番茄microtubule-associated RING E3 ligase 1之功能分析

Functional characterization of tomato microtubule-associated RING E3 ligase 1

蔡詠竹(Yung-Chu Tsai)

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(2012年)

https://doi.org/10.6342/NTU.2012.10623

摘要

我們先前發現一功能未知之番茄C3H2C3 type RING E3 ubiquitin ligase，將之命名為SlREL1 (Solanum lycopersicum RING E3 Ligase 1)。SlREL1可能參與番茄抗青枯病品系H7996之防禦反應，其表現在花與熟果較高，且其蛋白產物可能坐落在細胞骨架上。本研究旨在進一步探討SlREL1與其同源基因之蛋白質特性及功能。序列比對結果發現番茄有另一REL基因(SlREL2)；此兩蛋白質在其C端除了保守的RING domain外，也同時有一序列保守但功能未知的DAR1 domain (domain associated with RING 1)。SlREL1被預測具有一進核訊號與許多可能可被激酶進行磷酸化的區域。微管螢光標定物與微管聚合抑制劑試驗確認SlREL1坐落在微管網路上，且其胺基酸片段1-170為必要區段。In vitro self-ubiquitination assay證實SlREL1具有E3 ligase活性，且RING和DAR1 domains在其活性上具關鍵角色。SlREL1-RNAi轉殖番茄之青枯病抗性提高，且其花粉萌發情形較為遲緩，而過量表現SlREL1的轉基因菸草其青枯病病程發展則略為提早。根據短暫基因靜默與過量表現試驗結果初步推測SlREL1在乾旱、鹽害及氧化逆境反應也許不具關鍵功能。此外，阿拉伯芥有三個REL基因(AtREL1、AtREL2及AtREL3)，且其蛋白質產物在C端亦具有保守的RING domain與DAR1 domain；阿拉伯芥微陣列資料顯示AtREL1/2之轉錄表現在花粉中最高且會受許多病害與非生物性逆境因子調控，而AtREL3表現則較不受這些因子影響。此外，AtREL1也在細胞中呈現絲狀分布，且亦具有E3 ligase活性。這些結果指出SlREL1為一坐落在微管上之E3 ligase，其RING與DAR1 domain皆參與其酵素活性之維持，且阿拉伯芥AtREL1亦具有與SlREL1類似的蛋白質特性。SlREL1可能參與花粉萌發與青枯病反應。

關鍵字

泛素化系統 ； E3 ligase ； 微管 ； 青枯病 ； 花粉

並列摘要

Previously, an uncharacterized tomato C3H2C3 type RING E3 ubiquitin ligase was identified and namely SlREL1 (Solanum lycopersicum RING E3 Ligase 1). SlREL1was suggested to be involved in tomato response to bacterial wilt (BW), abundantly expressed in tomato flowers and mature fruits, and its protein product co-localized with the cytoskeleton. This study aimed to further investigate the protein properties and the biological functions of SlREL1 and its homologs in tomato and Arabidopsis. A paralog of SlREL1 was identified in tomato and named SlREL2; In addition to the RING domain, these RELs contain a conserved DAR1 (domain associated with RING1) domain without known functions. Bioinformatic analysis suggested that SlREL1 may contain a nucleus localization signal and several phosphorylation sites. SlREL1 was found to be localized on the microtubule via an unknown domain located at the region of amino acid 1~170. In vitro self-ubiquitination assay verified that SlREL1 conferred E3 ligase activity, and its RING and DAR1 domains were important for the activity. SlREL1-RNAi transgenic tomato plants conferred enhanced resistance to BW and its pollen germination was delayed, while transgenic Nicotiana benthamiana plants over-expressing SlREL1 displayed slightly increased susceptibility to BW. Furthermore, transient gene silencing and overexpression assays suggested that SlREL1 might not play an important role in tomato response to drought, salinity and oxidative stress. three Arabidopsis RELs were identified and named AtREL1, AtREL2 and AtREL3. Available Arabidopsis microarray data revealed that AtREL1/2 abundantly expressed in pollen grains and could be induced by various biotic and abiotic stresses, while AtREL3 did not exhibit similar tissue-specific and stress-inducible expression patterns. In addition, AtREL1 shared similar sub-cellular localization pattern and enzyme properties with SlREL1. Together, these results point out that SlREL1, as a novel microtubule-localized E3 ligase, is involved in pollen development and BW response.

並列關鍵字

ubiquitination ； E3 ligase ； microtubule ； bacterial wilt ； pollen

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