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研究生:謝佩珊
研究生(外文):Pei-Shan Hsieh
論文名稱:以生物資訊及分子生物法選殖乳牛與豬SMCX/SMCY基因
論文名稱(外文):In Silico and Molecular Cloning of the Bovine and Porcine SMCX/SMCY Loci
指導教授:薛佑玲戴謙戴謙引用關係
指導教授(外文):Yow-Ling ShiueChein Tai
學位類別:碩士
校院名稱:國立成功大學
系所名稱:生物科技研究所碩博士班
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:2002
畢業學年度:90
語文別:中文
論文頁數:74
中文關鍵詞:SMCX選殖表現序列標幟SMCY生物資訊
外文關鍵詞:SMCXSMCYbioinformaticsESTclone
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本研究利用生物資訊學 (bioinformatics) 與分子生物學的方法選殖在牛與豬的Y染色體上具有性別特異性 (sex-chromosome specific loci) 表現的基因SMCY (Selected Mouse cDNA on the Y chromosome , H-Y antigen),俾作為進一步應用分子免疫法進行選性繁殖 ( sex pre-selection ),以利控制家畜後裔之性別。試驗首先以生物資訊學之方法,利用以指數增加的各種DNA與蛋白質資料庫 (databases),計包括有genome sequences、expressed sequences tags (ESTs)、sequence tagged sites (STSs)、SWISSPROT等進行蛋白質序列層面的比對,繼而將所有牛、豬與人類SMCX/SMCY基因具有同源性的DNA片段擷取出來,進行contig的組合。序列組合所得到的contigs和人類SMCX/SMCY基因的cDNA全長相較之下,牛與豬的cDNA片段長分別為2,932bp與977bp,約為人類cDNA全長的63及21%。
為獲得牛與豬SMCX/SMCY基因全長cDNA序列,萃取牛與豬之白血球細胞中的total RNA作為模板進行聚合酶鏈反應 (polymerase chain reaction, PCR) 之方式進行選殖,再利用reverse transcriptase-PCR (RT-PCR)、5’or 3’ rapid amplification of cDNA ends (5’or 3’RACE) 的方式獲得全長cDNA序列。
牛與豬之SMCX核苷酸序列長度分別為5,607 bp與5,096 bp,其轉譯之蛋白質長度為1,528 aa與1,559 aa。所得到部分豬SMCY基因序列1,366核苷酸,轉譯之蛋白質為455個胺基酸。不同物種間的胺基酸序列之相似度都大於80%,且SMCX更是相似度達到90%以上。
The objective of this investigation is to clone and characterize the bovine and porcine sex chromosome specific locus SMCY ( Selected Mouse cDNA on the Y chromosome ) (H-Y antigen) that causes the antibody response from male individual when skin grafting even between inbred lines for further application on sex pre-selection system using immunological method in domestic mammals, cattle and pigs. Since SMCX (a SMCY homologous locus on the X chromosome) and SMCY are highly conserved within and across mammalian species. Databases searches (bioinformatics approaches) were first performed using human SMCY sequence and translated protein as DNA and protein probes to acquire any cattle or pig homologous cDNA , genomic DNA or amino acid fragments. Searched databases including genomic sequences [(e.g., GenBank), cDNA sequences]、expressed sequences [e.g., database for expressed sequence tags (dbEST)] and protein databases [e.g., SWISSPORT, Protein Information Resource (PIR)]. A total of fourteen cattle and six pig SMCX/SMCY homologous DNA sequences have been retrieved and further subjected to contigs assembly. Assembled bovine and porcine SMCX/SMCY contigs span 3,185 and 977 bp, comparing to human SMCX/SMCY cDNA. To obtain full-length bovine and porcine SMCX/SMCY cDNAs, we proposed further PCR-based cloning strategies. Thus, total RNAs from cattle and pig lymphocytes have been isolated and served as templates for reverse transcriptase-polymerase chain reaction (RT-PCR), 5'-rapid amplification cDNA ends (5'-RACE) and 3'-RACE. The full-length nucleotides of bovine and porcine SMCX are 5,607 bp and 5,096 bp, which encode 1,528 and 1,599 amino acids, respectively. Cloned partial porcine SMCY are 1,366 bp and encodes 455 amino acids.
