Gene characterization, analysis of expression and in vitro synthesis of dihydroflavonol 4-reductase from Citrus sinensis (L.) Osbeck
Gene characterization, analysis of expression and in vitro synthesis of dihydroflavonol 4-reductase from Citrus sinensis (L.) Osbeck
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DOI：
10.1016/j.phytochem.2006.01.025
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发表时间：
2006-04-01
期刊：
影响因子：
3.8
通讯作者：
Petrone, G
中科院分区：
文献类型：
--
作者：
Lo Piero, AR;Puglisi, I;Petrone, G
二氢黄酮醇 4-还原酶（DFR，EC 1.1.1.219）催化二氢黄酮醇还原为无色花青素，这是花青素生物合成中关键的“后期”步骤。在这项研究中，我们发现，与红橙（Tarocco）相比，非红橙品种（Navel 和 Ovale）的 DFR 表达显着降低，表明该酶可能与花青素生产的缺乏有关。因此，我们分离并比较了血液和金橙色dfrs的cDNA、基因组克隆以及启动子区域。我们的数据显示，着色和非着色橙色DFR的cDNA序列100%同源，包含1017 bp的开放阅读框，编码338个氨基酸残基的蛋白质，对应的分子量为38010.76 Da，理论等电点为5.96。此外，我们发现dfr序列的非编码区（内含子和5'上游区）没有显着差异。基因组 DNA 的 Southern 印迹分析表明 dfr 在两个品种中均以单拷贝基因形式存在。根据这些发现，金橙色 dfr 的低表达水平可能在从血液到金橙色的表型变化中发挥作用，被认为是控制 dfr 表达的调节基因可能发生突变的结果。此外，我们在此报道了橙色DFR cDNA的成功表达，产生了一种活性DFR酶，该酶将二氢槲皮素转化为无色花青素，从而证实了分离的基因参与花青素的生物合成。此外，据我们所知，这是第一篇关于果肉体外表达DFR的报道，其生化特性可能与其他植物器官DFR有很大不同。 (c) 2006 Elsevier Ltd. 保留所有权利。
Dihydroflavonol 4-reductase (DFR, EC 1.1.1.219) catalyzes the reduction of dihydroflavonols to leucoanthocyanins, a key "late" step in the biosynthesis of anthocyanins. In this study we showed that a strong reduction in DFR expression occurs in the non-red orange cultivar (Navel and Ovale) compared to that of the red orange (Tarocco) suggesting that the enzyme could be involved in the lack of production of anthocyanins. Therefore, we isolated and compared the cDNAs, the genomic clones, as well as the promoter regions of blood and blond orange dfrs. Our data revealed that the cDNA sequences of pigmented and non-pigmented orange DFRs were 100% homologous and contained a 1017 bp open reading frame which encodes a protein of 338 amino acid residues, corresponding to a molecular mass of 38010.76 Da, with a theoretical pI of 5.96. Moreover, we found that there were no significant differences in non-coding regions (introns and 5' upstream region) of dfr sequences. Southern blot analysis of genomic DNA indicated that dfr was present as a single copy gene in both cultivars. From these findings the low expression level of blond orange dfr which might play a role in the phenotypic change from blood to blond orange, is thought to be the result of a likely mutation in a regulatory gene controlling the expression of dfr. In addition, here we reported the successful expression of orange DFR cDNAs leading to an active DFR enzyme which converts dihydroquercetin to leucoanthocyanidin, thus confirming the involvement of the isolated genes in the biosynthesis of anthocyanins. Moreover, as far as we know, this is the first report concerning the in vitro expression of DFR from fruit flesh whose biochemical properties might be very different from those of other plant organ DFRs. (c) 2006 Elsevier Ltd. All rights reserved.