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  • 學位論文

由兩種不同病徵型茉莉花上所獲得之茉莉花T病毒全長基因體之解序與分析

Analyses of two sets of complete genome sequences of Jasmine virus T obtained from jasmine plants with different symptoms

指導教授 : 張清安
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摘要


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。

並列摘要


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.

參考文獻


力輕症病毒株系統之建築」,國立中興大學植物病理學系博士
林毅忠,「感染茉莉花之一種新Carlavirus之鑑定及其與黃化病徵
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被引用紀錄


張至超(2017)。馬鈴薯病毒多目標型檢測系統之改良及一種新紀錄之扁蒲種傳病毒之分子特性分析〔碩士論文,朝陽科技大學〕。華藝線上圖書館。https://www.airitilibrary.com/Article/Detail?DocID=U0078-2712201714433463

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