目錄
中文摘要.Ⅰ
英文摘要.Ⅱ
目錄.Ⅲ
表目錄.Ⅵ
圖目錄.Ⅶ
前言.1
文獻探討.3
1.家畜家禽之選性繁殖.3
1.1概述.3
1.2家畜之性別鑑定.3
1.3分離攜帶X-及Y-染色體精子技術.4
2.利用H-Y抗原進行選性繁殖之可行性.7
2.1H-Y抗原之概述.7
2.2H-Y抗原進行X-與Y-染色體精子之分離.8
2.3利用H-Y抗原進行選性繁殖之策略.9
3.哺乳類動物之SMCX與SMCY基因.9
3.1SMCY基因之H-Y抗原.9
3.2人類與小鼠的SMCY(Smcy)/SMCX(Smcx)基因.10
3.3SMCX及SMCY及之演化義意.12
4.利用表現序列表幟(ExpressedSequenceTag,EST)資料庫找尋新基因.13
材料與方法.15
1.材料.15
2.方法.15
2.1生物資訊法選殖基因-利用EST序列資料庫選殖牛與豬的SMCX/SMCYcDNA序列.15
2.2分子生物法選殖基因-荷蘭乳牛與台灣黑豬SMCX/SMCY基因片段之選殖與定序.18
2.3乳牛與豬SMCX/SMCY基因高保留區域之序列分析.29
2.4利用生物資訊法分析乳牛與豬SMCY基因之H-Y抗原胜肽序列.30
結果與討論.31
1.利用生物資訊法搜尋EST資料庫荷蘭乳牛與台灣黑豬SMCX/SMCY基因.31
2.分子生物法選殖荷蘭乳牛與台灣黑豬之SMCX/SMCY全長.36
3.荷蘭乳牛與台灣黑豬之SMCX/SMCY序列分析.40
4.預測SMCY基因之H-Y抗原.60
結論.63
參考文獻.64
附錄.70
附錄一、實驗試劑與試劑套組內容.70
附錄二、pGEM-TEasyvector.72
附錄三、ClontechSmartTMRACEcDNAamplificationkit原理圖73
自述.74

表目錄
表一、荷蘭乳牛與台灣黑豬之SMCX/SMCY基因引子對設計.19
表二、牛的SMCX基因同源性表現序列標幟.33
表三、豬的SMCX與SMCY基因同源性表現序列標幟.34
表四、豬與牛SMCX/SMCY蛋白質功能區域結構預測.59
表五、豬之SMCYH-Y抗原決定位置之預測.62

圖目錄
圖一、Insilicocloning流程.17
圖二、由BosTaurusEST序列組合SMCX-like之DNA與蛋白質序列.35
圖三、牛之SMCX/SMCYcDNA之部分RT-PCR電泳圖.38
圖四、豬SMCX與SMCYcDNA之5’RACEPCR電泳圖.39
圖五、荷蘭乳牛SMCXcDNA序列全長.41
圖六、牛SMCX全長胺基酸全長序列.45
圖七、台灣黑豬SMCXcDNA序列全長.46
圖八、豬SMCX全長胺基酸序列.49
圖九、人、牛、豬與小鼠之SMCX胺基酸序列多重序並列分析.51
圖十、台灣黑豬部分SMCYcDNA序列.54
圖十一、台灣黑豬部分SMCY蛋白質序列.55
圖十二、小鼠、人、牛及豬之部分SMCX/SMCY蛋白質之多重並列分析圖.56
圖十三、人、小鼠、豬與牛SMCX/SMCY之親緣演化關係圖.57
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