刺葡萄DFR基因克隆及生物信息学分析

根据葡萄DFR基因CDS序列设计刺葡萄开放阅读框(ORF)特异引物,利用RT-PCR技术克隆获得其DFR基因序列,并通过生物信息学方法分析其生物学特性。结果表明,刺葡萄DFR基因ORF序列全长1 014bp,编码337个氨基酸,命名为Vitis davidii dihydroflavonol 4-reductase gene(VdDFR),GenBank登录号为KF915803。刺葡萄DFR蛋白预测分子量为37 593.2Da,理论等电点pI为5.81,是一个跨膜亲水蛋白,无典型信号肽,不属于分泌蛋白,并且亚细胞定位主要位于细胞质中(70%);二级结构以无规则卷曲为主(52.82%),是一种mixed类蛋白;该蛋白有潜在的7个糖基化位点和16个磷酸化位点,具有NAD(P)结合位点,有NAD依赖型的表异构酶/脱氢酶的N端结构域,属于NADB_Rossmann超家族成员。核苷酸序列分析表明,刺葡萄DFR基因与美丽葡萄、山葡萄和酿酒葡萄的同源性为99%,与圆叶葡萄同源性为98%,与显齿蛇葡萄同源性为94%,进化上比较保守,利用DFR基因编码区碱基序列所建立的系统关系树与真实的植物进化基本一致。

Cloning and Bioinformatics of DFR Gene in Vitis davidii Foёx

The specific primers of open reading frame (ORF)of dihydroflavonol (DFR)gene in brier grapes (Vitis davidii Foёx.)were designed according to the CDS template of the gene.DFR gene sequence was cloned using RT-PCR,andsubsequently,the genetic characteristics analyzed by bioinformatics.The 1 014 bp full-length cDNA of DRF's ORF was thus obtained.It encoded 337 amino acids,and was named V .davidii DFR 4-reductase gene (VdDFR )with GenBank accession number of KF91 5803. The predicted molecular weight of VdDFR protein was 37 593.2 Da,theoretical pI is 5.81. As a transmembrane and a hydrophilic protein,it did not belong to secretory category,had no signal peptide,and was located largely in the cytoplasm (70%).The secondary structure ofthe mixed protein was mostly random coil (52.82%).The amino acids sequence of the gene possibly contained 7 glycosylation sites,1 6 phosphorylation sites,one NAD (P)binding site,and one NAD-dependent epimerase/dehydratase (N-terminal) domain,and the gene likely belonged to the NADB _ Rossmann superfamily.The nucleotide sequences of DFR from V . davidii,V . bellula,V . amurensis , and V . vinifera were 99%homogenous;those of V .davidii and V .rotundifolia, 98% homogenous; and, those of V .davidii and Ampelopsis grossedentata , 94% homogenous.These results indicated that the DFR gene coding region was evolutionally conservative.And,the phylogenetic tree constructed based on the sequence salso reflected the same evolutionary trait of these plants.