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標題: I.水稻OSCK基因靜默轉殖植物之分析 II.水稻OIP基因表現模式之分析
I. Analysis of OSCK-silenced transgenic rice. II.Characterization of OIP gene expression in rice.
作者: 童嬿融
Tung, Yen-Jung
關鍵字: CDPK;結鈣激活酵素
出版社: 生物科技學研究所
在植物中,細胞內鈣離子的濃度常會受到許多生物性或非生物性訊號調節,並藉由calcium-dependent calmodulin-independent protein kinases(CDPKs)可逆式的磷酸化受質蛋白,以傳遞鈣離子的訊號。CDPK為多基因家族,但特定的CDPK基因則經常具有專一性的基因表現模式或是專一的胞內分佈位置,此暗示其於多種生理反應中應扮演著重要的角色。OSCK1與OSCK2為水稻花粉專一性表現的CDPK基因,長久已知鈣離子於花粉發育過程中扮演著不可或缺的角色,但其訊號究竟如何傳遞仍毫無文獻探討過,因此先前實驗室利用酵母菌雙雜交法,以含帶OSCK1 kinase domain的蛋白為餌,已篩選出五個與OSCK1可能具有交互作用的蛋白,命名為OIP(OSCK1-interacting protein)。以blast程式搜查GenBank資料庫可知OIP1為一Hsp70/Hsp90 organizing-like protein (只有C半段區塊);OIP13 為一phosphatidylinositol phosphatidylcoline transfer protein(只得到C末端區塊);OIP30為RuvB-like DNA helicase(幾乎為全長);OIP18與OIP28則為兩個不同的CDPK基因。本實驗利用RT-PCR與Real-time PCR 分析水稻中上述五個OIP基因的表現模式,發現OIP1表現較不具專一性;OIP13、OIP18與OIP28主要於小穗花及成熟花粉中表現,惟OIP28在癒傷組織中亦有明顯訊號,OIP30則相反,其訊號僅大量出現在營養期樣品如癒傷組織、幼嫩稻穗與孕穗期的稻穗,在花粉中幾乎沒有訊號。其中OIP30推測為一DNA helicase,理論上應位於細胞核中;而OSCK1亦帶有一趨核訊號,若OIP30能與OSCK1 K domain形成蛋白複合體,則對於OSCK1在水稻中所扮演的生物功能,應可有更進一步的解釋與暸解。此外,利用RNA interference策略干擾OSCK1與OSCK2基因的表現。期望藉由OSCK1與OSCK2基因表現量之降低,觀察水稻花粉發育或萌發是否有異常之性狀,藉以瞭解OSCK基因在水稻花粉中所扮演的角色。經由觀察發現,轉殖株提早開花,並明顯有小穗軸軸距較長,掉穗率提高等性狀出現。

? In plants, intracellular Ca2+ levels are modulated in response to various biotic or abiotic signals. The calcium-dependent calmodulin-independent protein kinases (CDPKs) play critical roles in transducing calcium signals by reversibly phosphorylating substrate proteins. Encoded by a multigene family, CDPKs had been found expressed with distinct profiles or localized to specific subcellular locations, indicating their roles in various physiological responses. OSCK1 and OSCK2 are pollen-specific CDPK genes isolated from rice. As calcium was known to play crucial roles in pollen development, we had used the kinase domain of OSCK1 as a bait in yeast two-hybrid screens to peruse the OSCK1-interaction proteins (OIPs). Blast search of GenBank predicted that OIP1, 13, 30, 18 and 28 encoding proteins homologous to a Hsp70/Hsp90 organizing-like protein (C terminal half only), a phosphatidylinositol phosphatidylcholine transfer protein (C-terminal only), a RuvB-like DNA helicase (near full-length) and two CDPKs, respectively. In this study, analyses of gene expression profiles by RT-PCR suggested that OIP1 were expressed in various tissues. OIP13, OIP18 and OIP28 were expressed predominantly in spikelet and mature pollen, although OIP28 was detected also in callus. On the opposite, OIP30 was highly expressed in the vegetative tissues but surprisingly, barely detectable in the mature pollen. Moreover, Gene-transformed rice putatively interfere the endogenous OSCK1 and OSCK2 expressions resulted in early flowering, long rachilla and grain loss on rachis segment etc., if compared with vector-transformed rice.
Appears in Collections:生物科技學研究所

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