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

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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

Improvement Of A Potato Viruses Multiplex Detection System And Molecular Characterization Of A Seed-borne Tobamovirus Newly Found In Bottle Guard In Taiwan

張至超 , Masters　　Advisor：張清安　　

繁體中文

馬鈴薯；生物晶片；瓜類病害；病毒分類； Potato ； Bio-chip ； Melon disease ； Virus classification

Abstract │ Reference (59) │ Altmetrics

Abstract 〈TOP〉

本論文研究共包含二大主題，子題一乃針對過去本研究室所初步建立之馬鈴薯病毒多目標型增幅及晶片雜合檢測系統進行改良。此系統乃專為能同時快速檢測多種經濟重要性馬鈴薯病毒而設計，預期應用於馬鈴薯健康種薯之生產系統，對抗威脅馬鈴薯生產之病毒病害。傳統之病毒檢測法乃應用專一性抗體或PCR增幅引子對，針對標的病毒分別進行檢測。因此就感染同一作物之多種病毒必須分別進行多次檢測，以致耗費人力與耗材成本。過去建立之檢測系統乃針對五種關鍵馬鈴薯病毒(PVS、PVM、PVA、PVY及PVX)而設計，但測試結果發現所使用之一組Carlavirus屬廣效性引子無法如預期同時增幅PVS及PVM，而僅對PVS呈現較高的親和性。故本研究採取增加一組PVM專一性引子對之策略，並證實可改善兩病毒增幅上之差異。此外本研究也成功將另二個關鍵病原，馬鈴薯捲葉病毒(Potato leaf curl virus, PLRV)及馬鈴薯瘦薯類病毒(Potato spindle tuber viroid, PSTVd)，納入此多目標增幅系統中，以一組新設計之PLRV專一性引子(PL627)及另一組文獻上已發表之Pospiviroid屬類病毒廣效性引子與其他四組引子對混合進行增幅，成功整合後成為可同時檢測七種馬鈴薯病原之多目標增幅與晶片雜合系統。PCR增幅反應結果可利用電泳分析依據產物分子量之差異判別感染之病原種類，或以各病原之專一性探針在塑膠晶片上進行雜合反應，再以探針圖譜之呈色結果進行判別。研究中也已證實此檢測系統具有可信賴之重複性與再現性，且晶片雜合反應之偵測極限明顯高出電泳分析2-8倍以上。經田間臨床樣品實際測試後也確認可確實反應病毒感染狀況，而與單目標增幅系統所獲結果完全吻合。本論文另一個子題為針對本研究室過去由扁蒲上所分離之一種Tobamovirus屬病毒(WT2分離株)，進行全長基因體解序分析，以釐清其確實之分類地位。經以PCR增幅選殖其基因體九個不同區域片段並加以解序、整合、分析後確認此病毒之全長基因體乃由6498核苷酸所組成，由5’端起共包含4個開放轉譯架構(ORF)，ORF1可轉譯出一個128 KDa 的複製酶(replicase)，ORF2則涵蓋ORF1區域轉譯出一個185 kDa之複製酶(replicase)。ORF3及ORF4則分別轉譯出29 kDa之移動蛋白 (movement protein, MP)及18 kDa之外鞘蛋白(coat protein, CP)。比較特別的是MP之ORF3有部分序列與ORF2及ORF4有所重疊。此種基因組架構與轉譯策略與Tobamovirus屬特性完全符合。與所有登錄於GenBank之tobamoviruses比對後確認，與最相近的Cucumber mottle virus (CuMoV)間全長基因體核苷酸相同度只有80-85 %。根據International Committee on Taxonomy of viruses (ICTV)所訂定之Tobamovirus屬病毒之分類標準，同種病毒之全長基因序列相同度不得低於90%，因而確定WT2分離株應該是一個尚未記錄的新種病毒，乃根據其在扁蒲上所造成之斑紋病徵，定名為扁蒲斑紋病毒(Bottle gourd mottle virus, BGMoV)。藉由親緣樹分析(phytogenetic analyses)發現BGMoV與CuMoV及另一知名之瓜類種傳病毒胡瓜綠斑嵌紋病毒(Cucumber green mottle mosaic virus, CGMMV)乃屬於最相近之演化群組系統(cluster)。本研究也進一步證實BGMoV與上述感染瓜類的tobamovirus一樣都可以經由感病瓜果之種子傳毒到後代實生苗。

