大叶蛇葡萄类黄酮-3'-羟化酶基因(F3'H)的克隆及生物信息学分析  

Cloning and Bioinformatics Analysis of Flavonoid-3’-Hydroxylase(F3’H)Gene in Ampelopsis megalophylla

作　　者：李永华 蒲天珍 周佩娜 方佳慧 张秀桥 龚玲 Li Yonghua;Pu Tianzhen;Zhou Peina;Fang Jiahui;Zhang Xiuqiao;Gong Ling(College of Pharmacy,Hubei University of Chinese Medicine,Wuhan,430065)

机构地区：[1]湖北中医药大学药学院,武汉430065

出　　处：《分子植物育种》2021年第18期5984-5993,共10页Molecular Plant Breeding

基　　金：国家自然科学基金(31170335);武汉市科技局应用基础前沿专项项目(2018060401011308)共同资助。

摘　　要：类黄酮-3’-羟化酶(F3’H)是黄酮化合物生物合成过程中的关键酶之一,本研究从大叶蛇葡萄转录组数据库中筛选出类黄酮-3’-羟化酶基因(F3’H),利用RT-PCR等技术克隆了其全长序列,并分析F3’H基因编码蛋白的理化性质和结构特征。结果表明,该序列全长为1718 bp,开放阅读框(ORF)长度为1530 bp,相对分子质量为55.89 kD,编码509个氨基酸,等电点为7.3,分子式为C2(513)H3974N696O705S21,不稳定指数为38.26,为稳定蛋白;脂肪族指数为96.42,总平均疏水性为-0.004,为亲水性蛋白;具有多个磷酸化位点,有信号肽,存在一个跨膜区,亚细胞定位于内质网膜,二级结构以α-螺旋为主。系统进化分析和多重序列比对表明该序列与藤茶的亲缘关系最近。密码子偏性分析表明该基因以G/C结尾的密码子使用频率较高,最适合该基因的异源表达宿主是大肠杆菌。本研究结果为进一步研究大叶蛇葡萄F3’H蛋白功能,以及揭示该植物中具生物活性的黄酮类成分的生物合成机制提供了一定的基础。The Flavonoid-3’-hydroxylas(F3’H)is one of the key enzymes in the process of flavonoid biosynthesis.In this study,we screened the Flavonoid-3’-hydroxylase gene(F3’H)from the transcriptome database of Ampelopsis megalophylla,cloned its full-length sequence by RT-PCR,and analyzed the physicochemical properties and structural characteristics of the F3’H gene encoding protein.The results showed that the sequence of F3’H gene was 1718 bp,containing an open reading frame(ORF)of 1530 bp which encoded 509 amino acids.The relative molecular weight was 55.89 kD and isoelectric point was 7.3.The molecular formula was C2513 H3974 N696 O705 S21 and instability index was 38.26,which was a stable protein.F3’H was an hydrophilic protein with a aliphatic index of 96.42 and total average hydrophobicity of-0.004.The protein had multiple phosphorylation sites,signal peptide,and transmembrane region,which were localizated in endoplasmic reticulum,and the secondary structure was mainly consist inα-helix.Phylogenetic analysis and multiple sequence alignment showed that the sequence was closely related to Ampelopsis grossedentata.The codon bias analysis showed that the codon with G/C ending was frequently used,and the most suitable host for heterologous expression of the gene was Escherichia coli.The results lay a foundation for further study on the function of F3’H protein and reveal the biosynthesis mechanism of bioactive flavonoids in the plant.

关 键 词：大叶蛇葡萄(Ampelopsis megalophylla) 类黄酮-3’-羟化酶基因(F3’H) 基因克隆 生物信息学分析 

分 类 号：S567.19[农业科学—中草药栽培]