Wang Jingwen, Ming Feng, Han Yingying, Shen Daleng
Institute of Genetics, State Key Laboratory of Genetic Engineering, Research Center of Gene Diversity and Designed Agriculture, School of Life Science, Fudan University, Shangha 200433, China.
Biotechnol Lett. 2006 Mar;28(5):327-34. doi: 10.1007/s10529-005-5718-6.
A novel cDNA for the flavonoid-3',5'-hydroxylase (F35H) gene was cloned from petals of Phalaenopsis, and designated as Phf35h (accession number DQ148458 in GenBank/EMBL/DDBJ). The genomic clone of Phf35h was isolated by a PCR-based strategy. Nucleotide sequence analysis revealed that its genomic clone contains one intron and an open reading frame encoding a polypeptide of 507 amino acid residues. Southern hybridization analysis indicated the presence of a single gene coding for Phf35h. RT-PCR analysis showed that the Phf35h mRNA is transcribed in late phase of petal development, which is concomitant with the appearance of anthocyanins in petal tissue. The transcript is abundant in the purple petals but not in leaves or roots. The three-dimensional model of PhF3'5'H protein is classified into an alpha-domain which contains most of the alpha-helices with three small beta-sheets, a beta-domain that contains the larger beta-sheets with three small alpha-helices by homology modeling. The substrate-binding site for dihydrokaempferol on PhF3'5'H protein was determined by molecular docking algorithm. A highly conserved HPPTPLSLPH sequence was predicted to contact the aromatic ring of dihydrokaempferol.
从蝴蝶兰花瓣中克隆出一种新的类黄酮 -3',5'-羟化酶（F35H）基因的cDNA，并将其命名为Phf35h（GenBank/EMBL/DDBJ登录号为DQ148458）。通过基于PCR的策略分离出Phf35h的基因组克隆。核苷酸序列分析表明，其基因组克隆包含一个内含子和一个编码507个氨基酸残基多肽的开放阅读框。Southern杂交分析表明存在一个编码Phf35h的单一基因。RT-PCR分析表明，Phf35h mRNA在花瓣发育后期转录，这与花瓣组织中花青素的出现同时发生。该转录本在紫色花瓣中丰富，但在叶片或根中不丰富。通过同源建模，PhF3'5'H蛋白的三维模型被分类为一个α结构域，其中包含大部分α螺旋和三个小β折叠片，以及一个β结构域，其中包含较大的β折叠片和三个小α螺旋。通过分子对接算法确定了PhF3'5'H蛋白上二氢山奈酚的底物结合位点。预测一个高度保守的HPPTPLSLPH序列与二氢山奈酚的芳香环接触。
