中国水稻科学

中国水稻科学

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水稻种子脂氧合酶基因OsLOX1的原核表达、纯化及鉴定

汪仁1; 沈文飚1; 江玲1; 刘玲珑1; 翟虎渠2; 万建民1, 2, *   

  1. 1南京农业大学 作物遗传与种质创新国家重点实验室/江苏省植物基因工程研究中心， 江苏 南京 210095； 2中国农业科学院 作物科学研究所， 北京 100081； *通讯联系人， E-mail: wanjm@njau.edu.cn
  • 收稿日期:1900-01-01 修回日期:1900-01-01 出版日期:2008-03-10 发布日期:2008-03-10

Prokaryotic Expression,Purification and Characterization of a Novel Rice Seed Lipoxygenase Gene OsLOX1

WANG Ren , SHEN Wen-biao , JIANG Ling , LIU Ling-long , ZHAI Hu-qu , WAN Jian-min
  

  • Received:1900-01-01 Revised:1900-01-01 Online:2008-03-10 Published:2008-03-10
  • Contact: WAN Jian-min

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摘要： 脂氧合酶是动植物体内催化脂质降解的关键酶，也是茉莉酸合成途径的第一个关键酶。以先前克隆到的水稻种子脂氧合酶基因OsLOX1全长cDNA为模板，用含有特异酶切位点的P1、P2为引物，通过PCR方法将它构建到大肠杆菌表达载体pET30a(+)上并转化到大肠杆菌菌株BL21（DE3）中，获得相应的重组工程菌。经过20℃条件下的IPTG诱导，OsLOX1重组蛋白在BL21（DE3）菌株中得到表达，经生化特性分析，发现该重组蛋白具有LOX催化活性，其最适pH和温度分别为4.8和30℃。该重组子可进一步应用于体外生产茉莉酸和研究植物种子LOX结构与功能的关系。

关键词: 水稻种子, 脂氧合酶, 原核表达, 纯化, 鉴定

Abstract: Lipoxygenase(LOX,EC 1.13.11.12) is a key enzyme during the degradation of lipids in animals and even plants.It is also the first key enzyme responsible for the biosynthesis of jasmonate.In this paper,the expression,purification and characterization ofOsLOX1 were reported.The entire coding region of theOsLOX1 gene was inserted into an expression vector pET30a(+) and transformed into Escherichia coli: BL21(DE3).The fusion protein was successfully expressed induced by IPTG and the purified recombinant protein was obtained with His·Bind○R Kits.Further assay showed that the purified recombinant protein exhibited the LOX activity.The optimum pH and temperature of above enzyme were 4.8(acetate buffer) and 30℃,respectively.Thus,this recombinant might confer an available usage for the synthesis of jasmonate in vitro,and also provide a possibility for elucidating the inter-relationship between the primary structure of the plant seed lipoxygenase protein and its physiological functions.

Key words: rice seed, lipoxygenase, prokaryotic expression, purification, characterization