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研究生： 黎德君
Li, De-Jun
論文名稱： 扇形文心蘭重複性序列在中期染色體上的分布
The distribution of repetitive sequences on metaphase chromosomes of Erycina pusilla
指導教授： 張松彬
Chang, Song-Bin
學位類別： 碩士
Master
系所名稱： 生物科學與科技學院 - 生命科學系
Department of Life Sciences
論文出版年： 2016
畢業學年度： 104
語文別： 英文
論文頁數： 51
中文關鍵詞： 扇形文心蘭 、螢光原位雜合 、重複性序列 、核型
外文關鍵詞： Erycina pusilla, Fluorescence in situ hybridization (FISH), repetitive sequence, karyotype
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    • 蘭科(Orchidaceae)植物是高度演化並為顯花植物中最大的一科，具有優美多變的花型、花色與香氣，使其於高經濟園藝作物中佔有一席之地，特別是在東亞地區。扇形文心蘭(Erycina pusilla, 2n=10)是附生蘭中的一員，有多個優良的特性如基因組與染色體數目少、生殖世代短，成株個體較為矮小以及全年皆可開花的性質得以作為蘭科植物的模式物種。我們曾以全基因組片段的人工細菌染色體(Bacterial artificial chromosome, BAC)來做基因染色體定位(BAC chromosome mapping)，多發現在真染色質上非專一性的重複性訊號。所以，直至目前仍未有足夠的分子標誌得以辨明每一條染色體。本研究從 BAC 018-O-18 中分別找出三段不同的重複性序列(repetitive sequence, EpAG1-EpAG3)皆為扇形文心蘭 MADS 基因群的部分序列。利用螢光原位雜合(Fluorescence in situ hybridization, FISH)技術，發現多座落於所有染色體上的真染色質區域，並依據序列在每條染色體分布的不同，而建立出此蘭科模式生物的核型(karyotype)。將這些序列作為 BAC-FISH 的阻隔 DNA (blocking DNA)，雖然能夠隔絕大部分重複性序列的訊號，但仍無法於螢光原位雜合圖譜上獲得專一性信號。另外，
    我們以高度保守之 EpMADS8 M-domain 的部分序列作為探針，會以點狀訊號四散於染色體上。

    Orchidaceae is a highly evolved and largest angiosperm family which has a great diversity in floral morphology, color, size and fragrance being a valuable ornamental crop especially in eastern Asia. The orchid Erycina pusilla (2n=10) is an epiphytic Oncidiinae species and it has several advantageous properties, such as a relatively smaller genome size with low chromosome number, short generation time, small adult plant and blooming all year around, which makes it a potential model species in Orchidaceae family. So far there is no sufficient molecular markers to identify each chromosome. Here we report three repetitive sequences (EpAG1 - EpAG3) which were part of E. pusilla MADS gene family. With Fluorescence in situ hybridization (FISH) technique, we found those repetitive sequences scatter over euchromatic regions of all chromosomes. Based on the different patterns of the distribution of the studied repeats on all chromosome pairs, we constructed a karyotype for the chromosome complements of this potential orchid model. In addition, the pool of those repetitive sequences can be used as blocking DNAs to block repetitive DNA signals mostly but incapable of obtaining specific signals in BAC-FISH mapping. Besides, we used the partial length of highly conserved M-domain sequences of EpMADS8 gene as a probe in FISH. The hybridization signals of dots were scattered in every chromosome and this indicates E. pusilla MADS genes were located at many positions in all chromosomes.

    摘要....................................................I Abstract...............................................II 致謝..................................................III Index..................................................IV List of Tables.........................................VI List of Figures.......................................VII List of Supplements..........................................VIII Abbreviations..........................................IX Chapter 1 Introduction..................................1 1.1 Orchids.............................................1 1.1.1 Orchids...........................................1 1.1.2 Erycina pusilla...................................1 1.2 Cytogenetics studies on plants......................2 1.2.1 karyotyping.......................................2 1.2.2 Application of FISH in plant studies..............2 1.2.3 Cytogenetics studies on E. pusilla................3 1.2.4 FISH with BAC-based probes (BAC-FISH).............3 1.3 Repetitive sequences................................4 1.3.1 The fluorescenct signals of repetitive sequences by FISH....................................................4 1.3.2 The fluorescenct signals of repetitive sequences by FISH....................................................4 1.4 Purpose.............................................5 Chapter 2 Materials and Methods.........................6 2.1 Plant Materials.....................................6 2.2 Preparation of metaphase chromosome slide...........6 2.2.1 Liquid nitrogen method for chromosome prepaeation.6 2.2.2 Drop method for chromosome prepaeation............7 2.3 Preparation of DNA probe for FISH...................7 2.3.1 Extraction of Bacterial artificial chromosome (BAC) DNA.....................................................7 2.3.2 Extraction of plasmid DNA.........................8 2.3.3 Probe labeling with Nick translation kit..........8 2.3.4 Probe labeling with Polymerase chain reaction (PCR) method..................................................9 2.3.5 Quentify probe incorporation by DNA dot blot analysis...............................................10 2.4 Fluorescence in situ hybridization (FISH)..........10 2.5 Microscopy and image capture system................11 2.6 Subcloning of high repetitive sequences in BAC 018-O-18.....................................................12 2.6.1 Preparation of the plasmid vetcor................12 2.6.2 Preparation of highly repetitive sequences in BAC 018-O-18...............................................12 2.6.3 Ligation.........................................13 2.6.4 Transformation of heat-shock method..............13 2.7 Sequences analysis.................................13 Chapter 3 Results......................................14 3.1 Identification and subcloning of highly repetitive DNA sequences in BAC 018-O-18..........................14 3.2 FISH mapping of three repetitive sequences shared the same result that located at the whole range of euchromatic regions in all chromosomes.................14 3.3 The repetitive sequences could be used as blocking DNA....................................................15 3.4 BLASTn results of three repetitive sequences were high similarity with Erycina pusilla MADS genes........16 3. 5 Distribution of EpMADS gene family was globe location of chromosomes................................16 Chapter 4 Discussions..................................18 4.1 Benefits of using repetitive sequences for BAC-FISH in Erycina pusilla.....................................18 4.2 Three repetitive sequences (EpAG1, EpAG2 and EpAG3) showed similar distributed patterns in euchromatin regions................................................18 4.3 EpAG2-repeats, partial sequences of EpAG2 showed hybridization signals at the whole range of euchromatic regions in all chromosomes.............................19 4.4 MADS genes.........................................20 4.5 Conclusion.........................................21 Chapter 5 References...................................22 Chapter 6 Tables.......................................26 Chapter 7 Figures......................................33 Chapter 8 Supplements..................................46

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