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  • 學位論文

由馬利筋(Asclepias curassavica L.)嵌紋病分離之一種新potyvirus之特性研究

Characterization of a new potyvirus isolated from asclepias (Asclepias curassavica L.) showing mosaic symptom

指導教授 : 陳滄海

摘要


馬利筋(Asclepias curassavica L.)為自美洲引入台灣之蘿藦科(Asclepiadaceae)馬利筋屬(Asclepias)多年生草本植物,全株帶毒,人為栽培多作為誘蝶之蜜源植物。2009年本研究室自宜蘭地區取得葉片帶有嚴重嵌紋病徵之馬利筋插穗,其葉片粗汁液經由電顯觀察發現長度750-800 nm之絲狀病毒粒子,並經由機械接種於紅藜(Chenopodium amaranticolor)進行三次單斑分離後,回接於健康馬利筋上之後會產生相同的嵌紋病徵。該病毒之熱不活化溫度為55-60℃,耐稀釋度為10-4-10-5,生體外保毒能力於室溫及-75℃下分別可維持2-3天及4個月;將病毒接種於11科44種植物上,共計2科4種植物會被感染並產生病徵,包括蘿藦科之馬利筋與藜科(Chenopodiaceae)之紅藜、奎藜(Chenopodium quinoa)及綠藜(Chenopodium murle);以夾竹桃蚜(Aphis nerii)進行非永續型蚜蟲媒介傳播測試,蚜媒傳播成功率為16.7%;於試驗中未發現種媒傳播現象。以電顯觀察馬利筋病葉組織之超薄切片,可於細胞質中發現potyviruses所誘導之典型風車狀(pinwheels)及薄板狀(laminated aggregates)內含體,同時亦可觀察到細胞核及葉綠體等胞器之病變。以接種病毒之馬利筋葉片進行純化,經高低速交替離心及硫酸銫等密度梯度離心後可獲得純化病毒,每100 g馬利筋病葉之純化病毒收量約為2.6 mg;將純化病毒注射於紐西蘭白兔後製備出對應此病毒之抗血清,經環形介面法測定抗血清力價為4096倍,於瓊脂雙向擴散反應(SDS-agar gel double diffusion test)中該抗血清對本研究中分離之病毒產生專一性同源反應,而在間接式酵素連結免疫吸附反應(Indirect enzyme-linked immunosorbent assay,Indirect ELISA)與西方轉漬反應(Western blotting)中,純化後之免疫球蛋白,除對本研究中分離之病毒產生正反應外,亦與另外21種potyviruses產生正反應;以SDS-聚丙烯醯胺凝膠電泳(SDS-polyacrylamide gel electrophoresis, SDS-PAGE)與西方轉漬反應分析其鞘蛋白(Coat protein, CP)分子量約為30 kDa。以反轉錄-聚合酶連鎖反應(Reverse transcription- polymerase chain reaction, RT-PCR)選殖本病毒核酸3′端之序列,經由序列分析後,解得全長共1399個核苷酸(nucleotide, nt)之序列,經與已登錄於GenBank上之15種potyviruses之基因序列進行比對,CP與3′端非轉譯區(3′ non-coding region, 3′-NCR)之核苷酸序列相同度分別低於69.5%與48.6%,CP之胺基酸序列相同度則低於71.5%。綜合以上病毒粒子形態大小、基本物理特性、細胞病理學、血清學反應及核酸序列分析等證據,一致顯示本研究中所分離之病毒為一新種potyvirus,此為世界首次發現potyvirus感染馬利筋之紀錄,亦為世界首次自馬利筋上分離出病毒並完成其特性分析與核酸分子鑑定之記錄,因此暫將此一由台灣地區馬利筋嵌紋病所分離之新種potyvirus命名為馬利筋嵌紋病毒(Asclepias mosaic virus, AscMV)。

並列摘要


Asclepias (Asclepias curassavica L.) was a perennial herb plant of the family Asclepiadaceae, that was introduced from America area, and was toxic in whole plant. The diseased plants of asclepias showing mosaic symptom was found at Yi-Lan area, Taiwan in 2009. The flexuously filamentous viral particles from leaf saps of mosaic asclepias were observed by electron microscopy, and the length of viral particles was 750-800 nm. The flexuous virus was successfully isolated by mechanical inoculation on Chenopodium amaranticolor and the healthy seedlings of asclepias showed the same mosaic symptom after back inoculation. Thermal inactivation point of the virus was 55-60℃, longevity in vitro was 2-3 days at 25℃ and four months at -75℃, dilution end point was 10-4-10-5. It had a very narrow host range restricted to four species in chenopodiaceous and asclepiadaceous plants among 44 tested plant species belonging to 11 families. The virus was transmissible by aphis nerii as a vector with non-persistent type, and the transmission rate was 16.7%. Seed transmission was not observed in research. The typical pinwheel-shaped and laminated inclusion bodies of potyviruses infection were observed in diseased leaves of asclepias showing mosaic symptom by electron microscopy. An antiserum with a titre of 4096 by ring interface precipitin test was obtained by immunizing a New Zealand white rabbit with the purified virions. In sodium dodecyle sulfate (SDS)-agar gel double diffusion, western blotting and Indirect enzyme-linked immunospot assay the antiserum against the asclepias flexuous virus reacted strongly with its homologous antigens, and reacted with 21 other potyviruses in western blotting and Indirect enzyme-linked immunospot assay. Coat protein (CP) with a molecular weight of 30 kDa was detected by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting. In order to further characterize the virus at the molecular level, coat protein gene of the asclepias virus isolate was cloned , sequenced and analyzed. A sequence of 1399 nucleotides (nts) was obtained and the percentage of nucleotides identities of CP gene and 3′ non-coding region (NCR) were less than 69.5% and 48.6%, comparing to 15 potyviruses, respectively. Similarly, the percentage identity of CP amino acid sequence to those of 15 other known potyviruses were all below 71.5%. The virus was identified as a new potyvirus species on the basis of its particle morphology and size, physical properties, cytopathology, serological characteristics and identity of nucleotide and amino acid sequence of CP. The name Asclepias mosaic virus(AscMV) was provisionally proposed. It is the first report about a potyvirus infection on Asclepias curassavica in the world.

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