紫花苜蓿二氢黄酮醇还原酶基因(MsDFR)的克隆与分析

二氢黄酮醇还原酶(dihydroflavonol reductase,DFR)是缩合单宁生物合成途径中的关键酶,在单宁的合成中起着重要的作用。根据同源克隆的原理,利用RACE技术,从“中苜一号”苜蓿中克隆得到DFR基因(MsDFR),并对其进行了序列分析及不同胁迫条件下的表达模式分析。结果表明,MsDFR基因cDNA全长1 402 bp,包括开放阅读框1 023 bp,编码340个氨基酸,在N端存在1个NADP结合位点“VTGASGFIGSWLVMRLMERGY”,中部存在1个底物特异性结合的氨基酸基序“TLNVTEDQKPLWDESCWSDVEFCRRV”。实时荧光定量PCR结果表明,该基因在荚果中表达量较高,根中较弱;在NaCl和GA₃诱导下,MsDFR基因表达受到抑制;在黑暗条件下,该基因被诱导表达。由此推测“中苜一号”苜蓿中可能存在不依赖于GA₃信号的单宁合成途径。

Cloning and analysis of dihydroflavonol reductase(DFR) gene from Medicago sativa

Dihydroflavonol reductase(DFR) is a key enzyme in condensed tannin synthesis pathway.A cDNA of DFR gene is cloned from alfalfa(Medicago sativa) by using rapid amplification of cDNA ends(RACE) method.The expression pattern of MsDFR under different stresses was analyzed by qRT-PCR.The bio-informatical analysis showed that the full-length of cDNA sequence is 1 402 bp and includes a 1 023 bp open reading frame which encodes a 340-amino-acid polypeptide.A nicotinamide adenine dinucleotide phosphate(NADP) binding site "VTGASGFIGSWLVMRLMERGY" and a substrate specificity motif "TLNVTEDQKPLWDESCWSDVEFCRRV" were detected in the deduced amino acid sequence of MsDFR.The results of Real-time PCR indicate that the expression level of MsDFR gene is highest in pods,and least in root.The expression of MsDFR gene under stress of NaCl and GA3 is down-regulated.In dark environment,the MsDFR gene expression was induced.There is a gibberellin(GA) signaling-independent pathway of tannin synthesis in alfalfa.