水稻CAF1基因之功能分析-水稻CAF1基因的選殖、定性及表現

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

趙志豪(Chih-Hao Chao)

指導教授 ： 陸重安

國立中央大學/理學院/生命科學系/碩士(2006年)

摘要

一般來說，基因的表現可同時由轉錄階段 (transcriptional level) 和後轉錄階段 (post-transcriptional) 來調控；然而人們對於基因表現在轉錄階段的調控機制較為熟悉，對於後轉錄階段的調控較為陌生，而poly(A) tail的分解是絕大部分真核生物mRNA降解的速率決定步驟。從酵母菌的研究中得知，Caf1p (Ccr4p associated factor 1 protein) 與Ccr4p (carbon catabolite repression 4 protein) 具有deadenylase的功能，同時參與了mRNA的降解。然而我們並不了解CAF1基因在植物系統中是否具有相同的功能，因此在本論文中將進一步探討植物中CAF1基因的功能。
為了研究植物CAF1基因的功能，我們以單子葉模式植物-水稻為材料來分析。目前我們已經從水稻genomic DNA中成功的分離出6條與CAF1相似的基因-OsCAF1s，其中有4條OsCAF1s經由RT-PCR的分析得知可能為pseudo gene，而OsCAF1A和OsCAF1B為真實會表現的基因，利用北方墨點分析進一步觀察基因組中個別CAF1基因在不同組織和不同糖濃度下的表現模式，確認了這兩個基因的表現模式並不相同，其中OsCAF1A表現在leaves、sheaths與panicles，而OsCAF1B表現在roots、leaves、nodes、senesces leaves與panicles；另外，OsCAF1A是受糖抑制表現的基因，而OsCAF1B為受糖誘導表現的基因。此外我們也成功地利用E. coli表現系統將OsCAF1A、 OsCAF1B重組蛋白大量地表達出來，並分別分析重組蛋白OsCAF1A和OsCAF1B在細胞體外的生化活性，確定其都具有deadenylase的功能。最後我們也已經構築完成OsCAF1A 與 OsCAF1B overexpression 及RNAi的表達載體，並使用基因轉殖方法將OsCAF1A和OsCAF1B在水稻中大量表現，另一方面進行RNAi抑制水稻內生OsCAF1基因的表現，希望藉由這些水稻轉殖植株，可以進一步了解CAF1基因在水稻中所扮演的角色。

關鍵字

功能分析 ； 基因 ； 水稻

並列摘要

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.

並列關鍵字

rice ； deadenylase ； CAF1

參考文獻

Chan, M.T., and Yu, S.M. (1998a). The 3'' untranslated region of a rice alpha-amylase gene mediates sugar-dependent abundance of mRNA. Plant J 15, 685-695.

Chan, M.T., and Yu, S.M. (1998b). The 3'' untranslated region of a rice alpha-amylase gene functions as a sugar-dependent mRNA stability determinant. Proc Natl Acad Sci U S A 95, 6543-6547.

Chiba, Y., Johnson, M.A., Lidder, P., Vogel, J.T., van Erp, H., and Green, P.J. (2004). AtPARN is an essential poly(A) ribonuclease in Arabidopsis. Gene 328, 95-102.

Collart, M.A. (2003). Global control of gene expression in yeast by the Ccr4-Not complex. Gene 313, 1-16.

Couttet, P., Fromont-Racine, M., Steel, D., Pictet, R., and Grange, T. (1997). Messenger RNA deadenylylation precedes decapping in mammalian cells. Proc Natl Acad Sci U S A 94, 5628-5633.