臺灣博碩士論文加值系統

1995年本研究室於高屏地區發現茉莉花 (Jasminum sambac (Linn) Ait.) 葉片出現嚴重黃化嵌紋之病毒性病徵，經分離與鑑定證實病株中存在一種文獻尚未記載之馬鈴薯Y屬 (Potyvirus) 病毒，命名為茉莉花T病毒 (Jasmin virus T, 簡稱JaVT)。JaVT的各項生物及分子特性包括寄主範圍、感病細胞之病理變化、傳播方式及鞘蛋白 (CP) 基因之分子量與序列均已釐清；然而田間茉莉花病株上所呈現之嚴重黃化病徵是否即為JaVT所引起，由於沒有無病毒感染之茉莉花植株可供接種故至今無法確定。本研究室後續由嚴重黃化病徵之茉莉花上又分離獲得一Carlavirus屬病毒，經鑑定後證實為一尚未見諸報導之新病毒，命名為Jasmin virus C (JaVC)。在後續研究中發現JaVC只會發生在嚴重黃化嵌紋病徵之茉莉花植株中，而在少數呈現輕微嵌紋病徵之茉莉花上並未出現。本研究之目的在於釐清輕微與嚴重型病徵茉莉花植株中之JaVT在分子特性上是否有所差異，乃進行其全長基因體序列之解讀與分析。策略上利用已登錄於GenBank上之Potyvirus屬病毒基因體序列之保守區域配合JaVT已知之CP gene序列設計引子對進行此二不同病徵型茉莉花樣品中病毒全長基因體之增幅、選殖與解序。目前已分別獲得二組完整序列 (JaVT-M及JaVT-S)，其中來自輕微病徵樣品之JaVT-M全長基因體含9639核苷酸 (nt)，而來自嚴重型病徵之JaVT-S則含9660 nt。此乃目前已知感染茉莉花之potyviruses之首次全長基因體定序結果。依據此結果所為之病毒親緣性分析，證實印度於2008年登錄之Jasmine yellow mosaic potyvirus (JN807771.1) 與本研究室於2003年登錄之JaVT (EF535842) 應屬同種異名，故JaVT具命名優先性。我們比對JaVT-M與JaVT-S二基因體內各基因開放轉譯區 (open reading frame, ORF) 之核苷酸與胺基酸相同度 (identity)，除NIb外其餘各ORF之核苷酸與胺基酸之相同度均在98-100 % 間，幾近相同。分析發現兩者間NIb ORF之核苷酸與胺基酸相同度分別只有90及96 %，為所有基因區最低者。JaVT-M之NIb全長含526個胺基酸，較之JaVT-S之NIb之全長517個胺基酸於C端下游附近多出9個胺基酸之嵌入序列。此外JaVT-S之3’端非轉譯區 (UTR) 較JaVT-M含有較長的 poly A尾部。針對兩者在NIb C端區域序列之差異，本研究設計一組可以增幅JaVT病毒3’端涵蓋NIb C端約1000 bp區域之引子對，分別增幅輕微與嚴重病徵型之茉莉花樣品，結果證實唯有前者所增幅之產物在NIb C端具有該特異性插入片段。印證此片段乃造成輕微病徵之JaVT所獨有。根據文獻報導NIb及3’端UTR之生物功能與病毒複製有關，因此推測JaVT-M基因體在NIb C端之特異插入片段與較短的3’端UTR有可能影響JaVT在組織內之複製，進而造成病徵之弱化。接續研究曾嘗試以即時定量real-time RT-PCR之方式比較二個不同病徵型茉莉花葉片組織內病毒複製量，但未獲得與上述推測吻合之結論。因此推測JaVT NIb與3’端UTR上所發現之差異序列可能尚涉及其他機制而造成感病茉莉花病徵之差異表現。此有待進一步之研究方能澄清。當然目前為止我們仍無法排除另一個茉莉花病毒JaVC可引起感病後加成效果，而造成病徵之加重。

A newly recognized potyvirus isolated from jasmine exhibiting foliar yellow mosaic symptoms was identified, characterized and reported as Jasmin virus T (JaVT) in 2003 by our laboratory. Biological and molecular properties including host range, cytological effect, mode of transmission and coat protein gene sequence of JaVT had been clarified. However, the etiology and specific symptom induced by JaVT on jasmine was still uncertain due to the lack of virus free jasmine plant materials for conducting inoculation test. In a recent study we further identified a carlavirus, provisionally named as Jasmin virus C (JaVC), associated closely with jasmine plants showing yellow mosaic symptom. While in some jasmine plants with only mild mottling, but not severe yellow mosaic symptom, JaVC was never detected. Therefore, this study attempted to reveal if the possibilities of molecular differences between JaVTs that might result differential expression of symptom severity in jasmine. The complete genome sequence of JaVTs from jasmine plants with severe yellow mosaic and mild mottling were separately amplified by RT-PCR, cloned and sequence analyzed. The strategy adapted to obtain complete JaVT genome sequence was by overlapping sequences of RT-PCR products amplified from different regions of JaVT genome. PCR primers for amplification were designed based on conservative sequences of potyviruses