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研究生:林宛萱
研究生(外文):Wan-HsuanLin
論文名稱:原生種蝴蝶蘭染色質甲基轉移酶基因之染色體定位與基因序列分析
論文名稱(外文):Chromosome localization of Chromomethylase gene and sequence analysis on Phalaenopsis species
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
論文種類:學術論文
論文出版年:2012
畢業學年度:100
語文別:中文
論文頁數:66
中文關鍵詞:染色質甲基轉移酶螢光原位雜合粗絲期蝴蝶蘭
外文關鍵詞:chromomethylasefluorescence in situ hybridizationpachytenePhalaenopsis species
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蝴蝶蘭為花卉產業中具有高度經濟價值的作物,廣泛使用組織培養的方式大量培育分生子代,在組織培養的過程中,可能會藉由甲基化(methylation)改變基因表現,造成體細胞變異(somatic variation),而導致植株型態的變異。甲基轉移酶相關酵素其作用為催化DNA甲基化,依蛋白質結構及作用位置的不同,可進一步將甲基轉移酶相關酵素分為三大基因族:甲基轉移酶(MET)基因族、染色質甲基轉移酶(CMT)基因族及重新發生甲基轉移酶(DRM)基因族。2005年Chen等人從蝴蝶蘭商業品種Phalaenopsis Little Mary選殖出一甲基轉移酶相關基因,由其胺基酸序列之功能區分析發現此基因包含chromodomain,推測此蝴蝶蘭甲基轉移酶相關基因,屬於染色質甲基轉移酶基因族的成員。本實驗室以P. Little Mary PlMET cDNA序列當作探針對實驗室姬蝴蝶蘭BAC基因庫進行篩選,選殖出PeCMT-E07,並以454高通量定序分析,分析結果得到序列長度不同之連續體(contig),以TAIL-PCR方式將兩段比對到部分CMT基因序列之連續體連接。分析來自BAC選殖株得到的CMT 基因序列,共有21個exon,譯碼區為2598 bp可轉譯出866個胺基酸。進一步比較CMT基因在姬蝴蝶蘭與臺灣阿嬤蝴蝶蘭序列上的差異,分別對兩物種進行擴增,分別命名為PeCMT及PaCMT。比對兩者胺基酸序列,相似度高達98.5%,皆具有BAH domain及chromodomain兩個功能區。由分子親緣關係分析顯示蝴蝶蘭CMT基因屬於單子葉CMT基因族群。比較四個不同亞屬原生種蝴蝶蘭兩功能區序列,結果顯示在不同亞屬八個原生種蝴蝶蘭CMT基因功能區胺基酸序列相似度高達95%以上。以PeCMT基因中1.9 kb序列為探針,利用螢光原位雜合技術(fluorescence in situ hybridization, FISH)定位CMT基因在兩種臺灣原生種蝴蝶蘭:姬蝴蝶蘭及臺灣阿嬤蝴蝶蘭染色體分佈位置,結果顯示CMT基因在兩種原生種蝴蝶蘭粗絲期染色體中皆具有單一訊號且位於染色體末端。比對實驗室建立的姬蝴蝶蘭核型,PeCMT基因分佈在第六號染色體長臂距末端1.23 μm。綜合以上研究結果,推測蝴蝶蘭中CMT基因功能區保守性極高,單一的CMT基因訊號可作為辨識姬蝴蝶蘭及臺灣阿嬤蝴蝶蘭單一染色體的專一標誌。
Phalaenopsis orchid is one of the top economical crops in floricultural industry. To propagate massive clones, tissue culture is widely and routinely used. However, some somatic variations were generated through the vegetative propagation process and the changed level of DNA methylation is supposed to be one of the mechanism leading the plant morphology alteration. Methyltransferase plays important roles in DNA methylation and can be further grouped into three families, methyltransferase (MET) family, chromomethylase (CMT) family and de novo methyltransferase (DRM) family, based on their protein structure and function. Chen et al. (2005) have cloned a methyltransferase gene from Phalaenopsis cultivar P. Little Mary and its chromodomain indicates that this gene is a member of CMT family. The cDNA fragment of this gene was used as probe to screen P. equestris BAC library and the CMT gene-containing BAC clone- PeCMT-E07 was further sequenced through 454 pyrosequencing. First, the short sequence derived from 454 pyrosequencing were assembled in two contigs and the two sequenced contigs are linked by using TAIL-PCR. The CMT gene cloned from P. equestris BAC library, PeCMT-E07, had 21 exons and the length of cDNA was 2,598 bp which can be translated into 866 amino acids. In addition, we also cloned this gene from the genomes of two species, P. equestris and P. aphrodite, named as PeCMT and PaCMT. The amino acid sequences between PeCMT and PaCMT show very high identity up to 98.5% and both contain functional domains, BAH domain and chromodomain. Then, the phylogenetic analysis based on amino acid sequences revealed that PeCMT and PaCMT were clustered together with members of CMT family of monocot species. Furthermore, the amino acid sequences of two functional domains, BAH domain and chromodomain, were sequenced and compared among eight Phalaenopsis species belonging to four different subgenera. The functional domain amino acid sequence identities are all over 95% and therefore they are highly conserved among eight Phalaenopsis species. Finally, fluorescence in situ hybridization was applied to elucidate the chromosome location of CMT gene in P. equestris and P. aphrodite using 1.9 kb DNA fragment of CMT as a probe. It showed single signal of CMT on a high-resolution pachytene chromosome of both Phalaenopsis species. According to the known P. equestris karyotype, the PeCMT was speculated to locate on the euchromatin region of the long arm of P. euqetris chromosome 6 and about 1.23μm away from the telemetric end. In conclusion, the functional domains of CMT were highly conserved among Phalaenopsis species, and single locus of CMT can be used as a marker for chromosome identification in Phalaenopsis orchids.
中文摘要.......................................................i
英文摘要......................................................ii
誌謝.........................................................iii
目錄..........................................................iv
表目錄.......................................................vii
圖目錄......................................................viii
附錄目錄......................................................ix
縮寫字對照表...................................................x

