博碩士論文 106821015 詳細資訊




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姓名 莊芳晴(Fang-Ching Chuang)  查詢紙本館藏   畢業系所 生命科學系
論文名稱 探討ETO1-LIKE1(EOL1)及EOL2參與阿拉伯芥幼苗光形態發育之功能
(Investigate the role of ETHYLENE OVERPRODUCER1-LIKE1 (EOL1) and EOL2 in seedling development during photomorphogenesis in Arabidopsis thalaiana)
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摘要(中) 在暗形態發育的期間,植物的幼苗呈現拉長的下胚軸及閉鎖的子葉。當光照時,幼苗會產生去白化現象,呈現光形態的發育,包括較短的下胚軸長度及子葉展開的情況。植物激素乙烯會抑制白化苗子葉的展開,但在光照後會促進子葉的綠化。先前研究得知ETHYLENE OVERPRODUCER1(ETO1)及ETO1-LIKE(EOL1/2)蛋白質為Cullin3 (CUL3)-based泛素連接酶,ETO1及EOL1/2藉由降解第2型ACS蛋白質,共同負向調控阿拉伯芥的乙烯生合成。在本研究中,我們發現在光暗轉換時,EOL1及EOL2在幼苗發育中扮演新穎的角色。在4天暗處理後,eol1及eol2單一或雙重突變株的白化苗呈現較高的子葉展開率,接著在轉移至2天全光照後,突變株幼苗則呈現較低的子葉綠化率。利用微矩陣法所進行的基因轉錄研究分析結果,我們發現在四天暗處裡下eto1、eol1及eol2白化苗中,數個參與光合作用的基因與野生種呈現顯著差異性的表現。其中,NADPH-原葉綠素酯氧化還原酶(PROTOCHLOROPHYLLIDE OXIDOREDUCTASE, POR) PORA及PORB為促進葉綠素合成的重要酵素,催化原葉綠素(protochlorophyllide, Pchlide)轉化成脫植醇葉綠素(chlorophyllide, Chlide)。我們發現在eol1及eol2中PORA/B的基因表現為下降,而且脫植醇葉綠素的量在eol1及eol2中亦隨之減少,但在eto1與野生種控制組並沒有顯著差異。此結果表示在光暗轉換時EOL1及EOL2促進白化苗的綠化,在光形態發生中扮演著異於ETO1的角色。為了探討EOL蛋白質在幼苗發育的新功能,我們利用酵母菌雙雜交法,由3天大白化苗所製備而成的阿拉伯芥之cDNA庫,篩選出與EOL1有交互作用的蛋白質。在數個可能的候選蛋白中,我們發現JASMONATE ZIM DOMAIN9(JAZ9)與EOL1有專一性的交互作用,JAZ蛋白質為茉莉酸訊息傳遞路徑中的負調控因子。進一步的研究結果顯示,同屬為第五型JAZ蛋白的JAZ3及JAZ9,皆能夠與ETO1、EOL1及EOL2在酵母菌雙雜交系統中有交互作用,而位於JAZ9之羧基端的Jas結構域呈現與EOL1蛋白質有專一的交互作用。本研究結果揭露,EOL蛋白質和茉莉酸之間可能存在著共同調控幼苗光形態發育訊息的交互作用。最後,我們提出一個假設模型説明EOL1和EOL2在幼苗發育時對光反應的可能調節機制。
摘要(英) When exposed to light, plant seedlings undergo de-etiolation development, termed photomorphogenesis, which exhibits reduced hypocotyl elongation and cotyledon opening followed by greening prepared for photosynthesis. The plant gaseous hormone ethylene inhibits cotyledon opening and reduces hypocotyl elongation in the dark-grown (etiolated) seedlings but promotes cotyledon greening during photomorphogenesis. The Arabidopsis ETHYLENE OVERPRODUCER1 (ETO1) and ETO1-LIKE (EOL1 and EOL2) proteins were previously identified as components of cullin3(CUL3)-based ubiquitin E3 ligase to negatively regulate ethylene biosynthesis by promoting protein degradation of type 2 ACC synthases (ACS). In this study, we reveal a novel role of EOL1 and EOL2 in seedling development during dark-to-light transition. We found that the 4-d-old etiolated seedlings of eol1 and eol2 single and double mutants showed a higher percentage of cotyledon opening but a lower cotyledon greening rate after being exposed to white light for 2 days. Microarray analysis of 4-d-old etiolated eto1, eol1 and eol2 seedlings showed several genes involved in photosynthesis, including the key enzymes essential for chlorophyll synthesis, PROTOCHLOROPHYLLIDE OXIDOREDUCTASE (POR) PORA and PORB that catalyze the conversion of protochlorophyllide (Pchlide) to chlorophyllide (Chlide), were repressed in eol1 and eol2 but not in eto1. In line with this finding, the accumulation of Chlide was reduced in the etiolated seedlings of eol1 and eol2 but not in eto1. Our data suggest that EOL1 and EOL2 may play a regulatory role in photomorphogenesis by facilitating greening of etiolated seedlings upon exposure to light. To investigate the functional role of EOL proteins, we sought to identify interacting proteins of EOL1 by screening an Arabidopsis cDNA library prepared from 3-d-old etiolated seedlings using the yeast two-hybrid (Y2H) system. Among several potential interacting proteins, we found that JASMONATE ZIM DOMAIN9 (JAZ9) interacts with EOL1. JAZ proteins act as negative regulators of JA signaling pathway in Arabidopsis. Further analysis indicated that the Jas domain at C terminus in JAZ9 is responsible for the specific interaction with EOL1 in yeast. In addition, JAZ3 and JAZ9, which belong to the group of type 5 JAZ proteins as JAZ9, can interact with ETO1, EOL1 and EOL2 by the Y2H assay. Our data suggest a potential relationship between EOL proteins and JA signaling pathway in seedling development responsive to light. Finally, we propose a hypothetical model to further study the underlying regulatory mechanisms of EOL1 and EOL2 in seedling development during photomorphogenesis.
