從酵母菌到人類都存在著CCR4-NOT複合體，這複合體對於基因表達是個整體的調節者。複合體中的各個蛋白質對於不同層面的mRNA 代謝都有關聯，其中包括transcription initiation、elongation和mRNA degradation。其中一種影響基因表現的調控是控制mRNA 的半衰期。大部分的情況下，調控真核細胞mRNA半衰期的主要路徑是先將poly(A) tail縮短。在酵母菌的研究得知，CCR4蛋白質是CCR4-NOT 複合體中的一部分，目前已經證實在細胞質中具有deadenylation的作用。然而CCR4基因在植物系統中的功能並不清楚，所以本篇論文將進一步探討CCR4基因在水稻中的功能。 首先，透過資料庫比對分析發現有5個和酵母菌CCR4同源性較高的OsCCR4蛋白質，同時也發現皆屬於Mg2+-dependent exonuclease蛋白質家族。並發現在缺糖條件下，5個OsCCR4基因表現量較含糖條件下高，對於含糖條件下的相關基因可能有調控的作用。OsCCR4基因在水稻中分別有其組織特異性，其中OsCCR4-2則對於所有組織都有其重要性。藉由GFP融合OsCCR4-1、OsCCR4-2，發現OsCCR4-1、OsCCR4-2在細胞核及細胞質中皆有表現，所以可能參與了細胞核及細胞質中mRNA的deadenylation。並且成功建立過量表達GFP融合OsCCR4-1及OsCCR4-2的轉殖水稻，之後可進一步分析OsCCR4-1與OsCCR4-2在水稻內的功能。在E. Coli 表達系統中，也成功的表達出soluble的OsCCR4-1、OsCCR4-2重組蛋白質，之後將透過In vitro deadenylase activity分析，確認OsCCR4蛋白質的生化功能。 The CCR4-NOT complex is a global regulator of gene expression that is conserved from yeast to human. The proteins of the complex are involved in several aspects of mRNA metabolism, including transcription initiation and elongation and mRNA degradation. One of the regulations of gene expression is influenced by the control of mRNA turnover. In most cases, the major pathway of mRNA turnover in eukaryotic cells are initiated by shortening of the poly(A) tail. In yeast, the CCR4 (carbohydrate catabolism repression 4) protein, as part of the CCR4-NOT complex, has been show to be responsible for cytoplasmic deadenylation. However, the function of CCR4 in plants is not clear yet. In this research, we will investigate the functions of CCR4 in rice. First, there are five OsCCR4 proteins have high homology with yeast CCR4 which compared with the database. And all belongs to Mg2+-dependent exonuclease protein family. The five OsCCR4 genes have higher expression under sugar starvation, so that they have the possibility to regulate the sugar-related genes. The OsCCR4 genes have their tissue specificity in rice. Besides, OsCCR4-2 plays a important role for all tissues in rice. Through the GFP fused with OsCCR4-1 and OsCCR4-2 separately, we observed that OsCCR4-1 and OsCCR4-2 express both in nucleus and cytoplasm. So the OsCCR4-1 and OsCCR4-2 may participate the mRNA deadenylation in nucleus and cytoplasm. The transgenic rice which overexpressed the GFP fused with OsCCR4-1 and OsCCR4-2 separately were successful established, and will be analyzed for the functions of OsCCR4-1 and OsCCR4-2 in rice subsequently. The soluble recombinant proteins of OsCCR4-1 and OsCCR4-2 were expressed in E. Coli. In vitro assay of the deadenylase activity will be performed to ensure the biochemical function of OsCCR4-1 and OsCCR4-2.
