关键词: 芜菁, 二氢黄酮醇4–还原酶基因, 基因克隆, 遗传转化, 功能鉴定

Abstract: Dihydroflavonol 4-reductase（DFR）is the critical catalyze enzyme in the later stage of anthocyanins biosynthesis，which belongs to a large redox enzyme superfamily that shares a Rossmann-fold NAD(P)H/NAD(P)(+) binding（NADB）domain. DFR catalyzes the reaction from dihydroflavonol to unstable leucoanthocyanidin. The roots of‘Tsuda’turnip and‘Yurugi Akamaru’turnip were irradiated with UV-A light for 24 h. Total RNA was isolated，and then BrDFR1 and BrDFR2 genes were cloned by RT-PCR method. The open reading frame（ORF）of BrDFR1 and BrDFR2 genes contained 1 158 bp and 999 bp encoding proteins of 385 and 332 amino acids respectively. Amino acid sequence analysis showed that BrDFR1 and BrDFR2 have high homology with DFR of Brassica rapa subsp. pekinensis. The FR_SDR_e domain was in the amino acid sequence from 8 to 302 of BrDFR1 and BrDFR2. The whole genome sequences of BrDFR1 and BrDFR2 had five introns with the same location and sequence. Southern blotting result showed that the copy of BrDFR1 and BrDFR2 in‘Tsuda’turnip and ‘Yurugi Akamaru’turnip genome is only one. Northern blotting result indicated that the expression of BrDFR1 could be induced by irradiation of UV-A，and the expression of the gene was correlated with light-exposure time. The induction of UV-A irradiation on the expression of BrDFR2 gene was indistinctively. The 42.8 kD and 37.5 kD proteins of BrDFR1 and BrDFR2 were successfully purified after BrDFR1 and BrDFR2 genes expressed in E.coli cell，respectively. The color of the flowers from BrDFR1 and BrDFR2 over-expressed tobacco plants was darker than that of wild type. The transgenic plants with light-colored flowers were obtained after the RNAi vector containing the DFR gene fragment of turnip was introduced. The present study will establish the experiment foundation for preliminarily clarifying the mechanism of light-dependent and light-independent anthocyanin biosynthesis.

Key words: turnip, dihydroflavonol 4-reductase gene, gene clone, genetic transformation, function identification