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language
eng
Author
Mst Momtaz, Sultana Department of Molecular and Functional Genomics, Interdisciplinary Center for Science 8 Research, Shimane University, Matsue, Japan
Amit Kumar, Dutta Department of Molecular and Functional Genomics, Interdisciplinary Center for Science 8 Research, Shimane University, Matsue, Japan
Tanaka, Yuji Department of Molecular and Functional Genomics, Interdisciplinary Center for Science 8 Research, Shimane University, Matsue, Japan
Mostafa, Aboulela Department of Molecular and Functional Genomics, Interdisciplinary Center for Science 8 Research, Shimane University, Matsue, Japan
Sugiura, Sayaka Department of Applied Biosciences, Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya, Japan
Niwa, Tomoko Department of Applied Biosciences, Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya, Japan
Maeo, Kenichiro Department of Applied Biosciences, Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya, Japan
Goto-Yamada, Shino Malopolska Centre of Biotechnology, Jagiellonian University, Krakow, Poland
Kimura, Tetsuya Department of Life Sciences, Graduate School of Bioresources, Mie University, Tsu, Japan
Ishiguro, Sumie Department of Applied Biosciences, Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya, Japan
Mano, Shoji Department of Cell Biology, National Institute for Basic Biology, Okazaki, Japan
Description
Fluorescent proteins are valuable tools in the bioscience field especially in subcellular localization analysis of proteins and expression analysis of genes. Fusion with organelle targeting signal accumulates fluorescent proteins in specific organelles, increases local brightness, and highlights the signal of fluorescent proteins even in tissues emitting a high background of autofluorescence. For these advantages, organelle-targeted fluorescent proteins are preferably used for promoter:reporter assay to define organ-, tissue-, or cell-specific expression pattern of genes in detail. In this study, we have developed a new series of Gateway cloning technology-compatible binary vectors, pGWBs (attR1-attR2 acceptor sites) and R4L1pGWB (attR4-attL1 acceptor sites), carrying organelle-targeted synthetic green fluorescent protein with S65T mutation (sGFP) (ER-, nucleus-, peroxisome-, and mitochondria-targeted sGFP) and organelle-targeted tag red fluorescent protein (TagRFP) (nucleus-, peroxisome-, and mitochondria-targeted TagRFP). These are available for preparation of promoter:reporter constructs by an LR reaction with a promoter entry clone attL1-promoter-attL2 (for pGWBs) or attL4-promoter-attR1 (for R4L1pGWBs), respectively. A transient expression experiment with particle bombardment using cauliflower mosaic virus 35S promoter-driven constructs has confirmed the correct localization of newly developed organelle-targeted TagRFPs by a co-localization analysis with the previously established organelle-targeted sGFPs. More intense and apparent fluorescence signals were detected by the nucleus- and peroxisome-targeted sGFPs than by the normal sGFPs in the promoter assay using transgenic Arabidopsis thaliana. The new pGWBs and R4L1pGWBs developed here are highly efficient and may serve as useful platforms for more accurate observation of GFP and RFP signals in gene expression analyses of plants.
Subject
promoter assay
organelle-targeted fluorescent protein
binary vector
plant 66 transformation
Gateway cloning.
Journal Title
Journal of biotechnology
Volume
297
Start Page
19
End Page
27
ISSN
0168-1656
ISSN(Online)
1873-4863
Published Date
2019-05-19
DOI
PubMed ID
Publisher
Amsterdam : Elsevier Science Publishers, c1984-
NII Type
Journal Article
Format
PDF
Text Version
著者版
OAI-PMH Set
Interdisciplinary Center for Science Research
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