三种花色黄芩中F3′5′H基因的克隆及表达分析

Cloning and Expression Analysis of F3′5′H Gene in Three Colors of Scutellaria baicalensis

类黄酮3′,5′-羟化酶(flavonoid 3′,5′-hydroxylase,F3′5′H)是花青素合成途径中催化形成蓝色飞燕草素的关键酶。本研究通过RT-PCR方法,从蓝紫花、玫红花、白花黄芩中克隆得到F3′5′H基因的全长序列,分别命名为SbF3′5′H-b、SbF3′5′H-r、SbF3′5′H-w。经序列比对,SbF3′5′H-r基因较SbF3′5′H-b/w缺失7 bp,导致蛋白翻译提前终止。生物信息学分析显示,三个SbF3′5′H蛋白均属于细胞色素P450家族,定位在内质网。SbF3′5′H-B/W蛋白具有典型的CYP450保守序列,包括富含脯氨酸基序“LPPGP”、底物识别位点SRS1~6、折叠锁定基序“E××R”、血红素结合基序“PFGAGRRICAG”及预测的羟化活性位点CR1和CR2。而SbF3′5′H-R蛋白由于序列缺失影响二级、三级结构,并导致CYP450结构域不完整,C端缺少多个维持酶活性的保守基序。基于F3′5′H蛋白序列的系统进化分析结果显示,黄芩与同为唇形科的丹参、一串红等亲缘关系最近,聚在一个分支,与单子叶植物亲缘关系较远。基因表达分析结果显示,SbF3′5′H在白花中整体表达最高,其次是蓝紫花,玫红花中整体表达最低;不同花发育阶段比较,SbF3′5′H基因均呈现出先升高后降低的趋势,在中蕾期表达量最高,其次是幼蕾期,盛花期表达量最低,蓝紫和白花黄芩不同花发育阶段间差异显著或极显著。本研究结果可为进一步研究不同花色黄芩中SbF3′5′H基因的功能差异、揭示黄芩花色变异的分子机理奠定理论基础。

Flavonoid 3′,5′-hydroxylase(F3′5′H)is a key enzyme catalyzing the formation of blue delphinidin in the anthocyanin biosynthesis pathway.The full-length sequence of F3′5′H genes were cloned from Scutellaria baicalensis with bluish-purple,rose-red and white flowers using RT-PCR method,and they were named as SbF3′5′H-b,SbF3′5′H-r and SbF3′5′H-w,respectively.Sequence alignment showed that compared with SbF3′5′H-b/w,SbF3′5′H-r gene owned 7-bp deficient resulting in early termination of protein translation.Bioinformatics analysis revealed that all the three SbF3′5′H proteins belonged to the cytochrome P450 gene family and were located in the endoplasmic reticulum.SbF3′5′H-B/W protein had a typical CYP450 conserved sequence,including proline-rich motif‘LPPGP',substrate recognition site SRS1-6,fold-lock motif‘E××R',heme-binding motif‘PFGAGRRICAG',and predicted hydroxylation active site CR1 and CR2.However,the sequence deletion of SbF3′5′H-R protein affected its secondary and tertiary structures,and leaded to incomplete CYP450 domain,lacking several conserved motifs that maintain enzyme activity at C terminus.The phylogenetic evolution based on F3′5′H protein sequence indicated that S.baicalensis was closely related to Salvia miltiorrhiza and Salvia splendens,which also belonged to Labiaceae family and were clustered in one branch,while was far related to monocotyledon plants.Expression analysis showed that the expression level of SbF3′5′H gene was the highest in white flowers,followed by bluish-purple flowers,and was the lowest in rose-red flowers.Comparing among different flower developmental stages,SbF3′5′H gene showed the trend of increasing firstly and then decreasing,and had the highest expression level at the middle bud stage,followed by the young bud stage,and had the lowest level at the full bloom stage.There were significant or extremely significant differences among different stages of flower development in bluish-purple and white flowers.These results would provide important guidance for further research on the functional differences of SbF3′5′H genes in S.baicalensis with different flower colors and revealing the molecular mechanism of color variation.