Development of series of gateway binary vectors, pGWBs, for realizing efficient construction of fusion genes for plant transformation

Tsuyoshi Nakagawa, Takayuki Kurose, Takeshi Hino, Katsunori Tanaka, Makoto Kawamukai, Yasuo Niwa, Kiminori Toyooka, Ken Matsuoka, Tetsuro Jinbo, Tetsuya Kimura

Research output: Contribution to journalArticle

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Abstract

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 β-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.

Original languageEnglish
Pages (from-to)34-41
Number of pages8
JournalJournal of Bioscience and Bioengineering
Volume104
Issue number1
DOIs
Publication statusPublished - Jul 1 2007

Fingerprint

Plant Genes
Gene Fusion
hygromycin-B kinase
Fusion reactions
Genes
Proteins
Cloning
Organism Cloning
Caulimovirus
Glucuronidase
Biotechnology
Green Fluorescent Proteins
Luciferases
Viruses
Gene expression
Purification
Epitopes
Plasmids
Experiments
Tissue

All Science Journal Classification (ASJC) codes

  • Biotechnology
  • Bioengineering
  • Applied Microbiology and Biotechnology

Cite this

Development of series of gateway binary vectors, pGWBs, for realizing efficient construction of fusion genes for plant transformation. / Nakagawa, Tsuyoshi; Kurose, Takayuki; Hino, Takeshi; Tanaka, Katsunori; Kawamukai, Makoto; Niwa, Yasuo; Toyooka, Kiminori; Matsuoka, Ken; Jinbo, Tetsuro; Kimura, Tetsuya.

In: Journal of Bioscience and Bioengineering, Vol. 104, No. 1, 01.07.2007, p. 34-41.

Research output: Contribution to journalArticle

Nakagawa, Tsuyoshi ; Kurose, Takayuki ; Hino, Takeshi ; Tanaka, Katsunori ; Kawamukai, Makoto ; Niwa, Yasuo ; Toyooka, Kiminori ; Matsuoka, Ken ; Jinbo, Tetsuro ; Kimura, Tetsuya. / Development of series of gateway binary vectors, pGWBs, for realizing efficient construction of fusion genes for plant transformation. In: Journal of Bioscience and Bioengineering. 2007 ; Vol. 104, No. 1. pp. 34-41.
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