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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 蘇鴻基(Hong-Ji Su),洪挺軒(Ting-Hsuan Hung) | |
dc.contributor.author | Ya-Chih Feng | en |
dc.contributor.author | 馮雅智 | zh_TW |
dc.date.accessioned | 2021-06-13T06:19:21Z | - |
dc.date.available | 2006-01-27 | |
dc.date.copyright | 2006-01-27 | |
dc.date.issued | 2006 | |
dc.date.submitted | 2006-01-25 | |
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/34631 | - |
dc.description.abstract | 香蕉苞葉嵌紋病(Banana bract mosaic)於1979年在菲律賓民答那峨島(Mindanao)的達浮澳(Davao)首次發現,至今僅有27年的發生歷史,為一新興的病毒病害,在東南亞等國包括菲律賓、印度、斯里蘭卡、西薩摩亞獨立國、越南等危害甚重,影響當地香蕉產業甚鉅。因罹病蕉株在其苞葉(bract)上會出現深紅褐色紡綞條紋及嵌紋病徵,因此而命名為”bract mosaic”。 此病由香蕉苞葉嵌紋病毒(Banana bract mosaic Potyvirus=BBrMV)所引起,BBrMV在病毒分類上隸屬於Potyviridae科,Potyviurs屬,基因體為(+) ssRNA。它通常可藉由香蕉的無性繁殖體(vegetative planting materials)如吸芽或組織培養苗來傳播,田間也可由數種蚜蟲以非永續性的方式進行傳播。為準確偵測BBrMV,本論文特研發BBrMV的One-step RT-PCR快速偵測法,配合以植物總核酸抽取法來萃取病毒核酸,及使用專一性引子對(BBrMV 902),可正確而敏感的測到BBrMV。觀察呂宋蕉罹病株(Latundan,AAB蕉)葉片上明顯可見淡黃綠色的紡綞型條斑產生,將此試驗病蕉株當為材料,以PEG純化法進行病毒純化,在電顯觀察下可見750 nm 的絲狀病毒顆粒;將呂宋蕉之病葉以蕉蚜傳播至14棵北蕉(Cavendish,AAA),在感染45天後可用RT-PCR法偵測到8棵有BBrMV訊號,3個月後增至11棵可偵側到,但至14個月後的偵測追綜結果只剩3棵可測到,顯示時間拖長後病毒在香蕉植株內的含量也跟隨著降低,有弱化的情形,同時這些受測植株在感染試驗期間皆無苞葉嵌紋病之典型病徵。在台灣田間蕉株苞葉嵌紋病的調查方面,首先自台灣香蕉研究所的保存品種園挑選50種具有經濟重要性的國內外不同香蕉品種,進行RT-PCR檢測。結果在外來品種Cocos (編號21)及國內原有的三尺蕉(編號81)、北蕉(編號85)等三個品種可測到BBrMV,但觀察這些植株亦無BBrMV的典型病徵出現。將此三樣本的RT-PCR增幅片段(即病毒之鞘蛋白基因)予以選殖及定序,與國外的BBrMV分離株進行比對,其核苷酸序列相似度皆逹94%以上,胺基酸序列相似度逹95.7%以上。再將此感染BBrMV的Cocos、三尺蕉和北蕉再以蕉蚜傳毒至Saba (BBB)、Kluai Khai (AA)、Nam Wa (ABB)及Cavendish (AAA)等不同genotype的品種,經6個月後,發現以Cocos病葉經蕉蚜傳播至Kluai Khai植株,及以北蕉病葉經蕉蚜傳播至Saba植株可偵測到訊號。觀察這些接種6個月後植株之病徵,只見葉片有黃化,並未有BBrMV典型的病徵。另外也在高屏地區、台南縣及南投等地的蕉園進行大量抽樣偵測,結果僅在恆春地區田間發現一棵三尺蕉,將其RT-PCR增幅片段予以選殖及定序後也確認為BBrMV。研究結果發現田間感染BBrMV的蕉株比例很低,既使是測有BBrMV的蕉株也都未見典型苞葉嵌紋病徵,顯示台灣主要栽種的北蕉可能相當耐病,或者在台灣的生態環境有別於東南亞感病疫區,不利於BBrMV之感染散播。本論文亦從事以大腸桿菌系統表現BBrMV鞘蛋白(coat protein)作為免疫抗原製備抗血清(多元抗體),所得之抗血清(命名為PcAb-BBrMV-1)以間接酵素標誌抗體法(Indirect ELISA)測試香蕉樣品,病組織與健康組織讀值差可逹三倍以上,並且只會和BBrMV有專一性反應,和其他重要香蕉病毒如Banana streak virus、Cucumber mosaic virus與Banana bunchy top virus並無交叉反應現象。為求較好的ELISA偵測效果,論文中總共測試了9種萃取緩衝液,發現0.5 M borate buffer(含0.5% skim milk,pH 6.8)效果最佳也最穩定。RT-PCR法與抗體偵測法的雙向開發將提供不同的BBrMV檢測方式,有助於防檢疫之應用。 | zh_TW |
dc.description.abstract | Banana bract mosaic disease was first discovered in Davao of the Philippines in 1979. It is a newly rising banana disease but it has caused a great impact to the banana industry in several southeastern Asian countries including the Philippines, India, Sri Lanka, Western Samoa and Vietnam. Based on the dark spindle to mosaic discoloration on the bract of the inflorescence, this disease was therefore named “bract mosaic”. It is caused by Banana bract mosaic virus (BBrMV), a positive-sense RNA virus which is a member of Potyvirus, Potyviridae. BBrMV may be transmitted by the vegetative propagating materials such as suckers and tissue-cultured plantlets, and it is also transmitted by the aphids in the field in non-persistent manner. The One-step RT-PCR assay with “total nucleic acid preparation” using a BBrMV-specific primer pair (BBrMV 902) was devised for the accurate detection of BBrMV in this study. Flexuous rod-shaped virus particles (c. 750 nm long) could be observed under a transmitting electron microscope through the PEG purification method of BBrMV from Latundan (PHI-1 isolate, Latundan, AAB). PHI-1 was transmitted to 14 Cavendish banana plantlets by banana aphids (Pentalonia nigronervosa). Eight plantlets were positive for the BBrMV RT-PCR assay 1.5 months later, 11 plantlets were positive 3 months later, and only 3 plantlets were positive 14 months later. All test plantlets did not show the typical or apparent symptoms