百合水仙嵌紋病相關病毒在台灣之首次報導及其分子選殖與特性分析

First identification and molecular characterization of a potyvirus associated with Alstroemeria in Taiwan

王淳藝(Chuen-Yi Wang)

指導教授 ： 張雅君

國立臺灣大學/生物資源暨農學院/植物病理與微生物學研究所/碩士(2006年)

https://doi.org/10.6342/NTU.2006.10367

摘要

於台灣大學人工氣候室之百合水仙植株葉片上觀察到疑似病毒感染引起的嵌紋病徵，將罹病植物的組織液接種至指示植物番杏(Tetragonia expansa)上，可造成褪綠的侷限性斑點，故用以進行三次單斑的分離，進而取得一病毒分離株TW。以間接式酵素連結抗體免疫吸附法(indirect ELISA)檢測罹病百合水仙以及接種的番杏植株，結果顯示兩者皆與anti-potyvirus group之單元抗體呈正反應，且在穿透式電子顯微鏡下可觀察到長度約750 nm之長絲狀病毒顆粒，顯示植物樣品有potyvirus感染。同時，利用本實驗室針對potyvirus所設計之廣效性引子對(degenerate primers)進行RT-PCR，得一長度為2 kb的片段。經選殖以及序列分析，以BLASTn程式與資料庫中的病毒序列進行比對，確定此序列為potyvirus所有，包含部分NIb基因，完整的鞘蛋白基因以及3’非轉譯區的序列，與日本學者Fuji 等人於2004年發表之百合水仙嵌紋病毒 V 分離株(Alstroemeria mosaic virus V isolate, AlsMV-V)核酸序列的相同度高達98%，顯示所分離的病毒為AlsMV，並命名為AlsMV-TW分離株。為了進一步獲得AlsMV-TW的完整序列，利用本實驗室針對已發表之potyvirus保守性胺基酸序列所設計的廣效性引子對，配合本研究所設計的專一性引子，分別增幅出涵蓋不同區域的五種基因體cDNA片段，再以分子選殖的方法獲得各個區域的選殖株，經解序後進行AlsMV-TW全長序列的分析。目前已得AlsMV-TW近全長之序列共9439個核苷酸，除3’端非轉譯區外，就目前已完成的序列進行預測，可轉譯岀3008個胺基酸的大蛋白(polyportein)，內含部分P1以及完整的HC-Pro、P3、6K1、CI、6K2、NIa、NIb、CP等九個蛋白。將AlsMV-TW序列與其他41種已發表之potyvirus序列進行排並比對，預測AlsMV-TW分離株的蛋白裂解位置，並進行全長核酸、胺基酸，以及各個基因的類緣關係分析。從全長胺基酸和核酸的類緣關係分析結果顯示，AlsMV-TW屬於馬鈴薯病毒Y (Potato virus Y, PVY)亞群 (subgroup)。而各個基因之類緣分析結果中，多顯示AlsMV-TW與PVY的親緣關係最為接近。在鞘蛋白基因的類緣分析方面，另外加入AlsMV-V、Amazon lily mosaic virus (ALiMV)、Pepper severe mosaic virus (PepSMV)、Pepper yellow mosaic virus (PepYMV)、以及Sunflower chlorotic mottle virus (SuCMV)等五個僅發表鞘蛋白及3’非轉譯區序列的病毒進行分析。分析結果顯示，除先前已發表之AlsMV-V分離株外，以PepSMV與AlsMV-TW之類緣關係最接近，鞘蛋白胺基酸序列的相同度高達81%。同時，在鞘蛋白的類緣關係比對中，除PVY外，包括AlsMV以及Pepper mottle virus (PepMoV)、Peru tomato mosaic virus (PTV)、Potato virus V (PVV)，Wild potato mosaic virus (WPMV)以及上述提到的ALiMV、PepSMV、PepYMV、SuCMV等發源於中南美洲的病毒皆成一群，顯示這些來自中南美洲的potyvirus在序列上有地緣的特殊性。
本研究亦同時利用anti-potyvirus group之單元抗體進行indirect ELISA，以及AlsMV-TW專一性引子對進行RT-PCR，以檢測市面販售百合水仙之病毒感染情形。結果顯示市面上所販售之百合水仙確有AlsMV感染，而就受檢樣品中統計，AlsMV在台灣的感染情形並不少見。另外，為了能更直接快速檢測AlsMV，本研究已製備出高專一性之AlsMV多原抗體(polyclonal antibody)，可有效應用在檢疫工作上。總而言之，AlsMV為國內進口植物之檢疫對象，此研究為首次在台灣發現AlsMV的存在，以及首次嘗試進行此病毒的全長序列選殖與分析。

