菜豆黃化嵌紋病毒鶴頂蘭分離株之特性分析與感染性選殖株之構築

Abstract

紅花鶴頂蘭 (Phaius tankervilliae) 是屬於急需復育的臺灣原生地生蘭。於台北縣中和山區鶴頂蘭葉片觀察到疑似病毒所引起的嵌紋病徵，將罹病葉片汁液以機械方式接種於指示植物(Nicotiana benthamiana) 及白藜 (Chenopodium quinoa)，可分別造成系統性感染及局限性斑點的病徵。因此於白藜植株進行三次單斑分離，得到一病毒分離株PT，並完成寄主範圍測試。以穿透式電子顯微鏡觀察罹病組織汁液，可見長約750 nm的長絲狀病毒顆粒；經由ELISA反應檢測原始罹病株與PT接種的植株，發現與anti-potyvirus group之單株抗體呈正反應。利用針對potyvirus所設計的廣效性引子對進行RT-PCR的分析，發現能增幅出1.8 kb大小的片段。將此片段選殖後進行解序，且與GenBank資料庫中的序列進行比對，確定其為potyvirus的部份序列，包含部份NIb基因、鞘蛋白基因及3’非轉譯區的序列，且與菜豆黃化嵌紋病毒 (Bean yellow mosaic virus, BYMV) 之核苷酸序列具97%的相同度。因此判定此病毒株為BYMV在鶴頂蘭所發現的分離株，命名為BYMV-PT。利用針對potyvirus所設計的廣效性引子及此病毒的專一性引子，配合RT-PCR及5’RACE，進行病毒其餘基因體部分的選殖，經過解序和序列分析，已獲得BYMV-PT完整的病毒序列。BYMV-PT的全長度基因體大小為9547個核苷酸(不包含poly A尾端)。除了5’非轉譯區為205個核苷酸以及3’非轉譯區有174個核苷酸之外，轉譯區是由3056個胺基酸所組成，可產生約347 kDa的大蛋白 (polyprotein)，經由蛋白酶裂解位置預測，BYMV-PT可以分成P1、HC-Pro、P3、6K1、CI、6K2、NIa-VPg、NIa-Pro、NIb、CP等十個成熟蛋白。將BYMV-PT序列與其他44種已發表之potyvirus序列排並比對，分析結果顯示，不論是全長度核酸、胺基酸序列或是各個基因的分析，BYMV的不同分離株均會形成獨立的一群，而親源關係最接近BYMV的是苜蓿黃脈病毒 (Clover yellow vein virus, ClYVV)。利用限制片段黏合及RT-PCR增幅的方式構築全長度cDNA株，並已獲得23個帶有T7啟動子的BYMV-PT全長序列cDNA選殖株，目前正待篩選出具有感染力的選殖株，以便進一步探討病毒基因在感染時所扮演的功能。將BYMV-PT回接健康鶴頂蘭，七天後利用ELISA進行檢測，結果顯示為正反應，證實了鶴頂蘭能夠被BYMV-PT感染，但目前尚未觀察到明顯病徵。

Phaius tankervilliae is an indigenous orchid of Taiwan. Owing to be an almost extinct species, the plant needs to be protected extremely. Virus-like mosaic symptoms were observed on the leaves of P. tankervilliae which were found in mountain area of Chung-ho, Taipei County. Systemic symptom and chlorotic lesions appeared when we mechanically inoculated the plant saps onto the leaves of Nicotiana benthamiana and Chenopodium quinoa, respectively. After three successive single lesion isolations in C. quinoa, one virus isolate was obtained and named PT. The host range test of PT isolate was performed. We observed the filamentous virus particles with length of 750 nm in crude plant extract via the transmission electron microscope (TEM). Indirect ELISA test showed that the originally infected and PT-inoculated plants reacted with anti-potyvirus monoclonal antibody positively. Furthermore, RT-PCR was performed by using degenerate primers for potyviruses. A 1.8-kb fragment was amplified and cloned. The sequence of the fragment was compared with the NCBI GenBank database. The result indicated that the 1.8-kb fragment of PT isolate containing the 3’-terminal region of potyvirus including partial NIb gene, CP gene and 3’ untranslated region (UTR). Moreover, it had 97% nucleotide identity with Bean yellow mosaic virus (BYMV). Therefore, the virus obtained from P. tankervilliae is an isolate of BYMV, and designated as BYMV-PT isolate. RT-PCR and 5’RACE techniques were employed to analyze the remaining sequence of BYMV-PT by utilizing potyvirus degenerate primers and BYMV specific primers. After cloning, sequencing and analysis, the genomic sequence of BYMV-PT has 9547 nucleotides in length excluding poly(A) tail. Besides the 205-nt 5’UTR and 174-nt 3’UTR, there is one large open reading frame which encodes a polyprotein of 3046 amino acids with an M(r) of 347 kDa. According to the proteinase cleavage sites, the polyprotein can be processed into P1, HC-Pro, P3, 6K1 and 6K2, CI, NIa-VPg, NIA-Pro, NIb, and CP. Through the whole genome comparisons of BYMV-PT with other 44 potyviruses, BYMV isolates clustered together, and the most closely related virus to BYMV is Clover yellow mosaic virus (ClYVV). In order to study the function of virus genes during infection process, we used positional cloning and RT-PCR amplification to construct BYMV-PT full-length cDNA clone and acquired 23 clones with T7 promoter. The biological activity test of the cDNA clones of BYMV-PT is still going on. By back inoculation of BYMV-PT to healthy P. tankervilliae, the ELISA value was positive for the inoculated plants 7 days after inoculation. It demonstrated that P. tankervilliae could be infected by BYMV-PT. However, no obvious symptom was observed yet.