山葡萄F3’5’H基因的克隆与表达及其启动子5′端缺失片段的功能分析  

Cloning and expression of F3’5’H gene in Vitis amurensis and functional analysis of its promoter 5’-end deletion fragment

作　　者：张宇[1,2] 刘海峰 ZHANG Yu;LIU Haifeng(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,吉林延吉133002 [2]吉林省农业科学院,长春130033

出　　处：《扬州大学学报（农业与生命科学版）》2022年第3期26-33,共8页Journal of Yangzhou University：Agricultural and Life Science Edition

基　　金：国家自然科学基金资助项目(31511140284)。

摘　　要：通过克隆山葡萄(Vitis amurensis)类黄酮-3’5’-羟化酶(flavonoid-3’,5’-hydroxylase,F3’5’H)基因及其启动子,并进行基因的原核表达和启动子不同缺失片段的瞬时表达,研究山葡萄不同种类花色苷的形成机制。克隆得到F3’5’H基因全长1527bp,分析其理化性质发现,‘双红’品种F3’5’H较‘双丰’品种有所差异,通过对F3’5’H重组蛋白体外表达条件的探究,在温度为25℃、培养时间为11h且IPTG浓度为0.8mmol·L-1的条件下,目的蛋白在上清样品中有与预测重组蛋白分子量大小一致且较高浓度的表达带。山葡萄F3’5’H基因启动子序列共1271bp,启动子序列中除具有核心启动子元件CAAT-box和TATA-box外,还有一系列光响应元件。采用启动子缺失技术研究不同长度缺失片段启动子在烟草中的瞬时表达,发现随着F3’5’H启动子缺失片段长度的增加,GUS酶活性呈先上升后下降的趋势。In order to explore the formation mechanism of different types of anthocyanins in Vitis amurensis,this experiment cloned the flavonoid-3’,5’-hydroxylase(F3’5’H)gene and its promoter of V.amurensis,and the prokaryotic expression of the gene and the transient expression of different missing fragments of the promoter were performed.The full length of gene was 1527 bp,analysis of its physical and chemical properties showed that the F3’5’H of‘Shuanghong’was different from‘Shuangfeng’.Through the construction of prokaryotic expression,it was found that when the F3’5’H recombinant protein was incubated at 30℃for 11h,and the IPTG concentration was under the condition of 0.8mmol·L-1,the target protein in the supernatant sample had the expression band of higher molecular concentration consistent with the predicted molecular weight of the recombinant protein.In addition to the core promoter elements CAAT-box and TATA-box in the promoter sequence,there were a series of light-responsive elements.The promoter deletion technology was used to study the transient expression of promoteres of different length deletion fragments in tobacco,and the results showed that with the increase of the length of the missing fragment of the F3’5’H promoter,the GUS enzyme activity showed a trend of increasing first and then decreasing.

关 键 词：山葡萄 类黄酮-3’ 5’-羟化酶 启动子 功能分析 

分 类 号：Q786[生物学—分子生物学] S663.1[农业科学—果树学]