适用于双向电泳的芒果叶片蛋白提取方法的建立

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...

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Bibliographic Details
Published in西南农业学报 Vol. 28; no. 6; pp. 2696 - 2700
Main Author 陈香玲 任惠 黄章保 苏伟强 刘业强 李果果 刘要鑫 王小媚
Format Journal Article
LanguageChinese
Published 农业部南宁南亚热带果树科学观测实验站,广西南宁530007%广西农业科学院园艺研究所,广西南宁530007 2015
广西作物遗传改良生物技术重点开放实验室,广西南宁530007
广西农业科学院园艺研究所,广西南宁530007
Subjects
双向电泳
芒果
蛋白提取
蛋白质组
Mango(Mangifera.Indica Linn)
双向电泳
蛋白提取
Proteomics
Protein extraction
芒果
蛋白质组
Two-dimensional electrophoresis
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ISSN1001-4829
DOI10.16213/j.cnki.scjas.2015.06.064

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Summary: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。
Bibliography: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 lo
ISSN:1001-4829
DOI:10.16213/j.cnki.scjas.2015.06.064