臺灣博碩士論文加值系統

由台中及屏東之火焰百合植株上發現葉片有黃化嵌纹型的病徵，經由非直接酵素結合抗體反應測試，台中與屏東火焰百合均對LMoV免疫球蛋白有反應；但屏東火焰百合卻對CMV免疫球蛋白亦有反應，顯示有複合感染之情形。透過電子顯微鏡，以陰染法觀察台中火焰百合病毒液，得到病毒顆粒長度約在760-860 nm之間，寬度約在10-15 nm之間。而屏東火焰百合經由三次單斑接種奎藜純化所得之病毒液，透過電子顯微鏡觀察到直徑約為29-31 nm大小之球形顆粒，而此純化後的病毒液在光度吸收曲線圖方面，最高在 258 nm，最低在 221 nm，Amax/Amin之比值為 4.71，A280/A260比值為 0.591。以病毒RNA電泳分析，發現三條明帶大小約在4、3.8、2.5 kb。以SDS-PAGE凝膠電泳分析，分子量約為27.5 kDa。將鞘蛋白基因定序，共有657個核苷酸。其所對應轉譯蛋白質可產生218個胺基酸，分子量經DNASIS軟體分析約為24.1 kDa。與許多不同品系的胡瓜嵌紋病毒之鞘蛋白基因經核苷酸與胺基酸序列比對，發現核苷酸相同度可高達92～97%，胺基酸最高可達97～100%。本研究顯示該病毒為胡瓜嵌紋病毒的火焰百合分離株，且本論文是國內首次從火焰百合分離出胡瓜嵌紋病毒的報告。此外，將病毒感染之台中火焰百合病葉以potyvirus純化方法純化，在離心管中僅見一乳白色帶。純化病毒之紫外光吸光值最高在 260 nm，最低在 245 nm， Amax/Amin 之比值為 1.1，A280/A260之比值為 0.83。病毒RNA經膠體電泳顯示其分子大小約為10 kb。以SDS-PAGE凝膠電泳分析，鞘蛋白次單位分子量約為28.3 kDa。將鞘蛋白基因定序，共有798個核苷酸。其所對應轉譯蛋白質可產生266個胺基酸，分子量經DNASIS軟體分析約為29.8 kDa。進行間接型酵素連結免疫分析法時，發現病葉會與Lily mottle vius（LMoV）之免疫球蛋白產生反應。但經核苷酸序列比對，卻發現與NCBI上所發表之LMoV鞘蛋白基因序列相同度並不高，於核苷酸卻僅有61%，胺基酸58% 之相同度，故推斷台中火焰百合上之病毒與LMoV並不同一種病毒；進一步以台中火焰百合病葉及LMoV感染病葉透過針對於火焰百合上之病毒與LMoV其CP片段所設計之引子對進行RT-PCR，結果亦顯示此病毒與LMoV為不同種病毒，故推測與LMoV可能僅有血清學上之親緣關係。以上結果認定本病毒為一新病毒，將其命名為火焰百合嵌紋病毒（Glory lily mosaic virus, GLMV）。

Plants of Gloriosa superba L. with yellow mosaic symptom which collected from Taichung and Pingtung showed positive reaction in indirect ELISA test with Lily mottle virus antibody. However, the diseased G. superba collected in Pingtung also reacted with Cucumber mosaic virus antibody in indirect ELISA indicatory the plants collected in Pingtung were mix infected with viruses. Under electron microscope in negatively stained of the virus of glory lily in Taichung, and the particles that were 760-860 nm in length and 10-15 nm in width. Under electron microscope purified virus with the single lesion of Chenopodium quinoa L. were infected by glory lily in Pingtung were only observed icosaheral particles with 29-31 nm diameter and the virus purification inoculated C. quinoa had a maximum and minimum absorption at 258 nm and 221 nm, respectively. Amax/Amin and A280/A260 ratios were 4.71 and 0.591, respectively. There were three bands, 4.0, 3.8 and 2.5 kb observed in the electrophoresis of double strand RNA of virus. The molecular weight of coat protein of the virus was estimated 27.5 kDa in the SDS-PAGE. Besides, with cDNA synthesis and PCR amplification, the gene of coat protein was cloned and sequenced. 657 nucleotides encoded a 24.1 kDa protein of 218 amino acid. When inquired the nucleotide and amino acid sequences of CP gene with NCBI database, it showed a high identity of 92～97% and 97～100% , respectively with other strains of Cucumber mosaic virus. Cucumber mosaic virus isolated from Gloriosa superba was first report in Taiwan. Furthermore, the virus purified from diseased leaves of G. superba in Taichung with a white opaque circular band in the centrifuge tube. The purified virus had a maximum and minimum absorption at 260 nm and 245 nm, respectively. Amax/Amin and A280/A260 ratios were 1.1 and 0.83, respectively. Electrophoretic analysis revealed that a 10 kb size of viral RNA of virus purified from G. superba. The molecular weight of coat protein of the virus was estimated 28.3 kDa in the SDS-PAGE. Besides, with cDNA synthesis and PCR amplification, the gene of coat protein was cloned and sequenced, which was found to contain 798 nucleotides encoded a 29.8 kDa protein of 266 amino acid residues. In indirect ELISA test, the LMoV antiserum reacted with the leaves of virus-infected G. superba. When inquired the nucleotide and amino acid sequences of CP gene with NCBI database, it showed the nucleotide and amino acid identities of 61% and 58% with LMoV (NC_005288), respectively. The result indicated that the virus of G. superba in Taichung and LMoV were different viruses; and throught RT-PCR which were amplified from total RNA of diseased leaves with primers which designed by CP sequences of the virus and LMoV, respectively. The result also indicated this virus and LMoV were different viruses,and the virus may only have serological relationship with the LMoV. Since the evidence showed that this virus was a new virus on glory lily named as Glory lily mosaic virus（GLMV）temporaily.

