- 學位論文
落葵皺葉嵌紋病毒與感染藤三七之分離株之分子選殖與特性分析
Molecular cloning and characterization of Basella rugose mosaic virus and the isolate infecting Anredera cordifolia
摘要
自台北縣新店市發現藤三七(Anredera cordifolia)葉片上有疑似病毒引起的輪點狀病徵，將罹病材料以汁液機械接種於指示植物白藜及紅藜上，分別造成系統性的感染及局限性斑點的病徵。於紅藜植株進行三次單斑分離，得到一病毒分離株N。在寄主範圍測試方面，將分離株N機械接種於八科二十二種的植物，其中除了紅藜白藜外，僅在菸草(Nicotiana benthamiana) 以及番杏(Tetragonia expansa) 葉片上分別造成嵌紋和褪綠單斑的病徵，回接健康藤三七，則能在新葉上產生褪綠病徵，以穿透式電子顯微鏡觀察病毒分離株，可見長約650 nm的長絲狀病毒顆粒，經由酵素連結免疫吸附反應檢測 (ELISA)，發現與anti-potyvirus group之單元抗體呈正反應。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，經由BLASTn程式，與資料庫中的病毒序列進行比對，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，且與先前本實驗室所發現的落葵皺葉嵌紋病毒(Basella rugose mosaic virus, BaRMV)之核苷酸序列相同度達98%。因此判定此病毒株為落葵皺葉嵌紋病毒在藤三七上所發現的分離株，名為BaRMV-N。 利用本實驗室針對potyvirus所設計的廣效性引子及此病毒的專一性引子，配合RT-PCR或RACE進行病毒其餘基因體部分的選殖。經過解序以及序列分析，已獲得BaRMV-N完整的病毒序列。因為感染落葵和藤三七之BaRMV基因體序列的相同度極高，選用適合的引子對從罹病落葵中選殖出BaRMV-J的序列片段並定序之。從序列分析結果可知BaRMV-N與BaRMV-J具有相同的長度，皆為9804個核苷酸，除5’和3’非轉譯區外，全長序列可轉譯出包含P1、HC-Pro、P3、6K1、CI、6K2、NIa、NIb及CP九個蛋白共3079個胺基酸的大蛋白 (polyprotein)，經過和其他已發表的38種potyvirus的序列進行排並比較，可以推測出這九種蛋白裂解的位置，在BaRMV-N與BaRMV-J之間，這些蛋白的胺基酸相同度分別為95%、98%、97%、96%、97%、96%、98%、99%、98%。 BaRMV各個病毒蛋白的胺基酸序列、大蛋白胺基酸序列和全長核苷酸序列，也分別與這38種potyvirus進行排並比較以及類緣關係分析。在鞘蛋白部分，結果顯示其胺基酸序列相同度在各種病毒間最高為68%，而最低為44%；在病毒基因體全長部分，其核苷酸相同度最高則為62%。依現行國際病毒分類委員會(ICTV)分類標準，BaRMV為一新種的potyvirus而與之類源關係最為相近的是甜菜嵌紋病毒(Beet mosaic virus)。經由ELISA檢測，約有八成的受測檢體出現正反應，證實BaRMV 普遍存在於台灣的藤三七上。
並列摘要
Anredera cordifolia planted in Sindian city exhibited virus-like ringspot symptom on leaves. Leaf saps were mechanically inoculated on indicator plant Chenopodium amaranticolor and C. quinoa. Systemic symptom developed on C. quinoa but only local lesions appeared on inocualted C. amaranticolor. Through three times of successive single lesion isolation on C. amaranticolor, a virus isolate N was obtained. In host range test, in addition to C. amaranticolor and C. quinoa, the virus isolate can only infect Nicotiana benthamiana and Tetragonia expansa and develope mosaic and chlorotic local lesion on leaves, respectively, in 20 species of 8 families plants. When performing backinoculations, newly formed leaves of A. cordifolia exhibit chlorotic foliar symptom. With electron microscopy inspection, the virus particles were found to be filamentous and about 650 nm in length. Indirect ELISA indicated the viruses in the symptomatic leaves reacted with anti-potyvirus monoclonal antibody. Subsequently, the degenerate primers designed for detecting potyviruses were used for further confirmation. The amplified fragments, about 1.8 kb in size, were cloned and sequenced. The sequencing data were analysed with BLAST program with other viruses in the database. The result clearly indicated that the amplified fragments were derived from a potyvirus and contained partial NIb gene, coat protein gene and 3'UTR. Furthermore, these virus clones share 98% nucleotide identity with Basella rugose mosaic virus (BaRMV) previously identified by our laboratory. Therefore, the virus isolate was named as BaRMV-N. By means of RT-PCR or RACE method utilizing potyvirus degenerate primers and BaRMV-N specific primers designed from sequencing data, the remaining genomic fragments of BaRMV-N were cloned and sequenced. Because of the high identity between the two virus isolates, the full length of BaRMV-J genome sequence was cloned and sequenced by appropriate BaRMV-N primers. Both BaRMV-N and BaRMV-J are composed of 9804 nucleotides, excluding the 3’ terminal poly(A) tail, and contain an open reading frame of 9237 nt, encoding a polyprotein of 3079 amino acids. Nine putative proteinase cleavage sites were predicted by analogy with genome arrangements of other potyviruses. The nine mature viral proteins and the amino acid identity between BaRMV-N and BaRMV-J are P1 (95%), HC-Pro (98%), P3 (97%), 6K1 (96%), CI (97%), 6K2 (96%), NIa (98%), NIb (99%) and coat protein (98%). The amino acid identity of viral proteins, polyprotein and the identity of complete nucleotide sequence when comparing with 38 different species of potyvirus were calculated. Additionally these data were used for constructing phylogenetic trees. The highest amino acid identities of CP and highest nucleotide sequence identity of whole genome of BaRMV-N and other potyviruses were 68% and 54%, respectively. Therefore, in terms of species demarcating criteria in the genus Potyvirus, BaRMV is considered as a new potyvirus and the most closely related virus is Beet mosaic virus (BtMV). In detection by ELISA, there were about 80% sample positively reacting with BaRMV antiserum so we presumed that BaRMV exist in Taiwan commonly.
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