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| 題 名 | Molecular and Serological Characterization of a Distinct Potyvirus Causing Latent Infection in Calla Lilies=感染彩色海芋之新種Potyvirus病毒之分子及血清學特性 |
|---|---|
| 作 者 | 陳金枝; 徐惠迪; 鄭櫻慧; 黃春惠; 廖吉彥; 蔡惠婷; 張清安; | 書刊名 | Botanical Studies |
| 卷 期 | 47:4 民95.10 |
| 頁 次 | 頁369-378 |
| 分類號 | 435.43 |
| 關鍵詞 | 海芋; Potyvirus病毒; 核酸序列分析; 鞘蛋白表現; 血清分析; Call lily; Coat protein expression; Potyviruses; Sequence analysis; Serology; |
| 語 文 | 英文(English) |
| 中文摘要 | 本研究自彩色海芋(Zantedeschia spp.)分離到一個具有獨特分子及血清學特性之新種Potyvirus屬病毒,分離株代號為Ca-M19。Ca-M19具典型長絲狀病毒顆粒,可經由機械接種於奎藜(Chenopodium quinoa Willd.)葉片造成局部病斑。利用簡併式Potyvirus屬廣效性引子對可以穩定感染Ca-M19之罹病組織中以反轉錄聚合?鏈鎖反應增幅出大小約1.3kb之核酸片段。經解序分析後證實此片段符合Potyvirus屬病毒3’端基因體特性含有鞘蛋白基因(coat protein, CP)及3’端非轉錄區(3’-NCR)此序列已正式登錄於GenBank中(AF469171)。與已知Potyvirus屬病毒之CP基因及其3’-NCR序列比對後顯示,與Ca-M19親緣最相近之病毒為Soybean mosaic virus (SMV)及Watermelon mosaic virus 2 (WMV 2),然而其核酸序列與Ca-M19間之相同度僅達80%。由於利用傳統純化方法自接種Ca-M19之奎藜葉片所獲之純化病毒顆粒量不足以製備抗血清,因此本研究將選殖之Ca-M19鞘蛋白基因構築於蛋白表現載體pET-28b(+)上,再轉型於Escherichia coli寄主內以誘導其大量表現Ca-M19之CP,利用純化之細菌表達已成功製備出對應Ca-M19之專一性抗體。此抗體可應用於Indirect ELISA及SDS-免疫擴散法中以偵測Ca-M19之感染,且發現Ca-M19與Bean common mosaic virus, Blackeye cowpea mosaic virus, Melon vein banding mosaic virus, Passionfruit mottle virus, Passionfruit crinkle virus, Passionfruit woodness virus, Soybean mosaic Virus, Watermelon mosaic virus 2及Zucchini yellow mosaic virus等病毒具有血清類緣關係。綜合病毒型態、寄主反應、鞘蛋白序列比對與血清關係,Ca-M19顯然為Potyvirus屬中的一種新種病毒,小命為Calla lily latent virus (簡稱CLLV)。另外,由田間採樣標本檢測結果顯示,田間呈現明顯病徵之海芋若偵測到Ca-M19之感染時,該等植株均被證實同時感染到其他海芋病毒。然而只偵測到Ca-M19感染之植株則被證實於整個生長季節中均不會呈現任何可辨識病徵。 |
| 英文摘要 | A virus (isolate: Ca-M19) capable of inducing local lesions on Chenopodium quinoa Willd. was isolated from calla lilies (Zantedeschia spp.). Subculture of Ca-M19 was easily maintained in C. quinoa, but a back inoculation from single lesion of C. quinoa to calla lilies has so far not been successful. Typical potyvirus-like flexuous particles were consistently detected in Ca-M19 infected plants, and a 1.3-kb DNA fragment was amplified from these plants by reverse-transcription polymerase chain reaction (RT-PCR) using potyvirus degenerate primers. The PCR product was cloned and its sequence analyzed (AF469171). The amplicon was revealed to correspond to the 3 terminal region of a potyviral genome. After comparing this sequence with known potyviurs sequences in the GenBank, we considered the virus a new species of Potyvirus based on the uniqueness in its coat protein gene (CP) and the 3’ non-coding region (NCR). Comparative studies showed that Soybean mosaic virus (SMV) and Watermelon mosaic virus 2 (WMV 2) were the two most similar potyviruses with Ca-M19, but they shard only 80% of nucleotide identities in CP and NCR with Ca-M19. Attempts to purify a sufficient quantity of Ca-M19 from C. quinoa for preparation of antibodies were unsuccessful. Alternatively, Ca-M19 CP was expressed by the vector pET28b and purified from E. coli culture, and polyclonal antibodies were prepared in rabbits. The antibody was applied in ELISA, Western blotting, SDS-immunodiffusion and immuno-spcific electron microsocopy for the detection of Ca-M19 in calla lilies. It did not react with at least five calla lily infecting potyviruses, including Dasheen mosaic virus, Bean yellow mosaic virus, Konjak mosaic virus, Turnip mosaic virus, and Zantedeschia mild mosaic virus. Indirect ELISA and SDS-immunodiffusion tests showed that Ca-M19 was serologically related, but distinct from Bean common mosaic virus (BCMV), Black cowpea mosaic virus (BICMV), Melon vein banding mosaic virus (MVbMV), Passionfruit mottle virus (PaMV), Passionfruit crinkle virus (PCV), Passionfruit woodness virus (PWV), Soybean mosaic virus (SMV), Watermelon mosaic virus 2 (WMV 2), and Zucchini yellow mosaic virus (ZYMV). Besides serological techniques, a primer pair (M19u/M19d) and a DNA probe were designed which could also specifically detect and differentiate Ca-M19 from other viruses. By the use of specific antibodies in ELISA, Ca-M19 was frequently detected in calla lily plants collected from several major calla lily production townships in Taiwan. Among 86 filed samples positively reacting to the antibody, 77 of them exhibited evident systemic mosaic symptoms, but these symptomatic plants were confirmed to be infected simultaneously by other viruses. Nine plants were found to be infected by Ca-M19 alone. These plants were confirmed to have remained symptomless throughout a 6-month observation period. Therefore, we propose naming this isolate Calla lily latent virus (CLLV) for its inability to develop any visible symptoms on calla lily. |
本系統中英文摘要資訊取自各篇刊載內容。