Development of series of gateway binary vectors, pGWBs, for realizing efficient construction of fusion genes for plant transformation.
We developed a new series of binary vectors useful for Gateway cloning to facilitate transgenic experiments in plant biotechnology. The new system, Gateway Binary Vectors (pGWBs) realized efficient cloning, constitutive expression using the cauliflower mosaic virus (CaMV) 35S promoter and the construction of fusion genes by simple clonase reaction with an entry clone. The reporters employable in this system are beta-glucuronidase (GUS), synthetic green fluorescent protein with S65T mutation (sGFP), luciferase (LUC), enhanced yellow fluorescent protein (EYFP), and enhanced cyan fluorescent protein (ECFP). The tags available are 6xHis, FLAG, 3xHA, 4xMyc, 10xMyc, GST, T7-epitope, and tandem affinity purification (TAP). In total, 13 kinds of reporter or tag were arranged and were almost applicable to both N- and C-fusions. The pGWBs could be used for many purposes, such as promoter::reporter analysis, observation of subcellular localization by the expression of proteins fused to a reporter or tag, and analysis of protein-protein interaction by copurification and immunodetection experiments. The pGWBs were constructed with modified pBI101 containing a CaMV35S promoter-driven hygromycin phosphotransferase (HPT) gene as the second selection marker. We also constructed pGWBs with the marker HPT driven by the nopaline synthase promoter. By using the pGWB system, the expression of tagged proteins, and the localization of GFP-fused proteins were easily analyzed. Moreover, tissue-specific and inducible gene expression using a promoter was also monitored with pGWBs. It is expected that, the pGWB system will serve as a powerful tool for plasmid construction in plant research.
Nakagawa
Tsuyoshi
T
Department of Molecular and Functional Genomics, Center for Integrated Research in Science, Shimane University, 1060 Nishikawatsu, Matsue, Japan. tnakagaw@life.shimane-u.ac.jp
Kurose
Takayuki
T
Hino
Takeshi
T
Tanaka
Katsunori
K
Kawamukai
Makoto
M
Niwa
Yasuo
Y
Toyooka
Kiminori
K
Matsuoka
Ken
K
Jinbo
Tetsuro
T
Kimura
Tetsuya
T
D000595:Amino Acid Sequence; D019677:Artificial Gene Fusion; D001483:Base Sequence; D017796:Caulimovirus / Q000235:genetics; D002460:Cell Line; D003001:Cloning, Molecular / Q000379:methods; D050939:Gene Fusion; D017930:Genes, Reporter; D005822:Genetic Vectors / Q000235:genetics*; D005966:Glucuronidase / Q000032:analysis / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000235:genetics; D008969:Molecular Sequence Data; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000032:analysis / Q000235:genetics; D010944:Plants / Q000737:chemistry / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000235:genetics*; D014170:Transformation, Genetic
D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D017853:Phosphotransferases (Alcohol Group Acceptor); C063242:hygromycin-B kinase; D005966:Glucuronidase