台灣鳳梨介殼蟲萎凋－相關病毒－1之初步鑑定

The Preliminary Identification of Pineapple mealybug wilt-associated virus-1 on Pineapple in Taiwan

廖吉彥(Jye-Yann Liao) ； 胡仲祺(Chung-Ci Hu) ； 張清安(Chin-An Chang) ； 鄧汀欽(Ting-Chin Deng)

《台灣農業研究》 57卷1期 (2008/03) Pp. 1-14

https://doi.org/10.6156/JTAR/2008.05701.01

摘要

從彰化縣社頭及二水等地鳳梨[Ananas comosus (L.) Merr.]栽種區採集罹患萎凋病的台農2號鳳梨植株（代號：Pa1）及果實，經電子顯微鏡觀察、電泳分析、病毒鞘蛋白核酸與胺基酸序列比較、鞘蛋白基因之表現蛋白製作抗血清及其西方轉漬反應，證實在鳳梨萎凋病的病株中有屬於鳳梨介殼蟲萎凋－相關病毒－1(Pineapple mealybug wilt-associated virus-1, PMWaV-1)。而其果實上分離到的介殼蟲經鑑定爲鳳梨嫡粉介殼蟲[Dysmicoccus brevipes (Cockerell)]。利用電子顯微鏡以陰染法觀察，病毒顆粒爲長絲狀，大小約1250-1300×12-15 nm。從病毒RNA 3'端解序783個核苷酸屬於鞘蛋白基因，與GenBank中的PMWaV-1 (Access No. AF414119)比較鞘蛋白基因之核苷酸，其相同度達99.1%，而其胺基酸相似度則爲98.4%。將病毒鞘蛋白基因以限制酶切割後，再以DNA ligation kit粘接至pET28b(+)載體，轉殖於DH5α菌株，並於Escherichia coli BL21 (DE3)菌株表現蛋白，經電泳分析該表現蛋白之分子量約爲27 kDa。經注射白兔製備抗血清，以西方轉漬反應顯示自製的抗體與病毒鞘蛋白基因表現蛋白及侵染Pa1植株的病毒鞘蛋白有同源(homologous)反應。因此利用病毒鞘蛋白基因的表現蛋白所製備的抗體即可作檢測之用，本報告爲台灣地區鳳梨萎凋病的病原PMWaV-1之鑑定首篇紀錄。

關鍵字

鳳梨介殼蟲萎凋－相關病毒－1 ； 鞘蛋白基因 ； 表現蛋白 ； 序列比對

並列摘要

A naturally infected pineapple (Ananas comosus) sample (Pa1) was collected from major pineapple-growing area in Sheto and Ershui, Changhua County, Central Taiwan. The diseased plant showed typical symptoms of tip dieback, reddening and downward curling of leaf margins, and early stages of green bumps on leaves. The fruits of pineapple were infested with mealybug (Dysmicoccus brevipes) and sooty mold. By electron microscopic observation, immunosorbent electron microscopy, coat protein electrophoresis, Western blot analysis, and nucleotide sequencing, the virus was identified as an isolate of Pineapple mealybug wilt-associated virus-1 (PMWaV-1). Electron microscopic observations of diseased tissues revealed the presence of flexuous-shaped virus particles, 1250-1300×12-15 nm in dimension. The virus consisting of coat protein subunit with an estimated molecular weight of 27 kDa was found. The 783 3'-terminal nucleotides of this virus were sequenced and compared with corresponding sequences of PMWaV-1 (Access No. AF414119) listed in public databases. We found that this virus is most closely related to the PMWaV-1 with nucleotide and amino acid sequence similarities of 99.1% and 98.4%, respectively. The coat protein (CP) gene was further cloned into the vector pET28b (+), then used to express proteins in Escherichia coli BL21 (DE3) and to produce specific antiserum for future diagnostic purposes. In Western blot, the prepared antiserum reacted with the expressed protein of CP gene and PMWaV-1 antigens in infected pineapple plant. This is the first report of detection and identification of PMWaV-1 associated with infected Ananas comosus in Taiwan.

並列關鍵字

Pineapple mealybug wilt-associated virus-1 ； CP gene ； Expressed protein ； Sequence comparison