阿拉伯芥E3 ligases REDs 在生殖功能之研究

Function of Arabidopsis E3 ligases REDs in reproduction

李憶敏(Yi-Min Li)

指導教授 ： 鄭秋萍

國立臺灣大學/生命科學院/植物科學研究所/碩士(2017年)

https://doi.org/10.6342/NTU201700514

摘要

泛素化系統是生物體內相當重要之後轉譯修飾，在植物的許多功能皆是相當重要的調控機制，其中與目標蛋白結合之E3泛素接合酶 (E3 ligase) 更扮演極重要之角色，然而目前絕大多數之E3 ligase尚未被研究。本研究室先前發現在番茄與阿拉伯芥中有一群坐落於微管且功能未知之RING-H2 type E3 ligase，將其命名為RING-type E3 ligase with an uncharacterized DAR1 domain (REDs)；番茄SlRED1參與植物抵抗病原菌，且在花粉管萌發扮具正向調控功能；阿拉伯芥有三個REDs基因，其中AtRED1與AtRED2主要在花粉與精細胞表現。本研究旨在探討阿拉伯芥AtRED1/2之蛋白質特性、在生殖功能所扮演的角色及與微管相關蛋白之交互作用，並尋找其作用蛋白質，以利探討其功能與調控機制。實驗結果顯示，AtRED1/2皆具有E3 ligase活性並與E2具蛋白質交互作用，且RING domain之保守性胺基酸皆在其中扮演關鍵角色；AtRED1/2可形成homodimer與heterodimer且可能具有自我或彼此泛素化之能力。AtRED1與AtRED2之單基因突變株無生殖缺陷，但雙基因減量表現之植株其花粉粒活性與花粉管萌發率下降、果莢變短、種子發育不良、未受精胚珠比例提高，故有功能冗餘 (functional redundancy) 特性。本研究發現AtRED1/2可能與tubulins無直接交互作用，但與+TIPs AtEB1a/b具交互作用且AtEB1a/b可能為其泛素化對象，但目前尚未了解與其交互作用相關之生理功能。此外，以酵母菌雙雜交系統篩選AtRED1/2交互作用對象，但測試數個與生殖相關蛋白質後卻未能找到確切交互蛋白質。以上結果指出AtRED1/2為植物生殖功能之關鍵正向調控者，且可能藉AtEB1a/b間接座落於微管並泛素化AtEB1a/b。未來須進一步搜尋AtRED1/2之交互作用蛋白以釐清其作用機制。

關鍵字

RED ； 泛素化系統 ； E3 ligase ； 微管 ； EB1 ； 植物生殖

並列摘要

Protein ubiquitination is an important post-translation modification and involved in various plant functions. The E3 ligases play critical roles in specific recognition of the targets; however, most E3 ligases are not characterized. In previous studies, we identified a group of uncharacterized plant microtubule-localized RING-H2 type E3 ligases, named RING-type E3 ligase with an uncharacterized DAR1 domain (REDs). SlRED1 was involved in plants resistance to pathogens and played a positive role in pollen germination. There are three RED homologs in Arabidopsis and microarray analyses revealed predominant expression of AtRED1 and AtRED2 in pollens and sperm cells. This study aimed to further determine properties of AtRED1/2, their roles in reproduction and their interactions with microtubule-associated proteins. The results revealed an essential role of the conserved residue in the RING domain for AtRED1/2 E3 activity and their interactions with the E2 ligase. In addition, AtRED1/2 may form homodimer and heterodimer, and may self-ubiquitinate or ubiquitinate each other. Furthermore, double-knockdown lines of AtRED1 and AtRED2, but not single gene mutants, displayed significant reduction in pollen viability, pollen tube germination, siliques length and seed production, suggesting functional redundancy of AtRED1/2. Moreover, AtRED1/2 may not interact with tubulins, but interacted with AtEB1a/b, a group of microtubule plus end tracking proteins and may ubiquitinate AtEB1a/b. However, the physiological function of the AtRED1/2-AtEB1a/b interactions remaine to be determined. In addition, a yeast-two-hybrid systemic screening for AtRED1/2-interacting proteins failed to identify candidates related to reproduction. Together these results uncover the critical positive role of AtRED1/2 in regulating reproduction and reveal the AtRED1/2-AtEB1a/b interactions. Further identification and study of AtRED1/2 interacting proteins would help to elucidate the underlying regulatory mechanisms for their functions.

並列關鍵字

RED ； ubiquitination ； E3 ligase ； microtubule ； EB1 ； reproduction

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