冬凌草1-脱氧-D-木酮糖-5-磷酸合成酶基因克隆与表达分析  

Cloning and expression analysis of 1-deoxy-D-xylulose 5-phosphate synthase gene in Isodon rubescens

作　　者：朱畇昊 苏秀红[1,2] 董诚明[1,2] 陈随清[1,2] 邵远洋 张风波[1] 

机构地区：[1]河南中医药大学药学院,郑州450006 [2]河南中医药大学呼吸疾病诊疗与新药研发河南省协同创新中心,郑州450046

出　　处：《广西植物》2016年第12期1476-1482,共7页Guihaia

基　　金：国家自然科学基金(81173486);河南中医学院创新人才项目(2011XCXRC02);河南省高等学校青年骨干教师计划项目(2011GGJS-089)~~

摘　　要：1-脱氧-D-木酮糖-5-磷酸合成酶(1-deoxy-D-xylulose 5-phosphate synthase,DXS)是植物萜类代谢通路中2-C-甲基-D-赤藓糖醇-4-磷酸(MEP)途径的第一个关键酶,在植物萜类物质的生物合成中发挥重要的作用。为了研究该基因在冬凌草二萜类成分合成中的作用,该研究在冬凌草转录组测序结果的基础上设计一对特异性引物,采用RT-PCR方法得到冬凌草Ir DXS基因c DNA全长序列,并对其蛋白进行理化性质分析、信号肽预测、亚细胞定位预测、蛋白质二级结构、三级结构预测分析及跨膜域分析等生物信息学分析,同时利用实时荧光定量PCR的方法检测Ir DXS基因在冬凌草不同部位中的表达情况。结果表明:从冬凌草叶片中分离得到了一条编码DXS的全长基因,通过生物信息学软件分析发现,该基因编码全长2 169 bp,编码722个氨基酸,分子量为77.7 k D。多序列比对发现该基因编码的蛋白和其他植物中已知的DXS蛋白序列具有较高的同源性,N端均包含了一段质体转运肽序列,并均具有一个保守的焦磷酸硫胺素结构域和与吡啶结合相关的DRAG结构域。序列进化树分析显示,Ir DXS基因属于植物DXS2家族。DXS基因在冬凌草根中表达量最高、愈伤组织中最低。该研究首次获得了Ir DXS基因的全长c DNA序列,并揭示了其在不同组织中的表达差异,为后续的深入研究Ir DXS基因在冬凌草二萜类成分合成途径中的功能奠定了基础。1-Deoxy-D-xylulose-5-phosphate synthase（ DXS） catalyses the first committed step of the 2C-methyl-D-erythritol-4-phosphate（ MEP） pathway,and its expression level affects the contents of terpenoid. This study was aimed to clone the 1-deoxy-D-xylulose 5-phosphate synthase（ DXS） gene from Isodon rubescens and analyze the bioinformatics and expression of the gene.The primers were designed according to the transcript sequence of Ir DXS from I. rubescens transcriptome database. The physical and chemical characteristics,signal peptide,subcellular localization,secondary structure of Ir DXS protein were analyzed and predicted by the tools of bioinformatics. In this work,a DXS c DNA（ Ir DXS,KT831764） was isolated from I. rubescens. The full-length c DNA of Ir DXS encoded 722 amino acid residues with a predicted molecular mass of 77.7 KD. Sequence alignment showed that Ir DXS had high homology to known DXS proteins from other plant species and contained the conserved N-terminal plastid transit peptide,the N-terminal thiamine binding domain and pyridine binding DRAG domain. Phylogenetic analysis indicated that Ir DXS belonged to the plant DXS2 cluster. Quantitative real-time PCR analysis showed that the expression of Ir DXS was tissue-specific,and accumulation of transcripts was greater in roots and leaves. This study provides valuable information for future experiments on the molecular mechanisms underlying the isoprenoid biosynthesis in I. rubescens.

关 键 词：冬凌草 DXS基因 生物信息学分析 荧光定量PCR 二萜 

分 类 号：Q943.2[生物学—植物学]