臺灣博碩士論文加值系統

篩選自行政院農委會屏東種苗研究中心所栽植之孤挺花嵌紋病罹病株來純化病毒。此病毒可在奎藜、紅藜及千日紅等植物引起局部性病斑，且以ELISA檢定呈陽性反應。疑似孤挺花病毒(Hippeastrum flexuous virus, HiFV)是由具嵌紋病徵的孤挺花葉片的粗汁液以機械摩擦方式接種於奎藜上，再經奎藜三次單斑分離而得。病毒的生物特性分析，病毒粒子呈長絲狀，長度約為700-1,800 nm為單股的RNA病毒，基因組長度約20 kb左右。病毒純化結果A260/A280為1.712。熱不活化溫度為55℃，耐稀釋度測定為10-3，耐保存性測定為48小時。利用cDNA合成技術及PCR技術，增幅孤挺花病毒基因組片段來分析孤挺花病毒的3'端基因序列。Hippeastrum flexuous virus近3'端基因序列有4153個核苷酸，包括類poly A tail序列，其鹼基組成包含1344個A (32.36%)、882個C (21.23%)、885個G (21.30%)、1042個T (25.09%)。近3'端基因組RNA有五個開放轉譯架構，從5'到3'端方向依序可產生的蛋白質為8.1 kDa (71 aa)、13.2 kDa (116 aa)、12.7 kDa (108 aa)、5.6 kDa (52 aa) 和9.8 kDa (87 aa)。在Genbank上也沒有與HiFV的核酸序列有相近的植物病毒，就目前而言，HiFV沒有與之同源性高的植物病毒，是一新發現的病毒。

Hippeastrum hybridum plants with mosaic symptom were obtained from Taiwan Seed and Seedling Research Station Pintung Center, and used for virus purification. The virus caused local lesions on C. quinoa, C. amaranticolor and Gomphrena globosa and symptom on Hippeastrum hybridum which was positive in the ELISA test. Hippeastrum flexuous virus (HiFV) was isolated from local lesions of Chenopodium quinoa inoculated mechanically from mosaic symptom amaryllis leaves. HiFV has a length of 700-1,800 nm with positive-sense and single-stranded (ss) RNA genomes of up to 20 kb. The virus was purified with a A260/A280 ratio is 1.712. The thermal inactivation point is 55℃, dilution end point is 10-3, and longevity in vitro end point is 48 hours, respectively. The cDNAs of the 3' proximal HiFV genome were amplified by cDNA synthesis technology and polymerase chain reaction, then the cloned 3' proximal genes of the HiFV genome were sequenced and analyzed. The 3' proximal genes of the HiFV had 4153 nucleotides, including the poly(A) tail, and the base compositions were 1344 adenine (32.36%) ; 882 cytosine (21.23%) ; 885 guanine (21.30%) ; 1042 uracil (25.09%), respectively. From the 5' to 3' end, the 3' proximal genomic RNA of HiFV contained five open reading frames (ORFs), which encoded proteins of 8.1 kDa (71 aa), 13.2 kDa (116 aa), 12.7 kDa (108 aa), 5.6 kDa (52 aa) and 9.8 kDa (87 aa), respectively. The amino acid sequences of the conserved motifs in all proteins are observed and specific nucleotide sequences are also observed. In Genbank, neither closer nucleotide sequences nor high homologous of plant viruses with HiFV were found.

中文摘要…………………………………………………………… I
英文摘要…………………………………………………………… II
壹、前言…………………………………………………………… 1
一、孤挺花簡介………………………………………………….. 1
二、孤挺花之病毒病…………………………………………….. 2
三、Closteroviruses的特性分析………………………………5
貳、材料與方法…………………………………………………… 10
一、病毒的來源…………………………………………………. 10
二、病毒分離…………………………………………………….. 10
三、寄主範圍測定……………………………. .............10
四、病毒之物理性質測定…………………………...........10
（一）耐稀釋度測定…………………………………………… 10
（二）病毒之熱不活性測定…………………………………… 10
（三）耐保存性測定…………………………………………… 11
五、電子顯微鏡觀察…………………………………………….. 11
六、病毒的純化萃取…………………………………………….. 11
七、病毒之酵素結合抗體檢定法分析………………………….. 12
八、Hippeastrum flexuous virus之SDS-PAGE之膠體電泳分析………………………………………………………………....13
九、Hippeastrum flexuous virus病毒鞘蛋白之西方墨點分析..14
十、病毒核酸之萃取與電泳分析…………………………….…. 14
(一) Hippeastrum flexuous virus之病毒核酸萃取.…….14
(二) Hippeastrum flexuous virus之核酸電泳分析.…….15
十一、Hippeastrum flexuous virus基因核苷酸序列之定序.15
(一) SuperScriptTM III RNase H- Reverse Transcription的技術.16
1.第一股cDNA的合成……………………………………….......16
2.聚合酶連鎖反應(PCR)……..……………………………......16
3. PCR產物之電泳膠體分析…………………………..….......21
(二)回收PCR產物…………………………………………….......21
(三)連結(Ligation)……………………………………………....21
(四) DH5α E. coli勝任細胞(competent cells)的配置………22
(五)轉形作用(Transformation)…………………………………..22
(六)質體(plasmid) DNA的萃取…………………………….......23
(七)挑選正確的質體DNA……………………………….…….......23
1.質體DNA之電泳膠體分析………………………………..........23
2.聚合酶連鎖反應(PCR)……………………………….…….......24
(八)基因核酸序列之譯讀……………………………………... ....24
(九)基因序列之比對…………………………………………........25
參、結果……………………………………………………..…….....26
一、Hippeastrum flexuous virus感染植物之病徵…….……...26
二、寄主範圍測定…………..……..…………………………….....26
三、病毒之物理性質測定…………..……..……………….……....26
(一)、耐稀釋度測定…………..…..…………………………….....26
(二)、病毒之熱不活性測定…………..……..………….………....26
(三)、耐保存性測定…………..……..………………………….....26
四、病毒形態之電子顯微鏡觀察…………..……..…………….....30
五、病毒的純化…………..……………………………………….....30
六、病毒之酵素結合抗體檢定法分析…..……………………….....30
七、Hippeastrum flexuous virus之SDS-PAGE之膠體電泳與西方墨點分析…………………………………………….....................34
八、病毒核酸之萃取與電泳分析……………………………….. .....34
九、Hippeastrum flexuous virus病毒基因核苷酸序列之分析與比對………………………………………………………...............34
(一)反轉錄聚合酶連鎖反應產物之電泳膠體分析……………........34
(二)用PCR挑出正確的質體DNA之電泳膠體分析…………...........40
(三) Hippeastrum flexuous virus近 3'端基因組cDNA的合成..40
(四) Hippeastrum flexuous virus基因核苷酸序列之譯讀分析……………………………………………………………............40
肆、討論…………………………………………………………….......50
伍、參考文獻……………………………………………………….......54

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