三色堇花色素合成途径中部分结构基因的克隆及生物信息学分析  

Cloning and bioinformatics analysis of three structural genes involved in anthocyanin synthesis of Viola×wittrockiana

作　　者：龚胜[1] 孙海燕[1] 张珂[1] 曾媛[1] 李婧[1] 王健[1] 李新国[1] 

机构地区：[1]海南大学园艺园林学院,热带作物种质资源保护与开发利用教育部重点实验室,海南海口570228

出　　处：《西北农林科技大学学报（自然科学版）》2015年第7期133-142,共10页Journal of Northwest A&F University(Natural Science Edition)

基　　金：国家自然科学基金项目(31060265,31260488);海南大学中西部计划学科建设项目(ZXBJH-XK008)

摘　　要：【目的】对三色堇花色素合成的3个结构基因VwCHS、VzvCHI和VwF3H进行克隆以及生物信息学分析。【方法】利用RAcE／PcR技术克隆得到VwCHS、‰cHf和VzvF3H基因的全长序列，并通过在线分析软件对其生物信息学进行分析。【结果】获得VwCHS、VwCHI和VwF3H的cDNA全长分别是1481，975和1361bp；经多重序列比对发现这3个基因编码的氨基酸与其他植物相关氨基酸一致性较高，主要集中在62％～90％，并且在CHS氨基酸序列中有4个催化活性位点（Cys171、Phe222、His310、Asn343），在CHI氨基酸序列中有4个催化活性位点（Thr50、Tyr108、Asn115、Ser192），在VwF3H氨基酸序列中有5个催化活性位点（Asp218、His253、Arg215、Arg284、Ser286）；VwCHS、VwCHI、VwF3H的蛋白质分子量分别为9．91，5．37，4．1ku，等电点为5．02，5．16，5．44；二级结构预测发现，vwCHs和VwCHI二级结构主要是0t螺旋，分别占43．39％和41．89％，VwF3H的蛋白质二级结构主要是无规则卷曲，占42．30％；聚类分析发现VwCHS、VwCHI和VwF3H分别归属于各自的聚类簇。【结论1VwCHS、VwCHI和VwF3H是不存在信号肽的非跨膜蛋白。[Objective] Cloning and bioinformatics analysis were conducted for three genes,VwCHS, VwCHI and VwF3H,which are involved in anthocyanin synthesis of pansy. [Method] The full-length cDNA of VwCHS ,VwCHI and VwF3H were cloned by RACE/PCR,and bioinformatics analysis was carried out through online software. [Result] The full-length cDNA of VwCHS ,VwCHI and VwF3H were 1 481, 975 and 1 361 bp, respectively. Multiple sequence alignment revealed that the amino acids encoded by the three structural genes had high identity compared to other plants （62%-90%）. There were four active sites （Cys171 , Phe222 , His310 , and Asn343） in the amino acids sequences of CHS, four active sites （Thr50 , Tyr108 , Asn115 , and Ser192） in C HI, and five active sites （Asp218 , His253 , Arg215 , Arg284, and Ser286） in F3 H. Their molecular weights were 9.91,5.37 and 4.1 ku,and their pI values were 5.02,5.16 and 5.44,respec-tively. The secondary structure prediction revealed that the secondary structures of VwCHS and VwCHI protein mainly contained a helix with ratios of 43.39% and 41.89%respectively, while VwF3H protein was mainly random coil with a ratio of 42.30%. Phylogenic analysis showed that each of the three proteins belonged to the corresponding protein family cluster. [Conclusion] VwCHS, VwCHI and VwF3H were non-transmembrane proteins without signal peptide.

关 键 词：三色堇 查尔酮合成酶(CHS) 查尔酮异构酶(CHI) 黄烷酮-3-羟化酶(F3H) 生物信息学 基因克隆 

分 类 号：Q785[生物学—分子生物学]