documented in the GenBenk. As for the terminal sequences in the 5’ non-translated region (NTR) of JaVTs were obtained using commercial kit of GeneRacerTM. Two complete genome sequences were thus separately obtained from jasmine plants with severe yellow mosaic (JaVT-S) and mild mottling (JaVT-M) symptoms. The former contained 9660 nucleotides (nt) while the latter had a shorter sequence of 9639 nt. These were the first two complete genome sequences ever elucidated from potyviruses infecting jasmine. Phylogenetic studies on these sequences with other known potyvirus genome sequences confirmed that JaVT was indeed a unique Potyvirus species. A potyvirus described as Jasmine yellow mosaic virus (JN807771.1) in India in 2008 was confirmed having 100% identities in amino acid sequence of coat protein with JaVT (EF535842) reported in 2003 by our laboratory. This result confirmed JaVT having the naming precedence of this viral pathogen. Comparative studies on the sequences between JaVT-S and JaVT-M found that besides the open reading frame (ORF) in NIb of both sequences, which had lowest identities in amino acid (96%) and nucleotide (90%) among all ORFs, the other ORFs between these two sequences were almost identical to each other. The NIb sequence of JaVT-M contained 526 amino acid (a.a.) residues, while the JaVT-S had only 517 a.a. residues. These 9 a.a. residues were expressed as an inserted fragment located nearby the C-terminal region of NIb. Moreover, it was found that the 3’-NTR of JaVT-S contained a longer poly A tail (64 nt) than that (16 nt) of the JaVT-M. To confirm the existence of the 9 a.a. inserted sequence in JaVT-M, a primer pair was designed to amplify the 3’ end region covering the C-terminal of NIb in JaVT genome and used to react with total RNA separately extracted from jasmine plants with mild mottle and severe yellow mosaic symptoms. The sequence result of the amplicons confirmed that only the one from mild mottling, but not from the severe yellow mosaic, contained the specific 9 a.a. inserted sequence in NIb. Based on literature, NIb and poly A tail of potyvirus genome might involve the replication of virus in infected tissue. Therefore, we speculated that the sequence differences in NIb and 3’-NTR of JaVT might affect the efficiency of virus replication and accordingly resulting differential expression of symptom severity. However, a real-time quantitative RT-PCR technique used to compare the accumulated virus genome between the two symptomatic jasmine tissues did not correlate to our speculation. Therefore, there might be other mechanism of the sequence diversity in JaVT involved to effect the differential symptom expression. Certainly, the present result still could not rule out the possibility of JaVC, another virus existing in severe yellow mosaic symptom, might have synergistic effect with JaVT to promote symptom severity in jasmine.

