茶树HMG-CoA还原酶基因全长cDNA克隆及序列分析

香气是茶叶的重要品质之一,萜类物质不仅香气好,而且沸点普遍较高,是构成茶叶香气的重要物质基础,决定着茶叶的香气品质,也可作为茶叶香型划分的依据。在植物中,倍半萜、多萜醇等通过胞质中的甲瓦龙酸(MVA)途径合成。HMG-Co A还原酶(HMGR)催化HMG-Co A(3-羟基-3-甲基戊二酸单酰辅酶A)生成甲瓦龙酸,是依赖MVA萜类合成途径的关键限速反应。为了有助于理解茶树萜类合成的分子遗传机制,通过RACE-PCR方法从茶树中克隆了一个编码HMG-Co A还原酶的c DNA全长序列(命名为Cs HMGR1),该序列由1 979 bp组成,包含一个1 722 bp的完整开放阅读框,编码573个氨基酸。其推定的编码蛋白与橡胶树、旱莲木、人参、荔枝、西洋参、丹参、罗汉果及龙眼的同源蛋白具有80%~82%的序列一致性。利用Cs HMGR1和其它物种HMGR同源蛋白的催化区域构建系统发育树,表明其属于真核生物I类HMGR家族。结构分析表明,Cs HMGR1含有两个跨膜区,推测其与其它真核生物同源蛋白类似地定位于内质网上;含有两个HMG-Co A结合位点、两个NADPH结合位点、四个保守的催化活性残基及一个磷酸化位点,说明磷酸化/去磷酸化很可能也是其活性调节的重要方式。表达分析表明,Cs HMGR1在"大叶龙"叶芽、母株叶芽及花芽都有较强的表达。其表达调控及生理活性对茶叶品质可能有重要影响,并在其功能解析的基础上,有可能作为茶叶品质鉴定及育种的一个依据。

Cloning and sequence analysis of HMG-CoA reductase full-length cDNA from tea(Camellia sinensis)

Aroma is an important character of tea,and terpenoids,with preferable fragrance and relatively high boiling point,are substantial substances contributing to tea aroma,which not only determine the aroma quality of tea,but also provide a classification basis of scent types. In plants,sesquiterpenes and dolichol and so forth are synthesize via the cytoplasmic mevalonate-dependent pathway. The enzyme HMG-CoA reductase(HMGR)catalyzes the conversion of 3-hydroxy-3-methylglutaryl coenzyme A(HMG-CoA)to mevalonate,which is the committed step in the synthesis of isoprenoids via the MVA pathway. To help understand the molecular and genetic mechanisms underlying terpenoid synthesis of tea,a full-length cDNA encoding HMGR was cloned from tea(Camellia sinensis(L.)O. Kuntze)by using the RACE-PCR technique(designated as CsHMGR1). It comprised 1 979 bp,with a 1 722 bp intact open reading frame encoding a 573-amino-acid protein. The deduced protein showed 80% to 82% similarities to homologs from rubber tree(Hevea brasiliensis),common camptotheca fruit(Camptotheca acuminate),ginseng(Panax ginseng),litchis(Litchi chinensis),American ginseng(Panax quinquefolius),rooted salvia(Salvia miltiorrhiza),Momordica grosvenori(Siraitia grosvenorii),and longan(Dimocarpus longan). The phylogenetic tree,constructed with the catalytic domained of CsHMGR1 and homologs from other species,indicated that CsHMGR1 belonged to the eukaryotic class I HMGR family. CsHMGR1 consisted of two transmembrane domains,implying that it may be localized to endoplasmic reticulum(ER)similarly to other eukaryotic homologs. It also contained two HMG-CoA binding sites,two NADP(H)-binding sites,four conserved catalytic active residues and a phosphorylation site,indicating that phosphorylation/dephosphorylation is likely a crucial mode of regulation of its biochemical activity. Tissue expression analysis indicated that CsHMGR1 was expressed comparatively in the leaf buds of C. sinensis cv. Dayelong and in both leaf buds and floral buds of the mother plants. The regulation of expression and physiological activity of CsHMGR1 are likely to impact greatly on tea quality,and CsHMGR1 may provide a basis of the quality evaluation and breeding of tea given that its function is further resolved.