与酵母单杂交系统匹配的植物瞬时表达分析载体的构建  

Construction of Plant Transient Expression Analysis Vectors Matching with Yeast One-hybrid System

作　　者：张媛 黄芳[1] 马燕林[1] 马建忠[1] Zhang Yuan;Huang Fang;Ma Yanlin;Ma Jianzhong(College of Life Science and Engineering,Lanzhou University of Technology,Lanzhou,730050)

机构地区：[1]兰州理工大学生命科学与工程学院,兰州730050

出　　处：《分子植物育种》2021年第8期2616-2620,共5页Molecular Plant Breeding

基　　金：国家自然科学基金项目(31560073,31860063)资助。

摘　　要：为了在植物细胞/原生质体中验证酵母单杂交系统解析的植物转录因子及其关键结构域,本研究构建了一套与酵母单杂交系统相匹配的植物瞬时表达分析系统,并在拟南芥叶片原生质体中进行了瞬时表达分析。结果显示,以AtDPBF4的转录激活区(CR Ⅱ)构建的效应载体可在拟南芥原生质体中激活报告基因GUS的转录。这一结果与AtDPBF4的CR Ⅱ在酵母细胞中的结果一致。报告载体中,含有6个UASGal1的杂合启动子可引发下游报告基因GUS转录的酶活性达0.96 nmol 4-MU·mg^(-1)·min^(-1);含有3个UASGal1的杂合启动子可引发GUS基因的酶活性达0.73 nmol 4-MU·mg^(-1)·min^(-1)。含有6个UASGal1的杂合启动子的强度明显强于含有3个UASGal1的杂合启动子,但其活性并不是3个UASGal1杂合启动子的2倍,仅比其活性高了31.5%。由此表明,在报告基因中增加GAL4结合位点(UASGal1)的重复序列可以提高下游报告基因的表达,但是这种活性升高趋势并不与UASGal1的重复数量呈线性关系。In order to verify the plant transcription factors and their key domains resolved by yeast One-hybrid system in plant cells/protoplasts,a set of plant transient expression analysis system matched with yeast One-hybrid system was constructed,and transient expression analysis was carried out in Arabidopsis leaf protoplasts.The results showed that the reporter gene GUS could be activated in Arabidopsis protoplasts by the effector vector constructed from the transcriptional activation region(CR Ⅱ) of AtDPBF4.This result is consistent with the result of CR Ⅱ of AtDPBF4 in yeast cells.In the report vector,six UASGall in the promoter of the report gene could induce the GUS transcription of the downstream report gene with an enzyme activity of 0.96 nmol 4-MU·mg^(-1)·min^(-1);three UASGal1in the promoter could induce GUS gene with enzyme activity of 0.73 nmol 4-MU·mg^(-1)·min^(-1).The heterozygous promoter with 6 UASGal1 was stronger than that with 3 UASGal1,but its activity was not twice as high as that of3 UASGal1,only 31.5% higher than that of 3 UASGal1.This indicates that increasing the GAL4-bound sequences(UASGal1) in the reporter gene can increase the expression of the downstream reporter gene,but this increase in activity does not have a linear relationship with the number of repeats of UASGal1.

关 键 词：酵母单杂交系统 植物瞬时表达分析系统 载体构建 

分 类 号：Q943.2[生物学—植物学]