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研究生:吳佳春
論文名稱:結球白菜的超氧化歧化酵素與過氧化氫酵素基因轉移至甘藍及結球白菜之研究
論文名稱(外文):Transformation of superoxide dismutase(sod) and catalase(cat) genes of chinese cabbage into cabbage(brassica oleracea L. var. capitata L.)and chinese cabbage(Brassica campestris L. ssp. Pekinensis
指導教授:曾夢蛟
學位類別:碩士
校院名稱:國立中興大學
系所名稱:園藝學系
學門:農業科學學門
學類:園藝學類
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:124
中文關鍵詞:超氧化歧化酵素過氧化氫酵素農桿菌基因轉移法葉綠體
相關次數:
  • 被引用被引用:5
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超氧化歧化酵素 (superoxide dismutase) 及過氧化氫酵素 (catalase),在植物抗氧化逆境上扮演重要角色。本研究室已自結球白菜中篩選出銅鋅超氧化歧化酵素 (sod62) 及過氧化氫酵素基因 (cat78)。由於增加植物抗氧化酵素之活性,能提高其抵抗逆境之功能,且葉綠體是許多逆境最初受傷害的胞器,所以本研究嘗試以農桿菌法,將以rbcS啟動子驅動之攜帶大豆葉綠體的transit peptide的sod62及cat78基因,單一或共同基因轉移至'初秋´甘藍及'濱綠´結球白菜之下胚軸。其目的為探討利用基因轉移之技術,提高葉綠體之同源Cu/Zn-SOD或/與CAT酵素活性之可行性及增加植物對耐氧化逆境之能力。
試驗結果顯示甘藍植株再生率約為16%,但經kanamycin篩選後,其單一基因轉殖率約為3%,而共同轉殖率則約為1.25%;結球白菜之再生率為15.0 ~ 21.6%之間,但經kanamycin篩選後,單一基因轉殖率為3.5%,而共同轉殖率則約為1.63%。轉殖甘藍及結球白菜再生植株以PCR、南方墨點雜交及北方墨點雜交分析之結果顯示sod62或/及cat78基因存在於轉殖植株的染色體內,並能轉錄RNA。本試驗共分別獲得8及10株sod62/cat78共同轉殖之甘藍及結球白菜。電泳膠片酵素活性染色分析之結果顯示,轉殖植株之SOD62及CAT78條帶,均較未轉殖植物之條帶濃。轉殖植株的葉片或葉綠體之SOD及CAT酵素活性均較未轉殖之對照組顯著增加,且sod/cat共同轉殖植株,無論在葉綠體或是葉片,所增加之SOD或CAT酵素活性的量都比單獨轉殖sod62或cat78增加的多。轉殖之75 ~ 80%以上之SOD或CAT酵素可藉葉綠體transit peptide轉移到葉綠體中,並具有酵素活性。轉殖植株以400或600 ppb之SO2進行燻蒸處理4天,對照組出現嚴重白化、乾枯之受危害癥狀;sod62或cat78轉殖植株呈現中度耐SO2危害;sod/cat共同轉殖植株則具高度耐SO2危害能力。
為了揭露抗壞血酸過氧化酵素 (cytosolic ascorbate peroxidase, CAP) 及過敏性誘導反應 (hypersensitive induced reaction, HIR) 相關基因在植物耐逆境之分子層次上所扮演的角色,本研究亦利用RT-PCR之技術增幅玉米之CAP及HIR基因。定序及表現分析之結果顯示:增幅之CAP基因具有完整的752 bp,並可表現出一個27.3 kDa蛋白;增幅之HIR基因具有完整的845 bp,並可表現出一個31.4 kDa蛋白。
目 錄
中文摘要........................................................................................................................ 1
英文摘要......................................................................................................................... 3
前言.................................................................................................................................. 5
前人研究…………………………............................................................................... 8
一、植物抗氧化防禦機制之研究....................................…..................... 8
二、SOD基因於轉殖植物之研究............................................................ 17
三、CAT基因於轉殖植物之研究……....…………................................ 20
四、APX基因於轉殖植物之研究............................................................ 22
五、蛋白質運轉至葉綠體的機制與研究.........……............................. 23
材料與方法…............................................................................................................... 25
結果................................................................................................................................. 42
A. 結球白菜sod62及cat78基因轉殖至甘藍及結球白菜之研究.... 42
一、構築結球白菜之銅/鋅超氧化歧化酵素 (sod62) 及過氧化氫酵素基因 (cat78) 的轉殖載體............................................................ 42
二、sod62及cat78轉移至甘藍與結球白菜後,轉殖植株之再生情形....... 43
三、sod62與cat78基因轉殖甘藍及結球白菜植株之篩選與分析... 44
四、酵素活性之定性及定量分析............................................................. 47
五、酵素活性定量分析............................................................................... 48
六、以SO2燻蒸處理轉殖再生植株......................................................... 50
B. 玉米之CAP及HIR基因之合成及其表現分析................................ 51
討論.................................................................................................................................. 104
參考文獻......................................................................................................................... 113
附錄.................................................................................................................................. 124
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