适于番茄叶片蛋白质组分析的双向电泳优化体系的建立  

Establishment of Two-dimensional Electrophoresis Optimization System for Proteomic Analysis of Tomato Leaves

作　　者：王强[1] 王娟[2] 李宁[1] 唐亚萍[1] 杨涛[1] 王柏柯[1] 杨生保[1] 帕提古丽[1] 余庆辉[1] 

机构地区：[1]新疆农业科学院园艺作物研究所/新疆园艺作物生物技术与分子育种重点实验室,乌鲁木齐830091 [2]新疆农业科学院经济作物研究所,乌鲁木齐830091

出　　处：《新疆农业科学》2016年第4期610-616,共7页Xinjiang Agricultural Sciences

基　　金：新疆维吾尔自治区自然科学基金项目(2014211B035)~~

摘　　要：【目的】建立适于番茄叶片蛋白质组分析的双向电泳（2-DE）技术,为开展番茄蛋白质组学研究奠定技术基础。【方法】对番茄叶片蛋白质提取方法、蛋白质裂解液、上样量、胶条p H范围等关键步骤进行优化。【结果】采用三氯乙酸/丙酮法提取番茄叶片总蛋白质,含有7 mol/L尿素,2 mol/L硫脲,4%CHAPS,30 mmol/L Tris-HCl（p H 8.0）裂解缓冲液,上样量为100 mg,以p H 4~7、18 cm的IPG胶条在12%SDS-PAGE凝胶浓度下进行双向电泳,得到了蛋白点均匀分布、低峰度蛋白点较为清晰,蛋白点数目多且分辨率高的2-DE图谱。【结论】建立了一套适于番茄叶片蛋白质分析的双向电泳技术体系,为下一步在蛋白组学水平上分析番茄逆境胁迫等相关蛋白提供了技术支持。[ Objective] To establish a two -dimensional gel electrophoresis （2 -DE） protocol for pro- teomic analysis of tomato leaves, this system aims to make a foundation for tomato proteomic research. [ Method] The method of protein extraction,reagent of lysis buffer,the sampling amount, pH gradient of IPG strip and other factors were optimized. [ Result] The results showed that optimal tomato leaf protein extraction was obtained by the improved TCA/aeetone method,with lysis buffer containing 7 mol/L Urea,2 mol/L Thiourea, 4% CHAPS,30 mmol/L Tris- HCI（pH 8.0）, a protein loading amount of 100 mg. Under pH 4 -7,18 em IPG strip and 12% concentrations of the SDS - AGE gel. The protein spots we got were evenly distributed and the low protein kurtosis were clearer, with more numbers of protein and higher resolution two - dimensional e- lectrophoresis. [ Conclusion] This established experiment is suitable for the tomato leaf protein analysis sys- tem of two - dimensional electrophoresis, which might provide technical support for the next step proteomics level analysis of tomato stress conditions and other related proteins.

关 键 词：番茄叶片 蛋白质组 双向电泳(2-DE) 

分 类 号：S188[农业科学—农业基础科学] S641.2