Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/35856
標題: 評估OsCPK26抑制花粉萌發的機制與尋找水稻花粉中CDPKs的下游受質蛋白
Elucidate mechanism of OsCPK26 in inhibition of pollen germination and hunt for downstream substrates of pollen-predominant CPKs in rice
作者: 詹益維
Jan, Yi-Wei
關鍵字: 結鈣蛋白
CDPKs
出版社: 生物科技學研究所
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摘要: 水稻中有七個專一表現於花粉的OsCPK蛋白質,利用OsCPK26研究OsCPK在花粉萌發所扮演的角色,發現在百合花花粉大量表現OsCPK26會抑制花粉萌發。為了找出OsCPK26抑制花粉萌發的機制,將OsCPK26突變後,發現只有一個胺基酸差異的OsCPK26(G2A)不會抑制花粉萌發,這個結果指出荳蔻酸訊號可能是抑制花粉萌發的原因。domain swapping指出抑制花粉萌發可能為26V domain所造成的現象,為了找出26V domain的功能區,將26V domain縮減,發現26V(45-67)可能具有抑制花粉萌發的能力,且26V(1-82)中的α-helix結構具有一個特別的AAAAA motif,懷疑這可能與抑制花粉萌發有關。利用點突變26V(1-82, A56V) 和26V(1-82, A56F)分析,結果發現維持α-helix結構即可抑制萌發,說明26V中的α-helix對抑制現象可能很重要,但五個連續的alanine則非必要;最後以gain-of-function實驗進一步確認,證實26V(40-70)就會抑制花粉萌發。由於pollen tip具有大量的phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2)累積,且花粉萌發與花粉管延長皆需藉由PI(4,5)P2傳遞訊號,先前提到荳蔻酸修飾與抑制花粉萌發有關,所以利用PIP array觀察有或無荳蔻酸修飾的OsCPK26、大幅縮短的OsCPK26V和和突變型的OsCPK26(G2A) 與各種磷脂質的結合能力,發現myristoylated OsCPK26偏好與phosphatidylinositol 4-monophosphate (PI(4)P) 結合,而OsCPK26(G2A)則不會。這結果指出OsCPK26 V domain與荳蔻酸修飾基會與PI(4)P結合,並參與細胞中的訊號傳遞,可能進而調控花粉萌發。 儘管鈣離子與結鈣激活酵素在花粉萌發及延長上扮演重要的角色,目前只找到少數花粉專一的OsCPK下游受質蛋白。水稻中在花粉表現的七個OsCPK蛋白可因相似度分為兩型,兩型之間序列相似度為58%、同型內為70%,兩型中表現量較高的分別為第一型的OsCPK2和第二型的OsCPK29。為了解兩型OsCPK蛋白之功能,分別以E. coli大量表現融合(His)6標籤序列之OsCPK2和OsCPK29後,利用親和性管柱Ni-NTA結合融合蛋白,作為釣餌抓取水稻花粉蛋白。而為了避免OsCPK磷酸化後快速釋放其受質蛋白,故預先以腺酸水解酶(apyrase)去除花粉的內生性ATP,使受質蛋白黏附於OsCPK並充分洗除雜蛋白後,依磷酸激酶的生化特性,利用ATP洗提出可能的受質蛋白。接著進行SDS-PAGE將蛋白依照分子量分群,再使用LC-MS/MS分析,並扣除在控制組也出現的蛋白後,得到數個可能的受質蛋白,包含exopolygalacturonase、ATP synthase與villin-3,同樣實驗已重複操作數次,以確認受質蛋白的多樣性與再現性。
In rice, calcium-dependent protein kinases (CDPKs) contain 29 members. Seven of them show pollen-predominant expressions. Transient overexpression of OsCPK26 but not other OsCPK members severely impeded lily pollen germination, suggesting a unique phenomenon only attributed to OsCPK26. A G2A mutation abolished the myristoylation, membrane-binding, and impeditive role of OsCPK26 all together, indicates that the N-myristoylation mediated membrane-binding is essential for the inhibitory role of OsCPK26 on pollen germination. Domain-swapping and C-terminal serial deletions further defined the critical region to the N-terminal 46-67 amino acids (aa), comprising a 13-aa (46-58) alpha-helix. Point mutants of OsCPK26 (A56V and A56F) excluded the essentiality of the five consecutive alanine residues resided within the alpha helix. Conversely, the alpha-helix motif seems to be critical as the 40-70 aa fragment alone exhibited inhibition activity when fused to the N-terminal 1-7 aa myristoylation signal. Searching of lipid-binding preference revealed that OsCPK26 was specifically retained by PtdIns(3)P, PtdIns(4)P, and PtdIns(5)P but not other phospholipids. Moreover, abolishment of myristoylation of OsCPK26 reduced its binding with PtdIns(4)P specifically. The metabolic equilibrium among phosphoinositides, especially PtdIns(4)P, may be disturbed by binding with OsCPK26, therefore affect signal transduction, vesicle trafficking, etc., and cause inhibition of pollen germination. Although calcium signaling is known to play critical roles in pollen germination and tip growth, only a few proteins had been identified as substrates downstream to pollen-specific CDPKs so far. The seven pollen-expressed rice CDPKs can be grouped into two types, sharing 70% sequence similarity within group but just 58% between groups, thus may play distinct roles in pollen. OsCPK2 (type I) and OsCPK29 (type II), both accumulate relatively abundant transcript in pollen, are chosen for study in this report. Using Ni-NTA bound OsCPK2-(His)6 or OsCPK29-(His)6 as prey protein, we employed affinity-purification for bait substrate. Extracts of rice pollen, pretreated with apyrase to deplete endogenous ATP, were incubated with resin-bound OsCPKs. After extensive wash, candidate proteins were eluted by ATP that theoretically triggered kinase reaction so to release the phosphorylated substrates. The eluted proteins were then fractionated by SDS-PAGE and identified by LC-MS/MS. Three proteins including ATP synthase, villin-3, and exopolygalacturonase, were absent in the control resin but appeared in the CDPK-retained fractions. Experiments were repeated to confirm and expand profiles of the candidate substrates.
URI: http://hdl.handle.net/11455/35856
其他識別: U0005-1508201312365300
文章連結: http://www.airitilibrary.com/Publication/alDetailedMesh1?DocID=U0005-1508201312365300
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