for banana bract mosaic disease through whole experimental period. On the other hand, the field survey was conducted in the thesis to identify whether BBrMV existed in Taiwan. Totally 50 banana cultivars were collected from “banana germplasm orchard” in Taiwan Banana Research Institute. Cultivar Cocos (serial No. 21), San Chi Chiao (81) and Pei Chiao (85) tested positive in the RT-PCR assay for BBrMV, but they did not show symptoms. Their amplified 902-bp fragments (coat protein gene) of RT-PCR were further cloned and sequenced. The nucleotide sequences of three BBrMV isolates from Taiwan were about 94% identical to foreign published sequences of coat protein gene, and their translated amino acid sequences were about 95.7% identical. These domestic BBrMV isolates were then aphid-transmitted to Saba (BBB), Kluai Khai (AA), Nam Wa (ABB) and Cavendish (AAA) cultivars. Only 2 plantlets (one Saba and one Kluai Khai) tested positive for BBrMV 6 months after inoculation, and they did not show apparent symptoms. In addition, lots of banana foliar samples were collected for another survey from the fields of Pingtung, Kaohsiung, Tainan and Nantou counties. Only one sample of the San Chi Chiao (San-1) cultivar collected from southern Pingtung tested BBrMV-positive. The results of field survey demonstrated that few cases showed positive for BBrMV. It seems that the most banana cultivars grown in Taiwan are tolerant or resistant to BBrMV, or ecological factors in Taiwan are not suitable for multiplication and spreading of BBrMV. The antiserum against BBrMV was also prepared in the thesis for more economic and efficient BBrMV detection. The E. coli expressed coat proteins of BBrMV were prepared and used for the antigens of immunization. A BBrMV-specific antiserum (named PcAb-BBrMV-1) was obtained for the application in ELISA tests, and PcAb-BBrMV-1 showed good results in the BBrMV detection. An ideal extraction buffer (0.5 M borate buffer with 0.5% skim milk,pH 6.8) was selected from the buffer tests, and this buffer can give the best result in ELISA with PcAb-BBrMV-1. The devised RT-PCR and immunological assays can provide different detection methods for BBrMV detections, which should be helpful to inspection and quarantine of BBrMV in Taiwan. | en |
dc.description.provenance | Made available in DSpace on 2021-06-13T06:19:21Z (GMT). No. of bitstreams: 1 ntu-95-R92633007-1.pdf: 2283594 bytes, checksum: 124b2567aaccf5516fe8b3551585b5a0 (MD5) Previous issue date: 2006 | en |
dc.description.tableofcontents | 中文摘要 …………………………………………………………...……....Ⅰ
英文摘要 …………………………………………………………………...III 目錄 ………………………………………………………………………..Ⅴ 壹、 前言 ………………………………………………………………….…1 貳、 前人研究 ……………………………………………..…….………..…4 一、香蕉苞葉嵌紋病毒之發生歷史、病害發生地區及其重要性………..4 二、香蕉苞葉嵌紋病之病徵表現………………………………..…………5 三、香蕉苞葉嵌紋病毒之形態及其基因體………………………………..5 四、香蕉苞葉嵌紋病毒之傳播特性與寄主範圍…………………………..6 五、香蕉苞葉嵌紋病毒之偵測方法………………………………………..7 参、 材料方法……………………………………………………………..…9 一、試驗植物之準備…………………………………………..……………9 二、病毒顆粒之電子顯微鏡(TEM)觀察……………………………….10 1.病毒純化法……………………………………………................... 10 (1) 微量純化法(minipreparation)....................................................10 (2) PEG純法......................................................................................11 2電顯觀察.............................................................................................11 三、應用反轉錄聚合酶鏈鎖反應(RT-PCR)技術研發香蕉苞葉嵌紋 病毒之快速偵測………………………………………………...……12 1. BBrMV專一性引子對之設計………………………………….…12 2. 病毒RNA之萃取………………………………………………….12 3. RT-PCR增幅……………………………………………………….13 4. RT-PCR增幅反應中不同黏合溫度之比較.....................................15 5. PCR產物電泳膠體分析...................................................................15 四、香蕉苞葉嵌紋病毒(BBrMV)之媒介蚜蟲傳播試驗………………….15 1. 