枫香C4H基因的克隆及生物信息学分析  

Cloning and Bioinformatics Analysis of C4H Gene of Liquidambar formosana

作　　者：刘雄盛[1] 尹国平 肖玉菲[1] 王仁杰 黄荣林[1] 王勇[1] LIU Xiongsheng;YIN Guoping;XIAO Yufei;WANG Renjie;HUANG Ronglin;WANG Yong(Guangxi Forestry Research Institute/Guangxi Key Laboratory of Superior Trees Resource Cultivation,Nanning,Guangxi 530002,China)

机构地区：[1]广西壮族自治区林业科学研究院/广西优良用材林资源培育重点实验室,广西南宁530002

出　　处：《热带农业科学》2022年第6期45-52,共8页Chinese Journal of Tropical Agriculture

基　　金：广西优良用材林资源培育重点实验室自主课题(No.2019-A-03-02);2021年自治区林业科技推广示范项目(No.桂林科研[2021]26号)。

摘　　要：肉桂酸-4-羟基化酶(cinnamate4-hydroxylase,C4H)是花色素苷合成过程第二步中的重要酶。以变色期枫香叶片为试验材料,根据前期研究获得的枫香叶片RNA-Seq数据库中筛选的C4H基因序列设计一对引物,利用RT-PCR技术克隆C4H核苷酸序列,命名为LfC4Hb;将基因上传至GenBank数据库获得枫香C4H基因的登录号(OL871170),并对其进行生物信息学分析。结果显示:枫香LfC4Hb基因长度为1 551 bp,包含一个1 518 bp的完整开放阅读框,编码505个氨基酸;保守结构域分析显示,LfC4Hb基因编码的蛋白包含一个p450家族蛋白的结构域,分子量为58.00 kDa,理论等电点为9.13,为亲水性的不稳定蛋白质,定位在内质网,存在多个磷酸化位点,无信号肽,不含有跨膜结构域,蛋白的二级结构主要为α-螺旋和无规则卷曲,LfC4Hb基因在氨基酸水平与莲同源性最高(94.5%);进化分析结果表明,LfC4Hb与青脆枝亲缘关系相对较近。本研究成功克隆了枫香C4H基因,并分析了LfC4Hb的蛋白质结构功能,对下一步利用基因工程技术进行枫香叶色调控及新品种培育具有重要意义。Cinnamate 4-hydroxylase(C4H)is the second important enzyme in anthocyanin synthesis.A pair of primers were designed based on the C4H gene sequence screened from the RNA-Seq database of Liquidambar formosana leaves obtained in previous studies and then the nucleotide sequence of C4H was cloned by RT-PCR and named LfC4Hb.The gene was uploaded to GenBank database to obtain the gene login number(OL871170)of L.formosana C4H,and the bioinformatics analysis was conducted.The results showed that the length of LfC4Hb gene was 1551 bp,which contained a complete open reading frame of 1518 bp and encoded 505 amino acids.The conserved domain analysis showed that there was a p450 family protein domain.The molecular weight was 58.00 kDa and the theoretical isoelectric point was 9.13.The protein encoded by LfC4Hb was an unstable hydrophilic protein located in the endoplasmic reticulum.There were multiple phosphorylation sites,there was no signal peptide and no transmembrane domain.The secondary structure of the protein was mainlyα-helix and random coil.The homology of LfC4Hb gene with Nelumbo nucifera was the highest(94.5%).The phylogenetic analysis showed that LfC4Hb was relatively close to Nothapodytes nimmoniana.In this study,C4H gene of L.formosana was successfully cloned and the protein structure and function of LfC4Hb were analyzed.It will be of great significance for further application of genetic engineering technology in leaf color regulation and breeding of new varieties of L.formosana.

关 键 词：枫香 肉桂酸-4-羟基化酶 基因克隆 生物信息学分析 

分 类 号：S533[农业科学—作物学]