Premium
Gateway‐compatible vectors for plant functional genomics and proteomics
Author(s) -
Earley Keith W.,
Haag Jeremy R.,
Pontes Olga,
Opper Kristen,
Juehne Tom,
Song Keming,
Pikaard Craig S.
Publication year - 2006
Publication title -
the plant journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.058
H-Index - 269
eISSN - 1365-313X
pISSN - 0960-7412
DOI - 10.1111/j.1365-313x.2005.02617.x
Subject(s) - biology , computational biology , epitope , expression vector , cloning (programming) , target protein , proteomics , flag tag , protein tag , multiple cloning site , green fluorescent protein , tandem affinity purification , plasmid , agrobacterium tumefaciens , immunoprecipitation , gene , genetics , recombinant dna , transformation (genetics) , fusion protein , biochemistry , affinity chromatography , computer science , antibody , programming language , enzyme
Summary Gateway cloning technology facilitates high‐throughput cloning of target sequences by making use of the bacteriophage lambda site‐specific recombination system. Target sequences are first captured in a commercially available ‘entry vector’ and are then recombined into various ‘destination vectors’ for expression in different experimental organisms. Gateway technology has been embraced by a number of plant laboratories that have engineered destination vectors for promoter specificity analyses, protein localization studies, protein/protein interaction studies, constitutive or inducible protein expression studies, gene knockdown by RNA interference, or affinity purification experiments. We review the various types of Gateway destination vectors that are currently available to the plant research community and provide links and references to enable additional information to be obtained concerning these vectors. We also describe a set of ‘pEarleyGate’ plasmid vectors for Agrobacterium ‐mediated plant transformation that translationally fuse FLAG, HA, cMyc, AcV5 or tandem affinity purification epitope tags onto target proteins, with or without an adjacent fluorescent protein. The oligopeptide epitope tags allow the affinity purification, immunolocalization or immunoprecipitation of recombinant proteins expressed in vivo . We demonstrate the utility of pEarleyGate destination vectors for the expression of epitope‐tagged proteins that can be affinity captured or localized by immunofluorescence microscopy. Antibodies detecting the FLAG, HA, cMyc and AcV5 tags show relatively little cross‐reaction with endogenous proteins in a variety of monocotyledonous and dicotyledonous plants, suggesting broad utility for the tags and vectors.