Construction of expression vectors based on the rice actin 1 (Act1) 5' region for use in monocot transformation.
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.
McElroy
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Field of Botany, Cornell University, Ithaca, NY 14853.
Blowers
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Jenes
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Wu
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D000199:Actins / Q000235:genetics*; D000595:Amino Acid Sequence; D001483:Base Sequence; D005786:Gene Expression Regulation / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics*; D005966:Glucuronidase / Q000235:genetics; D007438:Introns / Q000235:genetics; D008969:Molecular Sequence Data; D009029:Mosaic Viruses / Q000235:genetics; D012275:Oryza / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000235:genetics; D014176:Protein Biosynthesis / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D014170:Transformation, Genetic / Q000235:genetics; D003313:Zea mays / Q000235:genetics
D000199:Actins; D011993:Recombinant Fusion Proteins; D005966:Glucuronidase