摘要 ………………………………………………………… Ι
英文摘要 …………………………………………………… IV
致謝 ………………………………………………………… VII
目錄 ………………………………………………………… VIII
表目錄 ……………………………………………………… XII
圖目錄 ……………………………………………………… XIII
壹、前言 …………………………………………………… 1
貳、前人研究 ……………………………………………… 4
一、茉莉花屬之分類 ………………………………… 4
二、茉莉花之病蟲害記錄 …………………………… 4
三、茉莉花屬之病毒害種類記錄 ……………………… 5
(一)、Jasmin chlorotic ringspot virus (JCRV) … 6
(二)、Potato virus Y (PVY) ………………………… 6
(三)、Carlavirus ……………………………………… 7
(四)、Jasmin virus T (JaVT) ………………………… 7
(五)、Jasmin virus C (JaVC) ………………………… 8
四、馬鈴薯Y屬病毒蛋白功能介紹 ……………………… 8
參、材料與方法 …………………………………………… 16
一、田間調查 …………………………………………… 16
(一)、田間採樣 ………………………………………… 16
(二)、病毒之來源與分離 ……………………………… 16
(三)、茉莉花樣品之病毒檢測 ………………………… 17
(四)、植物全量RNA之純化 …………………………… 17
二、病毒序列之分析 ……………………………………… 18
(一)、廣效性引子 (degenerate primers) 之運用…… 18
(二)、反轉錄聚合酶鏈鎖反應 (RT-PCR) ……………… 19
(三)、電泳膠體分析 …………………………………… 19
(四)、DNA 電泳膠體回收 ……………………………… 20
(五)、序列片段選殖 …………………………………… 21
(六)、選殖株篩選 ……………………………………… 21
(七)、Plasmid DNA純化 ………………………………… 22
(八)、選用EcoRⅠ酵素切位 …………………………… 23
(九)、序列定序與分析 ………………………………… 23
三、5’RACE (rapid amplification of cDNA ends) 技術
之運用 ……………………………………………… 23
(一)、Dephosphorylating RNA ………………………… 24
(二)、Removing the mRNA Cap structure …………… 24
(三)、Ligating the RNA Oligo to Decapped mRNA … 25
(四)、Reverse Transcribing mRNA …………………… 26
(五)、Gene-Specific Primers (GSP) 設計 ………… 27
(六)、PCR ………………………………………………… 27
(七)、Nested PCR ……………………………………… 28
(八)、基因片段選殖與定序 …………………………… 28
四、專一性引子對之設計 ……………………………… 29
五、親源分析 ……………………………………………… 29
六、Real-Time PCR測試 ………………………………… 30
肆、結果 …………………………………………………… 32
一、田間調 查及採樣 …………………………………… 32
(一)、田間樣品採集與病徵察 ………………………… 32
(二)、病毒之來源與分離檢測 ………………………… 32
(三)、檢測茉莉花樣品之病毒類型 …………………… 32
二、兩種不同病徵型茉莉花上之JaVT病毒全長基因體序列之
分析 …………………………………………………… 33
(一)、病毒基因序列解序策略與過程 ………………… 33
(二)、核苷酸架構分析 ………………………………… 34
(三)、核苷酸序列比對 ………………………………… 36
三、5’RACE (rapid amplification of cDNA ends) … 37
四、NIb區域中差異性序列之確認 ……………………… 38
五、親緣分析 ……………………………………………… 38
六、Real-Time RT-PCR定量分析 ………………………… 40
