Smith C R, Tousignant M E, Kaper J M
Microbiology and Plant Pathology Laboratory, U.S. Department of Agriculture, Beltsville, Maryland 20705.
Anal Biochem. 1992 Feb 1;200(2):310-4. doi: 10.1016/0003-2697(92)90471-i.
Total RNA extracted from cucumber mosaic virus (CMV) strains WT, with its associated satellite CARNA 5 (CMV-associated RNA 5), was successfully electroporated into isolated tomato protoplasts. At various time intervals samples were extracted for total nucleic acids and analyzed by semidenaturing polyacrylamide gel electrophoresis (PAGE). Sequence-specific hybridization probes were used for the detection of viral and satellite RNAs following Northern transfer. The resulting PAGE patterns and/or autoradiographs depict the proportional presence of viral and satellite RNAs in the extracts over time and have been referred to as "replication footprint profiles" (RFPs) of specific CMV/CARNA 5 combinations. The effective isolation and infection of tomato protoplasts, combined with the ability to follow virus/satellite titers during the infection by RFP analysis, yield results similar to those of infected plants and reduces experiments of 21 or more days in whole plants to less than 72 h in protoplasts.
从黄瓜花叶病毒（CMV）野生型毒株及其相关卫星RNA CARNA 5（CMV相关RNA 5）中提取的总RNA，成功电穿孔导入分离的番茄原生质体。在不同时间间隔提取样品用于总核酸分析，并通过半变性聚丙烯酰胺凝胶电泳（PAGE）进行分析。Northern转移后，使用序列特异性杂交探针检测病毒和卫星RNA。所得的PAGE图谱和/或放射自显影片描绘了提取物中病毒和卫星RNA随时间的相对含量，这些被称为特定CMV/CARNA 5组合的“复制足迹图谱”（RFP）。番茄原生质体的有效分离和感染，结合通过RFP分析在感染过程中跟踪病毒/卫星滴度的能力，产生的结果与受感染植物相似，并将在整株植物中进行的21天或更长时间的实验减少到原生质体中的不到72小时。
