十字花科黑腐病菌分泌蛋白质双向电泳条件的构建及优化  

Establishment and Optimization Analysis of Two-Dimensional Electrophoresis System for the Secreted Proteins of Xanthomonas campestris pv.campestris

作　　者：岑卫健 成春燕[3] 杨丽超[3] 王凛[3,4] 姜伯乐[1,3] 

机构地区：[1]亚热带农业生物资源保护与利用国家重点实验室,南宁530004 [2]广西理工科学实验中心,南宁530004 [3]广西大学生命科学与技术学院,南宁530004 [4]桂林医学院,桂林541004

出　　处：《基因组学与应用生物学》2015年第2期403-407,共5页Genomics and Applied Biology

基　　金：国家自然科学基金项目(31260443);广西创新研究团队项目(2014GXNSFFA118005);广西自然科学基金项目(2013GXNSFAA019097;2013GXNSFBA019088)共同资助

摘　　要：本研究旨在建立一套适合于十字花科黑腐病菌（Xanthomonas campestris pv.campestris 8004,Xcc8004）分泌蛋白质的双向电泳技术（two-dimensional electrophoresis,2-DE）,以便更好的利用蛋白质组学技术鉴定Xcc 8004的分泌蛋白质。我们就MME和XCM2两种培养基对分泌蛋白质的诱导能力、蛋白质提取方法以及样品上样量等方面对2-DE的影响进行了比较,结果表明,XCM2培养基对Xcc 8004分泌蛋白质的诱导能力比MME强。用TCA/丙酮法沉淀蛋白质得到的双向电泳图谱背景清晰,分辨率高。分别采用400μg、300μg、250μg和150μg四个不同的上样量进行双向电泳,结果显示在上样量为250-300μg时（IPG pH 3-10,24 cm）,电泳图谱分辨率最好。综上可知,XCM2培养基比较适合用于Xcc 8004分泌蛋白质的诱导,TCA/丙酮法提取分泌蛋白质为较优方法,250-300μg是较为合适的上样量。In order to establish a suitable and efficient two-dimensional electrophoresis（2-DE） technique for identifying the secreted proteins of Xanthomonas campestris pv. campestris 8004, the extracellular proteins on inducing media, MME and XCM2, different protein extraction methods, and loading amount on 2-DE profile were compared in this research. The results showed that XCM2 is more suitable for inducing extracellular proteins than that of MME, and clearer 2-DE maps can be observed while the proteins were precipitated by the TCA/acetone treatment. Furthermore, different loading amount（400 μg, 300 μg, 250 μg and 150 μg） of the proteins extracted by the method of TCA/acetone for 2-DE were used to optimize the conditions of sample preparation. As a result, the resolution and the quality of the 2-DE maps were most improved for proteins loading amount of 250μg to 300 μg separated in 24 cm IPG strips（pH 3-10）. To sum up, XCM2 is suitable for inducing extracellular protein,TCA/acetone precipitation is much more suitable for protein extraction and 250-300 μg is the optimum loading amount of 2-DE for secreted proteins of Xanthomonas campestris pv. campestris.

关 键 词：十字花科黑腐病菌 双向电泳 分泌蛋白质 蛋白提取方法 

分 类 号：S432.4[农业科学—植物病理学]