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研究生:許薾勻
研究生(外文):Erh-yun Hsu
論文名稱:以轉殖大豆及阿拉伯芥種子表現含降血脂活性胜肽之蛋白
論文名稱(外文):Production of bioactive peptides with hypotriglyceridemic action in seeds of transgenic soybean and Arabidopsis
指導教授:李佩芳李佩芳引用關係
指導教授(外文):Pei-Fang Lee
學位類別:碩士
校院名稱:輔英科技大學
系所名稱:生物技術系碩士班
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:2009
畢業學年度:97
語文別:中文
論文頁數:64
中文關鍵詞:轉殖大豆轉殖阿拉伯芥降血脂胜肽農桿菌媒介法
外文關鍵詞:Arabidopsis transformationAgrobacterium-mediated transformationhypotriglyceridemicactive peptidessoybean transformation
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大豆是世界上極重要之農作物,亦是最豐富的植物性蛋白來源,其飽和脂肪量低,不含膽固醇,可降低血中總膽固醇及三酸甘油脂,為具發展濳力之保健食品。目前己知飲食之蛋白質若存有降血脂胜肽VVYP (Val-Val-Tyr-Pro)序列,即可有效抑制腸道對三酸甘油脂的吸收,因此本研究擬發展大豆成為生物反應器,以生產含降血脂胜肽之蛋白於成熟種子中,為探討以成熟種子表現含降血脂胜肽蛋白之可行性,另以轉殖阿拉伯芥進行測試;研究中由中興大學林金和教授實驗室中承接轉殖載體並進行植物轉殖,轉殖載體中已改造大豆glycinin基因為富含VVYP序列,並以具種子專一性之大豆啟動子(GmPM9)驅動其表現;實驗結果顯示,阿拉伯芥轉殖植株經聚合酶連鎖反應及GUS組織化學染色分析確認後,由西方墨點法分析之結果得知外源蛋白可表現於成熟種子中;於轉殖大豆方面,爲有效提高轉殖大豆之效率,使用成熟子葉節、未成熟子葉節及胚尖分別做為培植體,經農桿菌進行大豆轉殖,結果得知以胚尖為培植體,可得穩定之再生及轉殖率,另於轉殖過程中加入SL-77及超音波震盪等因子以提高轉殖率,據以建立高雄選10號大豆之轉殖流程,並已得到經GUS 組織化學染色分析確認之大豆轉殖植株,日後將可進一步分析轉殖種子中之含降血脂胜肽蛋白之表現。本研究之實驗結果顯示,以成熟種子表現含降血脂胜肽蛋白為可行之策略。
Soybean is one of the most important crops for food, feed, and oil industrial applications. Val-Val-Tyr-Pro (VVYP) is a bioactive peptide with hypotriglyceridemic activity decreasing serum triglyceride level effectively. In this study, we intended to develop the transgenic plants as bioreactor for production of hypotriglyceridemic peptides. We generated transgenic soybean and Arabidopsis lines that expressed modified forms of the Gy1 and Gy5 of soybean seed storage protein glycinin containing tandem repeats of the bioactive peptide (VVYP). Segregation analyses of independently transformed Arabidopsis lines were performed by PCR and monitored by immunoblottings. These results indicated that the foreign genes were integrated with the genome of transformed Arabidopsis, and expressed modified glycinin in mature seeds. For improving the efficiency of Agrobacterium-mediated transformation of soybean (Glycine max L. cv. Kaohsiung sel. 10), we established the transformation system by using embryonic tips as explants, adding SL-77 as well as carborundum in co-culture medium and wounding explants by sonication. The resistant transgenic soybeans were performed by GUS histochemical staining and showed GUS gene expression. These result demonstrated the feasibility of production of bioactive peptides with hypotriglyceridemic action in transgenic plants. In conclusion, the goal of this study expected to establish the platform for high value-added soybeans and develop the health food with hypotriglyceridemic action.
目 錄

