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研究生:姜智益
研究生(外文):Zhi-Yi Jiang
論文名稱:蝴蝶蘭基因PhFYF1與PhFYF2異位表現於阿拉伯芥中之功能性分析與影響
論文名稱(外文):Characterization and functional analysis of PhFYF1 and PhFYF2 genes of Phalaenopsis in Arabidopsis
指導教授:楊長賢楊長賢引用關係
指導教授(外文):Chang-Hsien Yang
口試委員:李勇毅王強生
口試委員(外文):Yung-Yi LiChiang-Sheng Wang
口試日期:2017-07-20
學位類別:碩士
校院名稱:國立中興大學
系所名稱:生物科技學研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2017
畢業學年度:105
語文別:中文
論文頁數:46
中文關鍵詞:阿拉伯芥蝴蝶蘭乙烯老化脫落
外文關鍵詞:Arabidopsis thalianaPhalaenopsisEthyleneSenescenceAbscissionFOREVER YOUNG FLOWER
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已知MADS-box基因FOREVER YOUNG FLOWER (FYF)為一阿拉伯芥中調控花朵老化脫落的轉錄抑制子,當FYF在阿拉伯芥中進行異位表現時,會抑制乙烯訊息傳遞路徑下游基因EDFs (ETHYLENE RESPONSE DNA BINDING FACTOR),和離層形成相關基因BOP1/2 (BLADE-ON-PETIOLE)與IDA (INFLORESCENCE DEFICIENT IN ABSCISSION)的表現,導致阿拉伯芥基因轉殖株的花朵延遲老化凋落。本研究利用AtFYF胺基酸序列作為模版進行tblastn,從蝴蝶蘭V3大白花中找到序列相似的兩個AtFYF同源基因PhFYF1/2。在V3蝴蝶蘭中,PhFYF1/2主要表現在1公分的小花苞中。為了進一步探討PhFYF1/2的主要功能,利用35S 啟動子在阿拉伯芥中異位表現PhFYF1/2、PhFYF1/2+SRDX(含強抑制區域)和PhFYF1/2+VP16(含強激活區域)。結果發現異位表現PhFYF1/2、PhFYF1/2+SRDX和PhFYF1/2+VP16之阿拉伯芥基因轉殖株都會促進開花基因FT (FLOWER LOCUS T)表現而提早開花。並且在35S::PhFYF1/2、35S::PhFYF1/2+SRDX阿拉伯芥基因轉殖株中都有花朵延緩老化脫落的性狀出現。即使在經過外源性乙烯處理後,基因轉殖株仍然具有延緩花朵老化脫落的性狀,表示其為乙烯不敏感型植株。利用即時定量 PCR分析基因表現量後發現,在35S::PhFYF1/2 和 35S::PhFYF1/2+SRDX阿拉伯芥基因轉殖株中,乙烯訊息傳遞路徑下游與離層形成相關基因ERF1 (ETHYLENE RESPONSE FACTOR1)、EDF1/2/3/4、BOP1/2和IDA的表現量都有不同程度的抑制情形。從上述結果得知,PhFYF1/2在阿拉伯芥中應該和AtFYF一樣,作為一個轉錄抑制子去調控花朵的老化與脫落。未來會進一步探討PhFYF1/2在V3蝴蝶蘭中所扮演的角色,透過短暫大量表現和VIGS技術分析PhFYF1/2的功能與作用機制。藉由這些試驗,可以更了解PhFYF1/2在調控蘭花老化脫落過程中的主要功能。
MADS-box gene FOREVER YOUNG FLOWER (FYF) had been reported to be a transcriptional repressor that can regulate flower senescence and abscission. Ectopic expression of FYF significantly delayed flower senescence and abscission by repressing ethylene signaling pathway downstream gene EDFs (ETHYLENE RESPONSE DNA BINDING FACTOR), and abscission-associated genes BOP1/2 (BLADE-ON-PETIOLE) and IDA (INFLORESCENCE DEFICIENT IN ABSCISSION) in transgenic Arabidopsis. In this study, protein sequence of AtFYF was used for tblastn. Two AtFYF homologous PhFYF1/2 were identified from Phalaenopsis Sogo Yukidian V3. In P. Sogo Yukidian V3, PhFYF1/2 major expressed in the 1 cm flower bud. To explore the function of PhFYF1/2, 35S promoter was used to ectopic express PhFYF1/2, PhFYF1/2+SRDX (containing a strong