摘 要

蛋白质组学是指在大规模水平上研究蛋白质的特征，包括蛋白质的表达水平，翻译后的修饰，蛋白与蛋白相互作用等，由此获得蛋白质水平上的关于疾病发生，细胞代谢等过程的整体而全面的认识。蛋白质样品制备是蛋白质组学研究分析的前提，也是影响双向凝胶电泳有效分辨率的重要因素。叶片是植物光合作用重要组织，对叶片所含蛋白质的研究也非常重要。但是由于叶片中Rubisco蛋白的比例较高，而且对该蛋白研究得较成熟，在蛋白质组学研究中高丰度蛋白的存在会干扰低丰度蛋白质的呈现，干扰在双向凝胶电泳的分辨率并从而影响蛋白质鉴定的准确性。因此为了更好的进行杨树叶片蛋白质组学的研究，提高双向凝胶电泳的分辨率，剔除Rubisco蛋白是非常必要的。所以在从叶片总蛋白中提取蛋白质样品时，需要通过预处理分离出高丰度蛋白Rubisco再进行双向凝胶电泳等后续的分析，从而提升对低丰度蛋白检测的精确度。本实验采用PEG预分离法结合TCA/丙酮和PEG预分离法结合SDS/酚两种方法进行高丰度蛋白Rubisco的去除，并通过SDS-PAGE电泳和双向凝胶电泳图谱分析比较PEG预分离法结合TCA/丙酮和PEG预分离法结合SDS/酚去除高丰度蛋白质的效果，结果显示PEG预分离法结合TCA/丙酮有较好的去除效果。

关键词：蛋白质组学；蛋白质样品制备；杨树叶片；高丰度蛋白；双向凝胶电泳

The removal of high abundance of photosynthetic protein Rubisco in poplar leaves

ABSTRACT

Proteomics refers to the characteristics of the proteins in large scale level, including the level of protein expression, post-translational modification, protein protein interaction, thereby obtaining the protein level on the disease, whole cell metabolic process and comprehensive understanding. Protein sample preparation is a prerequisite for proteomics research, and is also an important factor affecting the effective resolution of the two-dimensional gel electrophoresis. Blade is an important organization of plant photosynthesis, protein of leaves is also very important. But, because of Rubisco in leaves of high proportion and research on Rubisco has matured. Besides, the exist of high-abundant protein also disturb the present of low abundance proteins and the accuracy of two dimensional gel electrophoresis resolution thus affect the identification of protein. So in order to do better study of poplar leaf proteomics, improve the two-dimensional gel electrophoresis resolution, excluding Rubisco protein is very necessary. So in the extraction of protein samples from the total leaf protein, we need to separate out the high abundance proteins by pretreatment before the subsequent Rubisco analysis such as two-dimensional gel electrophoresis, so as to improve the detect accuracy of low abundance proteins. The two methods of extract protein samples, PEG fractionation method combine with TCA/ acetone and PEG fractionation method combine with SDS/ phenol, are compared in this experiment, and through SDS-PAGE and the two-dimensional gel electrophoresis analysis we find that use the method of PEG fractionation combine with TCA/ acetone can be effectively separate the high abundance proteins.

Key words：Proteomics；Protein sample preparation；Poplar；high-abundant protein；two dimensional gel electrophoresis

目 录

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

1.1 植物蛋白质组学的研究目的及意义…………………………………………………………1

1.2 植物蛋白组学的研究方法……………………………………………………………………2

1.2.1 蛋白质组学的概念………………………………………………………………2

1.2.2 蛋白质样品的制备………………………………………………………………2

1.2.3 双向电泳技术……………………………………………………………………3

1.2.4 双向电泳技术在蛋白质组学中的应用…………………………………………4

2 正文………………………………………………………………………………5

2.1 实验材料……………………………………………………………………………5

2.2 方法…………………………………………………………………………………5

2.2.1 PEG预分离结合TCA丙酮的蛋白质提取法…………………………………………5

2.2.2 PEG预分离结合SDS/酚的蛋白质提取法…………………………………………5

2.2.3 蛋白质浓度的测定………………………………………………………………6

2. 2. 4 SDS-PAGE………………………………………………………………………………….

2.2.5 双向凝胶电泳……………………………………………………………………6

2.3 实验结果……………………………………………………………………………………………7

2.3.1 分光光度计测得蛋白质吸光值及浓度………………………………………………………7

2.3.2 SDS-PAGE………..............................................................................................5

2.3.3 双向凝胶电泳图谱............................................................................................................................

结论 ……………………………………………………………………………………………10

致谢 …………………………………………………………………………………………………11

参考文献 ……………………………………………………………………………………………12

1 文献综述

前言

在现今的蛋白质组学的研究中，由于细胞中高度拷贝的高丰度蛋白一般不参与基因调控、细胞生命活动调节而且研究相对比较成熟。相反一些研究得比较少的低丰度蛋白往往是参与细胞基因表达调控、细胞生命活动调节、信号传递等关键生命活动的重要蛋白质，所以低丰度蛋白是当下备受关注的研究对象。当人们在研究某一植物低丰度蛋白质在生命体内所发挥的功能作用时，常常会受到组织细胞中高丰度蛋白（如Rubisco蛋白）的干扰，进而影响对低丰度蛋白的分析。因此在进行蛋白质组学分析时，蛋白质提取是关键的一步，其影响着后续双向电泳图谱的质量和蛋白质的鉴定。

1.1 植物蛋白质组学的研究目的及意义