Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/63120
標題: Investigation of Roles of Osck1 and Its Downstream Substrate in the Mature Pollen of Rice
水稻激活酵素及其受質蛋白於調控花粉萌發之機制探討
作者: 呂維茗
關鍵字: 生物科學類;基礎研究;rice;水稻;花粉;結鈣激活酵素;解旋酵素;蛋白交互作用;pollen;CDPK;helicase;protein-protein interaction
摘要: 
結鈣激活酵素是植物與原生動物所特有一種蛋白,於高等植物中其基因家族成員高達近三十個,雖然如此,但各個成員分別受到不同的發育訊號或是環境刺激調控其表現,蛋白之濃度與胞內分布位置各不相同,加上對於鈣離子濃度的感受性與訊息傳導路徑之差異,因此在植物的生長發育與環境適應的調控上,結鈣激活酵素均扮演了重要的角色。本實驗室先前自水稻中選殖出一個僅於成熟花粉大量表現的結鈣激活酵素OSCK1,由於鈣離子早為人知是花粉萌發與花粉管延長不可或缺的重要元素,因此推測OSCK1 的角色可能與調控上述過程相關,本計劃即欲探討此一調控機制。首先以定點突變改變OSCK1 基因序列,包括(1) OSCK1-G2A (改變荳蔻酸化訊號使OSCK1 無法附著於膜),(2) NNLS (去除入核訊號使OSCK1 無法主動入核),(3) CI (Catalytically Inactive,使OSCK1失去磷酸化活性),與(4) CA (ConstitutivelyActive,使OSCK1 毋須鈣離子調節即具有活性),利用農桿菌將基因轉殖入水稻,並以花粉專一性啟動子使之大量表現,隨後分析T0 代植物花粉的性狀,佐以基因槍暫時性轉殖百合花粉的觀察結果,初步認為OSCK1 須附著於膜方能展現特定作用,OSCK1-G2A 即失去此作用;此外,OSCK1-CI 雖失去磷酸化活性,但卻具有dominant-negative 的效果,大量表現OSCK1-CI 即嚴重抑制花粉發芽與花粉管延長,暗示OSCK1-CI 可能與某種含量有限的未知蛋白具有複合體關係,而此未知的蛋白即有可能為OSCK1 的受質蛋白。另一方面, 本實驗室先前利用酵母菌的雙雜交系統, 經層層篩除過程獲得一個OSCK1-interacting protein,命名為OIP30,其與OSCK1 的表現模式高度重疊,經過in vitro far-westernhybridization 與in vivo pull-down assay 等實驗,已證實兩者明顯具有蛋白複合體關係,且in vitroOIP30 可被OSCK1 磷酸化,因此初步推論OIP30 是OSCK1 於水稻花粉中的受質蛋白。根據上述種種數據,我們提出OSCK1 的作用機制假說,亦即在成熟花粉中,OSCK1 大多附著於膜上,且與OIP30 形成蛋白複合體,當接受鈣離子訊號後,OSCK1 即磷酸化OIP30,使兩蛋白分離或共同離開胞膜,OIP30 即可移轉入核執行其DNA/RNA 觧旋酵素的功能。由於此假說雖已部分證實,但仍有數個關鍵待釐清,故本計畫針對其中的重點,提出五項最為迫切的實驗,擬以一年的時間完成驗證,並整理成兩篇報告發表。第一篇將以OSCK1 突變形蛋白為主角,探討OSCK1 於花粉萌發及花粉管延長的生理角色,此部分實驗由於觀察的對象是花粉,故必須釐清轉植株所攜帶轉殖基因的套數等遺傳關係,方能在大量表達OSCK1 突變形蛋白的T1 世代確認觀察並量化數據,本計畫擬分析其花粉外觀、花粉活性及其萌發能力;第二篇則將探討OIP30 是否即為OSCK1 的下游受質蛋白,以及OIP30 於花粉萌發的生理意義,此部分將釐清的重點首先為分析OSCK1 與OIP30 於胞內的分布位置,檢查此分布是否受到外在訊號刺激而改變,以及OIP30 是否具有解旋活性,其受質偏好為何等,均列為本計畫的工作重點。由於目前除了知道特定類型的CDPK 於基因槍轉殖之花粉大量表現時,會抑制花粉萌發外,尚無任何深入探討CDPK 調控花粉萌發機制或下游受質蛋白的研究,更缺乏以轉殖植物進行之分析,故評估本研究結果將提出較為接近原始生理意義的觀察分析。

CDPK (Ca2+-dependent calmodulin-independent protein kinase) gene family,constituted by ~30 members in higher plant, is unique to plants and protozoa. Controlledexpressions by various developmental or environmental signals, varied distributions andconcentrations of protein within cells, different responsiveness and downstream pathwaysto Ca2+ signaling, etc. all account for the uniqueness of each CDPK member in controllingplant development or defense responses. We have isolated OSCK1, a CDPK in rice, with adistinct pollen-predominant expression profile. As Ca2+ ion is known to play key roles inpollen germination and tube elongation, OSCK1 may regulate these processes in responseto Ca2+ ion. To investigate roles OSCK1 in pollen, four mutants were generated bysite-directed mutagenesis including (1) OSCK1-G2A (with the destroyed myristoylationsignal/ membrane-anchorage), (2) NNLS (No-NLS, with deletion of the nuclearlocalization signal), (3) CI (Catalytically Inactive, with mutation on the catalytic loop), and(4) CA (Constitutively Active, with mutation on the auto-inhibitory junction domain).Overexpression of the above OSCK1s driven by a pollen-specific promoter in transgenicrice, together with evidence from transient particle bombardment of lily pollen, weobserved that OSCK1 exhibit functions only when membrane-bound, in contrast to thecytoplasmically located OSCK1-G2A. Interestingly, OSCK1-CI, although catalyticallyinactive, impeded pollen germination and tube elongation when overexpressed intransgenic rice. Such a dominant-negative effect implicated that some limited proteinfactors, presumably trapped by the functionless OSCK1-CI, may be essential for OSCK1signaling.To search for downstream targets of OSCK1, yeast-two-hybrid screens were employedand one OSCK1-interacting protein, named as OIP30, passed all examinations and waschosen for further analyses. Sharing similar expression profiles with OSCK1,OIP30/OSCK1 complexes were detected by both in vitro far-western and in vivo pull-downanalyses. Moreover, in vitro phosphorylation assays demonstrated that OIP30 might be thesubstrate for OSCK1 in the mature pollen. We therefore hypothesize that OSCK1 maylocate on membrane and form complex with OIP30 originally. After perceiving the Ca2+signals, OSCK1 phosphorylate OIP30 and exhibit conformation changes to dissociateOIP30 from OSCK1, or to relieve both proteins from membranes. OIP30 then move intonucleus, alone or together with OSCK1, to execute its helicase activity. Although based onour previous observations, there are several critical arguments need to be clarified for theabove hypothesis. Five major experiments are proposed to be accomplished within one yearin order to finalize two manuscripts.The first manuscript will focus on addressing roles of OSCK1 in the mature pollen bycharacterization of the transgenic rice overexpressing mutated OSCK1. Genetic analysis forconfirming male sterility and physiological characterizations regarding pollen morphology,viability, germination ability, etc., will be performed on the T1 plants this year. The secondmanuscript will focus on elucidating roles of OIP30 as substrate for OSCK1 in pollen.Critical experiments regarding analysis of subcellular distributions of two proteins withinpollen will be performed. Moreover, signals that affect the protein localizations will besearched. The helicase activity and preferred substrate of OIP30 will also be investigated.We expect our research on CDPK be the first one to delineate its physiological roles inpollen germination and tube elongation.
URI: http://hdl.handle.net/11455/63120
其他識別: NSC97-2311-B005-004
Appears in Collections:生物科技學研究所

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