摘 要

蛋白质组学的目的是同时鉴定、定量、分析大量蛋白质的功能，因此，蛋白质分离的双向电泳技术和敏感快速的蛋白质鉴定技术相结合已成了植物功能基因分析的强有力工具。蛋白质组学的研究在IPG胶条、激光扫描技术以及双向电泳图谱数字化出现以后，得到了快速发展，特别是将荧光物质引入所产生的双向差异凝胶电泳(two dimension difference gel electrophoresis, 2D-DIGE)，将蛋白质组学推入了高速发展期。DIGE技术将荧光物质标记引入到传统的双向凝胶电泳（2-DE）体系中，比经典的双向凝胶电泳具有更高的动力学范围和灵敏性，该方法通过引入内标使得多个样品在一块胶上分离，进而减少了技术变异引起的误差。2-D DIGE结合质谱、生物信息学及高可信度的统计分析使得成功地定量、鉴定植物蛋白质成为可能。2-D DIGE技术使研究者能够对差异表达的蛋白质实现可靠的定量化分析。本文初步建立杉木种子蛋白质组的2D-DIGE技术系统，并对这一系统的可靠性、重复性进行评估，分析，为大规模的杉木种子蛋白荧光差异分析奠定了基础。

关键词：DIGE 蛋白质组 杉木种子

The establishment and evaluation of the system based on DIGE proteomic technology

ABSTRACT

The purpose of proteomics is to quantify, analyze and identify function of a large number of proteins at the same time. Therefore, the combination of protein separation of two-dimensional electrophoresis technology and sensitive rapid protein identification technology has become a powerful tool of plant functional gene analysis. The research of proteomics has had great development from IPG strip, laser scanning technology and two-dimensional electrophoresis. Especially, the idea of introducing fluorescent substances into the two-dimensional difference gel electrophoresis (two dimension difference gel electrophoresis, 2D-DIGE) pushes proteomics into a high-speed development period. DIGE introduces fluorescent material into the traditional two-dimensional gel electrophoresis (2-DE) system. It has higher dynamic range and sensitivity. The method separates the multiple samples in a piece of gel by introducing internal standard, which reduces the technical variation caused by the error. 2-D DIGE combined with mass spectrometry, bioinformatics and statistical analysis of high credibility that makes it possible to successfully quantitative, identification of plant protein. 2-D DIGE technology enables researchers to analyze the differentially expressed proteins to achieve reliable quantitative analysis. This paper preliminarily established Chinese fir seed protein group of 2-D DIGE technology system, evaluated its reliability and repeatability and laid the foundation for large-scale Chinese fir seed fluorescent differences in protein analysis.

Keywords: DIGE, proteomics, Chinese fir seed

目 录

1.文献综述……………………………………………....................……………….…………..….1

1.1 DIGE技术的产生.....................................................................................................................1

1.2 国内外同类研究概况..............................................................................................................1

1.3 DIGE发展预期.....................................................................................................................2

2.正文............................................................................................................................................3

2.1 前言......................................................................................................................................3

2.2 材料与方法..........................................................................................................................4

2.2.1材料..................................................................................................................................4

2.2.2 仪器.................................................................................................................................4

2.2.3 相关试剂.........................................................................................................................4

2.2.4 蛋白质提取.....................................................................................................................4

2.2.5 蛋白质定量.....................................................................................................................4

2.3 2-DE重复性评价.................................................................................................................5

2.3.1 等电聚焦 (IEF)..............................................................................................................5

2.3.2 IPG胶条的平衡..............................................................................................................6

2.3.3第二向SDS-PAGE..........................................................................................................6

2.4 荧光标记评价......................................................................................................................7

2.4.1 染料评价.........................................................................................................................7