關鍵字(中) ★ 阿拉伯芥
★ 光形態發育
★ 乙烯
★ 茉莉酸
關鍵字(英) ★ Arabidopsis thalaiana
★ Photomorphogenesis
★ Ethylene
★ Jasmonate
論文目次 目錄
中文摘要 i
英文摘要 ii
誌謝 iv
目錄 v
圖表目錄 vi
一、緒論 1
二、實驗材料與方法 16
三、實驗結果 16
1、鑑定eto1、eol1及eol2基因突變株 16
2、eto1、eol1及eol2白化苗呈現不同的下胚軸長度 16
3、在暗處理下,EOL1及EOL2參與調控子葉的展開 17
4、在光暗轉換處理下,EOL1及EOL2參與調控子葉的綠化 17
5、EOL1及EOL2調控PORA及PORB表現及葉綠素生成 18
6、利用酵母菌雙雜交法篩選與EOL1交互作用的蛋白 19
7、JAZ9140-267與EOL1有專一性之蛋白交互作用 20
8、全長JAZ3及JAZ9與ETO1、EOL1及EOL2有蛋白交互作用 20
9、JAZ9利用Jas區域與EOL1進行蛋白質交互作用 21
四、討論 23
五、參考文獻 27
六、附錄 59



圖表目錄
表一、基因功能分類(Gene Ontology)分析受EOL1及EOL2調控的基因分群 33
表二、基因功能分類分析受EOL1及EOL2調控的基因列表 34
表三、利用酵母菌雙雜交法篩選所得與EOL1有蛋白交互作用的基因列表 42
圖一、鑑定eto1, eol1 及 eol2的同型合子基因突變株 45
圖二、eto1, eol1及eol2突變株在三天暗處理下所呈現的下胚軸長度 46
圖三、eol1及eol2突變株在四天暗處理下所呈現子葉展開的性狀 47
圖四、eol1及eol2突變株在光暗轉換的處理下所呈現子葉黃化的性狀 48
圖五、利用基因微矩陣法(Microarray)分析四天暗處裡之eol1, eol2及eto1幼苗基
因表現變化 49
圖六、即時聚合酶鏈鎖反應(Real-time PCR)分析PORA、PORB及PORC在四
天暗處裡之eol1及eol2突變株中基因表現的變化 50
圖七、利用酵母菌雙雜交法篩選與EOL1交互作用的蛋白質 51
圖八、利用酵母菌雙雜交法檢測EOL1、EOL2及 ETO1與JAZ9140-267的蛋白質
交互作用 52
圖九、阿拉伯芥JAZ家族蛋白之親緣樹狀圖 53
圖十、利用酵母菌雙雜交法檢測ETO1、EOL1及EOL2與全長JAZ9及JAZ3之
蛋白質交互作用 54
圖十一、阿拉伯芥JAZ9與JAZ3及JAZ4的胺基酸序列比對 55
圖十二、利用定點突變法建構不同JAZ9蛋白結構域之突變序列 56
圖十三、利用酵母菌雙雜交法發現JAZ9利用Jas區域與EOL1進行專一之蛋白質
交互作用 57
圖十四、建立EOL1/2、JAZ3/4/9與PORA/B調控子葉開闔與綠化之假設模型 58
附錄一、測量帶有eol1或eol2背景突變株之Pchlide及Chlide含量 59
附錄二、ETO1、EOL1及EOL2具有高度相似的蛋白質結構 61
附錄三、質體pGBKT7-ETO1 62
附錄四、質體pGBKT7-EOL1 63
附錄五、質體pGBKT7-EOL2 64
附錄六、質體pGADT7-JAZ9 65
附錄七、植物體內的葉綠素生成途徑 66
附錄八、EIN3/EIL1和PIF1在調控子葉綠化中的作用模型 67
附錄九、ETO1、EOL1及EOL2之T-DNA插入位點圖 68
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指導教授 羅椀升 吳少傑(Wan-Sheng Lo Shaw-Jye Wu) 審核日期 2019-7-29
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