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研究生:周筱琦
研究生(外文):Hsiao-ChiChou
論文名稱:原生種蝴蝶蘭EFS基因之染色體定位與基因序列之分析
論文名稱(外文):Chromosome localization of early flowering in short days (EFS) gene and sequence analysis on Phalaenopsis orchids
指導教授:吳文鑾
指導教授(外文):Wen-Luan Wu
學位類別:碩士
校院名稱:國立成功大學
系所名稱:生命科學系碩博士班
學門:生命科學學門
學類:生物學類
論文出版年:2012
畢業學年度:100
語文別:中文
論文頁數:63
中文關鍵詞:蝴蝶蘭EFS基因粗絲期螢光原位雜合
外文關鍵詞:Phalaenopsis speciesearly flowering in short days (EFS)pachytenefluorescence in situ hybridization
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開花為植物繁衍後代,延續生命週期的重要階段。在模式植物阿拉伯芥(Arabidopsis thaliana)研究中,開花過程由四條途徑所控制:光週期、春化作用、吉貝素及自發性調控途徑,此外還有FRI (FRIGIDA)調控途徑,EFS (early flowering in short days)和其它蛋白形成PAF1相似蛋白複合體,透過組織蛋白甲基化的表觀調控方式共同影響目標基因FLC的表現,影響開花時間。蝴蝶蘭為國內重要經濟花卉之一,但其幼年期長,業界常利用催花方式調控花期,因此研究蝴蝶蘭開花基因將有助於產業應用。本實驗室利用BAC end定序,發現BAC選殖株Pe-NCKU-HBAC-1031M24末端序列具有阿拉伯芥EFS基因相似之片段。將此BAC DNA做為探針,利用螢光原位雜合技術(fluorescence in situ hybridization, FISH)進行定位。在姬蝴蝶蘭及台灣阿嬤蝴蝶蘭粗絲期染色體中皆具有單一的訊號且靠近於染色體長臂末端。EFS基因訊號與姬蝴蝶蘭染色體端點間的距離為1.13 μm;台灣阿嬤蝴蝶蘭則為1.28 μm。接下來以454高通量定序分析(454 pyrosequencing),得到14個序列長度不同之連續體(contig),利用軟體連接得到姬蝴蝶蘭PeEFS (Phalaenopsis equestris EFS)基因全長約為51 kb,包含17個exon,譯碼區為5,802 bp,可轉譯出1,934個胺基酸。以此PeEFS基因序列設計引子對,於臺灣阿嬤蝴蝶蘭選殖定序後得到譯碼區5,799 bp及1,933個胺基酸,命名為PaEFS (Phalaenopsis aphrodite EFS)。比較此兩物種胺基酸,序列相似度高達97.1%,皆具有CW、AWS、SET及Post-SET 四個domain。將姬蝴蝶蘭PeEFS與已發表之EFS基因族系比對,相似程度只有22.5%-25.4%。分子親緣關係之分析顯示PeEFS、PaEFS及水稻SDG8位於同一個分類枝(clade)。由於EFS基因cDNA序列在台灣阿嬤及姬蝴蝶蘭間相似度極高,進一步分析蝴蝶蘭屬內不同亞屬且不同季節開花之蝴蝶蘭功能性區域相似度分析,結果顯示無法將不同季節開花之蝴蝶蘭進行分群。另外,分析蝴蝶蘭屬EFS胺基酸序列與阿拉伯芥SDG功能性家族分子親緣關係,顯示蝴蝶蘭屬皆歸類在阿拉伯芥EFS基因所屬SDG功能性家族class Ⅱ中。綜合以上,EFS基因序列在屬內相似度是非常保守的,而單一的EFS基因訊號可做為辨識姬蝴蝶蘭及台灣阿嬤蝴蝶蘭的染色體標誌。
Flowering is a crucial stage in plant life cycle for reproduction. Previous studies in Arabidopsis thaliana have revealed four major pathways to regulate flowering: the photoperiod, vernalization, gibberellins dependent and autonomous pathways. Besides the four major pathways, FRI (FRIGIDA) pathway may also participate in flowering regulation. In Arabidopsis FRI pathway, PAF1-like (RNA polymerase II associated factor 1) complex and histone methyltransferase EFS (early flowering in short days) can induce FLC gene expression and further delay flowering. Phalaenopsis orchid is one of the most important commercial floral crops in Taiwan, but usually has a long juvenile period. Therefore, studying the flowering genes of orchid will contribute to orchid industry for flowering time regulation. In previous research, a partial sequence from BAC end sequence in P. equestris BAC clone Pe-NCKU-HBAC-1031M24 was with high similarity to Arabidopsis EFS gene. To investigate the chromosomal location of EFS gene in P. equestris and P. aphrodite subsp. formosana, EFS gene-containing BAC DNA together with either 45S rDNA or telomere DNA were used as probes in fluorescence in situ hybridization (FISH). The FISH results showed that EFS gene had a single signal which was close to telomere on both P. equestris and P. aphrodite subsp. formosana chromosome. The distance between EFS signal and telomere was about 1.13 μm and 1.28 μm in P. equestris and P. aphrodite subsp. formosana, respectively. These results suggested that EFS can be served as a chromosome-specific marker in P. equestris and P. aphrodite subsp. formosana karotyping. Furthermore, EFS gene-containing BAC clone was sequenced by 454 pyrosequencing and assembled into 14 contigs. The structure of EFS gene is composed of 17 exons in both P. equestris and P. aphrodite subsp. formosana. The EFS cDNA sequences derived from the BAC clone and P. equestris genome are both 5,802 bp in length, encoding 1,934 amino acids, while the length in P. aphrodite is 5,799 bp, encoding 1,933 amino acids, named PaEFS. The identity of EFS amino acid sequence derived BAC clone with that of P. equestris and P. aphrodite subsp. formosana was 99% and 97.1%, respectively. The published EFS gene amino acid sequences of other species were 22.5-25.4% identical with that of P. equestris. Molecular phylogenetic analysis revealed that PeEFS was clustered together with other monocot species. All amino acid sequences of EFS gene contained the conserved CW, AWS, SET and post-SET domains. Phylogenetic analysis based on amino acid sequences of EFS functional domains among eight Phalaenopsis species showed that the clustering was in accordance with subgenus. Moreover, EFS genes of eight Phalaenopsis species were clustered in SDG family class II by comparison of Arabidopsis SET domain group family. In conclusion, EFS gene was highly conserved among Phalaenopsis species and single locus of EFS gene can be used as a marker for chromosome identification in Phalaenopsis species.
中文摘要 i
英文摘要 ii
誌謝 iii
目錄 iv
表目錄 vi
圖目錄 vii
附錄目錄 viii
縮寫字對照表 ix

