Molecular Cloning and Functional Characterization of a Dihydroflavonol 4-Reductase from Vitis bellula.
Molecular Cloning and Functional Characterization of a Dihydroflavonol 4-Reductase from Vitis bellula.
复制标题
葡萄二氢黄酮醇 4-还原酶的分子克隆和功能表征
DOI：
10.3390/molecules23040861
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发表时间：
2018-04-10
期刊：
影响因子：
--
通讯作者：
Xie DY
中科院分区：
文献类型：
--
作者：
Zhu Y;Peng Q;Li K;Xie DY
葡萄属白铃葡萄是我国南方葡萄新品种。本种的浆果富含抗氧化花青素和原花青素。本研究克隆了一个编码V.bellula二氢黄酮醇还原酶（VbDFR）的cDNA，并对其功能进行了分析。从葡萄叶片中克隆了一个长1014 bp的cDNA，其开放阅读框（ORF）编码337个氨基酸，与葡萄DFR（VvDFR）高度相似。绿色荧光蛋白融合和共聚焦显微镜分析确定了VbDFR在植物细胞中的胞质定位。从大肠杆菌中诱导并纯化了可溶性重组VbDFR。coli进行酶活性测定。在NADPH存在下，重组酶催化二氢山奈酚（DHK）和二氢槲皮素（DHQ）生成相应的无色花青素。通过农杆菌介导法将VbDFR cDNA导入烟草植株。VbDFR的过表达增加花色素苷的产量。花色素苷水解和色谱分析表明，转基因花产生天竺葵素和飞燕草素，这在对照花中没有检测到。这些数据表明VbDFR的过表达产生了新的烟草花青素。综上所述，所有数据表明，VbDFR是一个有用的基因，为葡萄作物和其他作物中花青素和原花青素的代谢工程提供三种类型的底物。
Vitis bellula is a new grape crop in southern China. Berries of this species are rich in antioxidative anthocyanins and proanthocyanidins. This study reports cloning and functional characterization of a cDNA encoding a V. bellula dihydroflavonol reductase (VbDFR) involved in the biosynthesis of anthocyanins and proanthocyanidins. A cDNA including 1014 bp was cloned from young leaves and its open reading frame (ORF) was deduced encoding 337 amino acids, highly similar to V. vinifera DFR (VvDFR). Green florescence protein fusion and confocal microscopy analysis determined the cytosolic localization of VbDFR in plant cells. A soluble recombinant VbDFR was induced and purified from E. coli for enzyme assay. In the presence of NADPH, the recombinant enzyme catalyzed dihydrokaempferol (DHK) and dihydroquercetin (DHQ) to their corresponding leucoanthocyanidins. The VbDFR cDNA was introduced into tobacco plants via Agrobacterium-mediated transformation. The overexpression of VbDFR increased anthocyanin production in flowers. Anthocyanin hydrolysis and chromatographic analysis revealed that transgenic flowers produced pelargonidin and delphinidin, which were not detected in control flowers. These data demonstrated that the overexpression of VbDFR produced new tobacco anthocyanidins. In summary, all data demonstrate that VbDFR is a useful gene to provide three types of substrates for metabolic engineering of anthocyanins and proanthocyanidins in grape crops and other crops.
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影响因子：
4.3
作者：
Shi, Ming-Zhu;Xie, De-Yu
通讯作者：
Xie, De-Yu
影响因子：
7.4
作者：
Robbins, MP;Bavage, AD;Morris, P
通讯作者：
Morris, P
影响因子：
7.2
作者：
Xie, DY;Sharma, SB;Dixon, RA
通讯作者：
Dixon, RA
影响因子：
3.8
作者：
Xie, DY;Dixon, RA
通讯作者：
Dixon, RA
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影响因子：
4.6
作者：
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通讯作者：
Ono M