砂藓总蛋白质提取方法的选取及双向电泳体系的建立  

Establishment of Total Protein Extraction Methods and Two-Dimensional Electrophoresis System for Racomitrium canescens

作　　者：沙伟[1,2] 李雯煜 张丽丽 张梅娟[1,2] 马天意[1,2] Sha Wei;Li Wenyu;Zhang Lili;Zhang Meijuan;Ma Tianyi(College of Life Sciences,Agriculture and Forestry,Qiqihar University,Qiqihar,161006;Heilongjiang Provincial Key Laboratory of Resistance Gene Engineering and Protection of Biodiversity in Cold Areas,Qiqihar University,Qiqihar,161006)

机构地区：[1]齐齐哈尔大学生命科学与农林学院,齐齐哈尔161006 [2]齐齐哈尔大学,抗性基因工程与寒地生物多样性保护黑龙江重点实验室,齐齐哈尔161006

出　　处：《基因组学与应用生物学》2020年第6期2659-2665,共7页Genomics and Applied Biology

基　　金：国家自然科学基金项目(31570207);黑龙江省教育厅基本业务专项理工面上项目(135209262);黑龙江省省属高等学校基本科研业务费科研项目植物性食品加工技术特色学科专项(YSTSXK201876)共同资助。

摘　　要：为进行砂藓(Racomitrium canescens)蛋白质组学的双向电泳工作,首要前提是获得质量较好的砂藓总蛋白质,并建立一套完整高效的适用于砂藓的双向电泳体系。本研究以砂藓为研究对象,比较了Tris-酚抽提法、TCA-丙酮法和试剂盒法3种蛋白质提取方法获得砂藓总蛋白质的浓度和完整性,并对砂藓中蛋白质双向电泳的运行条件进行了优化和比较。结果表明:在3种砂藓总蛋白质提取方法中,Tris-酚抽提法获得的砂藓总蛋白质浓度最高,SDS-PAGE电泳结果中条带清晰且较完整,双向电泳后获得的蛋白质点最多,是3种方法中提取砂藓蛋白质浓度最高完整性最好的方法;基于Tris-酚抽提法的基础上进一步对砂藓总蛋白质双向电泳体系进行优化,结果显示:利用pH值范围为pH 4~7的IPG胶条,蛋白质上样量为1 000μg的条件下,获得的砂藓蛋白质双向电泳图谱蛋白质点最多,图谱中可分辨的蛋白质点共(872±15)个,且低丰度蛋白质点清晰。本研究筛选出的方法获得了蛋白质点分布均匀、背景清晰、分辨率高、质量较高的双向电泳图像,建立了砂藓蛋白质组学研究体系的较好方法。本研究结果为砂藓蛋白质组学研究提供了良好基础。In order to carry out the two-dimensional electrophoresis works of Racomitrium canescens proteomics,the prerequisite is to obtain the high-quality total protein and establish a complete and efficient two-dimensional electrophoresis system suitable for R. canescens. In this study, R. canescens was used as the research material,three protein extraction methods, including Tris-phenol extraction method, TCA-acetone method and Solarbio kit method, were compared by the total protein concentration and integrity of each method, and two-dimensional electrophoresis parameters were also optimized for R. canescens total protein. The results showed that the concentration of R. canescens total protein obtained by Tris-phenol method was with the highest concentration among the three methods, the protein bands in the SDS-PAGE electrophoretogram were the clearest and most integrated,and the protein spots in two-dimensional electrophoretogram were the most, which was the best method for extracting R. canescens total protein in the three methods. Based on the Tris-phenol extraction method, the two-dimensional electrophoresis system of R. canescens total protein was furtherly optimized. The results showed that using the condition of IPG strips of pH 4~7, protein loading amount of 1 000 μg performed the twodimensional electrophoresis pattern of the R. canescens protein with the most protein spots with the number of(872±15), meanwhile the low-abundance protein spots were clear. The method selected in this study obtained a two-dimensional electrophoretogram with good distribution of protein spots, clear background, high resolution and high quality, established a good method for R. canescens proteomics research system. The results of this study lay a good foundation for R. canescens proteomics researches.

关 键 词：砂藓(Racomitrium canescens) 蛋白质提取 蛋白质组学 双向电泳 

分 类 号：Q946.1[生物学—植物学]