摘要
建立绿盲蝽取食胁迫下赤霞珠冬芽防御反应的蛋白质组学双向电泳技术体系,旨在为后续筛选和分析差异蛋白相关研究提供参考。选取赤霞珠葡萄冬芽,分别设置绿盲蝽取食胁迫和空白对照试验,于24 h后采集样品用液氮带回,-80℃冻存;利用TCA-丙酮法、TCA-丙酮法+酚抽提法提取粗蛋白干粉;利用CBB G-250染色法进行蛋白质浓度测定;确定上样量及凝胶染色技术。扫描得到凝胶图像,进行对比分析。结果显示,利用不同蛋白质提取方法均可获得双向电泳所需粗蛋白,采用TCA-丙酮法获取的粗蛋白量明显多于TCA-丙酮法+酚抽提法;2种方法所提取的全蛋白浓度差异较大,TCA-丙酮法操作更为简便,蛋白丢失少。不同上样量对双向电泳图谱效果的影响差异显著,400μg蛋白上样量比800μg所得的凝胶图片上的蛋白点清晰,易于分离。考马斯亮蓝染色结果表明,R-350更适用于葡萄冬芽全蛋白双向凝胶电泳技术。利用TCA-丙酮法抽提蛋白,400μg蛋白上样,pH值4~7 NL 17 cm的IPG胶条,G-350热染色双向电泳技术体系得到的赤霞珠冬芽全蛋白凝胶图谱,符合绿盲蝽取食诱导赤霞珠防御反应产生防御蛋白的检测要求。
In order to establish a proteomic 2-DE system for the defense response of Cabernet sauvignon winter buds under Apolygus lucorum feeding stress,Cabernet sauvignon grape winter buds were used as materials to compare the effects of different protein extraction methods,sample loading and gel staining techniques on the 2-DE chromatogram.It provided reference for subsequent studies on screening and analyzing differential proteins.Winter buds of Cabernet sauvignon grape were selected,and feeding stress and blank control experiments were conducted,respectively.After 24 h,the samples were taken back with liquid nitrogen and frozen at-80℃.Crude protein powder was extracted by TCA-acetone and TCA-acetone+phenol extraction.Protein concentration was determined by CBB G-250 staining.Sample loading and gel staining techniques were determined.Gel images were obtained by scanning for comparative analysis.Crude protein for 2-DE could be obtained by different extraction methods.The amount of crude protein obtained by TCA-acetone method was significantly higher than that obtained by TCA-acetone+phenol extraction method.The concentrations of total protein extracted by the two methods were different,but both of them met the requirements of two-dimensional gel electrophoresis.TCA-acetone method was easier to operate and less protein loss.The influence of different loading amount on the effect of 2-DE was significant.Compared with 800μg protein,400μg protein showed clear protein spots and was easy to separate.The results of CBB staining showed that R-350 was more suitable for total protein two-dimensional gel electrophoresis of grape winter bud.Using TCA-acetone method,400μg protein sample,17 cm pH value 4-7 IPG dry adhesive strip,G-350 thermal staining 2-DE system to obtain the total protein gel map of Cabernet sauvignon winter buds met the requirements of further test.
作者
高素红
葛长虹
周国娜
姚淑娟
路常宽
高宝嘉
GAO Suhong;GE Changhong;ZHOU Guona;YAO Shujuan;LU Changkuan;GAO Baojia(College of Forestry,Hebei Agricultural University,Baoding 071001,China;College of Agronomy and Biotechnology,Hebei Normal University of Science&Technology,Changli 066600,China;Hebei Key Laboratory of Crop Stress Biology(in Preparation),Changli 066600,China)
出处
《华北农学报》
CSCD
北大核心
2022年第1期181-187,共7页
Acta Agriculturae Boreali-Sinica
基金
河北省重点研发计划农业关键共性技术攻关专项(20326507D)
河北省专业学位研究生教学案例库开发项目(KCJSZ2020096)
河北省科技厅科普专项(20557504K)。
关键词
赤霞珠葡萄
绿盲蝽
蛋白质组学
双向电泳体系
诱导抗性
Cabernet sauvignon
Apolygus lucorum
Proteomics
Two-dimensional electrophoresis
Inducible resistance
