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

摘要

1-Deoxy-D-xylulose-5-phosphate synthase ( DXS) catalyses the first committed step of the 2C-methyl-D-eryth-ritol-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 IrDXS from I. rubescens tran-scriptome database. The physical and chemical characteristics, signal peptide, subcellular localization, secondary struc-ture of IrDXS protein were analyzed and predicted by the tools of bioinformatics. In this work, a DXS cDNA ( IrDXS, KT831764) was isolated from I. rubescens. The full-length cDNA of IrDXS encoded 722 amino acid residues with a pre-dicted molecular mass of 77.7 KD. Sequence alignment showed that IrDXS 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 IrDXS belonged to the plant DXS2 cluster. Quantitative real-time PCR analysis showed that the expression of IrDXS was tissue-specific, and accumulation of transcripts was greater in roots and leaves. This study provides valuable information for future experiments on the mo-lecular mechanisms underlying the isoprenoid biosynthesis in I. rubescens.%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IrDXS基因cDNA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IrDXS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2169 bp,编码722个氨基酸,分子量为77.7 kD.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,IrDXS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IrDXS基因的全长cDNA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IrDXS基因在冬凌草二萜类成分合成途径中的功能奠定了基础.