method. The expression of IrHMGS was analyzed by fluorescence quantitative PCR characteristic. The cDNA of IrHMGS gene is 1 382 bp in length and encodes a protein of 460 amino acids. The sequence analysis showed that the amino acid sequence of the gene contains the N-terminal and C-terminal conserved domains of hydroxymethylglutaryl coenzyme A synthetase. Fluorescent quantitative PCR showed that the expression level of IrHMGS in the leaves and roots of the tissue culture seedling was significantly higher than that in the flower, stem and callus. This study lays the foundation for the follow-up study on the function of IrHMGS in the pathway of diterpenoid synthesis of Rubia cordata.