Gene Expression Regulation by the 5' Regulatory Sequences of the Rice rubi3 Gene in Transgenic Rice Plants

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Title: Gene Expression Regulation by the 5' Regulatory Sequences of the Rice rubi3 Gene in Transgenic Rice Plants
Author: Lu, Jianli
Advisors: Rongda Qu, Committee Chair
Abstract: Plant biotechnology has found extensive applications in agricultural production, environmental protection, and molecular farming. A promoter of known expression pattern and regulation mode is of primary importance for these applications in that it helps tailor transgene expression in plants. The promoter of the rice polyubiquitin gene rubi3 was isolated in our laboratory. In transient assays, its expression was strongly enhanced by its 5' UTR intron and further elevated to a high level by sequences from the coding region of the rubi3 gene. In this thesis research, I analyzed the expression pattern of the rubi3 promoter, and studied the effects of its 5'UTR intron as well as the first 27 bp of the rubi3 coding sequence on its expression pattern and strength in transgenic rice plants. The expression pattern of the rubi3 promoter was studied by using both GUS and GFP as reporter genes to obtain more reliable information. Histochemical GUS localization and GFP visualization revealed that the rubi3 promoter, with its 5' UTR intron, was active in all the tissues and cell types examined, suggesting that it is a constitutive promoter although the expression levels among the tissues were not uniform. Its activity was especially strong in root apical meristems, leaf mesophyll cells, embryos, and parenchyma cells in the vascular system, including the vascular cylinders of roots and the vascular bundles of stems. The 5'UTR intron substantially enhanced the expression level of the rubi3 promoter without altering its expression pattern, but the magnitude of enhancement showed profound difference among various tissues. The increase in GUS enzyme activity was 3.1-fold in leaf, 23.6-fold in root, and 48-fold in callus tissue. Differential enhancement was also observed in other tissues of the GUS transformants and in the GFP transgenic plants. For example, enhancement was higher in embryos and aleurone layers, but obviously lower in stigma tissues. This report is the first one to demonstrate the tissue-dependency of intron-mediated enhancement of gene expression (IME) in stably transformed monocot plants. Moreover, differential enhancement was also found in different tissues at GUS mRNA level. The increase was 4.2-fold in leaf, 11.1-fold in root, and 12.1-fold in callus tissue. Further analysis broke down the enhancement effects of the rubi3 5' UTR intron into a pre-translational phase and a translational phase. The intron enhanced GUS gene expression mainly by elevating mature mRNA level in all the three tissues examined, suggesting that it stimulated GUS gene expression primarily at the pre-translational phase. However, the magnitude of enhancement in GUS activity was different from that at mRNA level, suggesting that the intron affected GUS gene expression at the translational phase as well. A fusion of the first 27 bp of the rubi3 coding sequence to the GUS gene further enhanced GUS activity over that conferred by the intron-containing rubi3 promoter without changing its expression pattern. The magnitude of the enhancement by this sequence also varied by tissue types. Analysis indicates that the enhancement effect took place completely at the translational phase without affecting mRNA accumulation. The results suggest that the 5' regulatory sequences of the rice rubi3 gene, including the promoter, the exon 1, the 5' UTR intron, and the exon 2 sequence, can be combined to direct strong and constitutive transgene expression in transgenic monocot plants. The observation also helps gain insight on the mechanisms of gene expression regulation, especially IME, in monocot plants.
Date: 2006-08-06
Degree: PhD
Discipline: Crop Science

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