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目的:对乌拉尔甘草3-羟基-3-甲基戊二酰辅酶A还原酶(3-hydroxy-3methylglutary CoA reductase,HMGR)的cDNA克隆并进行序列分析。方法:根据NCBI数据库中的豆科其他物种HMGR的cDNA保守区设计引物,利用同源扩增和cDNA末端快速扩增技术从甘草根中获得目的基因;利用BLAST进行序列比对,ORF Finder寻找开发阅读框,Prosite分析蛋白质的基本结构域,Clustal x比对已有HMGR的氨基酸序列,并构建进化树。结果:得到1个全长为1 842 bp的HMGR的cDNA序列,含有1 722 bp的开放阅读框(open reading frame,ORF),编码573个氨基酸,具有HMGR家族的特异序列,推测的氨基酸序列与豌豆、蒺藜苜蓿的氨基酸序列一致性分别为84%,76%。结论:对甘草HMGR基因的cDNA进行了克隆,为进一步研究3-羟基-3-甲基戊二酰辅酶A在甘草酸生物合成途径中的作用提供了理论依据。
OBJECTIVE: To clone the cDNA of 3-hydroxy-3-methylglutaryl CoA reductase (HMGR) from Ural licorice and analyze its sequence. Methods: According to the conserved region of HMGR in NCBI database, primers were designed and the target genes were obtained from Glycyrrhiza glabra by homologous amplification and rapid amplification of cDNA ends. The sequences were aligned by BLAST and ORF Finder was searched for and developed Reading frame, Prosite analyzes the basic domain of a protein, Clustal x matches the amino acid sequence of an existing HMGR, and constructs a phylogenetic tree. Results: The cDNA sequence of HMGR with a total length of 1 842 bp was obtained, which contained a 1 722 bp open reading frame (ORF) encoding 573 amino acids with specific sequence of HMGR family. The deduced amino acid sequence was The amino acid sequence identities of pea and Medicago truncatula were 84% and 76%, respectively. Conclusion: The cDNA of Glycyrrhiza uralensis HMGR gene was cloned, which provided a theoretical basis for further study on the role of 3-hydroxy-3-methylglutaryl coenzyme A in glycyrrhizin biosynthesis pathway.