【作者】 王旭；

【导师】 郝东云；

【作者基本信息】 吉林大学 ， 生物化学与分子生物学， 2007， 硕士

【摘要】 样品制备是植物蛋白质组学研究中的重要环节,针对双向电泳的样品制备发展了聚乙二醇分级沉淀方法,设置四个聚乙二醇浓度梯度(8%,16%,24%,40%),共得到五个组分,同时将植物中存在的高峰度蛋白Rubisco特异性地集中于一个组分之中,提高了低丰度蛋白质的检测。经过双向电泳的分离和凝胶图像软件分析,所有组分在凝胶图像上共检测到5077个不同的蛋白点,与常规的TCA/丙酮沉淀方法相比其中80%的点是新检测出的,可分析的蛋白点数目提高了5倍,这为使用双向电泳技术研究植物蛋白质组学提供了新的方法。为了探索植物低温适应的分子基础,我们应用上述方法,以拟南芥(Arabidopsis thanliana, ecotype Col-0)为实验材料,对其在不同时间低温(4℃)胁迫的全细胞蛋白进行了差异蛋白质组学分析。凝胶分析后发现蛋白质丰度差异变化在2倍以上的点67个,对其中的57个蛋白点进行了质谱鉴定,有42个点得到了有效鉴定结果,包括9个已知的低温胁迫响应蛋白和在本实验发现的低温应答蛋白。这些蛋白43.3%定位于叶绿体中,在代谢、能量、防御反应等方面发挥作用。更多还原

【Abstract】 Poor detection of low-abundant proteins is a common problem in two-dimensional electrophoresis (2-DE) for separation of proteins in a proteome analysis. This is attributed partially, at least, to the existence of high-abundant proteins, e.g. ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) in plants. They engage a large proportion of the whole-cell proteins and thus prevent low-abundant proteins from being up-taken by immobilized pH gradient (IPG) strip, consequently making the latter poorly detectable by 2-DE. In this work, we report a straightforward protocol for preparation of whole-cell proteins through differential polyethylene glycol (PEG) precipitation aiming at elimination of Rubisco from plant protein samples. In comparison with 2-DE analysis of protein samples prepared using a conventional TCA/acetone method, a relatively high reproducibility of proteins was achieved using a PEG fractionation protocol in terms of protein yield and protein species. As expected, the large subunit of Rubisco was precipitated predominantly in the 16% PEG fraction. This allowed proteins of the Rubisco-containing fraction to be analyzed separately from those of other PEG fractions. After taking into account the overlapping protein spots among 2-DE gels of all fractions through image and statistical analyses, we detected with this protocol a total 5077 protein spots, among which about 80% are proteins undetectable with the TCA/acetone method, while the rest of proteins exhibited a significant increase in their abundance. This protocol was developed using Arabidopsis as a source of protein and thus may also be applicable to protein preparations of other plants. A proteomic analysis was carried out aiming at understanding of the molecular adaptation mechanisms of cold stress in Arabidiopsis (Arabidopsis thanliana, ecotype Col-0). The seedlings were exposed to 4℃for 8 and 24 h respectively, together with the controls which were grown at normal temperature for 8 and 24 h. In PEG fractionation, the whole-cell proteins were extracted from both control and stressed seedlings. Sixty-seven protein spots were found to exhibit different dynamic patterns on 2-D gels in responding to the low temperature stress and fifty-seven of them were identified using MALDI-TOF MS. These cold responsive proteins, besides eight proteins of unknown function, are involved in regulation of metabolism, energy, cell defense, biogenesis of cellular component, etc. by MIPS function category, TAIR database and related references. Proteins (43.3%) were predicted to be located in the chloroplasts, implying that chloroplast proteins are likely involved in the cold stress adaptation in Arabidopsis. The physiological implications, which were derived from the experimental data, are discussed, suggesting that there is a complex metabolic network in plants responsive to cold stress.更多还原