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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。
Deoxy-D-xylulose-5-phosphate synthase (DXS), as the first key enzyme of MEP pathway, plays an important role in plant terpenoid synthesis. In order to clone the DXS gene of D. peony, we first obtained the DXS gene fragment from P. peony root transcriptome data analysis. After that, specific primers were designed according to the fragment obtained, then the complete DXS gene (Pd DXS) was obtained from the root bark of D. peony by RACE and RT-PCR. The full-length Pd DXS gene was 3 137 bp in length. The full-length gene contained an open reading frame (ORF) of 2151 bp. The open reading frame (ORF) encoded 717 amino acids. Based on the open reading frame (ORF) The phylogenetic tree and molecular phylogenetic tree deduced that the deduced protein of this gene was clustered with the DXS protein of grape, therefore, the DXS proteins of the two genes had higher similarity. After amino acid sequence alignment, we conclude that DXS has the chloroplast transit peptide, thiamine diphosphate binding site and transketolase domain. Semi-quantitative RT-PCR results showed that Pd DXS was expressed in roots, stems, leaves, flower buds and petals. This study provided the theoretical basis for determining the gene function of DXS in Dyslexia pendula and revealing the biosynthesis of terpenoids in Dyslexia.