滇牡丹1-脱氧-D-木酮糖-5-磷酸合酶(DXS)基因的克隆及功能分析  

Cloning and Functional Analysis of 1-Deoxy-D-Xylulose-5-Phosphate Synthase Gene in Paeonia delavayi

作　　者：赵能[1,2] 原晓龙[1,3] 陈中华[1,3] 杨宇明[1,3] 王娟[1,3] 王毅[1,3] 

机构地区：[1]云南省林业科学院云南省森林植物培育与开发利用重点实验室,昆明650204 [2]西南林业大学,昆明650224 [3]云南省林业科学院国家林业局云南珍稀濒特森林植物保护和繁育重点实验室,昆明650204

出　　处：《基因组学与应用生物学》2017年第7期2919-2925,共7页Genomics and Applied Biology

基　　金：云南省科技计划项目(2015A005);云南省应用基础研究重点项目(2013FA054);国家林业公益性行业科研专项项目(201204110)共同资助

摘　　要：1-脱氧-D-木酮糖-5-磷酸合酶(DXS),作为MEP途径的第一个关键酶,在植物萜类合成中起着重要作用。为了克隆得到滇牡丹DXS基因,我们根据滇牡丹根皮转录组数据分析结果,首先获得滇牡丹DXS基因片段。之后根据获得的该片段设计特异引物,再利用RACE和RT-PCR技术从滇牡丹根皮中获得完整的DXS基因(Pd DXS)。Pd DXS基因全长为3 137 bp,全长基因中含有一个长度为2 151 bp的开放阅读框(ORF),该开放阅读框编码了717个氨基酸,根据开放阅读框序列推导所得蛋白序列绘制分子进化树,分子进化树将该基因推断所得蛋白与葡萄DXS蛋白聚为一类,因此,两者的DXS蛋白有较高相似性。经过氨基酸序列比对后,推断滇牡丹DXS具有叶绿体转运肽,二磷酸硫胺结合位点以及转酮醇酶结构域。半定量RT-PCR结果显示Pd DXS在根、茎、叶、花芽及花瓣中均有表达。本研究为确定滇牡丹中DXS的基因功能以及揭示滇牡丹中萜类化合物的生物合成提供了理论基础。1-deoxy-D-xylulose-5-phosphate synthase is the first key enzyme of the MEP pathway, and it plays an important role in terpene biosynthesis of plants. To clone the DXS gene ofPaeonia delcwayi, we first obtained DXS gene fragments of Paeonia delavayi according to the results of Paeorda del（wayi root bark transcriptome data analysis. After that, the obtained fragments were arranged to design the special primers, and then RT-PCR and RACE technology were used to obtain the full length DXS gene （PdDXS） from root bark ofPaeonia delavayi. The full length of PdDXS was 3 137 bp, which contained a 2 151 bp open reading frame （ORF）, and the ORF encoded 717 amino acids. The molecular evolution tree were drew according to the deduced protein sequence from open reading frame. The molecular evolution tree grouped the protein deduced from the genes and the DXS protein of grape together. Therefore, the two DXS proteins had a high similarity. After alignment of amino acid sequence, wededuced that Paeonia delavayi DXS had chloroplast transit peptide, thiamine diphosphate binding sites and transketolase domain. Semi quantitative RT-PCR results showed that the PdDXS expressed in roots, stems, leaves, flower buds and flower petals. This study would provide theoretical basis for determining the function of DXS in Paeonia delavayi and revealing the biosynthesis ofterpenoid.

关 键 词：滇牡丹 DXS基因 基因克隆 表达分析 

分 类 号：Q943.2[生物学—植物学] S685.11[农业科学—观赏园艺]