适用于双向电泳的芒果叶片蛋白提取方法的建立  

Establishment of Protein Extraction Protocols for Mango Leaf Adaptable to Two-dimensional Electrophoresis

作　　者：陈香玲[1,2,3] 任惠[1,3] 黄章保[1,2] 苏伟强[1,3] 刘业强[1,3] 李果果[1,3] 刘要鑫[1,3] 王小媚[1,3] 

机构地区：[1]广西农业科学院园艺研究所,广西南宁530007 [2]广西作物遗传改良生物技术重点开放实验室,广西南宁530007 [3]农业部南宁南亚热带果树科学观测实验站,广西南宁530007

出　　处：《西南农业学报》2015年第6期2696-2700,共5页Southwest China Journal of Agricultural Sciences

基　　金：广西自然科学基金项目(2012GXNSFBA053046);广西农科院基本科研专项(桂农科2012YZ17;桂农科2013YF01);国家现代农业产业技术体系广西杧果创新团队桂南综合试验站(桂农发[2011]33号)

摘　　要：为建立一套适用于鉴别"四季芒"成花蛋白表达差异的双向电泳方法,摸索出合适的芒果叶片蛋白提取方案。以"四季芒"芒果叶片为材料,采用TCA-丙酮法、酚抽提乙酸铵沉淀法、改良酚抽提乙酸铵沉淀法等3种芒果叶片蛋白提取方法,进行SDSPAGE和双向电泳,比较这些方法的提取效果。结果表明,改良酚抽提乙酸铵沉淀法比一般酚抽提法获得更高质量的蛋白。对沉淀产物洗涤和干燥步骤的优化能有效提高TCA-丙酮法蛋白提取质量。提取液p H能明显影响改良酚抽提乙酸铵沉淀法所提蛋白的得率、2-DE凝胶蛋白点数量和分布。p H 8.5提取液所提蛋白获得最多的2-DE蛋白点,但在小分子区域蛋白分辨率比p H9.0提取液所提蛋白低。TCA-丙酮法所提蛋白在大分子区域2-DE蛋白点分辨率不如改良酚抽提乙酸铵沉淀法,但小分子和高等电点区蛋白点分辨率却明显好于后者。实验表明改良酚抽提乙酸铵沉淀法和TCA-丙酮法有很强的互补性,可以在双向电泳体系中组成双提取法提高蛋白分辨率。The purpose of the study was to develop 2-DE method suitable for Mango leaf proteome analysis in future. This study compared TCA-Acetone protocol,Phenol Extraction-Ammonium Acetate Precipitation method（ PE-AP） and Optimized Protocol of Phenol ExtractionAmmonium Acetate Precipitation（ OPE-AP） to examine their effectiveness on protein extraction of mango leaf tissue. The result indicates that OPE-AP gained better quality of protein than PE-AP. The optimization on the steps of washing and drying of the pellet can effectively improve the quality of protein extracted by TCA-Acetone protocol. The p H value of extraction buffer applied in OPE-AP can greatly affect protein yield,number of protein spots and their distribution on 2-DE gel. Proteins extracted by extraction buffer with p H 8. 5 result in the most number of 2-DE protein spots,but the protein spot resolution in the zone of small molecular weigh is lower than proteins extracted by p H9. 0 extraction buffer. Proteins extracted by TCA-Acetone protocol have lower protein spot resolution in the zone of high molecular weigh than proteins extracted by OPE-AP,but gain much higher protein resolution in zones where proteins have small molecular weigh and high value of isoelectric point than the latter one. There is a high complementary result in protein spot resolution between protocol of TCA-Acetone and OPE-AP,thus their combination can be applied in two-dimensional electrophoresis to improve protein resolution.

关 键 词：芒果 蛋白提取 双向电泳 蛋白质组 

分 类 号：S667.7[农业科学—果树学]