不同色彩矮牵牛DFR基因的克隆与生物信息学分析

Cloning and Bioinformatical Analysis of Dihydroflavonol 4-reductase Gene (DFR) from Petunia hybrida with Different Color

二氢黄酮醇4-还原酶基因(DFR)是花色素合成途径中的一个关键基因。以新疆种植的白、红和蓝色矮牵牛为试验材料,通过同源克隆的方法从花中克隆到3个完整的DFR基因的全长编码序列(CDS),与已知的矮牵牛DFR基因(GenBank登录号:X15537)序列的相似性分别为97.79%、96.59%和97.99%,分别命名为PhDFR1,PhDFR2和PhDFR3;3个基因编码380个氨基酸,同已知矮牵牛DFR基因编码的蛋白(GenBank登录号:CAA33544)的同源性分别是95.53%、94.21%和95.79%;生物信息学分析表明,3个蛋白均具有NADB-Rossmann家族中高度保守的NADPH结合位点、底物特异性结合位点。3个矮牵牛品种DFR都不具有信号肽,为亲水蛋白,定位于细胞质的可能性最高;均具有两个跨膜结构,α-螺旋和β-折叠是3个DFR的主要二级结构元件,并且形成了β-α-β-α-β的Rossmann折叠,整本上呈对称分布。利用同源建模分析3个DFR蛋白与已知葡萄的DFR晶体结构有很高的相似性。系统进化树分析表明,PhDFR1、PhDFR2、PhDFR3与已知矮牵牛DFR蛋白亲缘关系最近。

In this study,through homologous gene cloning technology,three complete CDSs encoding dihydroflavonol 4-reductase（DFR）,a key enzyme in the pathway of anthocyanin biosynthesis,were cloned from the corolla with white,red and blue color ofpetunia hybridplanted in Xinjiang respectively,and namedPhDFR1,PhDFR2andPhDFR3.Homology alignment indicated that the nucleotide similarity of the three DFRs is 97.79 %、96.59% and 97.99% with anotherDFRAgene fromPetunia hybrida（GenBank access number ：X15537）.All these DFR CDSs encoded a polypeptide composed of 380 amino acid residues.The amino acid residues similarity of the three DFRs is 95.53 %、94.21% and 95.79% with DFRA protein（GenBank access number ：CAA33544）.DFRs of the threePetunia hybridastrains with different flower color contain a highly conserved NADP（H）-binding site and substrate specificity site which belong to NADB-Rossmann superfamily.They are stable proteins without signal peptide and are hydrophilic proteins probably located in cytoplasm.All of them have two transmembrane domains.α-helix and β-sheet are primary secondary structural components of DFR.The β-α-β-α-β structures forming Rossmann folding are symmetrically distributed on the whole.The phylogenyetic analysis of DFR proteins from different species revealed that three DFRs have closer relationship with the DFR fromDianthus caryophyllusthan from the other plant species.