第一章 前言 1
一、開花時間訊息傳遞路徑之調控 1
二、FRI訊息傳遞路徑 1
三、EFS基因的表現特性分析 2
四、蝴蝶蘭屬植物之生長特性 4
五、蝴蝶蘭屬的細胞遺傳學研究 5
六、研究目的 6

第二章 實驗材料與方法 8
一、實驗材料 8
二、實驗方法 8
(一)蝴蝶蘭基因組DNA微量萃取 8
(二)BAC選殖株重組質體DNA萃取 9
(三)EFS基因引子對之設計 9
(四)EFS基因序列分析及比較 10
(五)螢光原位雜合 13

第三章 結果 18
一、EFS基因在兩種台灣蝴蝶蘭原生種染色體上的定位 18
二、姬蝴蝶蘭PeEFS基因之選殖與序列分析 19
三、蝴蝶蘭PeEFS親緣演化分析 20
四、蝴蝶蘭亞屬EFS基因功能性區域DNA序列及胺基酸比較 21
五、EFS基因族系SET domain親緣演化分析 22

第四章 討論 23
一、EFS基因在姬蝴蝶蘭及台灣阿嬤蝴蝶蘭染色體上的分佈 23
二、比較EFS基因在阿拉伯芥、水稻以及姬蝴蝶蘭染色體上的位置 24
三、45S rDNA在姬蝴蝶蘭及台灣阿嬤蝴蝶蘭染色體上的分佈 25
四、姬蝴蝶蘭PeEFS基因結構組成分析 26
五、蝴蝶蘭PeEFS及PaEFS親緣演化分析 27
六、蝴蝶蘭亞屬EFS基因功能性區域序列分析比較 28
七、阿拉伯芥SDG family與蝴蝶蘭EFS基因親緣演化分析 28
八、PeEFS 基因微衛星序列(microsatellite)搜尋 29
九、以生物資訊方法分析PeEFS基因5'端上游序列 30

第五章、未來展望 32

第六章 參考文獻 33

表目錄
表一、本實驗所使用之蝴蝶蘭原生種 39
表二、擴增EFS基因之聚合酶連鎖反應資料 40
表三、比較姬蝴蝶蘭及台灣阿嬤蝴蝶蘭EFS基因結構 41
表四、比較姬蝴蝶蘭PeEFS與其他物種EFS胺基酸序列相似度 42
表五、EFS基因連鎖SSR基因座在蝴蝶蘭屬內之跨物種可擴增性與多型性評估 43
表六、搜尋PeEFS基因上游序列之cis-elements 44

圖目錄
圖一、以螢光原位雜合技術定位PeEFS基因與45S rDNA於姬蝴蝶蘭粗絲期染色體 45
圖二、以螢光原位雜合技術定位PeEFS基因與telomere DNA於姬蝴蝶蘭粗絲期染色體. 46
圖三、以螢光原位雜合技術定位PeEFS基因、45S rDNA與telomere DNA於台灣阿嬤蝴蝶蘭粗絲期染色體 47
圖四、PeEFS基因結構、PeEFS cDNA以及胺基酸結構對照圖 48
圖五、PeEFS與PaEFS之EFS胺基酸序列全長比對 49
圖六、PeEFS、PaEFS、水稻、葡萄及阿拉伯芥之EFS胺基酸序列比對 51
圖七、蝴蝶蘭與其他植物EFS胺基酸序列間的親緣演化分析 54
圖八、蝴蝶蘭不同亞屬間EFS功能性區域胺基酸序列比對 55
圖九、蝴蝶蘭亞屬EFS功能性區域胺基酸序列親緣演化分析 56
圖十、EFS基因族系SET domain親緣演化分析 57
圖十一、以螢光原位雜合技術定位45S rDNA於姬蝴蝶蘭與台灣阿嬤 蝴蝶蘭粗絲期染色體 58
圖十二、EFS BAC-derived SSR基因座在原生種蝴蝶蘭間的可擴增性與多型性 59

附錄目錄
附錄一、阿拉伯芥調控開花時間之訊息傳遞途徑 60
附錄二、SDG家族中SET群的蛋白結構示意圖 61
附錄三、姬蝴蝶蘭粗絲期染色體核型分析結果 62
附錄四、姬蝴蝶蘭粗絲期染色體核型分析結果示意圖 63

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