利用Native-PAGE鉴定大豆Lox同工酶

Identification of Soybean Lipoxygenase Isozyme by Native-PAGE

以Lox缺失体五星1号、3号、4号和非缺失类型大豆中黄13种子为材料,采用非变性聚丙烯酰胺凝胶电泳(Native-PAGE),通过改变分离胶浓度、电泳电压、样品浓度、电泳时间和染色方法等,探索大豆Lox同工酶电泳最佳条件。结果表明:浓缩胶浓度5%,分离胶浓度13%,浓缩胶电泳电压90 V、分离胶电泳电压190 V,样品浓度40%,电泳时间7 h,采用pH8.0、pH6.5两种不同的酶染液结合热考马斯亮蓝染色,4种Lox同工酶(Lox1、Lox2、Lox3a和Lox3b)的分辨清晰度最高。该方法简便安全、经济有效,可用于大豆种质资源Lox缺失体的筛选鉴定。

There are some problems existed in the detection of soybean lipoxygenase（Lox）isozymes such as complex procedures,poor resolution,higher cost and highly toxic reagents on the human,exploring an improved methods to identify Lox isozyme of soybean is necessary.In this study,Lox deletion mutants（Wuxing No.1,Wuxing No.3 and Wuxing No.4）and non-deletion type（Zhonghuang No.13）were used to survey the optimal experimental condition for Lox ioszymes by changing the separation gel concentration,electrophoresis voltage,sample concentration,time and staining method with Native-PAGE.From the results we found that the four Lox isoenzyme of soybean（Lox1,Lox2,Lox3a and Lox3b）could be separated clearly when the concentration of stacking gel and separating gel were 5% and 13%,repectively;electrophoresis voltage in stacking gel and separation gel were 90 V and 190 V,respectively;sample concentration was 40%,and the electrophoresis time was 7 hours,dying with two different enzyme staining of pH8.0 and pH6.5 combined with heat Coomassie blue staining.In short,it is a simple,safe,economical and effective method that can be used for the identification and screening of lipoxygenase deletion mutants of soybean germplasm.