伍、討論 …………………………………………………… 41
陸、參考文獻 ……………………………………………… 47
柒、附綠 …………………………………………………… 89

表目錄
表一、感染Jasminum屬植物之病毒 ……………………… 58
表二、本研究增幅並解序二種不同病徵型茉莉花植株組織內茉莉花
T病毒 (Jasmin virus T, JaVT) 全長基因體所使用之專一性
引子對一覽表 ……………………………………… 59
表三、由嚴重黃化嵌紋及輕微斑紋病徵型茉莉花植株所分別獲得之
二組茉莉花T病毒 (Jasmin virus T, JaVT) 全長基因體序列
中各開放轉譯架構 (ORFs) 之核苷酸與胺基酸序列相同度比
較 …………………………………………………… 60
表四、二組來自不同病徵型茉莉花之茉莉花T病毒 (Jasmin virus
T, JaVT)鞘蛋白 (CP) 基因與其他九種親緣最接近之馬鈴
薯Y屬病毒鞘蛋白基因之核苷酸與胺基酸序列相同度比對
………………………………………………………… 61
表五、二組來自不同病徵型茉莉花之茉莉花T病毒 (Jasmin virus
T, JaVT)之全長基因體與其他八種親緣最接近之馬鈴薯Y
屬病毒全長基因體之核苷酸與胺基酸序列相同度比對
………………………………………………………… 63

圖目錄
圖一、彰化縣花壇鄉茉莉花田植株葉片幾乎呈現嚴重黃化嵌紋病徵
之情形 ……………………………………………… 65
圖二、中興大學校園內栽植之茉莉花植株葉片呈現輕微斑紋病徵之
情形 ……………………………………………… 65
圖三、田間茉莉花依病徵嚴重程度可區分為三種不同類型 … 66
圖四、利用對應茉莉花T病毒 (Jasmin virus T) 之專一性引子對A-
30進行RT-PCR檢測經接種輕微斑紋與嚴重黃化嵌紋之茉莉花
病葉之奎藜接種葉 ……………………………………… 67
圖五、利用兩組專一性引子對進行RT-PCR檢測呈現輕微斑紋病徵之
茉莉花樣品 ……………………………………………… 68
圖六、利用兩組專一性引子對進行RT-PCR檢測呈現嚴重黃化嵌紋病
徵之茉莉花樣品 ………………………………………… 69
圖七、利用11組引子對進行輕微斑紋及嚴重黃化嵌紋等兩種病徵型
茉莉花中茉莉花T病毒 (Jasmin virus T, JaVT) 全長基因體
之RT-PCR增幅之電泳分析圖及所獲產物序列套疊後於全長基
因體之相對位置示意圖 ………………………………… 71
圖八、增幅自輕微斑紋病徵之茉莉花T病毒 (Jasmin virus T,
JaVT) 之全長基因體 (JaVT-M) 核苷酸與胺基酸列 …… 76
圖九、增幅自嚴重黃化嵌紋病徵之茉莉花T病毒 (Jasmin virus T,
JaVT) 之全長基因體 (JaVT-S) 核苷酸與胺基酸序列 … 81
圖十、兩組分別增幅自輕微斑紋 (JaVT-M) 及嚴重黃化嵌紋
(JaVT-S) 病徵茉莉花之茉莉花T病毒 (JaVT) 全長基因體架
構 …………………………………………………………… 82
圖十一、兩組來自不同病徵型茉莉花所增幅之基因體 (JaVT-M及
JaVT-S) 中核內含體蛋白b (nuclear inclusion b) 轉譯架
構之胺基酸序列比對 …………………………………… 83
圖十二、兩組來自不同病徵型茉莉花所增幅之基因體 (JaVT-M及
JaVT-S)中3''-端非轉譯區 (3’UTR) 核苷酸序列之比對
…………………………………………………………… 84
圖十三、利用專一性引子P2013-U與A-30-D進行RT-PCR確認輕微斑紋
與嚴重黃化嵌紋病徵茉莉花樣品所增幅產物在核內含體b蛋白
C端區域序列之差異性 ………………………………… 85
圖十四、增幅自輕微斑紋型 (JaVT-M) 及嚴重黃化嵌紋型 (JaVT-
S) 病徵之茉莉花T病毒鞘蛋白基因之核苷酸序列與其他登錄
於GenBank上相同度最高的九種Potyvirus屬病毒相關序列之
親緣演化分析 ………………………………………… 86
圖十五、利用即時定量RT-PCR分析比較輕微斑紋及嚴重黃化嵌紋病
徵茉莉花樣品中茉莉花T病毒 (Jasmin virus T, JaVT) 鞘蛋
白累積量之差異 ………………………………………… 88

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