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標題: 探討水稻花粉OsCPK之功能與分離其下游受質蛋白
Analysis on Function of Pollen-Predominant OsCPKs & Isolation of their Downstream Substrates in Rice
作者: 方以辰
Fung, Yi-Chen
關鍵字: 水稻花粉
Rice pollen
出版社: 生物科技學研究所
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摘要: 水稻有29個OsCPK (Oryza sativa Calcium-dependent Protein Kinase )基因成員,其中有七個專一表現於花粉,但目前對此七個OsCPK的受質研究甚少,至今僅知其中的OsCPK26與其受質蛋白OIP30 (OsCPK26-interaction protein 30,為一RuvB-like DNA 解旋酶)皆主要表現在成熟花粉中,據此判斷兩者可能影響花粉後期發育或花粉管萌發,故以轉殖植物策略進行其功能性分析。由於大量表現不具活性的OsCPK26(CI) (catalytically inactive)突變型蛋白之轉殖水稻有雄不稔現象,推估是OsCPK26(CI)牽絆住其受質OIP30所造成。對此假說,設計以大量表現OIP30試圖挽救上述雄不稔情況,但並未能證明上述假說。另外,先前研究亦發現,大量表現OsCPK26(WT)野生型蛋白之轉殖植株也有雄不稔現象;相反地,大量表現OsCPK26(G2A) (肉豆蔻酸化訊號突變,導致OsCPK26蛋白無法附著於胞膜)的轉殖植株稔實卻正常,由此推測雄不稔可能與OsCPK26以肉豆蔻酸基附著於胞膜的現象相關。 水稻在花粉表現的七個OsCPK蛋白可因相似度而分為兩型,兩型之間蛋白彼此序列相似度為58% 而非同型內的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抓取可與OsCPK結合之水稻花粉蛋白,而為了避免OsCPK磷酸化後快速釋放受質,則以腺酸水解酶(apyrase)預先去除花粉的內生性ATP,再使用ATP洗提吸附於管柱中的蛋白,接著進行SDS-PAGE依照蛋白分子量分群後,使用LC-MS/MS分析,配合水稻基因組資料庫搜查,並扣除在控制組管柱也出現的蛋白,嘗試分離與OsCPK具有交互作用的蛋白,最後希望得知這些蛋白質的表現情況、與OsCPK的親和性或被磷酸化機制等特性後,鑑定其是否為OsCPK之受質蛋白,再進一步釐清兩型OsCPK蛋白在水稻花粉中的功能。
Thirty one members of OsCPK (Oryza sativa Calcium-dependent Protein Kinase ) genes are found in rice, and seven of them are expressed predominantly in pollen. However, almost no reports were focus on substrates of those 7 OsCPKs. Only a RuvB-like DNA helicase, named as OIP30 (OsCPK26-interaction protein 30), had been demonstrated to be expressed predominantly in the mature pollen grain and be the substrate for OsCPK26. To analyze their roles in pollen development and/or pollen tube germination, transgenic studies were employed. As OsCPK26(CI) (catalytically inactive) overexpressing rice was found to be male-sterile, we hypothesized that the overwhelmed OsCPK26(CI) may trap OIP30 without phosphorylation and that, co-overexpression of OIP30 may be able relieve such an effect. However, no rescued phenotype could be detected thus far. On the other hand, OsCPK26(WT) (wild type) overexpressing rice also cause male-sterile, opposed to the OsCPK26(G2A) (myristoylation mutant) transgenic rice. We suspect that the myristoylated membrane-bound OsCPK26 may disturb pollen function via unknown mechanism. According to protein sequence similarity, the seven pollen-expressed OsCPKs can be grouped into two types, sharing 70% similarity within group but 58% between groups, and thus may play different roles in pollen. OsCPK2 (type I) and OsCPK29 (type II), both with relatively high expressions in pollen, were chosen for study. To isolate their downstream substrates from pollen, an affinity-purification strategy was employed. His-tagged OsCPK2 or OsCPK29 were expressed by E. coli, bound by Ni-NTA resin, and used to trap their interacting proteins from fresh rice pollen. To avoid substrate release after catalysis by OsCPKs, endogenous ATP within pollen extracts was depleted by apyrase treatment and the resin-bound proteins were eluted by ATP. The eluents were then fractionated by SDS-PAGE and identified by LC-MS/MS. The identified rice pollen proteins, if absent in control resin, will be further characterized regarding their expression profiles, affinities with OsCPKs, protein characteristics, and phosphorylations conferred by OsCPKs, etc., so to investigate the specific roles of each types of OsCPKs in rice pollen.
其他識別: U0005-1708201219194900
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