油菜不同器官高质量总蛋白提取方法和双向电泳体系的优化

以显性核不育油菜Shaan-GMS不育株和可育株的花序和叶片为材料，采用TCA（三氯醋酸）-丙酮法提取油菜花蕾、花药及叶片总蛋白，对总蛋白提取方法、IPG（固定pH梯度）胶条种类、聚焦程序、凝胶浓度、上样量等环节进行了优化。结果表明，采用理想的蛋白质裂解液（7 mol/L 尿素，2 mol/ L 硫脲，4% CHAPS（3-3-(胆酰氨丙基)二甲氨基]丙磺酸内盐），65mmol二硫苏糖醇，0.5% IPG缓冲液 pH 3~10或pH 4~7，全蛋白酶抑制片剂 (片／10 ml)）裂解蛋白，以150μg上样，按IEF程序Ⅱ(30V，12Hr；200V，1Hr；500V，1Hr；1000V，0.5Hr；10000V，2Hr；10000V，80000VHr)进行等电聚焦，10%SDS-PAGE胶浓度进行第二向电泳，银染方法检测，可得到背景清晰、分辨率较高的油菜花蕾、花药及叶片蛋白质电泳图谱。高质量油菜蛋白的提取方法和蛋白质分离双向电泳体系的优化可为油菜蛋白质组学研究奠定技术基础。

Optimization of high quality total protein extraction and two-dimensional gel electrophoresis system for different Brassica napus organs

Proteins from buds,anthers and leaves were extracted using TCA (trichloroacetic acid)-acetone method from sterile and fertile plants of Brassica napus L.dominant GMS (genic male sterile) line Shaan-GMS.Optimized factors included extraction parameters, types of IPG strip,focusing procedure,gel concentration and loading quantity of samples.High quality 2-DE map with low background was obtained using the following optimized procedure: dissolving proteins with lysis solution (7mol/L Urea,2mol/L Thiourea,4% CHAPS,65mmol DTT,0.5% IPG buffer pH 3～10 or pH 4～7,10mL Complete Protease Inhibitor Coctail Tablets for each sample), loading 150μg protein sample, isoelectric focusing with IEFⅡprocedure (30V,12h;200V,1h;500V,1h;1 000V,0.5h;10 000V,2h;10 000V,8h),SDS-PAGE with 10% gel concentration, and finally detecting proteins using silver-taining method.The optimized system will be able to facilitate the proteomics research on B.napus.