cDNA末端快速扩增法克隆高山离子芥原叶绿酸酯氧化还原酶基因及其表达特异性分析

Cloning of NADPH: protochlorophyllide oxidoreductase by rapid amplification of c DNA ends and its developmental expression in Chorispora bungeana

用cDNA末端快速扩增技术从高山离子芥中克隆得到完整的原叶绿酸酯氧化还原酶(POR,EC:1.3.1.33)基因,命名为CbPORB,DNAMAN软件分析CbPORB和其他几种植物的POR基因,有很高的同源性,将其cDNA序列提交到NCBI数据库(序列接受号为FJ390503)。CbPORB基因全长1444bp,包含1个1209bp的开放阅读框(ORF),编码402个氨基酸的蛋白CbPORB(序列号为ACJ12925)。NCBI数据库在线软件计算高山离子芥CbPORB蛋白的等电点为9.43,推测其相对分子量为43.37kD。组织特异性表达分析显示:CbPORB在叶、茎中表达,根中不表达,具有器官特异性;干旱胁迫处理抑制CbPORB的转录水平;而表油菜素内酯(24-epibrassinolide;EBR)处理提高了CbPORB的转录水平。

A NADPH： protoehlorophyllide oxidoreductase （POR） gene was obtained by 5＇-and 3＇- RACE （rapid amplification of eDNA ends） PCR in Chorispora bungeana. The gene was termed as Cb-PORB （GenBank Accession No. FJ390503） and showed high homology to POR eDNA sequences from related species analyzed with DNAMAN software. The full-length eDNA sequence of CbPORB is 1 444 bp containing a 1 209 bp ORF, which encoded a predicted protein of 402 amino acid residues （GenBank Accession No. ACJ12925） with a theoretical molecular weight of 43.37 kD, and the isoelectric point is 9.43 examined using iPSORT. Furthermore, by semi-quantitative RT-PCR, CbPORB expression was detected in leaves and stems, but not in roots, thereby demonstrating that this gene is of organ-specific expression patterns; CbPORB transcript was inhibited by drought stress; but was induced by 24-epibrassinolide.