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中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志
中国生物工程杂志  2021, Vol. 41 Issue (8): 25-32    DOI: 10.13523/j.cb.2104030
研究报告     
番茄SlTpx原核表达蛋白的体外功能分析*
乔圣泰,王曼琦,徐慧妮()
昆明理工大学生命科学与技术学院 昆明 650500
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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摘要：

H2O2是一种重要的信号分子,参与植物体内多种生理代谢活动,但过量的H2O2破坏生物大分子,从而使细胞受到毒害。硫氧还蛋白过氧化物酶(thioredoxin peroxidase,Tpx)通过清除H2O2在保护植物免受氧化损伤方面起着重要作用。为进一步研究番茄Tpx基因(SlTpx)的功能,构建了番茄SlTpx原核表达载体,并诱导和纯化了SlTpx蛋白,发现该蛋白质大小约为21 kDa。为检测SlTpx的抗氧化功能,通过体外的混合功能氧化酶(MFO)实验、过氧化氢清除实验和SlTpx蛋白体外抗重金属和H2O2实验,证明SlTpx可以保护DNA不受有害活性氧切割,并且提高大肠杆菌抵抗重金属和H2O2胁迫的能力。为揭示SlTpx在植物中的功能和作用机制奠定基础。
关键词： 硫氧还蛋白过氧化物酶;  原核表达;  大肠杆菌;  氧化胁迫    
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
收稿日期: 2021-04-18 出版日期: 2021-08-31
ZTFLH:  Q816  
基金资助: * 国家自然科学基金资助项目(31760582)
通讯作者: 徐慧妮     E-mail: xuhn@kust.edu.cn
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引用本文:

乔圣泰,王曼琦,徐慧妮. 番茄SlTpx原核表达蛋白的体外功能分析*[J]. 中国生物工程杂志, 2021, 41(8): 25-32.

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.

链接本文:

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

图1  pET-28a-SlTpx编码氨基酸序列与蛋白质分子量大小及原核表达载体的构建
图2  重组SlTpx在大肠杆菌中的表达条件优化
图3  SlTpx蛋白纯化的SDS-PAGE分析
图4  重组蛋白的Western blotting分析
图5  混合功能氧化酶(MFO)分析法测定SlTpx保护的超螺旋DNA裂解
图6  重组SlTpx蛋白H2O2清除能力的测定
图7  SlTpx蛋白抗重金属能力的体外测定
图8  SlTpx蛋白体外抗氧化活性测定
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