repressor motif) and PhFYF1/2+VP16 (containing a activation domain) in transgenic Arabidopsis. Early flowering caused by promoting flowering gene FT was observed when ectopic expressed PhFYF1/2, PhFYF1/2+SRDX and PhFYF1/2+VP16 in transgenic Arabidopsis. Delaying flower senescence and abscission were observed in 35S::PhFYF1/2 and 35S::PhFYF1/2+SRDX transgenic Arabidopsis. After exogenous ethylene treatment, the delaying flower senescence and abscission were still found in transgenic Arabidopsis, It means the transgenic Arabidopsis were ethylene insensitive. The expression of ethylene signaling pathway downstream genes ERF1 (ETHYLENE RESPONSE FACTOR1), EDF1/2/3/4 and abscission associated genes BOP1/2 and IDA were repressed in 35S::PhFYF1/2 and 35S::PhFYF1/2+SRDX transgenic Arabidopsis by real-time PCR. These results indicated that PhFYF1/2 may play as a transcriptional repressor in regulating flower senescence and abscission in Arabidopsis. In the future, the role of PhFYF1/2 in P. Sogo Yukidian V3 and the mechanism will be further studied by transient expression assay and VIGS analysis. By these experiments, the function of PhFYF1/2 in regulating orchids flower senescence and abscission will be well understood.
摘要 i
Abstract ii
目錄 iii
前言 1
材料方法 4
結果 13
討論 17
參考文獻 21
圖表附錄 25
表1、本論文所使用之引子(Primer)列表 25
表2、異位表現PhFYF1/2、PhFYF1/2+SRDX和PhFYF1/2+VP16轉基因植物開花時間及葉片數統計 27
圖1. 阿拉伯芥(Arabidopsis thaliana)與蝴蝶蘭(Phalaenopsis Sogo Yukidian V3 )中FYF基因的親緣演化樹狀圖 28
圖2. 阿拉伯芥與V3蝴蝶蘭中FYF基因之胺基酸序列相似度比對 29
圖3. PhFYF1/2在V3蝴蝶蘭花朵各時期和各部位之表現情況 30
圖4. 目標基因構築流程電泳圖 31
圖5. 異位表現PhFYF1/2和PhFYF1/2+SRDX與PhFYF1+VP16導致基因轉殖株提早開花 32
圖6. 異位表現PhFYF1和PhFYF1+SRDX導致基因轉殖株出現花朵延緩老化脫落的性狀 33
圖7. 異位表現PhFYF2和PhFYF2+SRDX導致基因轉殖株出現花朵延緩老化脫落的性狀 34
圖8. 異位表現PhFYF1/2、PhFYF1/2+SRDX之基因轉殖株為乙烯不敏感型植株 35
圖9. 異位表現PhFYF1和PhFYF1+SRDX之基因轉殖株中,離層形成相關基因IDA和乙烯訊息傳遞路徑下游基因ERF1、EDF1/2/4表現量受到抑制 36
圖10. 異位表現PhFYF2和PhFYF2+SRDX之基因轉殖株中,離層形成相關基因BOP1/2和乙烯訊息傳遞路徑下游基因ERF1、EDF2/3/4表現量受到抑制 37
圖11. 異位表現PhFYF1/2、PhFYF+SRDX於阿拉伯芥時,乙烯訊息傳遞路徑下游基因ERF1、EDF基因群以及離層形成相關基因BOP1/2和IDA的表現情形會受到抑制而導致植株花朵延緩老化及脫落。 38
附圖1. 利用AtFYF胺基酸序列為模版,於蘭花基因資料庫Orchidstra 2.0進行tblastn找到之AtFYF相似序列PATC136427和PATC154491 39
附圖2. PhFYF1核苷酸及胺基酸序列 40
附圖3. PhFYF2核苷酸及胺基酸序列 41
附圖4. pGEM®-T Easy vector圖譜 42
附圖5. pEpyon-22K之圖譜 43
附圖6. pEpyon-2aK之圖譜 44
附圖7. pEpyon-2bK之圖譜 45
附圖8. Gen-KB DNA Ladder 46
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