苦荞黄烷酮3-羟化酶基因FtF3H及其启动子的克隆与功能分析

doi:10.16036/j.issn.1000-2650.202210202

摘要/Abstract

摘要： 目的: 克隆苦荞FtF3H基因及其启动子序列,探究该基因的生物学效应及启动子对环境因素与激素的响应。方法: 基于苦荞基因组和转录组数据克隆FtF3H基因及其启动子,对二者进行生物信息学分析,采用qRT-PCR技术检测FtF3H对MeJA、ABA和光处理的响应,进一步利用转基因拟南芥技术,分析FtF3H的过表达对黄酮合成的影响。结果: 苦荞FtF3H基因DNA序列长2 034 bp,包括2个内含子和3个外显子;其ORF序列为1 104 bp,推导的FtF3H蛋白含367个氨基酸残基,归类于2-酮戊二酸依赖性双加氧酶超家族且具有典型的植物F3H的分子特征。启动子P_FtF3H在核心启动子基序外,还含有数目众多的光应答元件和激素响应元件,qRT-PCR检测结果显示,P_FtF3H可强烈响应ABA、MeJA和光照的诱导。转基因拟南芥中,FtF3H的过表达可引起总黄酮含量显著增加（P<0.05）,黄酮合成相关酶基因,AtANS、AtDFR和AtF'3H等的表达量显著上调（P<0.05）,而拟南芥内源的AtF3H表达受到抑制（P<0.05）。结论: 苦荞FtF3H基因是一个典型的植物黄烷酮3-羟化酶,其表达受到激素和光的调控,在拟南芥中的过表达可促进黄酮的合成和积累。

关键词: 苦荞, 黄烷酮3-羟化酶基因, 启动子, 黄酮, 功能分析

Abstract: 【Objective】 In this study,we cloned tartary buckwheat FtF3H gene and its promoter sequence to explore its biological effects and its promoter response to environmental factors and hormones. 【Method】 The FtF3H gene and its promoter were cloned based on tartary buckwheat genome and transcriptome data. The biological information of FtF3H gene was analyzed. The effects of MeJA, ABA and light treatments on the expression of FtF3H were identified by qRT-PCR. 【Result】 The DNA sequence of tartary buckwheat FtF3H gene was 2 034 bp length,including 2 introns and 3 exons. Its ORF sequence is 1 104 bp,encoding a protein of 367 amino acid residues. FtF3H belongs to 2-oxoglutarate dependent dioxygenase superfamily. The promoter sequence contains the basic structure of the promoter and a large number of hormone responsive elements of MeJA and ABA. P_FtF3H can respond to ABA, MeJA hormones and light treatment. In addition,overexpression of FtF3H in Arabidopsis thaliana significantly increased the content of total flavonoids （P<0.05）,and up-regulated the expression levels of flavonoid synthesis related enzymes, such as AtANS, AtDFR and AtF'3H （P<0.05）,while down-regulated the expression of AtF3H （P<0.05）. 【Conclusion】 In conclusion,FtF3H gene in tartary buckwheat has typical molecular characteristics of plant flavanone 3-hydroxylase,and its gene expression was regulated by hormones and light,and it plays an important role in flavonoid anabolic pathway.

Key words: Fagopyrum tataricum, flavanone 3-hydroxylase gene, promoter, flavonoids, functional analysis

中图分类号:

S517

曹鑫娴, 石佳琪, 王霜, 吴琦, 赵海霞, 吴花拉, 李成磊. 苦荞黄烷酮3-羟化酶基因FtF3H及其启动子的克隆与功能分析[J]. 四川农业大学学报, 2023, 41(4): 582-591.

CAO Xinxian, SHI Jiaqi, WANG Shuang, WU Qi, ZHAO Haixia, WU Huala, LI Chenglei. Cloning and Functional Analysis of Flavanone 3-Hydroxylase Gene FtF3H and Its Promoter in Fagopyrum tataricum[J]. Journal of Sichuan Agricultural University, 2023, 41(4): 582-591.

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