摘要
克隆同色兜兰二氢黄烷酮醇-4-还原酶基因DFR,分析其序列特征,了解其在不同组织部位的表达情况。采用RT-PCR和RACE技术从花中克隆DFR的全长cDNA,用生物信息学方法分析序列特征,并用半定量RT-PCR研究其在不同组织的表达。分离到DFR,该cDNA全长1267bp,具有1个1074bp的完整开放阅读框,编码357个氨基酸。序列分析表明,该DFR蛋白含有1个NADB_Rossmann superfamily保守结构域,1个NADP(H)结合域和1个底物特异性结合域,其与文心兰、石斛兰和大花蕙兰等的DFR同源性在75%～80%。系统进化树显示,该DFR蛋白与兰科植物的DFR蛋白亲缘关系比较近。半定量RT-PCR表明,该DFR在成熟花和营养叶中的表达量最高,在子房、苞叶、萼片和花瓣中的表达量相当,在唇瓣中的表达量次之,在蕊柱中的表达量较唇瓣中的低,在花茎中的表达量最低,在根中几乎检测不到。原核表达结果表明,该DFR在大肠杆菌中获得表达。从兜兰中克隆到花色代谢途径中的关键酶基因DFR,该基因可能参与调控花色素的合成。
The aim of this study was to clone full-length cDNA of dihydroflavonol 4-reductase gene （DFR） from Paphiopedilum concolor Pfitz., to analyze the sequence characteristics and to investigate its expression in different tissues. The cDNA of DFR gene was cloned from flowers of P. concolor by reverse transcriptase-polymerase chain reaction （RT-PCR） and rapid amplification of cDNA ends （RACE）. Its sequence characteristics were analyzed by bioinformatics tools and its expression levels in different tissues were detected by semi-quantitative RT-PCR. The obtained DFR gene was 1267 bp in full-length, containing a 5＇ -untranslated region （5＇ -UTR） of 10 bp, a 3＇ -UTR of 183 bp, and an opening reading frame （ORF） of 1074 bp encoding a 357 predicted amino acids residues. The DFR protein contained a conserved NADB Rossmann superfamily domain, an NADP （H） binding domain and a substrate binding site domain. The DFR protein shared 75%-80% identities with DFR proteins of Oncidium Gower Ramsey, Dendrobium hybrid, and Cymbidium hybrid. Phylogenetic tree analysis clearly showed that the DFR protein was close to DFR proteins in orchidaceae plants. Expression analyses revealed that DFR showed different expression patterns in tissues examined. The transcripts of DFR were the highest levels in mature flowers and vegetative leaves,moderate levels in columns, bracts, sepals and petals, lower levels in ovaries than in lips, lowest levels in flower stems, and almost no detection in roots. Prokaryotic expression showed that recombinant plasmid was efficiently expressed in Eschericbia coli Rosetta-gami B （DE3）. DFR gene was firstly isolated and characterized from Paphiopedilum orchid, which might participate in regulation of anthocyanin biosynthesis.
出处
《中国农学通报》
CSCD
2012年第28期203-210,共8页
Chinese Agricultural Science Bulletin
基金
广州市科技计划项目"兜兰花色素苷代谢途径的分子调控机理研究"(12C14071654)
广东省农业科学院院长基金项目"低温诱导蝴蝶兰花芽分化与发育的分子基础研究"(201019)
关键词
兜兰
二氢黄烷酮醇-4-还原酶基因
RT-PCR
基因克隆
表达分析
Paphiopedilum
dihydroflavonol 4-reductase gene
reverse transcriptase-polymerase chain reaction
gene cloning
expression analysis
