苦荞FtF5H基因克隆及表达分析

阿魏酸-5-羟基化酶(Ferulate 5-hydroxylase)是调控S型木质素合成的关键酶,为研究其在苦荞木质素生物合成途径中的分子机制,该文从苦荞转录组数据中筛选获得一个F5H基因,命名为FtF5H(GenBank登录号:MW455111),采用生物信息学方法对苦荞F5H蛋白的理化性质、信号肽、跨膜结构、亚细胞定位、亲疏水性、蛋白质二级结构、蛋白质三级结构、氨基酸结构、系统进化树等进行分析和预测,并运用实时荧光定量PCR(qRT-PCR)技术分析FtF5H基因在厚果壳苦荞与薄果壳苦荞的叶、花、茎、果壳中的差异表达。结果表明:(1)FtF5H基因序列包含1395 bp的完整cDNA开放阅读框,编码464个氨基酸。(2)FtF5H蛋白具有P450超家族结构,为亲水性稳定酸性蛋白,不具有跨膜结构域,且为非分泌性蛋白。(3)FtF5H蛋白的二级结构主要由α-螺旋和无规则卷曲组成,三级结构预测显示FtF5H蛋白与5ylw.1.A的相似度较高。(4)系统进化分析显示FtF5H属于CYP84A亚家族。(5)qRT-PCR显示FtF5H基因在两种苦荞中的不同部位均有表达,且在厚果壳苦荞果壳中的表达量是薄果壳的5倍,表达具有极显著差异。该研究为进一步研究苦荞木质素合成的分子调控机制奠定了基础,对苦荞新品种的培育具有重要意义。

Cloning and expression analysis of FtF5H gene from tartary buckwheat(Fagopyrum tataricum)

Ferulate 5-hydroxylase is a key enzyme that regulates the synthesis of S-type lignin.To study the molecular mechanism of ferulate 5-hydroxylase in lignin synthesis pathway of Fagopyrum tataricum,a FtF5H(GenBank accession number:MW455111)gene identified from F.tataricum RNA-seq data was screened and analyzed by bioinformatics methods including the physicochemical properties,signal peptide,transmembrane structure,subcellular localization,hydrophilicity,protein secondary structure and tertiary structure,amino acid structure,as well as phylogenetic tree.In addition,the real-time quantitative PCR(qRT-PCR)was applied to analyze the expression pattern of FtF5H gene in leaves,flowers,stems and husks of thick husk tartary buckwheat and thin husk tartary buckwheat.The results were as follows:(1)FtF5H gene sequence included completed open reading frame with 1395 bp,coding for 464 amino acids.(2)The FtF5H protein was predicted to have P450 superfamily structure,and FtF5H protein,a non-secretory protein,was a hydrophilic,stable and acidic protein without transmembrane domain.(3)The FtF5H protein secondary structure was predicted to be mainly composed ofα-helix and random coil,and its prediction of tertiary structure showed a high similarity with 5ylw.1.A.(4)Phylogenetic analysis showed that FtF5H belonged to the CYP84A subfamily.(5)In addition,qRT-PCR showed that the FtF5H gene was expressed in different parts of the two kinds of tartary buckwheat,and the expression level in the thick husk tartary buckwheat was four times higher than that in the thin husk,with extremely significant differences.The research establish a foundation for further study on the molecular regulation mechanism of lignin synthesis in tartary buckwheat,and has important research significance for the cultivation of new tartary buckwheat varieties.