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.
出处
《西南农业学报》
CSCD
北大核心
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)
关键词
芒果
蛋白提取
双向电泳
蛋白质组
Mango（Mangifera.Indica Linn）
Protein extraction
Two-dimensional electrophoresis
Proteomics
