Site-directed mutagenesis and saturation mutagenesis for the functional study of transcription factors involved in plant secondary metabolite biosynthesis

Methods Mol Biol. 2010;643:47-57. doi: 10.1007/978-1-60761-723-5_4.

Abstract

Regulation of gene expression is largely coordinated by a complex network of interactions between transcription factors (TFs), co-factors, and their cognate cis-regulatory elements in the genome. TFs are multidomain proteins that arise evolutionarily through protein domain shuffling. The modular nature of TFs has led to the idea that specific modules of TFs can be re-designed to regulate desired gene(s) through protein engineering. Utilization of designer TFs for the control of metabolic pathways has emerged as an effective approach for metabolic engineering. We are interested in engineering the basic helix-loop-helix (bHLH, Myc-type) transcription factors. Using site-directed and saturation mutagenesis, in combination with efficient and high-throughput screening systems, we have identified and characterized several amino acid residues critical for higher transactivation activity of a Myc-like bHLH transcription factor involved in anthocyanin biosynthetic pathway in plants. Site-directed and saturation mutagenesis should be generally applicable to engineering of all TFs.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Biosynthetic Pathways / genetics*
  • Cell Separation
  • Cloning, Molecular
  • DNA Primers / genetics
  • Deoxyribonucleases, Type II Site-Specific / metabolism
  • Electroporation
  • Escherichia coli / genetics
  • Genetic Vectors / genetics
  • Luciferases / metabolism
  • Mutagenesis, Site-Directed / methods*
  • Plant Proteins / genetics*
  • Plant Proteins / metabolism*
  • Plasmids / genetics
  • Polymerase Chain Reaction
  • Protoplasts / cytology
  • Saccharomyces cerevisiae / genetics
  • Sequence Analysis, DNA
  • Tobacco / cytology
  • Tobacco / genetics
  • Tobacco / metabolism*
  • Transcription Factors / genetics*
  • Transcription Factors / metabolism*
  • Transformation, Genetic
  • beta-Galactosidase / metabolism

Substances

  • DNA Primers
  • Plant Proteins
  • Transcription Factors
  • Luciferases
  • endodeoxyribonuclease DpnI
  • Deoxyribonucleases, Type II Site-Specific
  • beta-Galactosidase