Johnsen M G, Rasmussen O F, Albrechtsen M, Borkhardt B
Danish Research Service for Plant and Soil Science, Lyngby.
J Gen Virol. 1991 Jun;72 ( Pt 6):1223-7. doi: 10.1099/0022-1317-72-6-1223.
The purpose of this work has been to investigate in vivo transcription and translation products from an open reading frame (ORF) located downstream of the coat protein (CP) cistron on RNA-2 of pea early browning virus (PEBV). This work was initiated as a step towards elucidation of the significance of this putative gene. Sequence data on RNA-2 suggest that a 29,600 Mr (29.6K) protein is translated from the ORF in question. Hybridization with ORF-specific probes on Northern blots with RNA from infected plants showed that PEBV synthesizes a subgenomic RNA encoding CP (RNA-2a) with a size of 3000 nucleotides (nt) and that a putative subgenomic RNA (RNA-2b) encoding the 29.6K protein appears to have a size of 1600 nt. This is 300 nt less than the size predicted from the sequencing data. For antibody production, a cDNA fragment harbouring 85% of the 29.6K ORF was cloned into the pUEX3 expression vector. The resulting plasmid expresses the 29.6K protein as a fusion protein with beta-galactosidase and this protein was used for raising antiserum containing specific anti-29.6K protein antibodies. By using these antibodies on immunoblots it was demonstrated that the 29.6K protein is expressed in infected plants.
本研究旨在调查豌豆早褐病毒（PEBV）RNA - 2上位于外壳蛋白（CP）顺反子下游的开放阅读框（ORF）的体内转录和翻译产物。这项工作是朝着阐明这个假定基因的意义迈出的一步。RNA - 2的序列数据表明，上述ORF可翻译出一种29,600道尔顿（29.6K）的蛋白质。用感染植物的RNA在Northern印迹上与ORF特异性探针杂交显示，PEBV合成了一种编码CP的亚基因组RNA（RNA - 2a），其大小为3000个核苷酸（nt），并且一种编码29.6K蛋白质的假定亚基因组RNA（RNA - 2b）似乎大小为1600 nt。这比测序数据预测的大小少300 nt。为了制备抗体，将包含29.6K ORF 85%的cDNA片段克隆到pUEX3表达载体中。所得质粒表达的29.6K蛋白质是与β - 半乳糖苷酶的融合蛋白，该蛋白质用于制备含有特异性抗29.6K蛋白质抗体的抗血清。通过在免疫印迹中使用这些抗体，证明了29.6K蛋白质在受感染的植物中表达。
