水稻二氢乳清酸脱氢酶基因的克隆与表达研究
首发时间:2005-12-22
摘要:二氢乳清酸脱氢酶(DHODH,dihydroorotate dehydrogenase, EC1.3.3.1)催化嘧啶从头合成途径中第四步反应,即二氢乳清酸氧化生成乳清酸。本研究利用电子克隆的策略和RT-PCR的方法克隆了水稻二氢乳清酸脱氢酶基因的全长cDNA 序列,命名为OsDHODH2(Oryza sativa dihydroorotate dehydrogenase 2)。该cDNA 序列全长为1632bp,编码469个氨基酸,与拟南芥二氢乳清酸脱氢酶基因氨基酸序列的一致性和相似性分别为81%和89%。构建了OsDHODH2的原核表达载体pET-30a(+)- OsDHODH2,转入大肠杆菌后得到了有效表达并具有酶活性,证明OsDHODH2是编码DHODH的功能基因。半定量RT-PCR表明,OsDHODH2在水稻不同组织中均表达,其中在成株期的根和穗中表达量相对较低;OsDHODH2基因受干旱和高盐胁迫处理的诱导表达,推测OsDHODH2基因可能参与植物的高盐和干旱的胁迫应答反应。
关键词: 二氢乳清酸脱氢酶 尿嘧啶从头合成途径 原核表达 环境胁迫 酶活性
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Molecular Cloning and Expression Analysis of OsDHODH2 Encoding dihydroorotate dehydrogenase in rice
Abstract:Dihydroorotate dehydrogenase (DHODH) catalyzes the fourth, and only redox step of pyrimidine de novo biosynthesis. Using the cDNA of Arabidopsis thaliana dihydroorotate dehydrogenase gene (acc. no. AY099561) from as a query probe , a highly homologous rice genomic contig was obtained from Huada rice genome database. The full-length cDNA sequence of rice dihydroorotate dehydrogenase was assembled by informatics based on the contig. Furthermore, with the two primers designed according to this assembled cDNA, the full-length cDNA of rice dihydroorotate dehydrogenase was cloned by RT-PCR and named as OsDHODH2. The cDNA was 1632bp in length and contained a complete open reading frame of 1407bp, encoding a protein of 469 amino acid residues. The deduced protein of OsDHODH2 showed 89% similarity and 81% identity with that from Arabidopsis thaliana. In this paper, using semi-quantitative RT- PCR, OsDHODH2 mRNA transcripted in every tissue and only expressed lowly in roots and spikes, induced by drought and salinity treatment. In addition, the expression vector pET30a-DH2 was constructed and transformed into BL21 cells. The results of enzyme assay shows that OsDHODH2 protein is able to reduce dihydroorotate in the presence of DICP as artificial electron acceptor.
Keywords: Dihydroorotate dehydrogenase, pyrimidine de novo biosynthesis, gene cloning, expression, rice
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