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系統識別號 U0026-1312201215463400
論文名稱(中文) HAC1與PIE1在阿拉伯芥以及蝴蝶蘭上對開花影響的功能性分析
論文名稱(英文) Functional characterization of HAC1 and PIE1 in Arabidopsis thaliana and Phalaenopsis aphrodite
校院名稱 成功大學
系所名稱(中) 熱帶植物科學研究所
系所名稱(英) Institute of Tropical Plant Sciences
學年度 101
學期 1
出版年 101
研究生(中文) 林慧玲
研究生(英文) Hui-Ling Lin
學號 z36991029
學位類別 碩士
語文別 中文
論文頁數 84頁
口試委員 指導教授-詹明才
召集委員-劉景煌
口試委員-郭瑋君
口試委員-廖麗貞
中文關鍵字 蝴蝶蘭  開花時間  HAC1  PIE1  逆境  H2O2  維生素C 
英文關鍵字 Phalaenopsis  flowering time  HAC1  PIE1  ROS  H2O2  Vitamin C 
學科別分類
中文摘要 在前人研究中發現阿拉伯芥MsrB7 (methionine sulfoxide reductase B7) 會參與抵抗巴拉刈 (methyl viologen, MV) 的相關機制 (Li et al., 2012)。當轉殖基因MsrB7 OX使表現量增加時發現轉殖株體內的ROS累積含量會降低,且有晚開花的外表型,從Affymatrix microarray資料中,顯示開花相關途徑中的開花負調控重要基因FLC (flowering locus C) 表現量高達79倍。我們在FLC的上游中找到兩個基因,分別為自我調控開花途徑中的HAC1 (HISTONE ACETYLTRANSFERASE OF THE CBP FAMILY 1) 和PIE1 (PHOTOPERIOD- INDEPENDENT EARLY FLOWERING 1) 都有明顯的受到ROS所調控,我們推測當ROS含量下降時,FLC表現量的上升是經由HAC1和PIE1所調控。當阿拉伯芥外加H2O2和維生素C時發現能有效影響植株的開花,表示ROS確實會影響植物的開花時間。 接著我們利用突變株來研究ROS對於開花的影響,從中發現pie1基因突變後需在短日照的環境下較有明顯的提早開花現象,當hac1突變後處理H2O2對開花的影響變敏感。 進一步我們在蝴蝶蘭 (Phalaenopsis) 中找到這兩個相關基因,命名為PaHAC1及PaPIE1,其cDNA全長分別為5714 bp 和7407 bp。 在親源演化分析顯示PaHAC1相似於其他物種的HAC1,而不是其他的HACs 同源性基因,PaPIE1則相近於單子葉植物的PIE1。從即時定量PCR偵測兩個基因在蝴蝶蘭各組織部位的表現狀況顯示,PaHAC1在開花後的葉片、花梗及花部位表現較高,可能和花梗的抽長和花苞的發育都有調控,PaPIE1在營養生長和生殖生長的表現量則無明顯差異。利用VIGS (virus induced gene silencing) 試驗降低基因在蝴蝶蘭中的表現,發現PaHAC1基因調控花梗的抽長和花苞的成熟相關,PaPIE1在外表型並沒有明顯的影響,或許PaPIE1在蝴蝶蘭中還有其他的功能,對於開花時間得調控並不是最主要的影響因子。綜合以上結果,我們證實了在阿拉伯芥HAC1對於ROS影響開花時間具有重要的調控,並且在蝴蝶蘭中HAC1參與開花時間的調控。
英文摘要 In Arabidopsis, Methionine sulfoxide reductase B7 (MsrB7) had been resistant to Methy viologen (MV) (Li et al., 2012). Arabidopsis plants overexpressing MsrB7 reduce the accumulation of reactive oxygen species (ROS), and delay the flowering time. Microarray assays were performed to examine MsrB7 regulated genes, the results showed that while overexpressing MsrB7 resulted in 79-fold increase in expression level of FLOWERING LOCUS C (FLC), which acts as a floral repressor. Two upstream gene of FLC, HISTONE ACETYLTRANSFERASE OF THE CBP FAMILY 1 (HAC1) and PHOTOPERIOD- INDEPENDENT EARLY FLOWERING 1 (PIE1), were obviously regulated by ROS. We expect that when the content of ROS is decreasing, the increasing expression level of FLC may be regulated by HAC1 and PIE1. We treated Arabidopsis plants with additional H2O2 and Vitamin C can be significantly affected with flowering time. We used the mutant line of HAC1 is difficult to treat with ROS to identify the relationship between ROS and flowering. The results showed that hac1 mutant line showed early flowering phenotype and more sensitive to H2O2 treatment as compared to wild type. We isolated HAC1 and PIE1 in Phalaenopsis , with 5714 bp and 7404 bp of full-length of cDNA , respectively. Phylogenetic showed that PaHAC1 was close related to HAC1-like genes in other species rather than its orthology gene, PaPIE1 was close related to PIE1-like genes of monocots rather than those of dicots. Real-time PCR analysis revealed that PaHAC1 showed stronger expression in floral organs and leaves after flowering, which may be due to PaHAC1 involved in development of floral buds and stalks. PaPIE1 did not show differentiation between vegetative growth and reproductive growth. Virus induced gene silencing (VIGS) assays were performed to decrease the expression of target gene, the results showed that PaHAC1 were involved in floral development by delaying flowering time, but the phenotype did not show obvious change in PaPIE1 transgenic plants, indicating that PaPIE1 may not be a major regulator of flowering time. In conclusion, Arabidopsis HAC1 involve in flowering time in Arabidopsis under ROS stress. PaHAC1 play some role in Phalaenopsis flowering time.
