非变性PAGE在鉴定大豆脂肪氧化酶类型中的应用  

Application of Native PAGE in Identification of the Heterogeneity of Soybean Lipoxygenases

作　　者：马志民[1] 蒋春志[1] 杨春燕[1] 马峙英[2] 张孟臣[1] 

机构地区：[1]国家大豆改良中心石家庄分中心,河北石家庄050031 [2]河北农业大学,河北保定071000

出　　处：《华北农学报》2007年第3期5-8,共4页Acta Agriculturae Boreali-Sinica

基　　金：河北省自然科学基金(C2005000605);国家科技支撑计划(2006BAD13B05);国家863计划(2006AA100104)

摘　　要：为解决脂肪氧化酶检测复杂、费用昂贵、剧毒试剂对人体影响等问题,研究出一种安全、快速而经济有效的大豆脂氧酶检测技术。以Suzuyutaka脂氧酶缺失近等基因系和非缺失类型大豆冀豆12为材料,利用聚丙烯酰胺凝胶电泳探索非变性PAGE分析大豆脂肪氧化酶,通过对不同样品浓度、电泳条件和分离胶浓度的比较,摸索鉴定大豆Lox缺失类型的最佳条件。通过电泳条件优化分析发现,电泳样品中预样品的浓度为30%、分离胶浓度为11%、电泳时间为6 h时,3种脂肪氧化酶分辨清晰度最高;通过重复电泳的相对迁移率分析,表明在上述电泳条件下的检测结果比较稳定且重复性好;通过3种脂肪氧化酶酶带与缺失酶带对照比较和分子量测定两方面验证了所得到的3条脂肪氧化酶酶带。非变性PAGE是一种简单、经济、快捷而安全的检测分析大豆脂肪氧化酶同功酶的方法,因而可以用于资源分析、良种繁育以及生产、收购和加工利用中的大豆脂氧酶缺失类型鉴定。To solve the problems of the complicated, expensive detection of poisonous chemical reagents of Lipoxygenases, a method examining the Lipoxygenases economically and efficiently was described in this paper. The optimal experimental conditions for the method were surveyed by comparing different concentrations of the material and electrophoresis condition of native-polyacrylamid gel electrophoresis （native PAGE） and ananalyzing soybean lipoxygenase with Native PAGE, with the near-isogenic lines of soybean lipoxygenase and a common soybean variety Jidou No. 12 as the material in the study. We find that these three lipoxygenases could be separated clearly in the condition that 0.3 mL extract in 1 mL electrophoresismaterial, the concentration of native polyacrylamide gel is 11%, the total electrophoresis time is 6 hours ; the result could be achieved repeatedly and stably in the conditions mentioned above by the analysis of the move ratio of three lipoxygenases＇ enzymatic bands; through comparing the different null lipoxygenase bands with the natural lipoxygenase bands, as well as the measure of molecular weight, both validated the 3 lipoxygenase enzymatic bands. It is a simple and economical method that can be used as useful tools for analyzing the resources, the cultivar breeding, and the identification of the soybean Lox-free type in the process of production, machining, purchasing and so on.

关 键 词：大豆 脂肪氧化酶鉴定 非变性PAGE 迁移率 

分 类 号：S565.101[农业科学—作物学]