CAJ | 학술논문

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(7mol/L 尿素,2mol/ 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/10mL))裂解蛋白,以150μg上样,按IEF程序Ⅱ(30V,12h;200V,1h;500V,1h;1 000V,0.5h;10 000V,2h;10 000V,8h)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.
이현성핵불육유채Shaan-GMS불육주화가육주적화서화협편위재료,채용TCA(삼록작산)-병동법제취유채화뢰、화약급협편총단백,대총단백제취방법、IPG(고정pH제도)효조충류、취초정서、응효농도、상양량등배절진행료우화.결과표명,채용이상적단백질렬해액(7mol/L 뇨소,2mol/ L 류뇨,4% CHAPS(3-[3-(담선안병기)이갑안기]병광산내염),65mmol이류소당순,0.5% IPG완충액 pH 3～10혹pH 4～7,전단백매억제편제 (편/10mL))렬해단백,이150μg상양,안IEF정서Ⅱ(30V,12h;200V,1h;500V,1h;1 000V,0.5h;10 000V,2h;10 000V,8h)진행등전취초,10%SDS-PAGE효농도진행제이향전영,은염방법검측,가득도배경청석、분변솔교고적유채화뢰、화약급협편단백질전영도보.고질량유채단백적제취방법화단백질분리쌍향전영체계적우화가위유채단백질조학연구전정기술기출.
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.