Combination of the ALCR/alcA ethanol switch and GAL4/VP16-UAS enhancer trap system enables spatial and temporal control of transgene expression in Arabidopsis
Combination of the ALCR/alcA ethanol switch and GAL4/VP16-UAS enhancer trap system enables spatial and temporal control of transgene expression in Arabidopsis
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DOI：
10.1111/j.1467-7652.2007.00255.x
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发表时间：
2007-07-01
影响因子：
13.8
通讯作者：
Vissenberg, Kris
中科院分区：
文献类型：
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作者：
Jia, Hongge;Van Loock, Bram;Vissenberg, Kris
在特定时间点对特定组织或细胞中基因表达的实验控制是分析基因功能的有用工具。GAL 4/VP 16-UAS增强子捕获系可用于在特定组织或细胞中选择性表达基因，乙醇诱导系统可帮助控制表达时间。在这项研究中，这两种方法的结合允许在时间和空间上成功调控基因表达。为此，构建了二元载体962-UAS：：GUS，其中ALCR激活剂和β-葡萄糖醛酸酶（GUS）报告基因分别置于上游激活序列（UAS）元件和alcA反应元件的控制下。转化拟南芥的三个不同的GAL 4/VP 16-UAS增强子捕获系，得到转基因植物，其中仅在乙醇诱导下并且仅在增强子捕获系的预测组织中检测到GUS活性。由于存在具有不同的绿色荧光蛋白（GFP）模式的不同增强子捕获系的文库，用其中GUS被另一个基因取代的类似载体转化将能够控制转基因表达的时间和地点。我们已经构建了两种载体以便于克隆感兴趣的基因，一种具有多接头位点，另一种与GATEWAY（TM）载体转化系统相容。当增强子捕获线可用时，该方法可以扩展到其他物种。
The experimental control of gene expression in specific tissues or cells at defined time points is a useful tool for the analysis of gene function. GAL4/VP16-UAS enhancer trap lines can be used to selectively express genes in specific tissues or cells, and an ethanol-inducible system can help to control the time of expression. In this study, the combination of the two methods allowed the successful regulation of gene expression in both time and space. For this purpose, a binary vector, 962-UAS::GUS, was constructed in which the ALCR activator and beta-glucuronidase (GUS) reporter gene were placed under the control of upstream activator sequence (UAS) elements and the alcA response element, respectively. Three different GAL4/VP16-UAS enhancer trap lines of Arabidopsis were transformed, resulting in transgenic plants in which GUS activity was detected only on ethanol induction and exclusively in the predicted tissues of the enhancer trap lines. As a library of different enhancer trap lines with distinct green fluorescent protein (GFP) patterns exist, transformation with a similar vector, in which GUS is replaced by another gene, would enable the control of the time and place of transgene expression. We have constructed two vectors for easy cloning of the gene of interest, one with a polylinker site and one that is compatible with the GATEWAY (TM) vector conversion system. The method can be extended to other species when enhancer trap lines become available.