Effects of two vectors on the expression of the NbNAC1 transcription factor and preparation of its polyclonal antibody

[摘要] The NAC (NAM, ATAF, and CUC) superfamily is one of the largest plant-specific families containing transcription factors. An increasing number of studies suggest that NAC1 is involved in plants response to different biotic and abiotic stimulis. Nicotiana benthamiana is a widely used system for evaluating plant-pathogen interactions. In order to study the biochemical function of NbNAC1, NbNAC1 protein and antibody are essential. Therefore, we focused on developing a prokaryotic expression system for producing the Nicotiana benthamiana NbNAC1 protein of in Escherichia coli and the preparation of its polyclonal antibody. Firstly, we constructed two different molecular weight prokaryotic expression vectors: pGE vector with GST tag (pGEX4T-1–NbNAC1) and pET expression vector with His tag (pET28a-NbNAC1). The NbNAC1 protein can be successfully expressed in both vectors. The His-tagged NbNAC1 proteins are insoluble, while the GST-tagged NbNAC1 proteins are partially soluble. We then successfully purified and enriched both proteins. The His-tagged NbNAC1 was chosen to immunize rabbits owing to an unknown protein accompanying the GST-tagged NbNAC1. The anti-NbNAC1 polyclonal antibody had good specificity and could be used in subsequent protein-related studies.

[学科分类] 仪器

[关键词] NbNAC1 transcription factor;Nicotiana benthamiana;Prokaryotic expression;Purification;Antibodies