Genetic Characterization and Molecular Cloning of Genes Regulating Developmental Phase Transition in Arabidopsis thaliana

Abstract

在擬南芥(Arabidopsis thaliana)中，我們分離並鑑定一個新的fld突變種對偶基因，命名為fld-2，而這個突變種表現出極度晚開花的性狀 (第三章)。fld-2基因突變所導致的晚開花性狀，可以透過低溫春化和GA處理方式來部份減輕其性狀表現，但是對於5-azaC的處理卻無影響(第三章)。經由遺傳雙突變體分析，顯示fld-2與其他五個晚開花突變種gi-1、ft-1、fwa-1、ld-1和fca-9均上位顯性於elf1、elf2和elf3三個早開花突變種 (第二章、第四章)。為了證明FLD基因在營養期到花序期及花序期到花器期所扮演的角色，我們選取這方面的突變種與fld-2突變種進行雙突變體的分析 (第四章、第五章)。由fld-2 tfl1雙突變體中減輕terminal flower 1 (tfl1)的性狀，和在fld-2 lfy、fld-2 ap1與fld-2 ap2雙突變體中加重leafy (lfy)、apetala1 (ap1) 與 apetala2 (ap2)的性狀，顯示FLD在花器的啟始及形成過程亦扮演著重要的角色 (第四章)。另外由fld-2 co-3雙突變體所產生的葉狀新型花器性狀，進一步提供證據支持FLD與CO這兩個晚開花基因不只參與營養期到花序期的移轉過程，並參與花器形成的階段 (第五章)。綜合這些實驗結果，強烈的表示FLD基因在調節莖頂的開花能力上扮演重要的角色，進而導致擬南芥中不同發育時期的移轉過程。FLD基因被定位在第三條染色體的上端，此區域完整的酵母(YAC)及細菌(BAC)人造染色體已被連結及鑑定出，現正進一步對於這些YAC及BAC clone的分析與互補試驗為選殖FLD基因的重要策略 (第六章)，而這方面的資訊將使我們更深入瞭解植物莖與花發育機制的調控過程。為了進一步探討早晚開花基因在擬南芥中啟始花器發育過程的調控機制，我們將AP1::GUS構築體送入不同的晚開花與emf1或emf2的雙突變體中以進行分析(第七章)。結果發現當GUS 的活性在emf1-1單一突變種中，可在植株萌芽後五天後被偵測到，而在雙突變體則表現上較微弱或是偵測不到。後續經由RT-PCR的實驗分析中顯示AP1與LFY基因在emf1-1單一突變種中大量表現，而在所有的晚開花與emf1-1雙突變種中的表現量明顯降低。此實驗結果證實AP1與LFY基因的啟動除了須有低的EMF活性外，還須受到晚開花基因的促進 (第七章)。A new fld mutant allele, fld-2, which significantly delayed flowering, was isolated and characterized in Arabidopsis thaliana (Chapter 3). The late-flowering phenotype of the fld-2 mutation could be partially overcome by both vernalization and GA treatment but it was not influenced by 5-azaC treatment (Chapter 2). Genetic double mutant analysis indicates that late-flowering mutants fld-2, gi-1, ft-1, fwa-1, ld-1 and fca-9 are epistatic to early flowering mutants elf1, elf2, elf3 in regulating the flower transition in Arabidopsis (Chapter 2 and 3). To confirm the role of FLD in the regulation of the rosette-to-inflorescence transition and inflorescence-to-flower transition, we constructed double mutant between fld-2 and genes responsible for establishing and maintaining the inflorescence and flower meristem (Chapter 4 and 5). The relief of the terminal flower 1 (tfl1) mutant phenotype in fld-2 tfl1 double mutants, and the enhancement of leafy (lfy) and apetala1 (ap1) and apetala2 (ap2) mutant phenotype in fld-2 lfy, fld-2 ap1, and fld-2 ap2 double mutants, suggest that FLD is also likely to be involved the floral initiation and floral formation (Chapter 4). The novel flower phenotype observed in fld-2 co-3 double mutants provides evidence to further support that FLD and CO are not only involved in rosette-to-inflorescence transition but also involved in the flower formation (Chapter 5). These results strongly suggest that the FLD gene plays a key role in regulating the reproductive competence of the shoot and results in different developmental phase transitions in Arabidopsis. FLD is map on the top arm of chromosome three. YAC and BAC contig containing YAC or BAC clones in this region was identified (Chapter 6). Further cloning of FLD by characterization of these BAC clones and complementation analysis is in progress and should lead to a deeper understanding of the mechanisms involved in shoot development. To further investigate the roles for flowering time genes in regulating the initiation of floral development in Arabidopsis, construct containing AP1::GUS was introduced into the different emf late flowering double mutants (Chapter 7). GUS activity was detected on shoot meristem of emf1-1 AP1::GUS single mutants 5 days after germination, but was weakly detected or stained negatively on various emf1-1 late-flowering double mutants. Further RT-PCR analysis indicated that AP1 and LFY were strongly expressed in emf1-1 single mutants and their expression was significantly reduced in all the emf1-1 late-flowering emf1-1 double mutants tested. Our results indicate that the expression of AP1 and LFY is dependent on the low EMF activity as well as on the activation of the late-flowering genes (Chapter 7).封面 目錄 第一章 緒言 －擬南芥的發育時期轉移過程 －開花基因在發育時期轉移的重要性 －已選殖出之晚開花基因之分子結構 －光週期調控的開花途徑與自發性調控的開花途徑 －開花起始過程之調控 －花器形成過程之調控 －植物發育時期的轉移與COPS理論 －參考文獻 第二章 早開花基因與晚開花基因在發育時期轉移過程的遺傳調控機制之探討 －摘要 －前言 －材料與方法 －結果 －討論 －參考文獻 第三章 晚開花突變fld-2之分離及特性分析 －摘要 －前言 －材料與方法 －結果 －討論 －參考文獻 第四章 晚開花基因FLD與其他調節開花過程基因間的遺傳調控機制制之探討 －摘要 －前言 －材料與方法 －結果 －討論 －參考文獻 第五章 FLD與CO兩個晚開花基因在營養期至花序與花器形成移轉過程間的遺傳調控機制之探討 －摘要 －前言 －材料與方法 －結果 －討論 －參考文獻 第六章 晚開花基因FLD之染色體定位與初步分子選殖 －摘要 －前言 －材料與方法 －結果 －討論 －參考文獻 第七章 EMF基因與晚間花基因共同作用以調控花器啟始基因的表現與功能 －摘要 －前言 －材料與方法 －結果 －討論 －參考文獻 第八章 結論 －未來展望 參考文