建立 GFP 氧化還原感應器用於測定水稻原生質體細胞質穀胱甘肽
的氧化還原變化

曾馨誼

doi:10.6342/NTU201701718

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建立 GFP 氧化还原感应器用於测定水稻原生质体细胞质谷胱甘肽的氧化还原变化

Establishment of GFP-Based Ratiometric Probes for Measurement of Cytosolic Glutathione Redox Status in Rice Protoplasts

曾馨谊 , 硕士　　导师：洪传扬　　

繁体中文 DOI： 10.6342/NTU201701718

GRX1-roGFP2 ；谷胱甘肽还原酶； GSH/GSSG ；茎延长；水稻； GRX1-roGFP2 ； Glutathione reductase ； GSH/GSSG ； Stem elongation ； Rice

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摘要 │ 参考文献 (123) │ 国际计量

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Cellular redox changes extensively impacted plant growth, development and stress tolerance. Glutathione (GSH) is an ubiquitous tripeptide thiol in the cells. The ratio of reduced GSH to oxidized GSH (GSSG) is an indicator of plant health. Therefore, maintaining cellular GSH redox homeostasis is important to plant growth, development and stress tolerance. The majority of glutathione redox research has been based on the chemical analysis of whole-cell extracts, which unavoidably destroy subcellular compartment-specific information. Recently, GFP based ratiometric redox-sensitive GFP (roGFP) protein was used to detect the redox status within different organelles by stable transformation. To generate a simple and rapid detection system, this study is aim to target a redox sensitive GRX1-roGFP2 to cytosol or plastids of rice protoplasts. A glutathione reductase 1 transit peptide was fused with GRX1-roGFP2 for chloroplast and mitochrondria targeting. After transforming GRX1-roGFP2, GFP fluorescence intensity was significantly changed by DTT (reductant) or H2O2 (oxidant) in rice protoplasts, which reflected the fluctuation of GSH redox status. The results showed that GRX1-roGFP2 could detect GSH redox changes in rice protoplast system. Application of protoplast system on the redox status of GSH in glutathione reductase 2 (GR2)-RNAi transgenic plants showed that GSH/GSSG ratio significantly decreased in cytosol by confocal microscopy and fluorescence microplate reader. Thus, transient expression of GRX1-roGFP2 in rice protoplasts for detecting GSH redox changes can be a convenient and efficient system for detecting the redox changes in plant organelles.

参考文献 ( 123 ) 〈TOP〉

Aller, I., Rouhier, N., and Meyer, A.J. (2013). Development of roGFP2-derived redox probes for measurement of the glutathione redox potential in the cytosol of severely glutathione-deficient rml1 seedlings. Front Plant Sci 4, 506.
连结：
Bashir, K., Nagasaka, S., Itai, R.N., Kobayashi, T., Takahashi, M., Nakanishi, H., Mori, S., and Nishizawa, N.K. (2007). Expression and enzyme activity of glutathione reductase is upregulated by Fe-deficiency in graminaceous plants. Plant Mol Biol 65, 277-284.
连结：
Belousov, V.V., Fradkov, A.F., Lukyanov, K.A., Staroverov, D.B., Shakhbazov, K.S., Terskikh, A.V., and Lukyanov, S. (2006). Genetically encoded fluorescent indicator for intracellular hydrogen peroxide. Nat Methods 3, 281- 286.
连结：
Bradford, M.M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72, 248-254.
连结：
Bratt, A., Rosenwasser, S., Meyer, A., and Fluhr, R. (2016). Organelle redox autonomy during environmental stress. Plant Cell Environ 39, 1909-1919.
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使用DOI作为永久连结

引用含有DOI的文献

DOI可强化引用精确性、增强学术圈连结，并给予使用者跨平台的良好使用经验，目前在全世界已有超过五千万个对象申请DOI。 如想对DOI的使用与概念有进一步了解，请参考 （ ） 。

DOI可强化引用精确性、增强学术圈连结，并给予使用者跨平台的良好使用经验，目前在全世界已有超过五千万个对象申请DOI。如想对DOI的使用与概念有进一步了解，请参考（）。