【目的】获得银杏叶绿体蛋白质的提取方法,在蛋白质水平探讨银杏光合作用对环境的响应机制。【方法】建立一种适合银杏叶绿体蛋白双向电泳分离的实验体系,采用Tris-平衡酚抽提法对银杏叶绿体蛋白质进行提取,并与三氯乙酸(TCA)-丙酮沉降法进行对比分析。【结果】Percoll密度梯度离心法适用于银杏叶绿体的提取,提取的叶绿体平均完整率在85%左右。单向电泳结果显示,与TCA-丙酮沉降法相比,在用Tris-平衡酚抽提法分离的叶绿体蛋白质泳道中,低分子质量蛋白质条带更多、更清晰。进一步的双向电泳结果表明,用Tris-平衡酚抽提法对银杏叶绿体蛋白质进行提取,蛋白质产量更高,图谱清晰,所分离的蛋白点更多,形态更好,条纹影响相对较小。【结论】Tris-平衡酚抽提法可有效地提取高质量的叶绿体蛋白质并进行双向电泳,可用于银杏叶绿体的蛋白质组学分析。
Abstract
【Objective】To explore the mechanism of the response of photosynthesis in ginkgo plants at protein level to environmental conditions, an extraction method for chloroplast proteins was investigated. 【Method】An experimental system was developed that was compatible with two-dimensional electrophoresis(2-DE)of ginkgo chloroplast proteins. Tris-phenol method was used for the extraction of ginkgo chloroplast proteins, and compared with trichloroacetic(TCA)-acetone precipitation method.【Result】The results indicated that Percoll step gradient centrifugation was suitable for ginkgo chloroplast isolation, and the mean intact rate of purified chloroplasts was approximately 85%. The results of one-dimensional PAGE indicated that protein bands of chloroplasts were more distinct at the low molecular weight region of the gel for the Tris-phenol extracti method compared with the TCA-acetone precipitation method. Moreover, comparison of this method with TCA-acetone precipitation for the extraction of ginkgo chloroplast proteins showed that the method resulted in higher protein yields and produced more protein spots on 2-DE gels with less streak interference. 【Conclusion】The above results revealed that the Tris-phenol extraction method could efficiently extract high-quality chloroplast proteins for 2-DE, and could be applied in proteomic analysis of ginkgo chloroplasts.
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基金
收稿日期:2016-04-20 修回日期:2017-02-26
基金项目:国家自然科学基金项目(31300572); 江苏省大学生实践创新训练计划(201410298089X); 江苏高校优势学科建设工程资助项目(PAPD)
第一作者:施大伟(sdw1982@njfu.edu.cn),讲师,博士。
引文格式:施大伟,何梅,梁霏霏,等. 适于双向电泳分析的银杏叶绿体蛋白质提取方法[J]. 南京林业大学学报(自然科学版),2017,41(3):51-56.
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