健康蕉蚜之來源及飼養……………………………………..….…15 2.香蕉苞葉嵌紋病之生態感染試驗………………………………16 五、BBrMV在田間感染率調查……………….…………………………16 六、BBrMV基因序列之選殖與定序.........................................................17 七、以大腸桿菌表現之BBrMV鞘蛋白(coat protein) 製備多元抗體.....18 1. 重組鞘蛋白表現質體之構築..........................................................18 (1)表現蛋白之載體pGEX-4T-3 vector之酵解反應.....................18 (2) TOPO® vector上鞘蛋白片段之酵解反應.................................19 (3) pGEX-4T-3 vector與鞘蛋白之黏合反應...................................19 (4) 轉型作用.....................................................................................20 2. GST(glutathione S-transferase)融合蛋白在大腸桿菌內之誘導表現 ...........................................................................................................20 3. GST融合蛋白之純化.......................................................................21 4. 蛋白質電泳分析..............................................................................22 (1)製膠.............................................................................................22 (2)電泳分析..................... ................................................................22 (3) Coomassie brilliant blue膠體染色..............................................22 5. 多元抗體之生產.............................................................................23 6. 多元抗體之測試在間接酵素標誌抗體法(indirect-ELISA)及西方 轉漬法(western blotting)之測試....................................................23 (1) 間接酵素標誌抗體法(Indirect Enzyme-Linked Immunosorbent Assay=Indirect ELISA)……………………………………23 1) 萃取緩衝液(extraction buffer)之測試................................23 2) Indirect ELISA…………………………………………….23 (2) 西方轉漬法(western blotting)..................................................24 肆、 結果………………………………………………………………..……26 一、屏東香蕉研究所品種保存園之調查…………………………………26 1. 病徵之觀察.................................................................................. 26 (1) 斯里蘭卡罹病蕉株病徵之觀察.................................................26 (2) 屏東香蕉研究所品種保存園內採集之香蕉病徵之觀察.........26 2. 屏東香蕉研究所品種保存園BBrMV偵測結果................………26 二、 病毒顆粒之電子顯微鏡(TEM)觀察.............………………………27 三、香蕉苞葉嵌紋病毒之快速偵測技術之研發..........................……27 1. 病毒RNA之萃取方法比較…......................................……27 2. RT-PCR增幅....................................................................….……28 3. One-step RT-PCR增幅反應中不同黏合溫度之比較......................28 四、香蕉苞葉嵌紋病毒(BBrMV)之媒介蚜蟲傳播試驗.......….......…28 五、BBrMV在田間感染率調查.......................................................……3 六、國外斯里蘭卡分離株與國內分離株之基因序列選殖及序列分析.....30 七、以大腸桿菌表現之BBrMV鞘蛋白(coat protein) 製備多元抗體......31 八、多元抗體之測試在間接酵素標誌抗體法(indirect-ELISA=I- ELISA) 及西方墨漬法(western blotting)之測試...............................................32 1. 間接酵素標誌抗體法之測試..........................................................32 2. 西方墨漬法(western blotting)之測試...........................…...……33 伍、討論………………………………………………………………………34 陸、參考文獻…………………………………………………………………39 柒、圖表…………………………………………………………………........45 捌、附錄.............................................................................................................84 | |
dc.language.iso | zh-TW | |
dc.title | 香蕉新病毒-香蕉苞葉嵌紋病毒之鑑定及分佈調查 | zh_TW |
dc.title | Identification and Survey of Banana bract mosaic potyvirus (BBrMV)-A New Banana Virus in Taiwan | en |
dc.type | Thesis | |
dc.date.schoolyear | 94-1 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 黃新川,葉信宏,楊宏仁 | |
dc.subject.keyword | 香蕉,香蕉苞葉嵌紋病毒,多元抗體, | zh_TW |
dc.subject.keyword | Banana,Banana bract mosaic vrius,polyclonal antibody, | en |
dc.relation.page | 88 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2006-01-26 | |
dc.contributor.author-college | 生物資源暨農學院 | zh_TW |
dc.contributor.author-dept | 植物病理與微生物學研究所 | zh_TW |
顯示於系所單位: | 植物病理與微生物學系 |
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