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研究生:蘇育良
研究生(外文):Yu-liang Su
論文名稱:構築與分析高繁殖力豬卵之互補基因庫
論文名稱(外文):Construction and analysis of high reproductive porcine oocyte cDNA library
指導教授:黃弘文黃弘文引用關係
指導教授(外文):Hurng-wern Huang
學位類別:碩士
校院名稱:國立中山大學
系所名稱:生物醫學科學研究所
學門:生命科學學門
學類:生物化學學類
論文種類:學術論文
論文出版年:2004
畢業學年度:92
語文別:中文
論文頁數:133
中文關鍵詞:cDNA基因庫表現序列標記
外文關鍵詞:cDNA libraryoocytePigExpressed Sequence Tags
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早期豬胚胎發育和分化基因的研究,由於材料取得不易發展有限。為了要鑑定促進與豬繁殖力相關的ESTs,必須建立高繁殖力豬卵的cDNA基因庫和ESTs資料庫。由行政院農業委員會台灣畜產試驗所從杜洛克豬,利用超排卵方式完成了而且得到成熟卵。從50顆成熟的卵分離出total RNA,反轉錄後再利用PCR方式將cDNA放大,放大後的cDNA大小範圍從0.4-5 kb。取得的cDNA與pCR2.1質體進行接合作用,這些接合的cDNA經過質體轉形後,有5.26×104個獨立單株的複雜性。總共有320個克隆挑選與定序,利用BLASTx與BLASTn分析後,可轉為蛋白質序列的分析之123個序列,發現了53個粒線體相關的蛋白質(43.07%),70個序列則對應到人類、老鼠、果蠅的同源性蛋白質(56.91%);在核酸層面中,101個對應到粒線體與核醣體相關基因(82.11%),22個對應到其他同源性基因(17.89%)。藉由PCR方式分析卵母細胞基因庫中,對於一些特異性基因是否有轉錄,這些基因包括同源性基因(2個表現量最高與2個表現量最低的基因)、管家基因(ACTβ與G3PDH)和發育中的基因(NEK2和ZP1)。然而,將來許多豬的新基因將被發現,並可能成為豬高繁殖力的候選基因。cDNA基因庫對於早期表現的活化基因是一個有價值資源。成功的從非常少量細胞分離量構築cDNA表現資料庫的技術,將來可以用在由卵母細胞的活組織檢查和由核轉移過程的樣品中分析表現基因。
The progress of studies on genes concerning the development and differentiation of early swine embryos have been delayed by limited paucity material. In order to identify the porcine ESTs associates with promoting its breeding efficiency, a cDNA library and ESTs database from oocytes of high reproductive swine is established. Oocytes were obtained from Duroc pig by superovulation which was performed by Taiwan Livestock Research Institute, Council of Agriculture. Total RNA was isolated from 50 mature oocytes, reverse transcription is then performed, followed by PCR based amplification of the cDNA. The amplified cDNA size ranges from 0.4 to 5 kb. The derived cDNA were ligated to a pCR2.1 vector, and the library has complexities of about 5.26×104 independent clones. A total of 320 clones was picked and sequenced. By BLASTx analysis, among the 123 sequences, more than 43.07%(53/123) mitochondrial proteins are found, 56.91%(70/123) of the sequence were homologous to known transcripts from human, mouse, Drosophila. In nucleotide level analysis, 82.11%(101/123) matched with the mitochondrial, ribosome genes and 17.89%(22/123)matched with other homologous genes by BLASTn. PCR analysis of the oocyte library for specific genes revealed transcripts for genes including homologous genes(2 pairs highly abundance and 2 pairs low abundance genes), housekeeping genes(ACTβ and G3PDH) and developmental genes(NEK2 and ZP1). However, novel genes of swine are supposed to be the candidates for high productive phenotypes of swine. The library is a valuable resource for the isolation of clones representing genes active at the early stage. The ability to construct cDNA expression library from a few cells will allow gene expression analysis from oocyte biopsies and derived by nuclear transfer procedures.
致謝
中文摘要...............................I
英文摘要...............................II
英文縮寫表.............................III
壹、前言...............................1
一、表現序列標記定義與應用.............1
二、研究背景...........................3
貳、研究目的...........................8
参、材料與方法.........................9
一、高繁殖豬卵檢體的來源及前處裡.......9
二、Total RNA的萃取與純化..............10
三、mRNA的萃取與純化...................11
四、第一股互補基因合成.................12
五、長距離之聚合酶連鎖反應放大互補基因.12
六、互補基因大小層析...................13
七、互補基因庫的建立...................14
八、菌種保存...........................15
九、質體的純化.........................15
十、限制酶的切割.......................16
十一、自動核酸定序儀...................16
十二、同源性引子的設計.................20
十三、特異性基因擴增...................21
十四、篩選豬卵cDNA基因庫...............22
十五、切膠純化.........................25
十六、加入3''A-Overhangs................26
十七、高繁殖力豬卵的EST分析............26
肆、結果...............................27
伍、討論...............................33
陸、參考文獻...........................37
圖.....................................43
表.....................................54
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