百子莲茎尖分生组织蛋白双向电泳体系的建立  

Establishment of Protein Two-Dimensional Electrophoresis System of Agapanthus Praecox Stem Tip Meristem

作　　者：张洁[1] 孙一凡[2] 张荻[1] 申晓辉[1] 

机构地区：[1]上海交通大学农业与生物学院,上海200240 [2]上海交通大学生命科学技术学院,上海200240

出　　处：《上海交通大学学报（农业科学版）》2015年第3期43-52,共10页Journal of Shanghai Jiaotong University(Agricultural Science)

基　　金：国家自然科学基金(31300580;31370695);中国博士后科学基金特别资助项目(2013T60451)

摘　　要：以‘蓝色大花’百子莲（Agapanthus praecoxssp.orientalis‘Big blue’）茎尖分生组织为材料,对比研究了不同提取与裂解方法制备蛋白样品以排除多酚、多糖及核酸物质对蛋白双向电泳体系（Two-dimensional gel electrophoresis,2-DE）的干扰。提取过程采用TCA/丙酮沉淀法和超声波破碎法;蛋白裂解过程中分别使用苯甲基磺酰氟（Phenylmethanesulfonyl Fluoride,PMSF）和乙二胺四乙酸（Ethylene Diamine Tetraacetic Acid,EDTA）、Cocktail（广谱性）蛋白酶抑制剂、核酸酶Nuclease Mix和2-D Clean-Up蛋白纯化试剂盒。制备的各蛋白样品经过7cm IPG（pH 3~10）胶条上样、电泳后发现,使用TCA/丙酮沉淀法提取蛋白,裂解液中加入Cocktail蛋白酶抑制剂裂解后,用2-D Clean-Up试剂盒所制备的样品其双向凝胶电泳图谱效果最好,获得蛋白点数量最多,且蛋白等电点pI主要分布于4~8之间。随后,应用分辨率更高的24cm IPG（pH 4~7）胶条进行电泳效果的验证,蛋白点在凝胶的2个方向上均得到了较好地分离,经过ImageMaster 2-D Platinum5.0软件分析后共检测到有效蛋白点820个。本研究初步建立了百子莲茎尖分生组织蛋白双向凝胶电泳体系,为后续开展百子莲属植物蛋白质组学研究提供了重要的技术保障。The stem tip meristem has vigorous division ability,and contains abundant polyphenol,polysaccharide and nucleic acid substances which can strongly interfere for protein two-dimensional electrophoresis system（2-DE）.In this study,the different protein extraction and pyrolysis methods were used for eliminating these interferences,and to establish the stem tip meristem 2-DE system in Agapanthus praecoxssp.orientalis.TCA/acetone precipitation and ultrasonication methods were performed for protein extraction.Additionally,different protease inhibitors including PMSF,EDTA,Cocktail protease inhibitors and nuclease Mix were added in to pyrolysis buffer to protect protein integrity and digest nucleic acid.Furthermore,2-D Clean Up Kit was then used to purify the protein samples.All protein samples wereloaded onto 7cm,pH value 3~10linear gradient IPG strip,and were performed IEF and the second dimension electrophoresis.The result showed that TCA/acetone precipitation was preferable method to extract the protein.Cocktail protease inhibitors and 2-D Clean Up Kit had the best effect on protein protection and eliminating the interfering substance,respectively.Most amounts of proteins were distributed between pI 4~8of the gel,accordingly,the higher resolution of 24 cm IPG（pH 4~7）strip was applied to verify the above results by the same method.The total protein was separated by 2-DE and more than 800 protein spots were detected by ImageMaster 2DPlatinum 5.0software.This study established the optimal protein 2-DE system of stem tip meristem in Agapanthus praecox,and provided an important technical support for future proteomics research in this species.

关 键 词：百子莲 茎尖分生组织 蛋白提取 蛋白酶抑制剂 双向凝胶电泳 

分 类 号：S682.32[农业科学—观赏园艺]