臺灣博碩士論文加值系統

柑桔破葉病毒（Citrus tatter leaf virus, CTLV），分類上隸屬於Capillovirus屬，基因體為(+)ssRNA，長650nm。CTLV主要引起柑桔破葉病(citrus tatter leaf )，病毒可經由嫁接及機械感染傳播，對多數之柑桔品種呈潛伏感染，通常無病徵；但當感染某些特殊柑桔品種如Kalpi lime （Citrus excelsa）或Rusk citrange (Poncirus trifoliata × C. sinensis )時，則會產生萎縮、畸形、斑紋及破裂的葉片，並也會在許多柑桔品種上造成砧木與接穗不親和的裂痕，不但影響植株發育且易受強風摧折。蘇等人曾利用指示植物（Rusk）做生物檢測進行台灣CTLV的罹病率調查，結果顯示樣本中約有70~80%的柑桔株帶有CTLV，分佈遍及全省。前年在嘉義農試分所柑桔原種保存園中，發現許多以枳殼或枳柚為砧木的植株呈現砧木與接穗不親和現象，以Rusk生物檢測法鑑定出是受到CTLV的感染。為了進一步了解此病毒，本論文即從生物性與分子性兩方面探討並鑑定不同的CTLV系統。生物性方面，由指示植物枳橙Rusk citrange上的病徵表現為最初鑑定依據，並且再經三代局部病斑分離接種於單斑寄主白藜(Chenopodium quinoa)葉片上，分置於高溫30℃及低溫22℃的溫室中觀察其病徵表現，結果可區分出台灣柳橙分離株（LCd-NA-1）、台灣金柑分離株（Kq-6-2-46）、浙江溫州柑分離株 (Sat-HY-2)與廣西四季桔分離株（Cal-KS-1）在生物性上的差異。為了解台灣CTLV系統之分子特性，將台灣柳橙及金柑分離株逕行基因體全長定序，並已登錄至GenBank。與國外已發表之Capillovirus屬病毒比對的結果發現，台灣兩代表性分離株之相似度達95%，與日本所發表的CTLV只有81%相似度，與其它幾個Capillovirus屬的病毒則約有80%的相似度。另外，因為CTLV過去一直沒有良好的檢測方法，所以利用RT-PCR技術建立一套標準靈敏度高的檢測方法。在多種RNA的萃取方法，得到四種可行良方，其中以TRIzol Reagent 法最符合經濟、靈敏及穩定度高等特點，故將之推薦為最佳CTLV-RNA之萃取法。另外以全長序列分析結果為基礎選出兩特異性引子對：CTLV 636，用於CLTV之普測；CTLV 527，專對CTLV台灣分離株的特異性偵測，再配合最佳條件化之one-step RT-PCR，建立RT-PCR快速偵測法。利用此研發之方法針對嘉義柑桔原種保存園進行檢測，發現甜橙類受害比例甚高，其餘品種受害也不少。由此保存園中收集到來自不同柑桔品種的CTLV分離株，再加上所收集到的國外分離株，以CTLV 636引子對進行RT-PCR增幅，並將這636 bp的增幅片段做定序，再予以序列比對，繪製出國內外所有不同CTLV系統之親源關係圖，由此樹狀圖獲知各分離株之分子歧異度受到地理位置的影響頗大。本論文以生物性及分子性的證據證實CTLV確實具有系統多樣性，此研究結果有助於柑桔破葉病發病生態之進一步瞭解；而論文中所建立的RT-PCR快速偵測法則可提供正確而敏感的CTLV檢疫技術，直接應用於我國柑桔健康種苗制度之建立上。

Citrus tatter leaf disease is one of the important systemic diseases of citrus. It is caused by Citrus tatter leaf virus (CTLV), which is categorized into the genus Capillovirus consisting of a single-stranded, positive-sense RNA genome packaged in circa 650 nm flexuous particles. CTLV is transmitted by vegetative propagation and / or mechanical inoculation. The virus is usually latent in most commercialized citrus cultivars, but it induces dwarfing, distortion, mottling and “tatter leaf” in the infected leaves of susceptible citrus hosts such as Kalpi lime (Citrus excelsa) and Rusk citrange (Poncirus trifoliate × C.sinensis). It may also incite the “bud-scion crease” symptom, which retards citrus growth and sometimes causes breakage at the bud union when the strong wind blows. Su et al. investigated the infection rate for CTLV in citrus plants cultivated in Taiwan. They discovered that approximately 70%~80% of the collected samples were positive for CTLV in the bioassay with “Rusk” indicator plants and CTLV has distributed throughout Taiwan. Recently, the “bud-scion crease” symptom was found in several citrus cultivars with citrange or citrumelo rootstocks grown in the citrus nursery foundation of Chiayi Agricultural Experiment Station (CAES), Taiwan Agricultural Research Institute. The results of bioassays demonstrated that they had been infected by CTLV. This study is dedicated to categorize the different CTLV strains