CONSTRUCTION OF EXPRESSION VECTORS BASED ON THE RICE ACTIN-1 (ACT1) 5' REGION FOR USE IN MONOCOT TRANSFORMATION
CONSTRUCTION OF EXPRESSION VECTORS BASED ON THE RICE ACTIN-1 (ACT1) 5' REGION FOR USE IN MONOCOT TRANSFORMATION
复制标题
DOI：
10.1007/bf00293832
复制
发表时间：
1991-12-01
期刊：
影响因子：
--
通讯作者：
WU, R
中科院分区：
文献类型：
--
作者：
MCELROY, D;BLOWERS, AD;WU, R
已有报道表明，水稻肌动蛋白1基因的5‘端区域(Act1)促进了β-葡萄糖苷酸酶报告基因(GUS)在转化水稻细胞中的高水平表达。在本文中，我们描述了用于单子叶转化的基于Act1的表达载体的构建。作为这些载体开发的一部分，我们评估了Act1第一内含子、Act1-GUS连接编码的N末端氨基酸以及Act1和GUS翻译起始点周围的序列背景对Act1-GUS基因在水稻和玉米细胞中表达的影响。我们发现，在花椰菜花叶病毒(CaMV)35S启动子控制下，将Act1内含子1添加到GUS报告基因的转录单元中，可以使转化水稻细胞中的GUS活性提高10倍以上。GUS翻译起始点周围的序列背景优化导致了GUS在转化水稻细胞中表达的4倍刺激。通过利用Act1内含子1和优化的GUS翻译起始点，CaMV 35S启动子在转化的水稻细胞中实现了GUS表达的40倍刺激，在转化的玉米细胞中也得到了非常相似的结果。综上所述，这些结果表明，这里描述的基于Act1的表达载体应该可以促进大多数(如果不是全部)转化的单子叶细胞中外源基因的表达，达到以前使用替代表达载体所不能达到的水平。
It has been previously reported that the 5' region of the rice actin 1 gene (Act1) promoted high-level expression of a beta-glucuronidase reporter gene (Gus) in transformed rice cells. In this paper we describe the construction of Act1-based expression vectors for use in monocot transformation. As part of the development of these vectors, we have evaluated the influence of the Act1 first intron, the Act1-Gus junction-encoded N-terminal amino acids, and the sequence context surrounding the Act1 and Gus translation initiation site on Act1-Gus gene expression in rice and maize cells. We have found that addition of Act1 intron 1 to the transcription unit of a Gus reporter gene under control of the cauliflower mosaic virus (CaMV) 35S promoter stimulated GUS activity more than 10-fold in transformed rice cells. Optimization of the sequence context around the Gus translation initiation site resulted in a 4-fold stimulation of Gus expression in transformed rice cells. By utilizing both the Act1 intron 1 and optimized Gus translation initiation site, a 40-fold stimulation in Gus expression from the CaMV 35S promoter has been achieved in transformed rice cells; very similar results were obtained in transformed maize cells. Taken together these results suggest that the Act1-based expression vectors described here should promote the expression of foreign genes in most, if not all, transformed monocot cells to levels that have not previously been attainable with alternative expression vectors.