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二氢黄酮醇4-还原酶(dihydroflavonol 4-reduetase,DFR)是花色素苷合成途径中的一个关键酶。该研究利用RT-PCR和RACE技术从朵丽蝶兰‘满天红’深红色花瓣中克隆获得一个DFR基因,命名为DtpsDFR。该cDNA序列全长1 286 bp,编码378个氨基酸。氨基酸序列分析表明,DtpsDFR编码的蛋白与Bromheadia fi nlaysoniana、文心兰、大花蕙兰、石斛兰等兰科植物的DFR蛋白同源性均在76%以上,含有1个FR_SDR_e特征结构域,存在NADPH结合基序和底物特异性结合基序,属于NADB_Rossmann超家族;系统进化树显示,DtpsDFR与Bromheadia fi nlaysoniana的DFR蛋白亲缘关系最近。实时荧光定量PCR分析结果显示,DtpsDFR基因的表达量随着花的发育逐渐降低,凋谢期微量表达;在花瓣、萼片中的表达量高于唇瓣,在叶片和根中微量表达。
Dihydroflavonol 4-reductase (DFR) is a key enzyme in the anthocyanin biosynthesis pathway. This study used RT-PCR and RACE techniques to clone a DFR gene named DtpsDFR from the flower petals of Prunus salicina. The full-length cDNA was 1 286 bp in length and encoded 378 amino acids. Amino acid sequence analysis showed that the DFR proteins encoded by DtpsDFR shared more than 76% identity with Bromheadia fi nlaysoniana, Oncidium, Cymbidium, Dendrobium and other orchidaceous plants, and contained one FR_SDR_e domain NADPH binding motif and substrate-specific binding motif belong to the NADB_Rossmann superfamily. Phylogenetic tree shows that DtpsDFR has the closest genetic relationship with DFR protein of Bromheadia fi nlaysoniana. The results of real-time quantitative PCR showed that the expression of DtpsDFR gene decreased gradually with the development of flower and slightly expressed in the withering stage. The expression level of DtpsDFR gene in petal and sepal was higher than that in lip, and was slightly expressed in leaves and roots.