水稻籽粒蛋白的提取优化及其蛋白组学研究体系的建立#
 

摘要：本实验以水稻籽粒为研究材料，为建立适用于水稻籽粒的蛋白组学研究体系，首先对比了3 种不同的提取方法：TCA/丙酮提取法、可溶性提取法、苯酚提取法；在最优提取法的基础上，本研究优化了自制管胶与GE 公司IPG 商业化胶条的双向电泳条件；最后为评价优化后的籽粒蛋白提取方法和双向电泳体系与蛋白质组学后续研究策略的兼容性，本研究通过3 个重要的蛋白质组学研究策略MALDI-TOF/MS、western- blot 和磷酸化蛋白组学方法对胶内蛋白质进行鉴定分析；结果表明，可溶性提取法效果最佳，获得的双向电泳图谱背景低，蛋白质点多，693 个，且得到有效的分离；自制管胶载体两性电解质pH 3-10 和pH 5-8的比例为1: 3 时2- DE图谱上的蛋白质点分布更为均匀，同时将IPG 胶条一向电泳1 000 V 2h的除盐程序时间适当延长为6 h，图谱上的蛋白质点聚焦更为充分；胶内蛋白质鉴定分析结果表明可溶性提取方法获取的样品适用于质谱鉴定、western-blot分析、与磷酸化蛋白鉴定。本研究初步建立了水稻籽粒的蛋白组学研究体系。
 

关键词：水稻；水稻籽粒；蛋白质组学；蛋白质双向电泳（2- DE）；蛋白磷酸化修饰

4 结论
本研究结果表明，可溶性提取法提取蛋白结果优于苯酚提取法和TCA/丙酮提取法，适合于水稻籽粒蛋白的提取，该方法获得的蛋白质2- DE 图谱蛋白质点多且清晰，背景干净，谷蛋白少，重复性好。同时优化了自制管胶和固化IPG 胶条电泳参数，得到了更优的2-DE图谱。通过对胶内蛋白质点进行质谱分析、磷酸化蛋白质组学分析和western-blot 免疫印迹试验，得到良好的鉴定分析结果，说明籽粒蛋白可溶性提取方法和优化后的双向电泳体系与蛋白质组学后续研究策略兼容，从而建立了水稻籽粒蛋白组学研究体系，这为水稻籽粒蛋白质组学研究奠定了坚实的基础，同时也为其他谷类作物籽粒蛋白质组学研究提供了参考依据。
 

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