关键词: 刺葡萄, 二氢黄酮醇4-还原酶, DFR基因, 克隆, 生物信息学

Abstract: 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 KF915803. 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, 16 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.

Key words: brier grape (Vitisdavidii Foё, x.), dihydroflavonol 4-reductase, DFR gene, cloning, bioinformatics