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標題: 自海芋及康乃馨分離之康乃馨斑駁病毒鞘蛋白基因之選殖,定序,及序列分析
Cloning, sequencing and phylogenetic analysis of the coat protein gene of Carnation mottle virus isolated from calla lily and carnation in Taiwan
作者: 林靜宜
Lin, Ching-yi
關鍵字: Carnation mottle virus
calla lily
出版社: 植物病理學系
摘要: 中文摘要 最近分別自田間栽種之海芋 (Zantedeschia spp.) 及康乃馨 (Dianthus caryophyllus L.) 植株葉片顯現黃化壞疽斑點或黃化斑駁等徵狀分離出二個病毒分離株。上述之海芋及康乃馨罹病組織由台中區農業改良場陳慶忠博士初步以電子顯微鏡觀察到粗汁液中含有直徑約為32-35 nm之球形病毒顆粒。以電泳分析 (SDS-polyacrylamide) 此二病毒分離株分別得到一分子量約40 KDa之鞘蛋白,以雙向免疫擴散反應及酵素聯結免疫分析等血清學方法分析顯示上述海芋及康乃馨所分離之病毒分離株與康乃馨斑駁病毒 (Carnation mottle virus, CarMV) 血清呈現正反應。兩分離株之罹病組織超薄切片以電子顯微鏡鏡檢於葉肉細胞之細胞質內可觀察到病毒顆粒呈分散或結晶狀排列,大小與陰染法之球形病毒顆粒相似。進一步針對CarMV鞘蛋白基因保守區域設計簡並式引子對,利用反轉錄聚合酶連鎖反應 (RT-PCR) 增幅海芋及康乃馨病毒分離株鞘蛋白基因並進行選殖及定序,將分別所得之1047-nt鞘蛋白基因核酸序列與基因庫 (GenBank) 中已發表之24個CarMV分離株鞘蛋白基因序列進行比對分析,發現海芋分離株與CarMV各分離株間具94.6-98.2%之核酸序列相同度 (nucleotide identity) 及94.8-96.8%的胺基酸序列相同度 (amino acid identity);康乃馨分離株與CarMV各分離株間具95.6~97.9%之核酸序列相同度及98.0~99.1%的胺基酸序列相同度;而此兩台灣分離株之間則具96.4%之核酸序列相同度及96.0% 的胺基酸序列相同度。在演化關係上則發現CarMV海芋及康乃馨分離株之親源性並不十分相近,海芋分離株屬第二基因型而康乃馨分離株則為第三基因型。綜合以上電子顯微鏡鏡檢、血清學上試驗結果及鞘蛋白基因選殖及定序,確認分離自引起台灣海芋黃化斑駁病徵之病毒確為CarMV的分離株之一,同時這是首次發現海芋可為CarMV所感染;此外,我們亦確認自引起台灣康乃馨黃化斑駁病徵之病毒確為CarMV的分離株之一,而CarMV感染康乃馨在台灣亦為首次記錄。
Abstract Two virus cultures were isolated from leaves of calla lily (Zantedeschia spp.) (Zan-12B) and carnation (Dianthus caryophyllus L.) (CarMV-C) showing viral disease-like symptoms of yellow necrotic spots and yellow mottle on leaves in the fields in central Taiwan. Isometric particles about 32-35 nm in diameter were found in crude saps of diseased tissues from calla lily and carnation under electron microscopic examination. Similar particles were also observed in the cytoplasms of the mesophyll cells in ultrathin sections prepared from infected D. caryophyllus, D. chinensis and Zantedeschia spp.. SDS-polyacrylamide gel electrophoresis showed that the two viral isolates each contains a structural polypeptide about 40 kDa. Serological tests with double immunodiffusion test and double antibody sandwich-enzyme-linked immunosorbent assay showed that the two isolates reacted positively with an antiserum prepared against Carnation mottle virus (CarMV). Using primers specific to coat protein (CP) gene of CarMV, an expected viral CP gene product of 1.05 kb was amplified by reverse transcriptase polymerase chain reaction from total RNA isolated either from CarMV-C-infected C. quinoa or CarMV Zan12B-infected N. benthamiana. Comparisons of the 1047-nucleotide CP genes form CarMV-C and CarMV Zan12B with those of 24 CarMV isolates available in GenBank showed that the calla lily isolate (Zan-12B) had 94.6-98.2% nucleotide identity and 94.8-96.8% amino acid identity and the carnation isolate (CarMV-C) shared 95.6-97.9% nucleotide identity and 98.0-99.1% amino acid identity. 96.4% nucleotide identity and 96.0% amino acid identity were found between carnation and calla lily isolates from Taiwan. The phylogenetic analysis of the coat protein amino acid sequences indicated that Zan-12B belongs to genotype II while CarMV-C was grouped into genotype III. Base on the results of the coat protein sequence analysis along with the serological assays and electron microscopic examination indicated that the virus Zan-12B isolated from calla lily is an isolate of CarMV and this is the fist report of CarMV infection in calla lilies. In addition, our results also demonstrate that the virus infecting carnation is an isolate of CarMV and this is the first report of CarMV infecting carnation in Taiwan.
Appears in Collections:植物病理學系



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