- 學位論文
阿拉伯芥 E3 ligases REDs 在有性生殖功能之研究
Functions of Arabidopsis E3 ligases REDs in sexual reproduction
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摘要
泛素化系統在許多生理功能具重要調控功能，而與目標蛋白高度專一結合的E3 泛素接合酶(E3 ligase) 更扮演關鍵角色，然而目前絕大多數之E3 ligases 皆未被研究。本研究室先前發現一群坐落於微管且功能未知的植物RING-H2 type E3ligases，將其命名為RING-type E3 ligase with an uncharacterized DAR1 domain(REDs)。阿拉伯芥AtRED1 與AtRED2 之RING domain 的保守位點為其E3 ligase活性及與E2 結合之必需片段，且AtRED1 與AtRED2 可能藉由其C 端形成homodimer 或heterodimer。此外，在atred2 null mutants 中以RNAi 方式將AtRED1表現靜默之 red1i/red2 轉殖植株之花粉粒活性下降、花粉管萌發率下降、果莢較短、種子發育不良、未受精胚株比例提高。另外，AtRED1 與AtRED2 可能藉由與微管相關蛋白+TIPs AtEB1s 之交互作用而坐落於微管上。本研究旨在了解AtRED1 與AtRED2 以及其交互作用蛋白質在有性生殖之功能。Promoter:GUS 分析結果顯示AtRED1 與AtRED2 表現在幼苗根部、蓮座葉、花萼及花瓣之維管束，且在成熟花粉有更高之表現。互交試驗結果顯示red1i/red2 植株其雄性與雌性配子發育皆有缺陷。然而，雙基因突變株red1/red2 之初步分析結果卻顯示突變株之種子缺陷與AtRED1/2 雙基因表現缺失可能並無一致相關性，此外，以red1i/red2-1 為材料進行AtRED1::AtRED1gDNA-GFP 互補試驗卻發現 AtRED1 之過量表現與植株之種子發育回復性狀並無一致相關性，故AtRED1 與 AtRED2 是否確實在有性生殖具有重要角色實有疑慮。而AtRED1 與AtRED2 之互作蛋白AtEB1a 與AtEB1b，本研究之初步結果推測AtEB1a/b 在轉錄層次之過量表現與其生殖性狀並無一致關聯性，但AtRED1/2 是否可能藉由調控 AtEB1a/b 之蛋白質累積而影響微管動態，並進而其他植物生理功能，則尚需進一步研究。此外，依據酵母菌雙雜交與雙分子螢光互補試驗結果推測選自阿拉伯芥花cDNA library 之數個生殖相關蛋白質可能與AtRED1/2 並無交互作用。此外，本研究搜尋到具序列高保守性之水稻與玉米之功能未知REDs。未來可利用目前所得之各式重要材料與資源，在各種條件下觀察植物各部位中細胞微管分布與動態，以釐清植物這些保守泛素化酵素之具體功能與調控機制。
並列摘要
Protein ubiquitination is a central regulatory mechanism in controlling key cellular 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, namely RING-type E3 ligase with an uncharacterized DAR1 domain (REDs). A conserved residue in RING domain is essential for the E3 activity of Arabidopsis thaliana RED 1 (AtRED1) and RED2 (AtRED2) and for their interaction with an E2 enzyme. AtRED1 and AtRED2 may form homodimer or heterodimer through the C terminus. In addition, AtRED1-RNAi transgenic plants in the background of AtRED2-knockout mutants (red1i/red2), but not the single-gene mutants, displayed significant reduction in pollen viability, pollen tube germination, siliques length and seed production. Furthermore, AtRED1 and AtRED2 are localized on the microtubule (MT), probably via their interactions with AtEB1s, a group of MT plus end tracking proteins. This study aimed to further determine functions of AtRED1 and AtRED2 and their interacting proteins in sexual reproduction. Results of spatiotemporal transcriptional analyses using promoter-GUS assays showed that AtRED1 and AtRED2 are ubiquitously expressed with at very high levels in mature pollens. In addition, red1i/red2 plants displayed defects in both male and female gametogenesis. However, the current data showed that the seed phenotypes of AtRED1/2 double mutants (red1/red2) and red1i/red2 plants overexpressing AtRED1 might not correlate with the expression levels of these genes. Therefore, further studies are required to determine whether AtRED1 and AtRED2 play a role in sexual reproduction. Moreover, since AtRED1/2 interacted with AtEB1a/b, and may thus modulate MT dynamics, whether AtRED1/2 are involved in other plant functions such as stress responses, as what revealed by our study on tomato SlRED1, remained to be investigated. Furthermore, RED homologs are identified in rice and maize. Further study on the MT dynamic in different tissues of our collected plants and under various conditions can help elucidate the functions and underlying regulatory mechanisms of these conserved REDs.
參考文獻
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