on the biological and molecular level for understanding of the pathological characters of CTLV. In the biologically experimental results, four distinct isolates including LCd-NA-1, Kq-6-2-46, Sat-HY-2 and Cal-KS-1 isolates can be differentiated based on their induced symptoms in the leaves of Rusk citrange. In addition, four CTLV isolates also caused different foliar symptoms in Chenopodium quinoa, the local lesion host grown in a greenhouse under temperature control at 30℃and 22℃. To study the molecular characteristics and genomic structures of two Taiwanese isolates, LCd-NA-1 (from Luchen sweet orange) and Kq-6-2-46 (from kumquat swingle), their full genomic sequences (totally 6496 bases) have been determined and posted in GenBank (accession number AY646511). Alignment of full nucleotide sequences among LCd-NA-1, Kq-6-2-46 and several relative Capilloviruses indicated that the sequence homology is 95% between LCd-NA-1 and Kq-6-2-46; it is about 81% between the Taiwanese CTLV and the Japanese CTLV；and it is about 80% between the Taiwanese CTLV and the other Capilloviruses. On the other hand, a reliable and rapid assay based on the reverse transcription-polymerase chain reaction (RT-PCR) technique was developed for CTLV detection. Four out of ten RNA extraction methods were selected for efficient RT-PCR assays, and the “TRIzol Reagent method” was proven to be the most economic, sensitive and reliable among four feasible methods. Two primer pairs, named CTLV 636 and CTLV 527, were selected to establish a rapid and accurate detecting technique for CTLV with optimized one-step RT-PCR assays. Primer pair CTLV 636, designed from the common region of CTLV coat protein gene, is suitable for the general CTLV RT-PCR detection; Primer pair CTLV 527, designed from the varible region of CTLV coat protein gene, is suitable for differentiating Taiwan CTLV strains from foreign strains. A 636-bp fragment could be amplified from the diseased samples above by RT-PCR with primer pair CTLV 636. Our RT-PCR assay was applied to detect CTLV in many citrus cultivars in the CAES foundation. CTLV was detected in Luchen sweet orange, Valencia sweet orange, Kumquat swingle, Murcott, Minneola tangelo and Ortanique tangor. These amplified fragments were further cloned and sequenced. A phylogenetic tree was obtained according to the alignment of 636 nucleotide sequences among several domestic and foreign CTLV isolates. The viruses in the same geographical regions have high degree of sequence identity. This study provides biological and molecular evidences to demonstrate that CTLV has diverse strains in nature, and is an important step toward understanding of citrus tatter leaf disease and the developed RT-PCR assay is absolutely helpful in the progress of pathogen-free nursery policy of citrus in Taiwan.

