阳春砂3-羟基-3-甲基戊二酰辅酶A还原酶基因的克隆及分析

Clone and Analysis of Gene Encoding 3-hydroxy-3-methylglutaryl Coenzyme A Reductase from Amomum villosum

目的克隆阳春砂萜类生物合成途径上游关键酶——3-羟基-3-甲基戊二酰辅酶A还原酶(3-hydroxy-3-methylglutaryl coenzyme A reductase,HMGR)(EC:1.1.1.34)的编码基因;分析基因的功能及其在阳春砂不同组织中的表达。方法用基于RT-PCR的方法从阳春砂叶片中获得编码HMGR的cDNA全长序列,克隆基因编码区;用生物信息学的方法对其编码蛋白进行相似性检索和功能分析;用半定量RT-PCR法比较基因在阳春砂不同组织中的表达差异。结果获得了全长2 023 bp的编码阳春砂HMGR的cDNA序列,命名为AvHMGR(GenBank登记号:FJ455511)。AvHMGR编码的蛋白与其他植物来源的HMGR有很高相似性,含有NADPH结合基序和底物HMG-CoA结合基序,N-端有两个跨膜结构域。保守功能结构域的分析结果表明AvHMGR属于3-羟基-3-甲基戊二酰辅酶A还原酶家族。AvHMGR在包括茎、根、果皮和种子团的广泛组织中表达,且在这些组织中的表达量均高于在叶片中的表达。结论从阳春砂中克隆了AvHMGR基因,为进一步鉴定基因功能、探明阳春砂萜类生物合成的基因调控机制打下基础。

Objective To clone the gene encoding 3-hydroding 3-hydroxy-3-methylglutaryl coenzyme A reductase（HMGR,EC：1.1.1.34）,the key enzyme of biosynthetic pathways of terpenoids,from Amomum villosum and analyze its function and expression in different tissues of Amomum villosum.MethodsMethod based on RT-PCR by using degenerate primers and rapid amplification of cDNA ends（RACE） was used to clone gene.Bioinformatic analysis was used to identify the gene.Semi-quantitative RT-PCR was carried out to analyze the expressional difference of the gene in different tissues.ResultsA cDNA encoding HMGR（designated as AvHMGR,GenBank accession number FJ455511） was isolated from leaves of Amomum villosum for the first time.The full-length cDNA of AvHMGR was 2023 bp containing an open reading frame（ORF） of 1713 bp encoding 571 amimo acids.Bioinformatic analysis revealed that the deduced protein AvHMGR had extensive sequence similarities to other plant HMGRs and was more homologous with HMGRs from monocotyledonous plants.AvHMGRs had two NADPH binding motifs and two HMG-CoA binding motif.Two transmembrane regions were found in its N-terminal region.Result of CDD search indicated that it belonged to the HMGR super-family.AvHMGR expressed in leaves,stems,roots,pericarps and seeds of Amomum villosum and lower transcription level was observed in leaves than that of other tissues.ConclusionAvHMGR encodes HMGR in Amomum villosum.