四倍体刺槐枝条韧皮部总蛋白质双向电泳体系建立及应用  

Establishment and Application of Two-dimensional Gel Electrophoresis for Proteome from the Phloem of Tetraploid Robinia pseudoacacia

作　　者：张胜[1] 赵忠[1,2] 刘昭军[1] 张博勇[1,2] 宗建伟[1] 张玲玲[1] 

机构地区：[1]西北农林科技大学林学院,杨陵712100 [2]西北农林科技大学西部环境与生态教育部重点实验室,杨凌712100

出　　处：《生物技术通报》2013年第1期116-122,共7页Biotechnology Bulletin

基　　金：国家自然科学基金项目(30972352)

摘　　要：采用TCA/丙酮法对四倍体刺槐枝段韧皮部全蛋白质进行提取,通过对IPG胶条pH梯度、分离胶浓度的选择,上样量、等电聚焦条件的优化,建立起四倍体刺槐枝段韧皮部蛋白质双向电泳体系。研究结果表明:采用TCA/丙酮法提取四倍体刺槐枝段韧皮部全蛋白质,选用pH4-7的17 cm IPG胶条,考马斯亮蓝染色上样量550μg,等电聚焦IEF聚焦总伏小时数从60 000 Vh提高到80 000 Vh,并采用12%的分离胶对四倍体刺槐枝段韧皮部全蛋白进行双向电泳,能得到背景清晰、蛋白质点数相对较多,分离度高且重复性好的电泳图谱。利用建立的体系进行双向电泳分离蛋白质,能直接挖点送质谱分析。采用该体系分析四倍体刺槐硬枝扦插生根愈伤组织阶段蛋白质表达差异,共筛选出83个差异蛋白质点,其中上调蛋白15个,新产生蛋白22个,下调蛋白22个,缺失表达24个。This paper used the method of TCA/acetone to extract all proteins of phloem in tetraploid Robinia pseudoacacia. A two- dimensional electrophoresis system for proteomic analysis of tetraploid Robinia pseudoacacia was established by optimizing the parameters including the pH gradient of IPG strip, the gel concentration, the loading quantity of sample and isoelectric focusing conditions. The results showed that ： Use the TCA/acetone method to extract all phloem protein of tetraploid Robinia pseudoacacia, choose 17 cm IPG strip with pH4-7, load protein samples of 550 lag, followed staining with colloidal Coomassie Brilliant Blue and use 12% SDS-PAGE for tetraploid Robinia pseudoacacia phloem total proteins in two-dimensional electrophoresis while isoelectric focusing IEF hours increased from 60 000 Vh to 80 000 Vh can get a clear background with more protein points, high resolution and good repeatability. Using the established system of two-dimensional electrophoresis can dug protein points from the gel directly and send them to mass spectrometry. Use this system to analysis differences of hardwood cuttings rooting callus phase protein expression in tetraploid Robinia pseudoacacia. Filter out 83 differential protein spots, among which, 15 were higher, 22 were novel, 22 were lower and 24 were lack of expression.

关 键 词：四倍体刺槐 韧皮部 双向电泳 优化方法 差异蛋白质分析 

分 类 号：S792.27[农业科学—林木遗传育种]