Molecular characterization of a member of a new species of grapevine viroid
Molecular characterization of a member of a new species of grapevine viroid
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葡萄类病毒新种成员的分子特征
DOI：
10.1007/s00705-009-0454-1
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发表时间：
2009-09-01
影响因子：
2.7
通讯作者：
Li, Shifang
中科院分区：
文献类型：
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作者：
Jiang, Dongmei;Guo, Rui;Li, Shifang
材料与方法2005年至2006年，从中国新疆自治区、沉阳和北京采集了来自不同葡萄品种的67个葡萄树样品的幼叶。根据Li等人的方法提取低分子量RNA[12]并使用地高辛 (DIG) 标记的 GYSVd-1 核糖核酸探针通过斑点印迹或 Northern 杂交进行检测。 RT-PCR 分析使用引物 GYSVd-P F（5′-TTGGATCCCACCTCGGAAGGCCGCC-3′，与 nts 36-52 同源）和 GYSVd-P R（5′-TTGGATCCTAACCACAGGAACCACA-3′，与 nts 18-35 互补）。 PCR扩增的循环参数包括94℃热变性5分钟1个循环，94℃30秒、56℃30秒、72℃30秒的30个扩增循环，以及72℃5分钟的最终延伸步骤。克隆后，使用自动 DNA 测序仪（ABI PRISMTM 3730XL DNA 分析仪）对选定的分离株进行测序，并通过 DNAMAN 5.2 版进行分析。 2.使用Clustal W（版本1.83）将序列与保存在GenBank数据库中的澳大利亚GYSVd-1和GYSVd-2序列（登录号分别为X06904和NC003612）进行比对。使用CLC RNA Workbench软件包（版本3.0.1，http://www.clcrnaworkbench.com/）计算可能的二级结构，并使用邻接（NJ）和最大简约（MP）方法（分子进化遗传学分析[MEGA]软件（版本4.0，http://www.megasoftware.net）进行系统发育分析。
Materials and methodsBetween 2005 and 2006, young leaves of 67 grapevine samples from different grape varieties were collected from the Xinjiang autonomous region, Shenyang and Beijing, China. Low-molecular-weight RNAs were extracted according to Li et al.[12] and detected by dot-blot or Northern hybridization using digoxigenin (DIG)-labeled GYSVd-1 riboprobes. RT-PCR analysis used the primers GYSVd-P F (5′-TTGGATCCCACCTCGGAAGGCCGCC-3′, homologous to nts 36–52) and GYSVd-P R (5′-TTGGATCCTAACCACAGGAACCACA-3′, complementary to nts 18–35). The cycling parameters for PCR amplification consisted of one cycle of heat denaturation at 94 C for 5 min, 30 amplification cycles of 94 C for 30 s, 56 C for 30 s, 72 C for 30 s, and a final elongation step at 72 C for 5 min. After cloning, the selected isolates were sequenced using an automated DNA sequencer (ABI PRISMTM 3730XL DNA Analyzer) and analyzed by DNAMAN version 5.2. 2. Sequences were aligned with the Australian GYSVd-1 and GYSVd-2 sequences deposited in the GenBank database (accession numbers X06904 and NC003612, respectively) using Clustal W (Version 1.83). Possible secondary structures were calculated using the CLC RNA Workbench package (version 3.0. 1, http://www. clcrnaworkbench. com/), and phylogenetic analysis was performed using neighbor-joining (NJ) and maximum-parsimony (MP) methods (the Molecular Evolutionary Genetics Analysis [MEGA] software (version 4.0, http://www. megasoftware. net).