不同花色黄芩中dfr基因的克隆及时空表达分析

Cloning and temporal-spatial expression analysis of dfr gene from Scutellaria baicalensis with different colors

摘要:

二氢黄酮醇-4-还原酶 (Dihydroflavonol-4-reductase，Dfr) 是类黄酮生物合成途径中调控花青素和原花青素合成的关键酶。为探究dfr基因在同一生态环境下不同花色黄芩中的差异，以白色、紫红色和紫色3种花色的黄芩花瓣cDNA为模板，基于同源克隆和RACE技术从中克隆得到3个完整的dfr基因全长序列，分别命名为Sbdfr1、Sbdfr2、Sbdfr3，并采用生物信息学软件对其相关结构进行分析。结果表明，3个Dfr蛋白均存在高度保守的NADPH结合位点和底物特异性结合位点。系统进化分析表明，三者与已知粘毛黄芩 (GenBank登录号：ACV49882.1) 聚为一簇，亲缘关系最近；关键结构域和3D模型分析发现3个Dfr蛋白表面催化活性区域均存在差异，其独特的结构特性为研究Dfr底物特异性提供了有利条件。qRT-PCR分析显示，dfr在3种花色黄芩盛花期除根外的其他组织中均有不同程度的表达；在花瓣发育进程中，dfr基因在白花和紫花黄芩中的表达量均呈上升趋势，在紫红花黄芩中的表达量先缓慢上升后下降，之后再迅速上升至最大值。研究结果为进一步深入探究Dfr底物选择性的机理及功能提供一定的理论基础，对阐明黄芩花色差异变化的分子机理具有重要的科学研究价值。

Abstract:

Dihydroflavanol-4-reductase (Dfr) is a key enzyme that regulates the synthesis of anthocyanin and proanthocyanidin in the flavonoid biosynthesis pathway. To investigate the difference of dfr gene in Scutellaria baicalensis Georgi with different colors in the same ecological environment, three complete full-length sequences of dfr gene were cloned from the cDNA of S. baicalensis with white, purple-red and purple colors using homologous cloning and RACE techniques. The three genes were named Sbdfr1, Sbdfr2 and Sbdfr3, respectively, and their corresponding structures were analyzed. The results showed that all three Dfr proteins have highly conserved NADPH binding sites and substrate-specific binding sites. Phylogenetic analysis showed that they are closely related to that of the known S. viscidula (ACV49882.1). Analysis of key structural domains and 3D models revealed differences in the catalytically active regions on the surface of all three Dfr proteins, and their unique structural characteristics may provide favorable conditions for studying the substrate specificity of different Dfr proteins. qRT-PCR analysis shows that dfr was expressed at different level in all tissues except the roots of S. baicalensis in full-bloom. During floral development, the expression level of dfr in white and purple-flowered Scutellaria showed an overall upward trend. In purple-red-flowered Scutellaria, the expression first slowly increased, followed by a decrease, and then rapidly increased to the maximum. This research provides a theoretical basis for further exploring the mechanism and function of Dfr substrate selectivity, and are of great scientific value for elucidating the molecular mechanism of floral color variation in S. baicalensis.