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研究生:宋佳貞
研究生(外文):Chia-Chen Sung
論文名稱:矮南瓜黃化嵌紋病毒與木瓜輪點病毒西瓜系統鞘蛋白基因轉殖洋香瓜之研究
論文名稱(外文):Studies on the transformation of muskmelon using coat protein genes of Zucchini yellow mosaic virus and Papaya ringspot virus type W
指導教授:王惠亮
指導教授(外文):Hui-Liang Wang
學位類別:碩士
校院名稱:國立高雄師範大學
系所名稱:生物科技系
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:2008
畢業學年度:96
語文別:中文
中文關鍵詞:木瓜輪點病毒矮南瓜黃化嵌紋病毒基因轉殖作物基因轉殖洋香瓜
外文關鍵詞:papaya ringspot virus (PRSV)zucchini yellow mosaic virus (ZYMV)transgenic plantstransgenic muskmelon
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洋香瓜是台灣重要的經濟作物,栽培期間常受到多種病毒的侵害,其中矮南瓜黃化嵌紋病毒(Zucchini yellow mosaic virus, ZYMV)和木瓜輪點病毒西瓜型(Papaya ringspot virus type watermelon, PRSV-W),此二病毒在台灣危害瓜類最嚴重,是限制栽培的重要因素。近年因為分子遺傳及植物基因轉殖技術的發展,得使用這些遺傳工程的技術來培育抗病品種,達到植物病毒病害防治目的。本研究首先測試不同品種之洋香瓜子葉再生率,將含有PRSV-W及ZYMV雙鞘蛋白基因的pCAMBIA2301-PZ質體,經農桿菌轉基因轉殖至洋香瓜,最終共得620個轉殖子葉培植體,經100 mg/L kanamycin MS培養基篩選後,獲得到14株存活的擬轉殖植株,抽取其genomic DNA及PCR檢測,測得一株具有PRSV-W及ZYMV雙鞘蛋白基因品系(line 8),及一株具有PRSV-W單鞘蛋白品系(line 14),以組織培養法繁殖此二品系(line 8 和line 14),獲得line 8分株(8-1, 8-2, 8-3, 8-4, 8-5),line 14分株(14-1, 14-2, 14-3, 14-4),將所有分株檢測PCR為正反應,RT-PCR與ELISA皆為負反應。將line 8-2及line 14-1進行病毒PRSV-W挑戰接種,以ELISA追蹤檢測,發現此二品系延後9天出現嚴重病徵的現象;將line 8-3品系進行病毒ZYMV挑戰接種,以ELISA追蹤檢測,發現此品系延後8天出現嚴重病徵的現象。本研究顯示轉殖病毒鞘蛋白的洋香瓜對於病毒感染有抗病之效果,可作為後續洋香瓜抗病品系育種之重要參考,以達到改善田間防治該病毒之目的。
Muskmelon is one of most important agricultural products in Taiwan. During the cultivation period, it is often infected by several viruses, especially the Zucchini yellow mosaic virus (ZYMV) and Papaya ringspot virus watermelon type (PRSV-W). In fact, these two viruses have caused serious damage to the cultivation of muskmelon, and have been the major limitation to muskmelon cultivation. Recently, because of the development of biotechnology on plant genetic transformation, we can apply this technique to buid up the level of disease-resistance in the plant. In this research, the regenerate rates of various cultivars of muskmelons by the cotyledon tissue culture were investigated. Vectors containing the CP genes of both ZYMV and PRSV-W, or PRSV-W alone were constructed. The constructed vectors were introduced into muskmelon by Agrobacterium-mediated transformation using 620 cotyledons as explants. After selecting from MS media with 100 mg/L kanamycin, 14 putative transgenic lines were obtained. Genomic DNA from these 14 transgenic lines were extracted and analyzed the insertion by PCR. One line (line 8) containing fragments of the CP coding regions of ZYMV and PRSV-W, and the other line(line 14) containing fragments of the CP coding regions of PRSV-W were obtained. After subcloning these two lines (line 8 and 14) by tissue culture, a series of line 8 ramet (8-1, 8-2, 8-3, 8-4, 8-5) and line 14 ramet (14-1, 14-2, 14-3, 14-4) were obtained. The existence and expression of the inserted CP genes were confirmed by PCR, RT-PCR and ELISA. Lines 8-2, 14-1 and 8-3 were mechanically inoculated with PRSV-W and ZYMV separately to evaluate the virus resistance and the infection was analyzed by ELISA and symptom development. For lines 8-2 and 14-1, the first and obvious symptoms can be observed 9-days later than that of non-transformed control. Line 8-3 showed 8-day delay each for the first and obvious symptom developed. In conclusion, this research revealed that the symptom development of the transgenic muskmelons with viral CP genes could be positively delayed. Thus, it can be an essential reference to breeding the resistant lines of the muskmelon, and an effective way to achieve the goal of preventing infection by the viruses.
目 錄

中文摘要............................................I
英文摘要............................................III
壹、前言及前人研究...................................1
ㄧ、洋香瓜的特性及面臨問題............................1
二、矮南瓜黃化嵌紋病毒之發生及特性.....................3
三、木瓜輪點病毒之發生及特性..........................4
四、瓜類病毒之防治...................................6
貳、材料與方法......................................17
ㄧ、蜜華品種洋香瓜...................................17
二、病毒之來源.......................................17
三、組織培養藥品配置..................................17
四、實驗菌株及質體...................................18
五、洋香瓜品種再生試驗................................19
六、農桿菌媒介基因轉殖蜜華洋香瓜........................20
七、PRSV及ZYMV鞘蛋白基因轉殖蜜華洋香瓜植株之篩檢.........21
八、繁殖已轉殖之植株..................................30
九、轉殖蜜華洋香瓜馴化.................................32
十、病毒挑戰接種R0轉基因蜜華洋香瓜......................32
参、結果.............................................33
ㄧ、PCR檢測農桿菌基因.................................33
二、不定芽再生測定....................................33
三、農桿菌轉殖蜜華品種洋香瓜...........................35
四、 PRSV及ZYMV轉殖蜜華洋香瓜之基因檢測................38
五、基因轉殖蜜華之植株RNA相關實驗......................41
六、基因轉殖蜜華品種洋香瓜蛋白質相關實驗................41
七、繁殖已轉殖之植株及馴化.............................45
八、轉殖蜜華洋香瓜分株之基因檢測........................45
九、基因轉殖蜜華之分株之RNA相關實驗.....................48
十、 基因轉殖蜜華品種洋香瓜分株蛋白質相關實驗.............48
十ㄧ、病毒挑戰接種R0轉基因蜜華洋香瓜.....................51
肆、討論..............................................59
伍、參考文獻...........................................64
附錄一................................................77
附錄二................................................78
附錄三................................................79
附錄四................................................80
附錄五................................................81
附錄六................................................82
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