华南农业大学学报
華南農業大學學報
화남농업대학학보
JOURNAL OF SOUTH CHINA AGRICULTURAL UNIVERSITY
2014年
3期
79-85
,共7页
赵芹%谢大森%彭庆务%郭巨先%周念
趙芹%謝大森%彭慶務%郭巨先%週唸
조근%사대삼%팽경무%곽거선%주념
节瓜%叶片总蛋白%蛋白质组学%双向电泳
節瓜%葉片總蛋白%蛋白質組學%雙嚮電泳
절과%협편총단백%단백질조학%쌍향전영
chieh-qua%total protein of leaf%proteomics%two-dimensional electrophoresis
【目的】建立适合节瓜叶片蛋白质组学研究的双向电泳体系。【方法】以节瓜品种“A37毛节瓜”为材料,对蛋白质抽提方法、上样量、凝胶浓度及IPG胶条pH范围等关键因素进行探索与优化。【结果和结论】与TCA/丙酮沉淀法及酚提取法相比,改良酚提取法抽提蛋白质总量较高,纯度最高,SDS-PAGE电泳条带明显、清晰,双向电泳图谱蛋白质点最多、清晰均匀、纵横条纹少。利用17 cm pH 4~7范围IPG胶条、120μg蛋白质上样量、12%凝胶浓度,硝酸银染色获得蛋白质点数丰富、分辨率高、背景清晰且重复性好的双向电泳图谱,检测到节瓜叶片蛋白质点1936个,相对分子质量主要分布于15000~100000之间。初步建立了一套适用于节瓜叶片蛋白质组学研究的双向电泳体系,为后续开展蛋白质组学研究及其相关工作奠定了基础。
【目的】建立適閤節瓜葉片蛋白質組學研究的雙嚮電泳體繫。【方法】以節瓜品種“A37毛節瓜”為材料,對蛋白質抽提方法、上樣量、凝膠濃度及IPG膠條pH範圍等關鍵因素進行探索與優化。【結果和結論】與TCA/丙酮沉澱法及酚提取法相比,改良酚提取法抽提蛋白質總量較高,純度最高,SDS-PAGE電泳條帶明顯、清晰,雙嚮電泳圖譜蛋白質點最多、清晰均勻、縱橫條紋少。利用17 cm pH 4~7範圍IPG膠條、120μg蛋白質上樣量、12%凝膠濃度,硝痠銀染色穫得蛋白質點數豐富、分辨率高、揹景清晰且重複性好的雙嚮電泳圖譜,檢測到節瓜葉片蛋白質點1936箇,相對分子質量主要分佈于15000~100000之間。初步建立瞭一套適用于節瓜葉片蛋白質組學研究的雙嚮電泳體繫,為後續開展蛋白質組學研究及其相關工作奠定瞭基礎。
【목적】건립괄합절과협편단백질조학연구적쌍향전영체계。【방법】이절과품충“A37모절과”위재료,대단백질추제방법、상양량、응효농도급IPG효조pH범위등관건인소진행탐색여우화。【결과화결론】여TCA/병동침정법급분제취법상비,개량분제취법추제단백질총량교고,순도최고,SDS-PAGE전영조대명현、청석,쌍향전영도보단백질점최다、청석균균、종횡조문소。이용17 cm pH 4~7범위IPG효조、120μg단백질상양량、12%응효농도,초산은염색획득단백질점수봉부、분변솔고、배경청석차중복성호적쌍향전영도보,검측도절과협편단백질점1936개,상대분자질량주요분포우15000~100000지간。초보건립료일투괄용우절과협편단백질조학연구적쌍향전영체계,위후속개전단백질조학연구급기상관공작전정료기출。
[Objective]To establish two-dimensional electrophoresis (2-DE) system suitable for chieh-qua leaf proteomics analysis .[Method]The effects of various protein extraction methods of 2-DE were inter-compared using the leaves of “A37maojiegua” as the materials, and the loading quantity of sample ,SDS-PAGE gel concentration and the pH range of IPG strips were also investigated and optimized .[Result and conclusion]The results showed that modified phenol extraction method was notably superior to TCA /ace-tone precipitation method and phenol extraction method in total protein quality and clarity of protein bands in SDS-PAGE electrophoresis , and the final 2-DE image map based on modified phenol extraction method had the maxium protein dots and clear background , slight streaking , more completely separated spots compared with the other methods .The further optimization of 2-DE system was carried out to obtain a 2-DE electrophoretogram abundant protein spots , high resolution , clear background and excellent repeat-ability with 17 cm IPG strips,120 μg protein loading quantity , SDS-PAGE with 12% gel concentration and IPG strips with pH 4-7, finally detecting proteins with modified silver nitrate staining .More than 1 936 protein spots were detected and their relative molecular mass ranged from 15 000 to 100 000 .The optimization of the experimental conditions of 2-DE system suitable for chieh-qua proteomics research has been constructed , which provides a basis for further investigation of chieh-qua proteomics researches .