小麦花药蛋白质组双向电泳技术体系的优化  

OPTIMIZATION OF TWO-DIMENSIONAL GEL ELECTROPHORESIS FOR PROTEOME FROM WHEAT ANTHERS

作　　者：陈蕊红[1] 张改生[1] 刘卫[1] 叶景秀[1] 牛娜[1] 

机构地区：[1]西北农林科技大学,陕西省作物杂种优势研究与利用重点实验室/小麦育种教育部工程研究中心,陕西杨凌712100

出　　处：《核农学报》2008年第4期404-409,共6页Journal of Nuclear Agricultural Sciences

基　　金：国家自然科学基金项目(301705760);国家“863”重大专项计划(2002AA207004);陕西省“13115”科技创新工程重大科技专项(2007ZDKG-020)

摘　　要：以小麦单核期花药为材料,比较了两种不同蛋白质提取方法TCA/丙酮法和酚提取法及不同的蛋白质裂解液组成对双向电泳的影响,并在蛋白质上样量和SDS-PAGE胶浓度等方面也进行了探索与优化。结果表明,采用TCA/丙酮提取法比用酚提取法提取的蛋白质所得的2-DE胶图谱上蛋白质点数增加,图谱背景也比较清晰;样品溶解于含有硫脲和TBP的蛋白质裂解液Ⅱ中,可显著提高蛋白质的溶解性,在2-DE图谱上可分辨出554个蛋白质点,比用蛋白质裂解液Ⅰ提取的蛋白多39个点。以TCA-丙酮法提取小麦花药组织中的蛋白,用蛋白质裂解液Ⅱ[7mol/L尿素、2mol/L硫脲、4%CHAPS、2%TBP、65mmol/L DTT、0.2%载体两性电解质（其中0.1%pH 3-10,0.1%pH4-6）]溶解蛋白,以pH4-7线性17cm的IPG胶条进行双向凝胶电泳,在上样量为800μg,13%SDS-PAGE胶浓度下,蛋白质得到了更好的分离,2-DE图谱上可分辨出631个蛋白点,图谱质量最佳。优化后的双向电泳技术体系,适合于小麦花药全蛋白质的双向电泳分析。The effects of different protein extraction methods （phenol extraction method and TCA-acetone precipitation method）, and different lysis buffer on two-dimensional electrophoresis （2-DE） gels were compared, the loading quantity of sample and the gel concentrations were also optimized for the analysis of 2-DE system on the monokaryotic stage anther of wheat. The results showed that, samples prepared by TCA/acetone precipitation had observed more protein spots than phenol extraction method, and clearer background and protein spots were obtained in the 2-DE map. The sample dissolved in the lysis buffer Ⅱ which contained thiourea and TBP, could enhanced the protein solubility, and about 554 protein spots were detected on the 2-DE gels, which was 39 protein spots more than the lysis buffer Ⅰ . The proteins extracted by the TCA/acetone method, dissolved protein with the lysis buffer Ⅱ, with pH 4 - 7 17cm IPG scip, 800μg loading quantity and 13% concentrations of the SDS-PAGE gel, the proteins were well separated, total 631 protein spots were detected. The optimized condition of 2-DE was suitable for proteomic analysis of wheat anthers.

关 键 词：小麦 花药蛋白 蛋白质提取方法 蛋白质裂解液 双向电泳技术 

分 类 号：S512.1[农业科学—作物学]