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适于双向电泳分析的银杏叶绿体蛋白质提取方法
引用本文:施大伟,何梅,梁霏霏,徐鸣,谢寅峰.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(3):51-56.
作者姓名:施大伟  何梅  梁霏霏  徐鸣  谢寅峰
作者单位:1. 南方现代林业协同创新中心,江苏 南京210037;南京林业大学生物与环境学院,江苏 南京210037;2. 南京林业大学生物与环境学院,江苏 南京210037;江西省林业科学院,江西 南昌 310032;3. 南京林业大学生物与环境学院,江苏 南京,210037
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
摘    要:【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。

关 键 词:银杏  叶绿体蛋白质组  蛋白质提取方法  双向电泳

An efficient extraction method of chloroplast proteins from ginkgo plants compatible for two-dimensional electrophoresis
SHI Dawei,HE Mei,LIANG Feifei,XU Ming,XIE Yinfeng.An efficient extraction method of chloroplast proteins from ginkgo plants compatible for two-dimensional electrophoresis[J].Journal of Nanjing Forestry University(Natural Sciences ),2017(3):51-56.
Authors:SHI Dawei  HE Mei  LIANG Feifei  XU Ming  XIE Yinfeng
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.Trisphenol 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.
Keywords:ginkgo  chloroplast proteomics  protein extraction method  two-dimensional electrophoresis
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