誌謝-------------------------------------------------- i
中文摘要---------------------------------------------- ii
英文摘要---------------------------------------------- iv
目錄-------------------------------------------------- v
表目錄------------------------------------------------ vii
圖目錄------------------------------------------------ viii
縮寫表------------------------------------------------ ix
第一章 緒論----------------------------------------- 1
第二章 文獻探討------------------------------------- 4
第一節 生物活性胜肽--------------------------------- 4
一 生物活性胜肽之介紹--------------------------- 4
二 大豆胜肽------------------------------------- 4
三 活性胜肽VVYP之重要性------------------------- 5
第二節 大豆基因轉殖--------------------------------- 6
一 基因槍轉殖法--------------------------------- 6
二 農桿菌轉殖法--------------------------------- 7
第三節 植物生物反應器------------------------------- 9
第三章 研究方法------------------------------------- 11
第一節 試驗材料------------------------------------- 11
一 植物材料------------------------------------- 11
二 培養基--------------------------------------- 11
三 農桿菌與載體--------------------------------- 12
四 轉殖質體------------------------------------- 12
五 緩衝溶液及試劑------------------------------- 12
第二節 試驗方法------------------------------------- 14
一 質體製備------------------------------------- 14
二 農桿菌轉型作用------------------------------- 14
三 大豆基因轉殖--------------------------------- 14
四 阿拉伯芥基因轉殖----------------------------- 15
五 轉基因植株之篩選及確認----------------------- 18
第四章 研究結果------------------------------------- 22
第一節 質體確認------------------------------------- 22
第二節 以轉殖阿拉伯芥表現含降血脂活性胜肽之蛋白----- 23
一 轉基因之確認--------------------------------- 23
二 GUS組織化學染色分析-------------------------- 23
三 西方墨點轉漬法分析--------------------------- 23
第三節 以轉殖大豆表現含降血脂活性胜肽之蛋白--------- 24
一 轉殖大豆流程之建立--------------------------- 24
第五章 討論與結論----------------------------------- 27
參考文獻 --------------------------------------------- 31
附錄一 pC2-Gy1 之質體構築示意圖--------------------- 62
附錄二 pC2-Gy5 之質體構築示意圖--------------------- 63
附錄三 pC2-Gy15 之質體構築示意圖-------------------- 64

表 目 錄
表一 不同策略之大豆胚尖轉殖方法------------------- 41

圖 目 錄
圖一 以聚合酶連鎖反應分析轉殖載體----------------- 42
圖二 以聚合酶連鎖反應分析轉殖載體----------------- 43
圖三 以聚合酶連鎖反應分析轉殖載體----------------- 44
圖四 以聚合酶連鎖反應分析轉殖載體----------------- 45
圖五 以聚合酶連鎖反應分析轉殖載體----------------- 46
圖六 以聚合酶連鎖反應分析TAG1轉殖植株------------- 47
圖七 以聚合酶連鎖反應分析TAG5之阿拉伯芥轉殖植株--- 48
圖八 以聚合酶連鎖反應分析TAG15之阿拉伯芥轉殖植株-- 49
圖九 阿拉伯芥轉殖植株之GUS化學組織染色分析-------- 50
圖十 以SDS-PAGE分析阿拉伯芥轉殖植株之種子蛋白質表現51
圖十一 以西方墨點轉漬法分析TAG1中之種子蛋白--------- 52
圖十二 以西方墨點轉漬法分析TAG5之種子蛋白----------- 53
圖十三 以西方墨點轉漬法分析TAG15之種子蛋白---------- 54
圖十四 以西方墨點轉漬法分析TAG15之種子蛋白---------- 55
圖十五 成熟大豆子葉節轉殖方法之流程----------------- 56
圖十六 大豆胚尖轉殖方法之流程----------------------- 57
圖十七 未成熟大豆子葉節轉殖方法之流程--------------- 58
圖十八 以大豆不同部位之培殖體,經轉殖後以GUS化學組織
染色分析之結果--------------------------------59
圖十九 以五種不同策略測試,經轉殖後之GUS染色率及再生
率之結果------------------------------------- 60
圖二十 大豆轉殖植株之GUS化學組織染色分析------------ 61
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