转HBsAg基因樱桃番茄组培苗叶片总蛋白双向电泳体系的建立  

ESTABLISHMENT OF TWO-DIMENSIONAL GEL ELECTROPHORESIS SYSTEM FOR LEAF PROTEINS OF HBsAg TRANSGENIC CHERRY TOMATO

作　　者：张超[1] 郭斌[1] 祁洋[1] 孙杰[1] 薛柯[1] 戴佳锟[2] 关正君[1] 尉亚辉[1] 

机构地区：[1]西北大学生命科学学院/西部资源生物与现代生物技术教育部重点实验室,陕西西安710069 [2]陕西省科学院酶工程研究所,陕西西安710600

出　　处：《核农学报》2012年第8期1111-1117,共7页Journal of Nuclear Agricultural Sciences

基　　金：国家自然科学基金青年基金(No.30900914);国家自然科学基金(No.310001444);陕西省生物技术重点实验室项目(No.08JZ72);西北大学自主创新类项目(No.10YZZ36)

摘　　要：建立转乙肝表面抗原(HbsAg)基因樱桃番茄组培苗叶片的蛋白质组双向电泳体系,为转基因番茄的蛋白质组学研究提供技术参数。比较转HBsAg基因的樱桃番茄与野生型在不同蛋白裂解液、不同蛋白上样量及不同等电聚焦程序下,叶片总蛋白的双向电泳结果差异。以裂解液Ⅲ制备叶片总蛋白,采用一向13cm、pH3-10L IPG胶条,二向12.5%的SDS-PAGE凝胶,单次上样120μg叶片总蛋白,聚焦程序Ⅲ,银染时,可以得到最理想的双向电泳分析结果。在此操作程序下,野生型樱桃番茄叶片蛋白双向电泳最多可识别699个蛋白点,而转基因型樱桃番茄叶片蛋白双向电泳最多可识别545个蛋白点,其中有368个点匹配,选取丰度百分比差异2倍以上蛋白点25个进行质谱鉴定,16个蛋白点成功鉴定。本研究建立的转HBsAg基因番茄植株叶片总蛋白的双向电泳体系可为以番茄试管苗为材料进行的蛋白质组学研究提供技术支持。Two-dimensional gel electrophoresis（2-DE） system was established for detection of the leaf proteins of HBsAg transgenic cherry tomato,which provided the experiment data for proteomics study of transgenic cherry tomato.This study compared the effects of different protein lysis buffers,protein loading amounts and isoelectric focusing conditions on the 2-DE maps of the transgenic plants.An optimum system was obtained as follows： dissolving total proteins of samples with the lysis buffer Ⅲ,separating the proteins with 13cm pH3-10L IPG strips,loading protein samples of 120μg followed by isoelectric focusing program Ⅲ,and staining the gels with silver staining after 12.5% SDS-PAGE.Under the above conditions,545 protein spots were identified from the transgenic plants and 699 protein spots from control plants,of which 368 protein spots were matched.A total of 25 protein spots were analyzed by MALDI-TOF MS and 16 proteins were identified by PMF.The 2-DE system for leaf proteins of HBsAg transgenic cherry tomato established in this work provides technical base for the studies of tomato proteomics.

关 键 词：双向电泳 转基因植物 口服植物疫苗 蛋白组 组培苗 

分 类 号：S641.2[农业科学—蔬菜学]