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中国农学通报

中国农学通报 ›› 2019, Vol. 35 ›› Issue (36): 127-132.doi: 10.11924/j.issn.1000-6850.casb18100002

所属专题： 生物技术； 水稻

• 生物技术科学 • 上一篇    下一篇

东乡野生稻过氧化物酶分离纯化及其酶学特性研究

肖伟, 徐梅珍, 谷丰, 蒋伦伟, 王义华   

  1. 江西农业大学生物技术与物理农业工程研究所,江西南昌
  • 收稿日期:2018-10-02 修回日期:2019-11-24 接受日期:2018-11-25 出版日期:2019-12-26 发布日期:2019-12-26
  • 通讯作者: 王义华
  • 基金资助:
    国家自然科学基金“冷胁迫下东乡野生稻根系特异性表达基因的克隆与功能鉴定”(30660087)，“冷胁迫下东乡野生稻特异性表达蛋白鉴 定及其功能研究”(31060172)；江西省教育厅科技项目“甲硫氨酸亚砜还原酶在植物抗逆中作用及其分子机理的研究”(赣教技字[2006]138 号)；第二 批“赣鄱英才555 工程”

Peroxidase Purification and Its Enzymatic Kinetics of Dongxiang Wild Rice

  • Received:2018-10-02 Revised:2019-11-24 Accepted:2018-11-25 Online:2019-12-26 Published:2019-12-26

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摘要： 过氧化物酶与植物体内活性氧的清除和逆境适应性反应密切相关，为了深入研究过氧化物酶在植物抗逆反应中的重要作用，本文以东乡野生稻叶片组织为材料，采用浸提、盐析、透析和柱色谱法分离纯化POD蛋白，采用聚丙烯凝胶活性电泳检测蛋白，利用愈创木酚法研究POD的活性和酶学特性。结果表明，经磷酸缓冲液浸提、40%~80%硫酸铵盐析、再经Sephadex G-100凝胶柱层析等步骤，从东乡野生稻叶片组织分离制备得到POD1同工酶组分，酶蛋白的纯化倍数为16.49，比活力为568.80 U/mg。聚丙烯凝胶活性电泳检测仅呈单一POD1同工酶带，属于单一组分。经测定，POD酶Km为1.26 mmol/(L?min)，最大反应速度Vmax为17.51 mmol/(L?min)，最适宜的pH为5.0，最适宜温度35℃~45℃，Cu2+和Zn2+对POD有较强的激活作用，而Fe3+对POD活性有一定的抑制作用，POD酶活性受20%的甲醇、乙醇和丙酮等有机试剂抑制。本研究为进一步探讨东乡野生稻POD抗氧化功能和东乡野生稻抗寒生理提供了基础材料和依据。

关键词: 水稻不育系, 水稻不育系, 抑制, 稗草, 化感作用, 竞争作用, 稳定性

Abstract: Peroxidase is closely related to the scavenging of reactive oxygen species (ROS) and the adaptive response to stresses in plants. In order to further explore the role of peroxidase in stress resistance mechanism of crops, POD protein was isolated from leaves of Dongxiang wild rice by extraction, salting out, dialysis and column chromatography, the purified protein was detected by active gel electrophoresis with polyacrylamide gel and the enzymatic characteristics of POD were studied by the guaiacol method. The results showed a component of POD1 isozyme with a specific activity of 568.80 U/mg was obtained about 16.49-fold from leaves of Dongxiang wild rice via extraction with phosphate buffer buffer, precipitation by ammonium sulfate of 40%-80% saturation, and separation by Sephadex G-100 chromatography, which was detected to be a homogeneous component by polyacrylamide gel electrophoresis (PAGE). The detection of enzyme kinetics showed that the POD enzyme had Km of 1.26 mmol/(L?min) and Vmax of 17.51 mmol/(L?min) with the optimum pH of 5.0 and optimum temperature of 35℃-45℃. Cu2+ and Zn2+ had strong activation effects on POD activity, while Fe3+ had inhibition effect, and POD activity was inhibited by 20% of methanol, ethanol and acetone. This study provided foundation for further insights on POD antioxidant function and cold resistance physiology of Dongxiang wild rice.