Sulfiredoxin（Srx）作為氧化還原酶家族的成員,在有能量（ATP）存在以及有有機硫化合物并以S-RH形式存在的情況下,特別是當(dāng)有機硫化合物為硫醇化合物（Cys-SH）時,能夠催化過氧化酶的半胱亞磺酸殘基向半胱氨酸殘基的轉(zhuǎn)化。Srx通過重新活化過氧化物酶而參與抗氧化物的代謝。當(dāng)peroxiredoxin（Prx）被過氧化抑制時,Srx通過重新還原次磺酸重新激活Prx參與抗氧化代謝。由于有ATP以及Srx存在,半胱氨酸（二硫化物）的這些較低的氧化態(tài)和亞磺酸（Cys-SOOH）等較高的氧化態(tài)都一樣易于逆轉(zhuǎn)。然而,人們對Srx在植物氧化應(yīng)激狀態(tài)中的作用了解甚少。本課題的目的是獲得重組表達(dá)的擬南芥（A.thaliana）的硫氧還蛋白Srx,進(jìn)行蛋白晶體生長條件篩選和優(yōu)化,為其結(jié)構(gòu)解析和活性功能分析打下基礎(chǔ)。本課題將Srx基因克隆到表達(dá)載體pET-22b（+）和pET-28a（+）中,并在大腸桿菌BL21（DE3）表達(dá)系統(tǒng)中進(jìn)行表達(dá)。采用鎳親和層析和凝膠過濾對目的蛋白進(jìn)行純化,用SDS-PAGE鑒定,獲得了純度較高和狀態(tài)均一的蛋白。通過晶體生長條件的篩選,在0.8 M NaH_... 

【文章頁數(shù)】：35 頁

【學(xué)位級別】：碩士

【文章目錄】：
摘要
abstract
Chapter 1 Introduction
    1.1 Oxidative stress in plants:Case of Arabidopsis thaliana
        1.1.1 Arabidopsis thaliana:Overview
        1.1.2 Oxidative stress in plants:Arabidopsis thaliana
        1.1.3 Reactive oxygen species(ROS)and their role in oxidative stress in A.thaliana
    1.2 Sulfiredoxin
        1.2.1 Structure of Sulfiredoxin
        1.2.2 Functions of Sulfiredoxin in A.thaliana
        1.2.3 Mutational variation of sulfiredoxin and their functions
Chapter 2 Materials and Methods
    2.1 Experimental materials
        2.1.1 Experimental strains and vectors
        2.1.2 Main reagents in the experiment
        2.1.3 Buffer and reagent preparation
        2.1.4 Laboratory instruments and equipment
    2.2 Clone construction
        2.2.1 Amplification of target fragment by PCR
        2.2.2 Double digestion of PCR products and vectors
        2.2.3 Ligation reaction
        2.2.4 Transformation of E.coli competent cells
        2.2.5 Recombination verification test
    2.3 Protein expression and purification
        2.3.1 Protein expression test
        2.3.2 Small-scale protein expression
        2.3.3 Large-scale protein
        2.3.4 Purification of protein by affinity chromatography
        2.3.5 Purification of protein by gel filtration chromatography
    2.4 Crystal Screen
Chapter 3 Results
    3.1 Clone construction
    3.2 Recombined protein expression and purification
    3.3 Crystal growth conditions of Srx
Chapter 4 Conclusion,discussion and prospect
    4.1 Experimental conclusion
    4.2 Discussion of recombinant protein Srx expressed on different vectors
    4.3 Discussion on the interaction between Srx and Prx
    4.4 Prospect of sulfiredoxin
References
Appendix
Acknowledgements


【參考文獻(xiàn)】：
期刊論文
[1]Molecular and functional characterization of sulfiredoxin homologs from higher plants[J]. Xian Peng Liu~(1,2) Xue Ying Liu~(1,2) Juan Zhang~(1,2) Zong Liang Xia~(1,2) Xin Liu~1 Huan Ju Qin~1 Dao Wen Wang~11 The State Key Laboratory of Plant Cell and Chromosome Engineering,Institute of Genetics and Developmental Biology,Chinese Academy of Sciences,Datun Road,Chaoyang District,Beijing 100101,China;2 Graduate University of Chinese Academy of Sciences,Yuquan Road,Shijingshan District,Beijing 100039,China.  Cell Research. 2006(03)



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