中文摘要 …………………………………………………………………II
英文摘要 …………………………………………………………………III
目錄 …………………………………………………………………………V
壹、 前言 ……………………………………………………………………1
貳、 前人研究 ……………………………………………………………4
一、 CTLV之寄主範圍及病徵學…………………………………… 4
二、 柑桔破葉病病毒（CTLV）之電子顯微鏡觀察 …………………6
三、 CTLV的傳播途徑 ………………………………………………6
四、 台灣田間柑桔植株的帶毒率調查 ………………………………7
五、 CTLV的偵測 ……………………………………………………7
六、 CTLV 的分類地位與分子特性 …………………………………8
参、 材料方法………………………………………………………………9
一、 試驗植物之準備……………………………………………………9
二、 CTLV分離株收集…………………………………………………9
三、 生物性鑑別試驗……………………………………………………10
1. 以Rusk枳橙為指示植物之生物鑑別法（bioassay）…………10
2. 白藜（Chenopodium quinoa, local lesion host）的病毒接種
與單斑分離 …………………………………………………10
四、 CTLV之分子特性分析……………………………………………10
1. 台灣兩個不同的CTLV分離株之基因體全長定序策略………10
2. RT-PCR增幅……………………………………………………11
(1). One-step RT-PCR增幅…………………………………11
(2). Two-step RT-PCR增幅……………………………………11
3. PCR產物電泳膠體分析…………………………………………12
4. RT-PCR產物之選殖與定序……………………………………12
五、 RT-PCR快速偵測法之研發與最佳化……………………………13
1. 各種RNA萃取方法之偵測效果比較…………………………13
(1). 迷你管柱純化法…………………………………………13
(2). TENS鹽析法………………………………………………14
(3). Total RNA Reagent法………………………………………14
(4). TRIzol Reagent法…………………………………………15 (5). 總核酸（total nucleic acids）抽取法……………………………15
(6). Tris-Guanidine法…………………………………………16
(7). Guanidine法………………………………………………16
(8). LiCl法………………………………………………………16
(9). CTAB-PVP法………………………………………………17
(10). ASGV法……………………………………………………17
2. 引子對之設計……………………………………………………18
(1). Common CTLV RT-PCR引子對設計………………………18
(2). Taiwan-specific CTLV RT-PCR 引子對設計………………18
3. Two-step RT-PCR與One-step RT-PCR增幅反應之比較………18
4. RT-PCR增幅反應中不同黏合溫度之比較……………………19
5. 不同植株採樣部位之效果比較…………………………………19
6. RNase抑制劑使用與否之效果比較……………………………19
肆、 結果……………………………………………………………………20
一、 不同CTLV分離株之生物性鑑別…………………………………20
1. 以枳橙Rusk為指示植物之生物檢定……………………………20
2. 以白藜（Chenopodium quinoa）為指示植物之生物檢定………20
二、 台灣兩代表性系統病毒之基因體全長定序………………………22
三、 基因體序列分析……………………………………………………22
1. 與全世界已發表之Capillovirus 全長核酸序列進行比對………22
2. 與全世界已發表之Capillovirus部分胺基酸序列做比對………23
四、 RT-PCR快速偵測技術之研發與最佳化………………………24
1. 各種RNA萃取方法之偵測效果比較…………………………24
2. 引子對之設計………………………………………………….24
(1). Common CTLV RT-PCR引子對- CTLV 636……………………24
(2). Taiwan-specific CTLV RT-PCR 引子對- CTLV 527……………25
3. RT-PCR增幅反應………………………………………………25
(1). Two-step RT-PCR與one-step RT-PCR增幅反應之比較………25
(2). One-step RT-PCR 增幅反應中不同黏合溫度之比較…………26
4. 不同植株採樣部位之效果比較…………………………………26
5. RNase抑制劑使用與否之效果比較……………………………27
6. RT-PCR快速偵測技術標準化…………………………………27
五、台灣柑桔原種保存園之CTLV感染調查與品種感病性比較……27
1. CTLV感染調查…………………………………………………27
2. 砧木接穗之感病性比較……………………………………28
六、國內外不同病毒系統之部分核酸序列分析………………………28
伍、討論………………………………………………………………………30
陸、參考文獻…………………………………………………………………35
柒、圖表…………………………………………………………………42

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