論文目次 中文摘要I
英文摘要II
誌謝IV
目錄V
表目錄VIII
圖目錄IX
前言1
1. 植物體內活性氧化物ROS對開花作用的影響2
2. 植物開花基因的調控3
3. FLC的上游基因HAC15
4. FLC的上游基因PIE1 6
5. ROS促進開花在花卉的應用6
材料與方法8
一、材料8
二、實驗方法9
1. Hydrogen Peroxide 染色9
2. 阿拉伯芥 total RNA 的純化9
3. RNA 電泳10
4. 反轉錄聚合酶連鎖反應 (Reverse transcription PCR) 10
5. 即時定量PCR (quantitative real-time PCR)11
6. 蝴蝶蘭 total RNA 的純化12
7. 蝴蝶蘭基因序列的選殖13
8. 序列分析16
9. 蝴蝶蘭之反轉錄聚合酶連鎖反應 (RT-PCR)16
10. 轉型試驗 (transformation) 17
11. 快速萃取質體DNA18
12. 蝴蝶蘭total DNA的萃取19
13. 南方轉漬法 (Southern blot)20
14. 蝴蝶蘭VIGS (virus induced gene sciencing) 試驗22
15. 統計分析25
結果26
在表達 MsrB7 OX及 MsrB7 RNAi植物中發現開花時間受到影響26
MsrB7 OX及 MsrB7 RNAi轉殖植物體內 ROS含量累積受到影響26
外加H2O2及維生素C影響阿拉伯芥開花時間26
利用 Affymatrix microarray分析 MsrB7OX和 MsrB7 RNAi及野生型 植株中開花時間相關的基因表現情形28
延緩開花的突變株hac1噴灑H2O229
外加20 mM H2O2及50 mM VitC會影響HAC1和FLC的表現29
蝴蝶蘭PaHAC1 cDNA的選殖30
蝴蝶蘭PaHAC1基因檢測30
蝴蝶蘭PaHAC1親源演化分析31
蝴蝶蘭PaPIE1 cDNA的選殖31
蝴蝶蘭PaPIE1基因檢測32
蝴蝶蘭PaPIE1親源演化分析32
蝴蝶蘭PaHAC1以及PaPIE1基因表現分析33
蝴蝶蘭PaHAC1以及PaPIE1基因進行VIGS33
討論35
MsrB7 OX及 MsrB7 RNAi會受到植物體內ROS含量而影響開花時間35
外加H2O2及VitC影響阿拉伯芥開花時間36
ROS會調控開花基因使誘導下游基因造成植物提早開花37
於hac1突變株噴灑H2O2會使植株提前開花,且對ROS更敏感38
外加H2O2及維生素C影響HAC1和FLC的表現39
蝴蝶蘭PaHAC1之基因選殖及特性分析39
PaHAC1在蝴蝶蘭內的親源演化分析40
PaHAC1基因表現分析及生物功能40
蝴蝶蘭PaPIE1之基因選殖及特性分析41
PaPIE1在蝴蝶蘭內的親源演化分析42
PaPIE1基因表現分析及生物功能42
蝴蝶蘭開花後續研究及未來展望44
圖表45
參考文獻72
附錄82
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