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研究生:蔡雯莉
論文名稱:火焰百合嵌紋病毒分子特性與檢測技術之研發
論文名稱(外文):Molecular characterization and development of detection methods for Glory lily mosaic virus
指導教授:王惠亮
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:2010
畢業學年度:98
語文別:中文
中文關鍵詞:火焰百合嵌紋病毒馬鈴薯Y屬病毒火焰百合
外文關鍵詞:Glory lily mosaic viruspotyvirusGloriosa spp.
相關次數:
  • 被引用被引用:3
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  • 下載下載:32
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中文摘要

火焰百合(Gloriosa superba L.)屬於秋水仙科嘉蘭屬,多年生攀緣性草本藥用花卉。2007年本實驗室自火焰百合植株呈現黃化嵌紋病徵的葉片,進行病原鑑定及生物特性分析,證實其為新病毒,將其命名為火焰百合嵌紋病毒(Glory lily mosaic virus, GLMV)。為了建立此病毒的序列資料,故利用已發表之Potyvirus屬保留性胺基酸所設計之degenerate primer,並配合本研究所設計之專一性引子及隨機引子,進行RT-PCR以及分子選殖和定序。加上本實驗室已解出包含GLMV之3' NIb、CP、3' UTR的序列,經Vector NTI 9程式將所得序列銜接,共獲得7383個核苷酸,包含3' HC-Pro、P3、6K1、CI、6K2、NIa、NIb、CP等基因及3' UTR。以Vector NTI 9程式預測其胺基酸序列,可找到Potyvirus所具有之保守性區域。將所獲得之CP基因胺基酸序列,分別與其他10種Potyvirus屬病毒進行比對,相似度大致由54至65%,但和Gloriosa stripe mosaic virus (GSMV)相似度有87%;然而,根據國際病毒分類委員會(ICTV)之分類標準,其GLMV與GSMV應為相同病毒之不同strain。本研究亦將純化之病毒注射於紐西蘭雌白兔,採血製得免疫球蛋白。此抗體可應用於indirect ELISA、SDS-免疫擴散法及免疫吸附反轉錄聚合酶連鎖反應中,以偵測火焰百合嵌紋病毒。以間接式酵素連結抗體免疫吸附法測試,結果顯示GLMV 抗血清具有高度的力價。此外,為研發GLMV之檢測系統,故分別比較indirect ELISA、RT-PCR、bead separation-RT-PCR、IC-RT-PCR四者之靈敏度;結果顯示,以IC-RT-PCR最為靈敏,比indirect ELISA靈敏15,000-100,000倍;bead separation-RT-PCR次之,比indirect ELISA靈敏1000倍;第三為RT-PCR,比indirect ELISA靈敏10-100倍;而indirect ELISA靈敏度則最低。利用IC-RT-PCR及bead separation-RT-PCR可直接從火焰百合球根中檢測出GLMV,故本研究所研發的火焰百合嵌紋病毒檢測方法,未來可應用於火焰百合球根病毒檢測,以協助篩選健康之火焰百合球根。
目錄

中文摘要………………………………………………………………I
英文摘要………………………………………………………………III
壹、前言………………………………………………………………1
一、火焰百合簡介……………………………………………………1
二、火焰百合之病毒病害……………………………………………3
三、火焰百合嵌紋病毒特性…………………………………………4
四、Potyvirus屬之病毒特性………………………………………5
五、火焰百合病毒病害之檢測法……………………………………9
(一)指示植物生物檢定法…………………………………………10
(二)電子顯微鏡觀察法……………………………………………11
(三)光學顯微鏡觀察法……………………………………………11
(四)血清免疫檢定法………………………………………………12
1.免疫擴散法…………………………………………………12
2.酵素連結抗體吸附反應……………………………………12
3.西方轉漬法…………………………………………………13
(五)分子生物學方法………………………………………………13
1.雙鏈型核醣核酸dsRNA圖譜之比較………………………13
2.互補核酸探針………………………………………………14
3.聚合酶連鎖反應與反轉錄聚合酶連鎖反應………………14
六、研究目的…………………………………………………………15
貳、材料與方法………………………………………………………16
一、病毒來源與初步鑑定……………………………………………16
(一)病毒的來源……………………………………………………16
(二)植物 total RNA之萃取………………………………………16
(三)反轉錄聚合酶連鎖反應………………………………………17
二、病毒的純化………………………………………………………17
三、病毒核酸之萃取與電泳分析……………………………………18