關鍵字

百合水仙 ； 嵌紋病 ； 病毒

並列摘要

Virus-like mosaic symptom was observed on alstroemerias cv. Sunny Rebecca growing in the phytotron of National Taiwan University. Chlorotic lesions appeared on mechanically inoculated leaves of Tetragonia expansa. After three successive single lesion isolations in T. expansa, a virus isolate named TW was obtained. By indirect ELISA, both symptomatic alstroemeria and T. expansa plants tested positive for potyviruses using an anti-potyvirus group monoclonal antibody. Flexuous filaments particles about 750 nm in length were observed in crude sap of the inoculated T. expansa leaves by transmission electron microscopy. Based on the morphology and size of virus particles, results of ELISA test and western blot, it revealed that the virus isolated from Alstroemeria spp. was assumed to should be a potyvirus. To further identify this unknown potyvirus, RT-PCR was performed by using degenerate primers at the conserved region of 3’–end sequence for potyviruses. A 2-kb fragment was amplified, cloned, sequenced and a BLAST search performed against the NCBI database. The results revealed that the sequenced fragment (accession number DQ295032) containing the 3’-terminal region of AlsMV including partial NIb gene, complete CP gene of 801 nt and 3’UTR of 412 nt excluding poly (A) tail. It has 98% nucleotide identity with the only published partial sequence of Alstroemeria mosaic virus V isolate (AlsMV-V, (Fuji et al., 2004; accession number AB158522) indicating that the virus obtained is an isolate of AlsMV, and so is designated as AlsMV-TW isolate.
To analyze the rest remaining sequence of AlsMV-TW, several degenerate primers were paired with specific primers designated in this study, and the full-length cDNA genome of AlsMV-TW was separately RT-PCR amplified to produce 5 overlapping cDNA fragments. After cloning and sequencing these fragments, the nearly whole genomic sequence of AlsMV-TW was determined which contains 9439 nt in length, excluding the poly (A) tract. The revealed genome contains a single long ORF encoding a large polyprotein which may generate P1 (partial of 231 aa), HC-Pro (456 aa), P3 (366 aa), 6K1 and 6K2 (52 aa), CI (634 aa), NIa-VPg (187 aa), NIa-Pro (244 aa), NIb (519 aa), and CP (267 aa) via proteolytic processing. Through pairwise comparisons on the whole genome, it revealed that AlsMV-TW is most closely correlated with PVY among 41 compared potyviruses with complete sequences. This result referred to suggested that AlsMV-TW belongs to the PVY subgroup. As for the phylogenetic analysis of CP and 3’ UTR, several South American potyviruses of incomplete sequences were collected to analyze, including AlsMV-V, Amazon lily mosaic virus (ALiMV), Pepper severe mosaic virus (PepSMV), Pepper yellow mosaic virus (PepYMV), and Sunflower chlorotic mottle virus (SuCMoV). Notably, these South America originated viruses including AlsMV itself clustered within the PVY subgroup with robust bootstrap values based on the CP genes, as previously described (Fuji et al., 2004). This phenomenon stands out that these South American viruses may be isolated and distinct from others by their geographical character.
The infection frequency of AlsMV on alstroemeria in Taiwan was investigated by means of indirect-ELISA experiment using potyviral monoclonal antibody as well as RT-PCR by using AlsMV-specific primers. The result demonstrated that infection of AlsMV on alstroemeria in Taiwan is frequently occurred. In Taiwan, AlsMV is in the list of quarantine pests, but the corresponding detecting strategy has never been developed. In order to detect and identify AlsMV in Taiwan, AlsMV-specific primers and polyclonal antibody were prepared for sensitively and efficiently detecting AlsMV by RT-PCR and ELISA. This is the first report of the identification and determination of complete genome sequence of AlsMV in Taiwan.

並列關鍵字

alstroemeria ； potyvirus

參考文獻

林瑋瑾. 2005. 光週、光強度、溫度及藥劑處理對水仙百合開花與開花枝品質之影響. 國立台灣大學園藝學研究所碩士論文: 1-5.

吳纯宜. 2004. 感染彩色海芋之蕪菁嵌紋病毒之分子特性分析及抗體製備. 國立台灣大學植物病理與微生物學研究所碩士論文: 23-24.

葉慈容. 2004. Potyvirus 快速鑑定晶片之研發. 國立台灣大學植物病理與微生物學研究所碩士論文: 44.

Adams, M. J., Antoniw, J. F., and Fauquet, C. M. 2005. Molecular criteria for genus and species discrimination within the family Potyviridae. Arch. Virol. 150: 459-479.

Aleman-Verdaguer, M. E., Goudou-Urbino, C., Dubern, J., Beachy, R. N., and Fauquet, C. 1997. Analysis of the sequence diversity of the P1, HC, P3, NIb and CP genomic regions of several yam mosaic potyvirus isolates: implications for the intraspecies molecular diversity of potyviruses J. Gen. Virol. 78: 1253-1264.