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作物杂志,2016, 第2期: 50–56 doi: 10.16035/j.issn.1001-7283.2016.02.009

• 遗传育种·种质资源·生物科技 • 上一篇    下一篇

作物叶片蛋白质组提取与酶切方法比较研究

苏小琴1,3,魏红茹2,3,段双梅1,赵明1,潘映红3   

  1. 1 云南农业大学龙润普洱茶学院,650201,云南昆明
    2 河北农业大学农学院,071000,河北保定
    3 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,100081,北京
  • 收稿日期:2015-10-27 修回日期:2016-03-04 出版日期:2016-04-15 发布日期:2018-08-26
  • 通讯作者: 赵明
  • 作者简介:苏小琴,在读硕士研究生,主要从事蛋白质组学研究
  • 基金资助:
    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)

Comparison of Preparation and Digestion for Crop Leaf Proteomes

Su Xiaoqin1,3,Wei Hongru2,3,Duan Shuangmei1,Zhao Ming1,Pan Yinghong3   

  1. 1 College of Longrun Puer Tea,Yunnan Agricultural University,Kunming 650201,Yunnan,China
    2 College of Agronomy,Hebei Agriculture University,Baoding 071000,Hebei,China
    3 Institute of Crop Science,Chinese Academy of Agricultural Sciences/National Key Facility for Crop Gene Resources and Genetic Improvement,Beijing 100081,China
  • Received:2015-10-27 Revised:2016-03-04 Online:2016-04-15 Published:2018-08-26
  • Contact: Ming Zhao

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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。

关键词: 蛋白质组, 样品制备, 蛋白酶切, 作物, 叶片

Abstract:

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 Tris- HCl 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.

Key words: Proteome, Sample preparation, Protein digestion, Crop, Leaf

图1

不同缓冲液提取物SDS-PAGE1.pH8.5 Tris-HCl酚Tris-HCl phenol;2.pH7.0 磷酸盐pH7.0 phosphate;3.pH9.0碳酸盐pH9.0 carbonate;4.尿素/硫脲Urea-thiourea;5.pH8.0三氨基甲烷pH8.0 Tris-HCl;6.pH4.5醋酸盐pH4.5 acetic acid salt;M.250kDa"

图2

不同缓冲液和预处理方法的蛋白得率 1.pH8.5 Tris-HCl酚Tris-HCl phenol;2.pH7.0 磷酸盐pH7.0 phosphate;3.pH9.0碳酸盐pH9.0 carbonate;4.尿素/硫脲Urea-thiourea;5.pH8.0三氨基甲烷pH8.0 Tris-HCl;6.pH4.5醋酸盐pH4.5 acetic acid salt"

图3

尿素/硫脲提取物SDS-PAGE1,2.水稻Rice;3,4.小麦Wheat;5,6.大豆Soybean;7,8.玉米Corn;M.250kDa"

图4

尿素/硫脲重复提取物的SDS-PAGE a.丙酮沉淀的水稻、小麦、大豆和玉米尿素/硫脲提取物(10%分离胶)Extracts of rice,wheat,soybean and corn precipited with acetone(10% resolving gel); 1.水稻Rice;2.小麦Wheat;3.大豆Soybean;4.玉米Corn;M.250kDa b.TCA-丙酮预处理水稻的尿素/硫脲提取物(12%分离胶)Proteins from rice pretreated with TCA-acetone(12% resolving gel); c.TCA-丙酮预处理小麦的尿素/硫脲提取物(11%分离胶)Proteins from wheat pretreated with TCA-acetone(11% resolving gel); d.大豆的尿素/硫脲提取物(10%分离胶)Proteins extracted from soybean (10% resolving gel); e：玉米的尿素/硫脲提取物(10%分离胶)Proteins extracted from corn (10% resolving gel)"

表1

传统酶切和微波辅助酶切牛血清白蛋白"

酶切方法Digestion 酶切时间(h)
Digestion time		 牛血清白蛋白覆盖率（%）BSA coverage
0漏切位点
0 missed cleavages		 1漏切位点
1 missed cleavages		 2漏切位点
2 missed cleavages
传统酶切Standard digestion 20.0 74.55 21.82 3.64
微波辅助酶切Microwave-assisted digestion 3.3 70.77 23.82 6.15

图5

两种方法酶切小麦总蛋白后的总离子流图（TIC）和质谱鉴定结果"

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