阿拉伯芥中與 ARGONAUTE 有潛在交互作用之蛋白質研究

Putative interacting proteins of ARGONAUTE in Arabidopsis

郭巡銓(Hsun-Chuan Kuo)

指導教授 ： 楊健志

國立臺灣大學/生命科學院/微生物與生化學研究所/碩士(2010年)

https://doi.org/10.6342/NTU.2010.10754

摘要

RNA 靜默作用對於植物的生長與發育都有重大的影響。RNA 靜默作用主要是由不同的小片段 RNA 與 ARGONAUTE (AGO) 家族蛋白質形成複合體來進行。阿拉伯芥中 AGO 蛋白質共有 10 種，AGO 複合體與不同的輔助蛋白質進行交互作用來產生不同的功能，包括對目標 mRNA 的降解、抑制轉譯作用、增加 DNA 和組蛋白質甲基化，以及對抗病蟲害等。本論文利用建立過量表現轉殖株、酵母菌雙雜合法和免疫共沉澱法等方式，尋找與 AGO1 或 AGO4 可能具有交互作用的蛋白質。由於載體上的設計錯誤，造成 frame-shift，本實驗得到兩種基因表現低下的突變株 ago1 與 ago4。藉由酵母菌雙雜合法，篩選到兩個與 AGO1 產生交互作用的 CHR 蛋白質，它們屬與需要 ATP 共同作用的染色質重組酵素。另外，以大腸桿菌表現 GST-AGO1 與 GST-AGO4 重組蛋白質作為釣餌，與阿拉伯芥葉片抽出的蛋白質粗抽液，進行免疫共沉澱測試，發現了數個候選蛋白質，進一步以 LC-MS/MS 進行分析，它們是分別是 clathrin binding protein (epsin)、F-box/FBD/LRR-repeat protein、short-chain dehydragenase/reductase、Ferredoxin-dependent glutamate synthase 1、Glutamine synthetase、ATP synthase subunit alpha和 Elongation factor 1-alpha。未來需進一步的分析，才能證實 AGO 與這些候選蛋白質間的交互作用。

關鍵字

阿拉伯芥 ； 蛋白質交互作用

並列摘要

Abstract
RNA silencing is important in the growth and development of plants. The major components of RNA silencing complex are different small RNA and ARGONAUTE (AGO) family. Ten AGOs are present in Arabidopsis. It is expected that different function, degrading the target transcript, inhibiting translation, methylating DNA or histone and defending against virus and insects, are achieved by AGOs in complex with different interacting proteins. In this study, putative interacting proteins of AGO1 or AGO4 were searched using the overexpression mutants, yeast two hybrid assay or co-immunoprecipitatin assay. Due to the presence of two extra bases in the vector pN-TAPa, two knock-down mutants, ago1 and ago4 were obtained instead of the expected overexpression. By yeast two hybrid, two CHR proteins, ATP-dependent chromatin remodeling enzymes, were found to interact with AGO1. Recombinant GST-AGO1 expressed in E.coli was used as bait to find interacting protein from crude extract of Arabidopsis using co-immunoprecipitation. The candidates found in this Co-IP experiment were analyzed by LC-MS/MS. They are clathrin binding protein (epsin), F-box/FBD/LRR-repeat protein, short-chain dehydragenase/reductase, Ferredoxin-dependent glutamate synthase 1, Glutamine synthetase, ATP synthase subunit alpha and Elongation factor 1-alpha. Further experiments are necessary to further investigate these putative interacting proteins.

並列關鍵字

ARGONAUTE ； Arabidopsis ； interactiing protein

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