作物叶片蛋白质组提取与酶切方法比较研究  

Comparison of Preparation and Digestion for Crop Leaf Proteomes

作　　者：苏小琴[1,2] 魏红茹 段双梅 赵明[1] 潘映红[2] 

机构地区：[1]云南农业大学龙润普洱茶学院,云南昆明650201 [2]中国农业科学院作物科学研究所/国家农作物基因资源与基因改良重大科学工程,北京100081 [3]河北农业大学农学院,河北保定071000

出　　处：《作物杂志》2016年第2期50-56,共7页Crops

基　　金：中国农业科学院科技创新工程(作物分子标记技术及其应用科研创新团队)

摘　　要：蛋白质组样品制备主要涉及蛋白质的提取和酶切处理,是蛋白质组学分析的限制环节之一。为了提高作物叶片蛋白质组样品制备的效率和重复性,系统比较了6种缓冲液(pH8.5 Tris-HCl酚、pH7.0磷酸盐、pH9.0碳酸盐、尿素/硫脲、pH8.0 Tris-HCl、pH4.5醋酸盐)和TCA-丙酮处理对水稻、小麦、大豆和玉米叶片蛋白质提取的影响,同时以小麦叶片总蛋白和牛血清白蛋白为样本,比较了传统方法和微波辅助方法的酶切效率。结果表明,TCA-丙酮处理的小麦和水稻样品采用尿素/硫脲方法能获得较高的蛋白得率,而玉米和大豆样品采用尿素/硫脲直接提取时蛋白质得率更高,同时,微波辅助酶切值得用于蛋白质组样品制备。本研究采用适当的蛋白质提取和酶切方法,有效提高了蛋白质的提取率和鉴定率,可为进一步深入开展作物叶片的蛋白质组学研究提供借鉴。The sample preparation,extraction and protein digestion,are key steps in proteomics analysis. In order to improve the efficiency and repeat ability of crop leaf proteome samples,the leaves of rice,wheat,soybean and corn were respectively extracted with six buffers（pH4.5 acetic acid salt,pH7.0 phosphate,pH8.0 Tris-HCl,pH8.5 TrisHCl phenol,pH9.0 carbonate,and urea-thiourea）,and pretreatment with TCA-acetone. And then,the proteins extracted from wheat leaves and bovine serum albumin were digested by standard trypsin digestion and microwave-assisted digestion. SDS-PAGE and protein extraction rates showed that TCA-acetone precipitation combined with urea-thiourea extraction was suitable for preparation of rice and wheat leaf protein,and urea-thiourea extraction without TCAacetone precipitation was suitable for preparation of corn and soybean leaf protein. LC-MS-MS analysis of digested peptides suggested that standard and microwave-assisted digestion were both effective in proteomics analysis. Together,the protocol for preparation of proteome samples from leaves of rice,wheat,soybeans and corn was developed. This study confirmed that,the method by using the appropriate protein extraction method combining microwave-assisted digestion,could effectively improve the coverage and efficiency of crop leaf proteome analysis.

关 键 词：蛋白质组 样品制备 蛋白酶切 作物 叶片 

分 类 号：Q946[生物学—植物学]