滇重楼3-羟基-3-甲基戊二酰辅酶A还原酶PpHMGR基因的克隆及分析

Cloning and Analysis of 3-hydroxy-3-methylglutaryl Coenzyme-A Reductase PpHMGR Gene in Paris polyphylla var. yunnanensis(Franch.) Hand.-Mazz.

为了克隆滇重楼编码3-羟基-3-甲基戊二酰辅酶A还原酶(HMGR)的基因,并分析其在滇重楼不同组织中差异表达。本研究采用RT-PCR的方法从滇重楼根茎中克隆到编码HMGR的基因全长序列,用生物信息学的方法对HMGR蛋白进行理化性质、蛋白结构及功能的预测分析,并对该蛋白进行相似性检索、构建进化树;利用实时荧光定量的方法检测该基因在滇重楼不同组织中的差异表达。结果获得了全长1 494 bp的ORF,编码497个氨基酸,命名为PpHMGR(GenBank登记号:MG601751)。PpHMGR编码的蛋白含有NADPH结合基序和底物HMG-CoA结合基序,属于3-羟基-3-甲基戊二酰辅酶A还原酶家族。PpHMGR在根茎的表达量明显高于叶片、茎和芽,其中在3年生根茎的表达量又明显高于1年和5年生根茎。本研究从滇重楼中克隆了PpHMGR基因,为进一步鉴定基因功能、探讨滇重楼皂苷的合成生物学研究提供了科学依据。

To clone the gene encoding 3-hydroding 3-hydroxy-3-methylglutaryl coenzyme A reductase(HMGR)from Paris polyphylla var.yunnanensis(Franch.)Hand.-Mazz.and analyze its differential expression in different tissues of Paris polyphylla var.yunnanensis(Franch.)Hand.-Mazz..In this study,the gene full-length sequence of encoding HMGR was cloned from the rhizome of Paris polyphylla var.yunnanensis(Franch.)Hand.-Mazz.by RT-PCR.Bioinformatic analysis was used to predicte the physical and chemical properties,protein structure and function of HMGR protein,carry out similarity retrieval,and construct evolutionary tree.Quantitative RT-PCR was carried out to detect the differential expression of the gene in different tissues of Paris polyphylla var.yunnanensis(Franch.)Hand.-Mazz..And an ORF with a total length of 1 494 bp was obtained,encoding 498 amimo acids and named PpHMGR(GenBank registration number:MG601751).PpHMGRs encoded protein had NADPH binding motifs and HMG-CoA binding motif,belonging to the 3-hydroxy-3-methylglutaric coenzyme A reductase family.The PpH-MGR expressed in rhizomes was significantly higher than that in leaves,stems,buds of Paris polyphylla var.yunnanensis(Franch.)Hand.-Mazz.,and the expression in 3-year-old rhizomes was significantly higher than that in 1-year-old and 5-year-old rhizomes.In this study,PpHMGR gene was cloned from Paris polyphylla var.yunnanensis(Franch.)Hand.-Mazz.,which would provide a scientific basis for exploring the gene function and discussing the synthetic biology of Paris polyphylla var.yunnanensis(Franch.)Hand.-Mazz..