中文摘要……………………………………………………………………… I
英文摘要……………………………………………………………………… III
壹、前言……………………………………………………………………… 1
一、火焰百合簡介…………………………………………………………… 1
二、可感染火焰百合的病毒種類…………………………………………… 3
三、病毒簡介………………………………………………………………… 4
(一)Potyvirus屬病毒特性………………………………………………… 4
(二)胡瓜嵌紋病毒簡介……………………………………………………… 6
四、鞘蛋白基因在分類上之應用………………………………………… 8
五、植物病毒鑑定與病害防治之發展現況………………………………… 9
六、研究目的………………………………………………………………… 11
貳、材料與方法……………………………………………………………… 12
一、病毒之來源與接種試驗………………………………………………… 12
二、病毒以間接酵素結合抗體檢定法初步鑑定…. ……………………… 12
三、寄主範圍之測定………………………………………………………… 13
四、病毒之分離與純化……………………………………………………… 13
(一)台中火焰百合及屏東火焰百合之長絲狀病毒……………………… 13
(二)屏東火焰百合球狀病毒……………………………………………… 14
五、電子顯微鏡觀察……………………………………………………… 16
六、火焰百合病毒之SDS-PAGE膠體電泳分析…………………………… 16
七、火焰百合病毒鞘蛋白之西方墨點分析……………………………… 17
八、病毒ssRNA與dsRNA之萃取與分子大小測定………………………… 18
(一)萃取病毒RNA………………………………………………………… 18
(二)病毒RNA分子大小測定……………………………………………… 19
(三)萃取病毒double strand RNA…………………………………… 19
(四)病毒double strand RNA分子大小測定………………………… 20
(五)萃取植物組織total RNA………………………………………… 20
九、病毒基因之定序……………………………………………………… 21
(一)SuperScript RNase H- Reverse Transcription技術 … 22
1.第一股cDNA的合成………………………………………………… 22
2.聚合酶連鎖反應(PCR)………………………………………… 22
3. PCR產物之電泳膠體分析………………………………………… 25
(二)回收PCR產物………………………………………………………… 25
(三)連結(ligation)…………………………………………………… 25
(四)E. coli DH5α勝任細胞(competent cells)的配置………… 26
(五)轉形作用(transformation)…………………………………… 26
(六)質體(plasmid) DNA的萃取……………………………………… 27
(七)挑選正確的質體DNA………………………………………………… 27
1.質體DNA之電泳膠體分析…………………………………………… 27
2.聚合酶連鎖反應(PCR)……………………………………………… 28
(八)基因核酸序列之譯讀……………………………………………… 28
(九)基因序列之比對…………………………………………………… 28
參、結果……………………………………………………………………… 32
一、病毒之來源與初步鑑定………………………………………………… 32
(一)病毒感染植物之病徵………………………………………………… 32
(二)間接ELISA檢定……………………………………………………… 32
二、寄主範圍……………………………………………………………… 32
三、病毒純化與分析……………………………………………………… 39
(一)病毒純化……………………………………………………………… 39
(二)吸光度曲線及吸光值………………………………………………… 39
四、病毒型態之電子顯微鏡……………………………………………… 40
五、病毒進行indirect ELISA測試…………………………………… 40
六、火焰百合之SDS-PAGE膠體電泳與西方墨點分析…………………… 45
七、病毒核酸之萃取與分子大小測定…………………………………… 51
八、GL–poty與GL–CMV病毒鞘蛋白核酸與胺基酸序列分析…………… 51
(一)反轉錄聚合酶連鎖反應產物之電泳膠體分析……………………… 51
(二)利用PCR篩選出正確的質體DNA之電泳膠體分析………………… 51
(三) GL–poty病毒CP基因核酸序列之譯讀分析…………………… 51
(四) GL–CMV病毒CP基因核酸序列之譯讀分析……………………… 56
(五)以RT-PCR確認台中火焰百合之病毒感染………………………… 56
九、GL–poty病毒基因序列與不同potyviruses之比較……………… 61
十、GL–CMV與不同品系CMV CP基因核酸與胺基酸序列比對………… 67
肆、討論……………………………………………………………………… 72
伍、參考文獻………………………………………………………………… 80

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