山茶属三个F3H基因的分子特性、系统进化及蛋白结构差异分析  

Comparison of Molecular Characteristics, Phylogeny and Structure of Three Camellia F3H Genes

作　　者：范晶[1] 黄明远[1] 吴苗苗[1] 叶红[1] 张西玉[1] 

机构地区：[1]乐山师范学院生命科学学院,乐山614004

出　　处：《基因组学与应用生物学》2016年第5期1195-1205,共11页Genomics and Applied Biology

基　　金：乐山师范学院科研项目(Z1201)资助

摘　　要：黄烷酮3-羟化酶基因(flavanone 3-hydroxylase,F3H)在植物花青素合成途径中发挥重要作用,可作为山茶花色遗传育种的候选基因。本研究在前期从Gen Bank数据库中筛选得到白花茶树CsF3H、黄花金花茶CnF3H和红花浙江红山茶CcF3H三个基因。然而,它们的分子遗传与变异信息仍然缺乏,不利于最大正向效应基因的选择利用。本研究系统探讨了CsF3H、CnF3H和CcF3H基因的分子特征、系统进化和蛋白三维结构。结果发现CsF3H、CnF3H和CcF3H三者之间存在高度序列多样性,共含37个核苷酸和9个氨基酸差异,且与CsF3H和CnF3H相比,CcF3H序列发生了更多的变异。系统进化结果表明CsF3H、CnF3H和CcF3H蛋白与猕猴桃AcF3H具有共同祖先,但在后期进化过程中,CcF3H率先与CsF3H和CnF3H二者发生分化。序列比对和保守结构域分析发现CsF3H、CnF3H和CcF3H蛋白均含有一个保守的依赖2-酮戊二酸和二价铁离子的双加氧酶超家族特征区域,该区域内的组氨酸H^(218)和H^(276)以及天冬氨酸D220是Fe^(2+)结合位点,精氨酸R^(286)和丝氨酸S^(288)是2-酮戊二酸的重要结合位点。本研究还发现了3个茶属F3H蛋白的保守结构域中存在一个差异位点,与CsF3H和CnF3H第200位异亮氨酸I200相比,CcF3H对应氨基酸变异为缬氨酸V^(200),Web logo3.4分析揭示V200在植物F3H蛋白中更为保守。空间结构分析显示CcF3H中氨基酸V~(200的替换导致它和赖氨酸K197的分子作用力增强。本研究结果表明CcF3H是更为保守的植物F3H蛋白,缬氨酸V200可能是一个重要的功能位点。上述研究也为山茶属F3H基因提供了新的信息,为今后选择CcF3H基因作为山茶花色育种最适候选基因提供了理论支撑。F3H, an ideal candidate gene for flow er color breeding of Camellia by genetic engineering, plays important roles in regulating plant anthocyanin biosynthesis. Three F3H genes including CsF3H from Camellia sinensis（white flower）, CnF3H from Camellia nitidissima（yellow flower） and CcF3H from Camellia chekiangoleosa（red flower） have been obtained from Gen Bank database in our previous study. However, the information about their molecular properties and genetic divergence are still very limited, which is unfavorable to the utility of F3H gene with the largest positive effect. In this study, molecular characteristics, phylogeny and structure of CsF3H,CnF3H and CcF3H were analyzed. High levels of sequence polymorphisms containing 37 different nucleotides and9 amino acids were found among the CsF3H, CnF3H and CcF3H, and CcF3H harbors more sequence variation in comparison with CnF3H and CsF3H. Phylogeny results showed that CsF3H, CnF3H and CcF3H proteins have the same ancestor with Ac F3H from Actinidia chinensis, and CcF3H was firstly separated from CsF3H and CnF3Hduring the evolution process. Results of sequence alignments and structure analysis showed all the three F3H contain a conserved 2-oxoglutarate and Fe（Ⅱ） dependent oxygenase superfamily domain, which contains 5 functional sites including three ferrous iron（Ⅱ） binding residues（H（218）, D（220）, H（276）） and two 2-oxoglutarate binding residues（R（286）, S（288））. An Isoleucine（I（200）） to Valine（V200） substitution was also observed at position 200 of CcF3H protein when compared with CsF3H and CnF3H, Web logo3.4 analysis showed V（200） is more conserved than I（200） in plant F3H proteins. Protein tertiary structure analysis showed the V（200） substitution strengthened inter-residue interaction with Lycine（K（197）） in CcF3H. All above results suggest CcF3H is more conservative as a plant F3H protein, the V200 is an important potentially functional site. This study also provides new insights

关 键 词：山茶属 F3H基因 系统进化 蛋白三维结构 2-酮戊二酸和二价铁离子双加氧酶超家族 

分 类 号：Q943.2[生物学—植物学]