第一章 前言....................................................1
ㄧ、蝴蝶蘭繁殖與體細胞變異..................................1
二、植物基因組甲基化........................................2
三、DNA甲基轉移酶...........................................3
四、DNA甲基化對植物生長發育之影響...........................5
五、蝴蝶蘭細胞遺傳之研究....................................6
六、螢光原位雜合技術之應用..................................7
七、甲基轉移酶相關基因於蝴蝶蘭之研究........................9
八、研究目的...............................................10

第二章 實驗材料與方法.........................................11
一、實驗材料...............................................11
二、實驗方法...............................................11
(一)以TAIL-PCR連接含有CMT基因序列兩個連續體.............11
1.引子設計.........................................11
2.TAIL-PCR試劑組成.................................12
3. TAIL-PCR條件....................................12
(二)微量萃取蝴蝶蘭基因組DNA.............................13
(三)CMT序列基因座專一引子對設計及擴增...................13
1.設計CMT序列基因座之專一引子對....................13
2.聚合酶連鎖反應...................................13
(四)選殖CMT基因序列.....................................14
1.由瓊脂凝膠回收CMT基因片段........................14
2.接合作用.........................................14
3.轉型作用.........................................15
4.塗碟.............................................15
(五)微量製備質體DNA.....................................15
1.養菌.............................................15
2.微量抽取質體.....................................15
(六)核酸定序及分析......................................16
(七)序列分析............................................16
(八)螢光原位雜合實驗....................................16
1.染色體細胞分裂時期鑑定.........................16
2.染色體製片.....................................17
3.探針標定.......................................17
4.染色體DNA與探針雜合............................18
5.清洗...........................................18
6.訊號偵測.......................................18
第三章 結果...................................................20
一、利用TAIL-PCR連接454基因體序列片段......................20
二、蝴蝶蘭CMT基因實質定位..................................20
三、姬蝴蝶蘭PeCMT及臺灣阿嬤PaCMT基因選殖與序列分析.........21
四、蝴蝶蘭CMT基因胺基酸序列及親緣演化分析..................22
五、不同蝴蝶蘭亞屬物種CMT基因功能區序列之擴增..............23

第四章 討論...................................................25
一、蝴蝶蘭CMT基因在染色體上實質定位........................25
二、蝴蝶蘭PeCMT及PaCMT基因結構與分子特性...................27
三、蝴蝶蘭PeCMT及PaCMT親緣演化分析.........................28
四、蝴蝶蘭亞屬間功能區序列分析比較.........................28
五、姬蝴蝶蘭CMT基因連鎖微衛星序列分析......................31
六、以生物資訊法搜尋PeCMT-E07 5'端上游序列.................33

第五章 未來展望...............................................34

第六章 參考文獻...............................................35
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