Functional Analysis of Prokaryotic Expression Protein of Tomato SlTpx <i>in Vitro</i>

doi:10.13523/j.cb.2104030

China Biotechnology

2021, Vol. 41

Issue (8): 25-32 DOI: 10.13523/j.cb.2104030

Functional Analysis of Prokaryotic Expression Protein of Tomato SlTpx in Vitro

QIAO Sheng-tai,WANG Man-qi,XU Hui-ni(

)

College of Life Science and Technology,Kunming University of Science and Technology,Kunming 650500,China

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Abstract

H₂O₂ is an important signaling molecule that participates in a variety of physiological metabolic activities in plants. However, excessive H₂O₂ destroys biological macromolecules, thus poisoning cells. Thioredoxin peroxidase (Tpx) plays an important role in protecting plants from oxidative damage by scavenging H₂O₂. The prokaryotic expression vector of tomato Tpx gene (SlTpx) was established, and SlTpx protein was induced and purified, and we found that the size of SlTpx protein was about 21 kDa. In vitro mixed functional oxidase (MFO) assay showed that SlTpx could protect DNA from harmful reactive oxygen species. In vitro experiments on SlTpx protein showed that SlTpx could improve the anti-heavy metal toxicity and antioxidant capacity of Escherichia coli. It laid a foundation for further study on the function and mechanism of SlTpx in plants.

Key words： Thioredoxin peroxidase Prokaryotic expression Escherichia coli Oxidative stress

Received: 18 April 2021 Published: 31 August 2021

ZTFLH:

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Corresponding Authors: Hui-ni XU E-mail: xuhn@kust.edu.cn

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Cite this article:

QIAO Sheng-tai,WANG Man-qi,XU Hui-ni. Functional Analysis of Prokaryotic Expression Protein of Tomato SlTpx in Vitro. China Biotechnology, 2021, 41(8): 25-32.

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https://manu60.magtech.com.cn/biotech/10.13523/j.cb.2104030 OR https://manu60.magtech.com.cn/biotech/Y2021/V41/I8/25

Fig.1 Amino acid sequence of pet-28a-sltpx and construction of prokaryotic expression vector (a)Amino acid sequence and molecular weight of SlTpx (b)Schematic diagram of gene expression vector (c)The PCR analysis of pET-28a-SlTpx vector

Fig.2 Optimization of expression conditions of recombinant SlTpx in E. coli with 0.5 mmol/L IPTG at 28℃ and 37℃ for 0, 2,4, 6 and 8 h M: Protein marker

Fig.3 SDS-PAGE analysis of the purified SlTpx M: Protein marker

Fig.4 Western blotting analysis of recombinant proteins in the E. coli BL21(DE3) cells with anti-His-tag antibody M: Protein marker

Fig.5 Determination of SlTpx protected supercoiled DNA cleavage in mixed-function oxidase (MFO) assay 1: pEGFP without incubation; 2: pEGFP + FeCl₃(10 μmol/L); 3: pEGFP + DTT (10 mmol/L); 4: pEGFP + MFO mix(10 μmol/L FeCl₃+10 mmol/L DTT); 5: pEGFP + MFO mix + 1 μg of SlTpx; 6:pEGFP + MFO mix + 4 μg of SlTpx; 7: pEGFP+MFO mix + 8 μg of SlTpx; 8: pEGFP + MFO mix + 16 μg of SlTpx; 9: pEGFP + MFO mix + 24 μg of SlTpx. OC DNA: Open circular plasmid DNA; CCC DNA: Covalentlyclosed circular DNA

Fig.6 Hydrogen peroxide-scavenging assay for the recombinant SlTpx proteins treated with H₂O₂ The absorbance at 560 nm was measured every minute until 20 min after H₂O₂ was added. Empty pET28a vector expressed protein were used as the negative control

Fig.7 In vitro metal stress tolerance assay of SlTpx protein (a)The growth of Escherichia coli in pET-28a-SlTpx experimental group and pET-28a control group stimulated by 0.1 and 1 mol/L heavy metals (b)Inhibition zone diameter of E. coli after H₂O₂ challenge Data was showed as mean ± SD (n=3).Error bars above the vertical bars indicated statistically significant differences between every intergroup by the asterisk ** P≤ 0.01

Fig.8 In vivo antioxidant activity assay using SlTpx protein (a)Bacteria transfected with only pET-28a vector were used as the negative controls; LB agar plates (LB with ampicillin) were inoculated with SlTpx protein. Filter discs treated with different concentrations of H₂O₂, including the concentration of H₂O₂ at 30%, 20%, 15%,10%, 6%, 3% (b)Inhibition zone diameter of E. coli after H₂O₂ challenge. Data was showed as mean ± SD (n=3). Error bars above the vertical bars indicated statistically significant differences between every intergroup by the asterisk ** P≤0.01


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