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研究生:陳慧珊
研究生(外文):Hui-Shan Chen
論文名稱:基因轉殖石斛蘭對喜姆比蘭嵌紋病毒抗病性之研究
論文名稱(外文):Study on transgenic Dendrobium resistant to Cymbidium mosaic virus
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:生物科學研究所
學門:生命科學學門
學類:生物學類
論文種類:學術論文
畢業學年度:93
語文別:中文
中文關鍵詞:基因轉殖石斛蘭喜姆比蘭嵌紋病毒抗病性
外文關鍵詞:Dendrobium Cymbidium mosaic virus CymMV
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石斛蘭(Dendrobium)屬於蘭科(Orchidaceae)單子葉之觀賞用作物,也是國內花卉產業中重要的經濟作物之一,主要應用於切花及盆栽花卉之用。蘭花在栽培的過程中極易受喜姆比蘭嵌紋病毒(Cymbidium mosaic virus, CymMV)及齒舌蘭輪斑病毒(Odontoglossum ringspot virus, ORSV)的侵襲,且在各種已知之病毒中對蘭花威脅最大的就是喜姆比蘭嵌紋病毒,CymMV在分類上屬於馬鈴薯X屬(genus Potexvirus),其顆粒大小約為13 × 490 nm。本研究乃是將喜姆比蘭嵌紋病毒鞘蛋白基因與載體pBin19所構築成之重組載體利用基因槍之技術轉殖入石斛蘭體內,經由聚合酶連鎖反應、反轉錄聚合酶連鎖反應及非直接酵素連結免疫分析法等相關步驟,以了解鞘蛋白基因是否成功轉殖進入蘭花體內,並了解植株之抗病機制。經由上述三種檢測分析方法,將所有基因轉殖之植株分為四大族群﹝ELISA(-)/Virus(-)/CP transgene(+)族群、ELISA(-)/Virus(+)/CP transgene(+)族群、ELISA(+)/Virus(+)/CP transgene(+)族群和ELISA(+)/Virus(+)/CP transgene(-)族群﹞;並且得知具有轉殖鞘蛋白基因而沒有病毒存在之植株﹝ELISA(-)/Virus(-)/CP transgene(+)族群﹞,並沒有鞘蛋白之表現;在具有轉殖鞘蛋白基因且具有病毒存在之植株﹝ELISA(+)/Virus(+)/CP transgene(+)族群﹞,病毒的增殖並沒有受影響,由ELISA(-)/Virus(+)/CP transgene(+)與ELISA(+)/Virus(+)/CP transgene(+)兩族群之比較得知,ELISA(-)/Virus(+)/CP transgene(+)轉殖基因蘭花對病毒的增殖產生了抑制作用。之後再利用多組檢測基因沉寂(gene silencing)之引子對所進行的反轉錄聚合酶連鎖反應結果,及在植株上病徵之表現得知,ELISA(-)/Virus(+)/CP transgene(+)族群的植株對喜姆比蘭嵌紋病之抗病機制乃是來自於RNA媒介抗病性的基因沉寂之結果。
Dendrobium orchid, a monocotyledon ornamental crop belonging to the Orchidacea, is an important commercial crop as cut flower and potted plant in the floral industry in Taiwan. For orchid, it is easily infected by viruses, Cymbidium mosaic virus (CymMV) and Odontoglossum ringspot virus (ORSV), during the process of propagation and cultivation. CymMV belongs to Potexvirus genus with the particle size of 13 × 490 nm. In this study, the recombinant pBin19 vector containing CymMV coat protein (CP) cDNA was transformed into Dendrobium by particle bombardment. PCR、RT-PCR and indirect ELISA were used to confirm the integration of the CP gene in the orchid genome and explore the resistance mechanism. All transformed plants were categorized as four groups﹝ELISA(-)/Virus(-)/CP transgene(+)group、ELISA(-)/ Virus(+) /CP transgene(+) group、ELISA(+)/Virus(+)/CP transgene(+) group, ELISA(+)/Virus(+)/CP transgene(-)group﹞.There is no expression of coat protein gene from ELISA(-)/Virus(-)/CP transgene (+) group. The transgenic orchid in ELISA(+)/Virus(+)/CP transgene(+) group showed that the multiplication of virus was not inhibited. Compared with the transgenic orchid in ELISA(+)/Virus(+)/CP transgene(+) group, ELISA(-)/Virus(+)/CP transgene(+) group showed the inhibition to the multiplication of CymMV. The results of the RT-PCR which using several specific primers to confirm the gene silencing and the symptom appearance showed that the resistance mechanism to CymMV in ELISA(-)/Virus(+)/CP transgene(+) group was RNA-mediated, through a post- transcriptional gene silencing mechanism(PTGS).
中文摘要……………………………………………………………… I
英文摘要…………………………………………………………… III
壹、前言及前人研究…………………………………………… 1
貳、材料與方法……………………………………………………… 17
一、喜姆比蘭嵌紋病毒RNA之來源………………………… 17
二、基因轉殖石斛蘭植株之培養……………………………… 17
三、喜姆比蘭病毒鞘蛋白基因轉殖石斛蘭植株之篩檢……… 18
(一)、基因轉殖石斛蘭植株DNA相關實驗之進行……… 18
1、抽取基因轉殖石斛蘭植株genomic DNA…………… 18
2、聚合酶連鎖反應(PCR)……………………………… 19
3、PCR產物之電泳膠體分析 …………………………… 19
(二)、基因轉殖石斛蘭植株RNA相關實驗之進行……… 23
1、抽取基因轉殖石斛蘭植株total RNA………………… 23
2、反轉錄聚合酶連鎖反應(RT-PCR)………………… 24
3、RT-PCR產物之電泳膠體分析 ……………………… 24
(三)、基因轉殖石斛蘭植株蛋白質相關實驗之進行……… 26
非直接酵素連結免疫分析法…………………………… 26
五、35S啟動子基因序列之偵測………………………………… 27
1、35S啟動子基因序列之核酸引子之選置…………… 27
2、增幅35S啟動子基因序列片段……………………… 27
3、PCR產物之電泳膠體分析………………………………28
參、結果……………………………………………………………… 30
一、非直接酵素連結免疫分析法……………………………… 30
二、基因轉殖石斛蘭植株DNA相關實驗之進行…………… 30
三、基因轉殖石斛蘭植株RNA相關實驗之進行…………… 43
四、35S啟動子基因序列之偵測…………………………… 59
肆、討論……………………………………………………………66
伍、參考文獻………………………………………………………… 72
陸、附錄……………………………………………………………… 80
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