Functional Analysis of the CAF1 Gene in Oryza sativa L.-Cloning, Characterization and Expression of the Rice CAF1

• Main Authors: Chih-Hao Chao, 趙志豪
• Other Authors: Chung-An Lu
• Format: Others
• Language: zh-TW
• Published: 2006
• Online Access: http://ndltd.ncl.edu.tw/handle/69301851030549250401

碩士 === 國立中央大學 === 生命科學研究所 === 94 === In general, gene expression is regulated at either a transcriptional level or a post-transcriptional level, or even both. Although regulation at both levels affects gene expression patterns, the mechanisms of transcriptional controlling gene expression are documented much more than mRNA degradation. In eukaryotes, the major mechanism of mRNA degradation involves a poly A tail deadenylation in the first step. The deadenylation is the rate-limiting step in many mRNA degradation events in a wide-range of organisms. In yeast, Caf1p and Ccr4p have been proposed that they play a prominent role as deadenylase in mRNA degradation. However, the function of CAF1 in plants is not known yet. Therefore, in this study we use the monocot model plant, rice, to further investigate the general role of plant CAF1 in mRNA degradation. First, we identified 6 CAF1-like genes in rice genomic DNA, OsCAF1s, and there were 4 OsCAF1s pseudo gene by RT-PCR analysis, the OsCAF1A and OsCAF1B were expressed gene. We also used northern blot analysis to address whether the rice OsCAF1s express in different organs and sugar concentrations. We confirmed that OsCAF1A express in leaves、sheaths and panicles, while OsCAF1B express in roots、leaves、nodes、senesces leaves and panicles. The expression of OsCAF1B was responded to sugar, while OsCAF1A were sugar independent. Second, we used E. coli expression system to get recombinant OsCAF1A and OsCAF1B proteins, which contained a deadenylation activity in vitro. Finally, we constructed overexpression and RNAi expression vectors of OsCAF1A and OsCAF1B to analyze the in vivo function of OsCAF1s.