(一)病毒核酸之萃取………………………………………………18
(二)病毒核酸電泳分析……………………………………………19
四、病毒基因體核酸序列之定序……………………………………19
(一)SuperScriptTM III RNase H- Reverse Transcription
的技術…………………………………………………………20
(二)回收PCR產物……………………………………………………21
(三)連結(Ligation)………………………………………………22
(四) DH5α E. coli勝任細胞(competent cells)的配製……22
(五)轉形作用(Transformation)…………………………………23
(六) plasmid DNA的萃取…………………………………………23
(七)挑選正確的質體DNA……………………………………………24
(八)基因體核酸序列之譯讀…………………………………………24
(九)基因體序列之比對………………………………………………25
(十)不同potyvirus屬病毒其鞘蛋白核苷酸及胺基酸序列之比
較…………………………………………………………………25
五、檢測技術之研發……………………………………………………26
(一)抗血清之製備……………………………………………………26
(二)抗血清力價測定…………………………………………………26
(三)抗血清與不同種類病毒之SDS免疫擴散反應…………………27
(四)免疫球蛋白(IgG)之製備………………………………………27
(五)不同濃度IgG之indirect ELISA反應試驗…………………28
(六)不同potyvirus屬病毒之indirect ELISA反應試驗………29
(七)火焰百合嵌紋病毒專一性引子對之專一性測試………………30
(八)免疫吸附反轉錄聚合酶連鎖反應………………………………30
(九)磁顆粒分離之反轉錄聚合酶連鎖反應…………………………31
(十)利用indirect ELISA、IC-RT-PCR、bead separation- RT-PCR檢
測火焰百合球根…………………………………………………33
六、Indirect ELISA、RT-PCR、IC-RT-PCR及bead separation-RT-PCR鑑定技術偵測法之靈敏度分析…………………………………………33
(一)純化病毒檢測之靈敏度分析………………………………………33
(二)組織液中純化病毒檢測之靈敏度分析……………………………35
(三)病葉組織中病毒之indirect ELISA、 IC-RT-PCR及bead
separation-RT-PCR偵測法的比較分析………………………36
七、種子帶毒測定…………………………………………………………37
参、結果……………………………………………………………………38
一、病毒以反轉錄聚合酶連鎖反應之初步鑑定…………………………38
二、病毒之純化……………………………………………………………38
三、病毒核酸之萃取與電泳分析…………………………………………38
四、病毒基因體核苷酸序列之定序………………………………………39
(一)火焰百合嵌紋病毒部份基因體序列之譯讀結果…………………39
(二)不同Potyvirus屬病毒其鞘蛋白核苷酸及胺基酸序列之比
較……………………………………………………………………40
五、檢測技術之研發………………………………………………………41
(一)抗血清力價測定……………………………………………………41
(二)抗血清與不同種類病毒之SDS免疫擴散反應……………………41
(三)免疫球蛋白之製備…………………………………………………42
(四)不同濃度免疫球蛋白之indirect ELISA反應試驗……………42
(五)不同Potyvirus屬病毒之indirect ELISA反應試驗…………42
(六)火焰百合嵌紋病毒專一性引子對之專一性測試…………………43
(七)免疫吸附反轉錄聚合酶連鎖反應之電泳分析……………………43
(八)磁顆粒分離之反轉錄聚合酶連鎖反應之電泳分析………………43
(九)利用indirect ELISA、IC-RT-PCR、bead separation - RT-PCR
檢測火焰百合球根……………………………………………………44
六、Indirect ELISA、RT-PCR、IC-RT-PCR及bead separation -RT-PCR鑑定技術偵測法之靈敏度分析…………………………………………44
(一)純化病毒檢測之靈敏度分析………………………………………44
(二)組織液中純化病毒檢測之靈敏度分析……………………………45
(三)比較indirect ELISA、 IC-RT-PCR及bead separation - RT-PCR
偵測法對病葉組織中病毒之靈敏度分析…………………………46
七、以bead separation-RT-PCR、indirect ELISA及 IC-RT-PCR對火焰百合幼苗進行檢測…………………………………………………………47
肆、討論……………………………………………………………………48
伍、參考文獻………………………………………………………………54
陸、圖表……………………………………………………………………63
伍、參考文獻
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