茭白线粒体蛋白双向电泳体系建立  

Establishment of two-dimensional electrophoresis system for mitochondrial proteins of Zizania latifolia

作　　者：杜传来[1] 罗海波[2] 彭昕[2] 王韦华[3] 郁志芳[3] 

机构地区：[1]安徽科技学院食品药品学院,安徽凤阳233100 [2]浙江医药高等专科学校食品学院,浙江宁波315100 [3]南京农业大学食品科技学院,南京210095

出　　处：《南方农业学报》2016年第3期332-336,共5页Journal of Southern Agriculture

基　　金：国家自然科学基金项目(31401612);浙江省自然科学基金项目(LY14C200005)

摘　　要：【目的】建立适合于茭白线粒体蛋白质组分析的双向电泳技术体系,为进一步开展茭白线粒体差异蛋白质组学研究提供参考。【方法】以茭白为试验材料,比较不同线粒体提取纯化方法、IPG胶条p H范围及蛋白上样量等条件对双向电泳图谱的影响。【结果】差速离心（3000~14000×g）联合Percoll不连续密度梯度离心（20%∶24%∶40%=2∶4∶2）对茭白线粒体的提取纯化效果优于仅采用差速离心。采用改良酚抽法提取茭白线粒体蛋白,17 cm、p H 3~10的IPG胶条,1.0 mg上样量,12%SDS-PAGE进行双向电泳,0.12%考马斯亮蓝G-250胶体考染法染色,茭白线粒体蛋白主要分布在p H 4~8范围内,p H 3~4和p H 8~10范围内蛋白点较少;进一步选用p H 4~7和p H 5~8的IPG胶条对茭白线粒体蛋白进行双向电泳,分离效果均明显提高,且p H 4~7范围内IPG胶条的双向电泳图谱清晰,蛋白点分辨率较高。分别采用0.8、1.0和1.2 mg 3个不同的上样量进行双向电泳,结果表明上样量为1.0 mg时,电泳图谱质量最好。【结论】通过差速离心联合Percoll不连续密度梯度离心提取纯化茭白线粒体,并控制好双向电泳体系的p H范围、上样量等因素,可获得蛋白质点清晰、分辨率高、重复性好的双向电泳图谱。【Objective】The present experiment was conducted to establish two-dimensional electrophoresis（2-DE） system suitable for analysing mitochondrial proteomics of Zizania latifolia, in order to provide reference for further researching its differential proteomics of mitochondria. 【Method 】Using Z. latifolia as material, the effect of mitochondrial extraction and purification methods, p H ranges of IPG strip, loading quantities, etc. on 2-DE map were compared and analyzed.【Result】Based on comparison of Z. latifolia mitochondrial extraction and purification effect, the differential centrifugation（3000-14000×g） combined with Percoll discontinuous density gradient centrifugation（20%∶24%∶40%=2∶4∶2） was superior to single differential centrifugation method. The protein spots were mainly distributed in the range of p H 4-8 using the following procedure： extracting mitochondrial proteins with modified phenol extraction method, 17 cm p H 3-10 IPG strip,loading 1.0 mg protein sample, SDS-PAGE with 12% gel concentration, and finally staining with 0.12% coomassie brilliant blue（CBB） G-250, but the protein spots were less distributed in the ranges of p H 3-4 and p H8-10. The separation effect of 2-DE on mitochondrial proteins was improved significantly by using p H 4-7 and p H 5-8 IPG strips, and p H4-7 was more suitable for separation of mitochondrial proteins because 2-DE map was more clear, and resolution of protein spots was higher. Furthermore, the 2-DE results of different protein loading quantities（0.8, 1.0, 1.2 mg） showed that,the quality of 2-DE map were the best at the loading quantity of 1.0 mg. 【Conclusion】The 2-DE map with clearer protein spots, higher resolution and good repeatability is obtained by extracting and purifying mitochondria with differential centrifugation combined with Percoll discontinuous density gradient centrifugation and controlling p H range and protein loading quantity.

关 键 词：茭白 线粒体 双向电泳 蛋白质组学 

分 类 号：Q81[生物学—生物工程]