水稻CAF1基因在水稻懸浮培養細胞之研究

Functional Analysis of OsCAF1 Genes in Rice Suspension Cells

方振丞(Jhen-Cheng Fang)

指導教授 ： 陸重安

國立中央大學/理學院/生命科學系/碩士(2011年)

摘要

mRNA降解作用為一個重要的基因表現調控機制，參與調控基因的表現，改變胞內狀態，以因應不同外在環境的變化。Poly(A) tail deadenylation為mRNA降解的速率決定步驟，而CCR4-associated factor 1s（CAF1s）在真核生物中催化deadenylation的一個主要酵素。水稻中含有四個不同的CAF1，分別為OsCAF1A、OsCAF1B、OsCAF1G、OsCAF1H。由北方墨點法與RT-PCR的分析顯示，OsCAF1s分別受到糖及不同的非生物逆境所調控。針對rOsCAF1A與rOsCAF1B重組蛋白進行胞外活性分析，結果指出其確實具有deadenylase的活性。先前的研究指出水稻受糖調控之α-澱粉降解酶的基因，αAmy3，其mRNA的半衰期會受到糖的調控。分析有糖及缺糖培養情況下之水稻細胞αAmy3 mRNA poly(A) tail長度，結果顯示在缺糖培養時其poly(A) tail長度較有糖培養長。有趣地是，在大量表現OsCAF1A轉殖水稻懸浮細胞中，發現其參與在αAmy3 mRNA的累積。由先前研究指出，OsMYBS1與OsMYBS2被證實參與αAmy3基因的表現，分別作為正向與反向的轉錄調控因子。由此分析大量表現OsCAF1A轉殖水稻懸浮細胞OsMYBS1與OsMYBS2的表現情況，結果顯示OsMYBS1表現會大量提升而OsMYBS2表現量會因此而下降。以上結果顯示OsCAF1A參與在OsMYBS1與OsMYBS2 deadenylation過程中，進而調控αAmy3 mRNA的累積。

關鍵字

糖 ； mRNA的降解 ； α-澱粉水解酶

並列摘要

One of central mechanisms to determine the expression level of genes for adjustment cellular status in response to various signals is messenger RNA (mRNA) degradation. Poly(A) tail deadenylation is a rate-limiting step of mRNA degradation. CCR4-associated factor 1s (CAF1s) is one of major enzymes to catalyze mRNA deadenylation in eukaryotes. Four members of CAF1 family, designated OsCAF1A, B, G and H, were analyzed and characterized in rice (Oryza sativa). Northern experiments and RT-PCR revealed diverse transcription patterns in response to sugar and various abiotic stresses among the four genes. Two recombinant proteins, rOsCAF1A and rOsCAF1B, had a deadenylation activity in vitro. It is known that mRNA half-life of αAmy3, which encodes a α-amylase, is regulated by sugar. Poly(A) tail analysis indicated that the length of poly(A) tail of αAmy3 mRNA was longer in the sugar-starved cells than in sugar-feasted cells. Interestingly, ectopic expression approach indicated that OsCAF1A was involved in αAmy3 mRNA accumulation. Previously, the OsMYBS1 and OsMYBS2 have been demonstrated to play a positive and negative regulator for αAmy3 expression, respectively. RT-PCR analysis indicated that ectopic expressed OsCAF1A caused the expression level of OsMYBS2 decreasing and the OsMYBS1 increasing. These results suggested that OsCAF1A may play a crucial role in mRNA deadenylation of OsMYBS1 and OsMYBS2 to regulate αAmy3 mRNA accumulation in rice.

並列關鍵字

OsMYBS2 ； OsMYBS1 ； α-amylase ； CCR4-associated factor 1s ； mRNA deadenylation ； mRNA degradation ； sugar

參考文獻

趙志豪，水稻CAF1基因之功能分析-水稻CAF1基因的選殖、定性與表現，碩士論文，民國九十五年。

Belostotsky DA (2008) State of decay: an update on plant mRNA turnover. Curr Top Microbiol Immunol 326:179-199.

Belostotsky DA, Sieburth LE (2009) Kill the messenger: mRNA decay and plant development. Curr Opin Plant Biol 12:96-102.

Bremer KA, Stevens A, Schoenberg DR (2003) An endonuclease activity similar to Xenopus PMR1 catalyzes the degradation of normal and nonsense-containing human beta-globin mRNA in erythroid cells. RNA 9:1157-1167.

Brown CE, Sachs AB (1998) Poly(A) tail length control in Saccharomyces cerevisiae occurs by message-specific deadenylation. Mol Cell Biol 18:6548-6559.