草斑点病毒通过结合3'RACE和RNA-Seq两种方法完成了基因组的确定和传染性cDNA的开发
Ina Balke1, Ivars Silamikelis2, Ilze Radovica-Spalvina3
1Plant Virus Protein Research Group, Latvian Biomedical Research and Study Centre, Riga, Latvia.
PloS one
|December 5, 2023
概括
研究人员成功地通过使用高通量测序 (HTS) 识别缺失的未翻译区域 (UTR) 来创建传染性莱格拉斯斑点病毒 (RGMoV) 补充DNA (cDNA). 这一突破使得病毒感染研究成为可能,并证明了HTS用于完整的病毒基因组测序.
科学领域:
- 病毒学 病毒学
- 分子生物学分子生物学
- 植物病理学 植物病理学
背景情况:
- 草斑点病毒 (RGMoV) 是一种Sobemovirus,它具有编码必需病毒蛋白质的阳性感单链RNA基因组.
- 以前试图创建传染性RGMoV补充DNA (cDNA) 的尝试失败了,这表明基因组序列不完整,特别是在未翻译区域 (UTR).
研究的目的:
- 识别和表征RGMoV基因组的完整的5'和3'未翻译区域 (UTR).
- 构建一种具有传染性的RGMoVcDNA克隆,可以在宿主植物中诱导病毒感染.
- 验证高通量测序 (HTS) 对于完整病毒基因组确定的实用性.
主要方法:
- 利用cDNA末端的3'端快速放大 (RACE) 来确定3' UTR序列.
- 使用高通量测序 (HTS) -RNA-Seq来识别5' UTR序列.
- 构建具有新发现的UTRs的传染性cDNA (icDNA) 克隆,用于体外转录和对麦植物的注射.
主要成果:
- 成功识别了RGMoV的完整的5'和3'UTR,并将其纳入了icDNA结构.
- 修改后的icDNA克隆在麦植物中诱导了典型的病毒感染症状,并允许后代病毒传播.
- 高通量测序 (HTS) 证明可靠地获得完整的病毒基因组序列.
结论:
- 完整的UTR对于RGMoV感染性和复制性至关重要.
- 3'RACE和HTS-RNA-Seq的协同使用对于解决不完整的病毒基因组序列是有效的.
- 这项研究确立了产生传染性RGMoV的可靠方法,并突出了HTS作为病毒学中的强大工具.
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