Parallel Abstract 〈TOP〉

This thesis contains two topics aiming at virus diseases of two different crops.  The first topic is to modify and improve the multiplex PCR and biochip hybridization system developed by our laboratory for simultaneous detection of five different viruses infecting potato.  The system is designed for quick detection of multiple potato viruses and to be applied in the production of virus-free seed potato, which is the most commonly used strategy against potato viral diseases.  Different from traditional virus detection techniques, our new system can simultaneously detect five commevcially important potato viruses, i. e. Potato virus Y (PVY), Potato virus A (PVA), Potato virus M (PVM), Potato virus S (PVS) and Potato virus X (PVX), in one single test and thus save time and labor cost.  However, repeated testing has shown the Carlavirus-specific primer pair used in the detection system was incapable of amplifying both PVM and PVS, instead it showed higher affinity only to PVS. This research has accomplished that by incorporated a PVM specific primer into the system, both carlaviruses could be detected.  The second improvement that we made was to further incorporate detection primers for another two potato viral pathogens, i.e. Potato leaf curl virus (PLCV) and Potato spindle tuber viroid (PSTVd).  As a result, our newly developed system has allowed us to detect all these seven potato viral pathogens in one test.  The result can be read either by traditional electrophoresis analyses of the amplification products or by processing further in biochips using viral specific probes to hybridize and capture the amplicons and followed by visualizing color reaction of probe diagram on biochip.  The whole detection process can be completed within six hour time.  We have also confirmed the newly developed system is reproducible in different laboratory conditions.  The system’s detection limit was found that biochip hybridization has at least 2-8 times higher sensitivity than the electrophoresis analyses.  When tested with the blind samples collected from potato fields, we confirmed that the system could actually reveal the real infection status in every sample tested.  Furthermore, the detection result obtained by this multiplex system was corresponded to those obtained by simplex detection system.  The second subtopic of this thesis is to conduct genome sequence analyses of a tobamovirus (WT2) newly isolated from bottle gourd.  By cloning and sequencing eleven different regions of the full length genome of WT2 isolate, a complete sequence with 6498 nucleotides was obtained.  The genome encodes four open reading frames (ORFs) that could be translated into four functional viral proteins.  The ORF1 containing 3501 bp can be translated into a putative 128 kDa replication protein.  A 185 kDa readthrough protein containing ORF1 and ORF2 also functions as replication protein of virus RNA.  The next ORF3 (789 bp) and ORF4 (489 bp) encode a 29 kDa movement protein (MP) and a 18 kDa coat protein (CP), respectively.  Interestingly, MP ORF region overlaps both the 185 kDa ORF2 and CP ORF.  This genome organization is the same as those of other reported tobamoviruses.  Comparing with the tobamovirus sequences documented in the GenBank, Cucumber mottle virus is the one with highe nt sequence similavity of 80-85% with WT2.  Based on the species demarcation criteria of tobamoviruses, sequence difference within the same species should be less than 10% so that WT2 is considered as a separate unique species of the genus Tobamovirus.  It is therefore designated as Bottle gourd mottle virus (BGMoV) based on its specific mottle symptom expressed in bottle gourd.  Phylogenetic tree analyses also confirmed that BGMoV is clustered together with two other tobamoviruses infecting cucurbits, i.e. CuMoV and Cucumber green mottle mosaic virus (CGMMV).  Similar to these closely related cucurbit tobamovirus, BGMoV was also confirmed to be seed-borne in our experiment.

Reference ( 59 ) 〈TOP〉

王信旻，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，碩士論文，朝陽科技大學生化科技研究所，台中(2014)
連結：
王瑞章、江汶錦、吳雅芳、林棟樑、孫文章、陳昇寬、彭瑞菊、鄭安秀、謝明憲、鍾瑞永，馬鈴薯栽培管理技術(2011)
連結：
林上湖、鍾文全、楊佐琦，台灣馬鈴薯產業 80 年之回顧與展望植物種苗 SEED&NURSERY (Taiwan)：1-23 (2012)
連結：
林宜螢，由茉莉花 (Jasminum sambac (L.) Ait) 分離一種新potyvirus之特性研究，碩士論文，國立屏東科技大學植物保護系碩士班，屏東(2003)
連結：
行政院農業委員會，台中區農業改良場台中區農情月刊 (2000)
連結：

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