鹅掌楸愈伤蛋白质提取方法比较分析毕业论文

 2021-04-12 11:04

摘 要

杂交鹅掌楸是我国重要的用材树种,有着重要的经济和生态价值。但由于种子的短缺限制了苗木的供应,通过体胚发生获取优质的种苗是保障林业可持续发展的重要的手段。外植体在特定的条件下脱分化进行细胞重编程来关闭、开启一些基因表达来获得胚性能力。胚性能力的获得是一个复杂的过程,体细胞胚胎发生需要跨越的第一步。目前关于愈伤组织胚性获得的分子机制还不清楚。蛋白质组学是功能基因组学的一部分,愈伤胚性能力获得的蛋白质组学将为有助于全面了解胚性愈伤的蛋白质调控网络。蛋白质样品制备是蛋白质组学研究的第一步,且关键的一步。理想的蛋白质提取方法将可重复捕获、溶解给定样品中的蛋白质,并减少污染、人为的修饰及蛋白质降解。由于蛋白质样品多态性、复杂性及蛋白质翻译后修饰,因此没有一种蛋白质提取方法可以捕获所有植物样品的蛋白质组。优化和选择适合的提取方法将提供全面的蛋白质组信息。

为建立适合于杂交鹅掌楸胚性愈伤蛋白质提取方案,本实验对比了四种蛋白质提取方法:Mg/NP-40—TCA/actone、TCA/actone、Lysis buffer—clean up、Lysis buffer—TCA/actone。蛋白质样品进行SDS-PAGE和双向电泳分析。SDS-PAGE图谱显示Lysis buffer—clean up、Lysis buffer—TCA/actone分离的高分子量蛋白质条带相对Mg/NP-40—TCA/actone、TCA/actone少;Mg/NP-40—TCA/actone和Lysis buffer—clean up低分子量提取、分离效果较差。在此SDS-PAGE基础上对Mg/NP-40—TCA/actone、TCA/actone提取的蛋白质样品进行双向电泳。实验结果表明:TCA/actone得到的凝胶图蛋白质点分布均匀、背景清晰且纵横条纹较少,且TCA/actone相对于Mg/NP-40—TCA/actone来说在小分子量蛋白质和碱性蛋白质分离方面有着优势。同时随机选取双向电泳分离的蛋白质进行质谱分析,结果显示TCA/actone较适合杂交鹅掌楸胚性愈伤蛋白质提取及后续分析。

关键词:愈伤组织 蛋白质提取方法 SDS-PAGE 双向电泳

Evaluation of sample extraction methods for proteomic analysis of Liriodendron callus

ABSTRACT

Liriodendron hybrid is an important timber species, with economic and ecological value in China. The expansion of plantations has been curtailed, however, by the inconsistent availability of high-quality seeds. Clone propagation through somatic embryogenesis is a potentially large-scale alterative strategy for reforestation. In specific condition, explants are dedifferention to turn off/on some genes expression to acquire embryogenic competence by cell reprogramming. The acquisition of embryogenic competence is a complex process, which somatic embryogenesis needs to go through the first step. The molecular basis of embryogenic competence in callus is obscure. Proteomics is a part of functional genomics, which will help to understand the regulatory metworks of embryogenic protein. Sample preparation of protein is first step, a critical step in proteomics research. The ideal protein extraction protocol should reproducibly capture and solubilize the full ensemble of proteins in a given sample, reducing contaminants and minimizing post-extraction artifacts such as protein degradation and modification. Due to the diversity, complexity of proteins and post-modification, no single protein extraction protocol will capture the full proteome for all types of plants, the chosen protocol should be optimized for the secific plant tissue.

To establish the suitable extraction program for hybrid Liriodendron embryonic callus, four protein extraction methods were evaluated: Mg/NP-40—TCA/actone, TCA/actone precipitation, Lysis buffer—clean up, Lysis buffer—TCA /actone were used to extract embryonic callus proteins. The extraction proteins were separated by SDS-PAGE and two-dimensional electrophoresis (2-DE). SDS-PAGE indicated that Mg/NP-40—TCA/actone combined method and TCA/actone precipitation are better than lysis buffer—clean up lysis buffer—TCA/actone in high molecular proteins. Mg/NP-40—TCA/actone and Lysis buffer—clean up have poor separation in low molecular proteins. On the basis of SDS-PAGE, Mg/NP-40—TCA/actone and TCA/actone precipitation were separated by two-dimensional electrophoresis (2-DE). The results show that spot distribution of TCA/actone precipitation was uniform, less vertical and horizontal stripes and clear background. The map of 2-DE showed that TCA/actone precipitation method is better than Mg/NP-40—TCA/actone combined method in the separation of small molecule proteins and alkaline protein. Protein spots were randomly selected for mass spectrometry identification. In general, TCA/actone precipitation method is more suitable for protein extraction, separation and mass spectrometry of hybrid Liriodendron embryonic callus.

Keywords: callus, protein extraction, SDS-PAGE, two-dimensional electrophoresis

目 录

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

1.1体细胞胚胎发生调控研究现状………………………………………………………………1

1.2鹅掌楸体胚发生的意义…………………………………………………………………………2

1.3 蛋白质组学研究…………………………………………………………………………………….3

1.4 蛋白质组学发展趋势………………………………………………………………………………...4

参考文献……………………………………………………………………………………………………..5

2正文………………………………………………………………………………7

2.1前言……………………………………………………………………7

2.2 材料与方法…………………………………………………………………………8

2.2.1实验材料……………………………………………………………………8

2.2.2仪器……………………………………………………………………………8

2.2.3相关试剂……………………………………………………………………………8

2.2.4愈伤蛋白质提取方法………………………………………………………………8

2.2.5蛋白质定量……………………………………………………………………………9

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