山葡萄F3'H基因及其启动子的克隆与表达分析  

Cloning and Expression Analysis of F3’H Gene and Promoter from Vitis amurensis

作　　者：张宇[1,2] 徐智慧 任邵琦 杨铭慧 陈蒙 刘海峰 ZHANG Yu;XU Zhi-Hui;REN Shao-Qi;YANG Ming-Hui;CHEN Meng;LIU Hai-Feng(College of Agriculture,Yanbian University,Yanji 133002,China;Jilin Academy of Agricultural Sciences,Changchun 130033,China)

机构地区：[1]延边大学农学院,延吉133002 [2]吉林省农业科学院,长春130033

出　　处：《农业生物技术学报》2021年第11期2099-2108,共10页Journal of Agricultural Biotechnology

基　　金：国家自然科学基金(31511140284)。

摘　　要：类黄酮-3’-羟化酶(flavonoid-3’-hydroxylase,F3’H)作为花青素合成路径第二阶段的关键酶,在花色苷的合成途径中至关重要。为探究F3’H在山葡萄(Vitis amurensis)花色苷形成中的作用机理,本研究以山葡萄品种’双红’为材料克隆得到F3’H基因及其启动子序列,对其进行了生物信息学和原核表达分析,并以F3’H基因不同长度缺失片段启动子转化烟草(Nicotiana tabacum)叶片进行瞬时表达。结果显示,F3’H基因ORF全长1530 bp,启动子序列长1051 bp。生物信息学分析发现,’双红’与’双丰’品种相比较,F3’H’的等电点、分子量和二级结构组成上有所差异。启动子顺式作用元件预测显示,启动子序列中除具有核心启动子元件CAAT-box和TATA-box外,还有光响应元件及一系列与逆境胁迫、激素调节相关的元件。不同长度缺失片段启动子转化烟草叶片的瞬时表达结果显示,F3’H启动子随着缺失片段启动子长度的增加,GUS酶活性呈现上升的趋势。通过原核表达载体的构建发现,F3’H重组蛋白在条件分别为温度30℃,培养时间11 h,异丙基硫代半乳糖苷(isopropylβ-D-thiogalactoside,IPTG)浓度0.8 mmol/L的条件下,目的蛋白在上清液样品均中有与预测重组蛋白相对分子质量大小一致且较高浓度的表达带。本研究可为后续深入研究山葡萄F3’H基因在花色苷合成过程中的调控机理及启动子转录作用机理提供基础资料。Flavonoid-3’-hydroxylase(F3’H)is the key enzyme in the second stage of the anthocyanin synthesis pathway,it is very important in the anthocyanin synthesis pathway.In order to explore the formation mechanism of F3’H in the formation of anthocyanins in Vitis amurensis,the F3’H gene and its promoter sequence were cloned from the’shuanghong’variety of V.amurensis,and the bioinformatics and prokaryotic expression analysis were performed on it,and the different length deletion fragment promoters of F3’H gene were transformed into tobacco(Nicotiana tabacum)leaves for transient expression.The results showed that the ORF of the F3’H gene was 1530 bp in length and the promoter sequence was 1051 bp in length.Bioinformatics analysis showed that the composition of the’Shuanghong’variety F3’H was different from that of the’Shuangfeng’variety in isoelectric point,molecular weight and secondary structure.The prediction of promoter cis-acting elements showed that in addition to the core promoter elements CAAT-box and TATA-box,there were a series of light-responsive elements,stress elements,hormone response elements in the promoter sequence.The transient expression results of different length deletion fragment promoters transformed into tobacco leaves showed that as the length of the deletion fragment promoter increases,the GUS enzyme activity of the F3’H promoter showed an upward trend.Through the construction of prokaryotic expression vector,it was found that the F3’H recombinant protein had a relative molecular weight of the predicted recombinant protein in the supernatant sample under the conditions of 30℃,culture time 11 h,and isopropylβ-D-thiogalactoside(IPTG)concentration 0.8 mmol/L,expression bands with consistent mass and high concentration.This study provides basic data for further study on the regulation mechanism of Vitis amurensis F3’H gene in the process of anthocyanin synthesis and the transcription mechanism of promoter.

关 键 词：山葡萄 类黄酮-3’-羟化酶(F3’H) 启动子 顺式作用元件 

分 类 号：S663[农业科学—果树学]