Gene modification by fast‐track recombineering for cellular localization and isolation of components of plant protein complexes
pmid: 31276249
pmc: PMC6852550
Gene modification by fast‐track recombineering for cellular localization and isolation of components of plant protein complexes
SummaryTo accelerate the isolation of plant protein complexes and study cellular localization and interaction of their components, an improved recombineering protocol is described for simple and fast site‐directed modification of plant genes in bacterial artificial chromosomes (BACs). Coding sequences of fluorescent and affinity tags were inserted into genes and transferred together with flanking genomic sequences of desired size by recombination into Agrobacterium plant transformation vectors using three steps of E. coli transformation with PCR‐amplified DNA fragments. Application of fast‐track recombineering is illustrated by the simultaneous labelling of CYCLIN‐DEPENDENT KINASE D (CDKD) and CYCLIN H (CYCH) subunits of kinase module of TFIIH general transcription factor and the CDKD‐activating CDKF;1 kinase with green fluorescent protein (GFP) and mCherry (green and red fluorescent protein) tags, and a PIPL (His18‐StrepII‐HA) epitope. Functionality of modified CDKF;1 gene constructs is verified by complementation of corresponding T‐DNA insertion mutation. Interaction of CYCH with all three known CDKD homologues is confirmed by their co‐localization and co‐immunoprecipitation. Affinity purification and mass spectrometry analyses of CDKD;2, CYCH, and DNA‐replication‐coupled HISTONE H3.1 validate their association with conserved TFIIH subunits and components of CHROMATIN ASSEMBLY FACTOR 1, respectively. The results document that simple modification of plant gene products with suitable tags by fast‐track recombineering is well suited to promote a wide range of protein interaction and proteomics studies.
Chromosomes, Artificial, Bacterial, Agrobacterium-Mediated Transformation, Molecular biology, Genome Evolution and Polyploidy in Plants, Green Fluorescent Proteins, Arabidopsis, DNA repair, Plant Science, Gene, Histones, Agricultural and Biological Sciences, Genes, Reporter, Biochemistry, Genetics and Molecular Biology, Escherichia coli, Genetics, Plant Molecular Farming, Recombineering, Homologous recombination, QH301 Biology / biológia, Molecular Biology, Biology, Recombination, Genetic, Elicitor Signal Transduction for Metabolite Production, Arabidopsis Proteins, Plant Secondary Metabolites, Production of Recombinant Pharmaceuticals in Plants, Life Sciences, Plants, Genetically Modified, Cyclin-Dependent Kinases, QK Botany / növénytan, Luminescent Proteins, Mutagenesis, Insertional, Nucleotide excision repair, Technical Advance, Transcription factor II H, FOS: Biological sciences, Genetic Engineering, Red Fluorescent Protein, Biotechnology
Chromosomes, Artificial, Bacterial, Agrobacterium-Mediated Transformation, Molecular biology, Genome Evolution and Polyploidy in Plants, Green Fluorescent Proteins, Arabidopsis, DNA repair, Plant Science, Gene, Histones, Agricultural and Biological Sciences, Genes, Reporter, Biochemistry, Genetics and Molecular Biology, Escherichia coli, Genetics, Plant Molecular Farming, Recombineering, Homologous recombination, QH301 Biology / biológia, Molecular Biology, Biology, Recombination, Genetic, Elicitor Signal Transduction for Metabolite Production, Arabidopsis Proteins, Plant Secondary Metabolites, Production of Recombinant Pharmaceuticals in Plants, Life Sciences, Plants, Genetically Modified, Cyclin-Dependent Kinases, QK Botany / növénytan, Luminescent Proteins, Mutagenesis, Insertional, Nucleotide excision repair, Technical Advance, Transcription factor II H, FOS: Biological sciences, Genetic Engineering, Red Fluorescent Protein, Biotechnology
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