草莓植株中病毒dsRNA的分离和鉴定.pdfVIP

中国农业科学 2006,39(1):145-152 Scientia Agricultura Sinica 草莓植株中病毒dsRNA的分离和鉴定 李 贺，代红艳，张志宏，高秀岩，杜国栋，张馨宇 （沈阳农业大学园艺学院，沈阳 110161 ） 摘要：【目的】病毒核酸分离是病毒基因组解析的基础，本研究旨在建立草莓病毒的 dsRNA 分离方法，获得 草莓主要病毒的部分基因组序列。【方法】采用改进的dsRNA分离方法，对草莓病毒指示植物和栽培品种中的病毒 dsRNA 进行分离，通过琼脂糖凝胶电泳和 RT-PCR 来鉴定。【结果】不同草莓品种中病毒 dsRNA 的含量不同，试管 苗叶片中 dsRNA 含量高于露地苗幼叶，而露地苗幼叶中 dsRNA 含量明显高于完全成熟叶。dsRNA 对 DNase Ⅰ具有 很强的耐性；当酶解缓冲液中含0.5 mol·L-1 NaCl时，dsRNA对RNase A具有较强的耐性。利用DNA 凝胶回收试剂 盒，能够有效回收凝胶中的 dsRNA 片段。应用 RT-PCR 技术，从凝胶回收的 dsRNA 及 DNase Ⅰ/RNase A 两酶酶解 处理后的dsRNA中分别扩增出草莓斑驳病毒和草莓轻型黄边病毒的特异片段。【结论】建立了从草莓植株中分离和 鉴定病毒dsRNA的完整技术体系，为草莓病毒中国分离物的基因组解析奠定了重要基础。 关键词：草莓；dsRNA；病毒；分离；RT-PCR Isolation and Identification of Virus dsRNA from Strawberry Plants LI He, DAI Hong-yan, ZHANG Zhi-hong, GAO Xiu-yan, DU Guo-dong, ZHANG Xin-yu （College of Horticulture, Shenyang Agricultural University, Shenyang 110161 ） Abstract: 【Objective 】The analysis of virus genome is based on nucleic acid isolation. The aim of this study was to develop the method of isolation and identification of virus dsRNA and elucidate the nucleotide sequences of strawberry virus. 【Method 】Using the modified method, the virus dsRNA was extracted from strawberry virus indicator plants and cultivated strawberry plants, and detected by employing agarose gel electrophoresis of EtBr staining and reverse transcription-polymerase chain reaction (RT-PCR). 【Result 】The quantity of virus dsRNA was different among strawberry cultivars. The quantity of dsRNA in microplants was higher than in the young leaves of field plants. And for the field plants, there was more dsRNA in the young leaves. The dsRNA was resistant to DNase Ⅰ, but it became evidently resistant to RNase A only in the presence of 0.5 mol·L-1 NaCl. The dsRNA in agarose gel c