An Improved Method for Isolating Closteroviral dsRNAs from Leafroll Affected Grapevines

葡萄捲葉毒素病病毒雙股核醣核酸分離方法之改進

陳慧璘(Huey-Lin Chen Tzeng) ； 陳脉紀(Moh-Jih Chen) ； 曾德賜(Dean Der-Syh Tzeng)

《植物病理學會刊》 5卷1期 (1996/03) Pp. 47-54

https://doi.org/10.6649/PPB.199603_5(1).0006

摘要

本研究旨在改進傳統上應用酚與月桂硫酸鈉(SDS)之核酸萃取方法，藉中藥磨粉機之配合應用，由捲葉病罹病葡萄韌皮組織中抽取複製型病毒雙股核醣核酸(dsRNA)，以供進一步研究應用。本研究發現，萃取液中添加2% SDS情況下，延長高速離心去雜前之靜置時間，對dsRNA收量影響極大。含酚之葡萄萃取液經40分鐘之攪拌，再經60分鐘離心前之靜置，比只經10分鐘攪拌、60分鐘靜置或經40分鐘攪拌而完全未經靜置所獲得之病毒dsRNA收量要高。當萃取液中SDS含量增加至5%時，葡萄Closterovirus病毒dsRNA的收量會提高。但當萃取液中添加不溶性Polyvinyl polypyrrolidone時，葡萄病毒dsRNA收量反而有輕微的減少。經過高速離心去雜後，葡萄病毒經吸附於CF-11纖維素管柱層析並以緩衝液多次清洗，汁液中所含褐化成分即可去除，而不影響病毒dsRNA之收量，因而不需擔心多酚化合物會干擾葡萄病毒dsRNA之分離。利用本研究所研擬之葡萄Closterovirus dsRNA萃取方法，大約用1g新鮮巨峰葡萄樹皮韌皮組織，即可獲得10-20 ng 分子量大小在23 kb的病毒核酸。同一萃取純化流程應用於柑桔南美立枯病(CTV)病毒基因體dsRNA之萃取純化已證實亦相當理想，此一方法改進之成功，已為葡萄捲葉病毒基因體之進一步選殖與遺傳操作建立一條可靠的途徑。

關鍵字

柑桔南美立枯病 ； Closterovirus病毒 ； 雙股核醣核酸分離 ； 葡萄捲葉毒素病 ； 技術改進

並列摘要

Improvement of the conventional phenol-sodium dodecyl sulfate (SDS) protocol for dsRNA extraction from plant tissue was attempted for the isolation of closterovirus dsRNAs from leafroll affected grapevines. Cortical tissues collected from dormant canes of diseased grapevines were pulverized by a Chinese herb grinding machine in the presence of liquid nitrogen. With 2% SDS in the extraction buffer, a higher yield of the viral dsRNAs was obtained with 40-min phenol extraction in a stirred condition followed by a 60-min settling time before sample clarification as compared to that with only 10-min phenol extraction or that without settling treatment. The increase of SDS content up to 5% (W/V) in the extraction buffer further improved the yield of viral dsRNAs, especially the genomic dsRNA. In contrast, the addition of insoluble polyvinyl polypyrrolidone in the extraction buffer slightly reduced the dsRNA yield. The browning effect generally observed during the sample homogenization was found easily removable, without affecting the dsRNA yield, in the subsequent high speed centrifugation and continuous column washing during CF-11 cellulose column chromatography. The concern of interference of phenolic compounds appeared to be unnecessary in the isolation protocol finally adapted. By the described method, approximately 10-20 ng of the genomic dsRNA, with the molecular size estimated at 23 kb, were consistently obtained from per gram fresh weight of stem cortical tissues of diseased Kyoho grapevines. The established method was proved to be also ideal for the isolation of viral genomic dsRNA from citrus tristeza virus affected tissues. The improvement in extraction method has paved a reliable way for further cloning and genetic manipulation of the grapevine leafroll associated virus genome.

並列關鍵字

Citrus tristeza virus ； closterovirus ； dsRNA isolation ； grapevine leafroll virus disease ； technique improvement