以RING E3蛋白質作為誘餌進行酵母菌雙雜合系統分析

Abstract

RING蛋白質在阿拉伯芥中是一個相當大的家族，它們具有相當廣泛的功能。許多RING蛋白質與泛素/26S蛋白酶體系統有關，其中有一部分為泛素黏合酶 (E3)，能夠利用RING 區塊直接和泛素銜接酶 (E2) 作用。同時，這些RING E3 也能夠專一性地辨認可被E2 泛素化的受質。在本篇論文，我們利用微陣列資料庫找出一些阿拉伯芥中非生物逆境相關的RING E3，並利用它們當做釣餌來對cDNA庫進行篩選。使用酵母菌雙雜合系統對阿拉伯芥花序組織cDNA 庫進行篩選時，發現其中一個RING E3－R2 (at5g27420) 可能和一個NAC 家族蛋白質－NTL11(At5g04410)－具有作用性。同時，當NTL11 蛋白質融合上GAL4-DNA binding 區塊，會表現自我活化；顯示NTL11 可能如前人研究所預測為轉錄活化子。進一步的研究也顯示，R2 可以和去NAC 區塊的NTL11 具有作用性，而本實驗室先前的研究也顯示AtMAPR4 能和去NAC 區塊的NTL11 具有作用性。R2、NTL11與 AtMAPR4 在阿拉伯芥扮演的生理角色，仍須進一步確認。

RING proteins are defined as quite a big family in Arabidopsis, and their functions are varied. Many of RING proteins are involved in ubiquitin/26S proteasome system, some of them are ubiquitin ligases (E3s) which can interact directly with ubiquitin-conjugating enzyme (E2) by their RING domains. These RING E3s can also specifically indentify substrates that are going to be ubiquitinated by E2. In this study, we chose RING E3s related to abiotic stress in Arabidopsis based on microarray database and use them as baits to screen Arabidopsis library cDNA. Employing Yeast-two hybrid technique for cDNA (CD4-30) library screening from influoroscence meristem, floral meristem and floral buds up to stage 8 or 9, we indentified a RING E3, R2 (at5g27420), may have interaction with a NAC family protein, NTL11 (At5g04410). NTL11 also showed self-activation when it was fused to GAL4-DNA binding domain, implicating NTL11 may be a transcription factor as previous predictions from other studies. In further test, R2 can interact with NTL11 truncation without NAC domain and so does AtMAPR4 from previous study. Their roles in Arabidopsis physiological pathway need more investigations.