Identification and Characterization of SisLOG Homologue from

发布时间：2023-03-19 02:54

　　

【文章页数】：134 页

【学位级别】：博士

【文章目录】：
Abstract
Abbreviations
1 Introduction
    1.1 Archaea: discovery, diversity and significance
        1.1.1 Characteristics of domain archaea
        1.1.2 Description and classification of Sulfolobus
        1.1.3 Implications of Sulfolobus as hosts of extrachromosomal genetic elements
    1.2 Archaeal DNA replication, repair, and recombination
        1.2.1 DNA replication origins in archaea
        1.2.2 Types of the DNA lesions
        1.2.3 Archaeal DNA repair pathways
        1.2.4 DNA processing protein A and their functions in prokaryotes
        1.2.5 Archaea and biotechnology
    1.3 Importance of LOG enzyme in plants and microorganisms
    1.4 Cytokinins
        1.4.1 Cytokinin biosynthesis in plants and bacteria
        1.4.2 Cytokinin signaling transduction pathway in plants and bacteria
        1.4.3 Implication of microbial derived cytokinins as virulence factor in plants
        1.4.4 Uncertainty in the role of human pathogen derived cytokinins
    1.5 Aim of this study
2 Bioinformatics analysis of SiRe₀427
    2.1 Materials and Methods
        2.1.1 Sequence alignment and databases
    2.2 Results and discussion
        2.2.1 Presence of conserved LOG motif within SiRe₀427 sequences
        2.2.2 Structural comparison of SiRe₀427 with known LOGs from bacteria
        2.2.3 Phylogenetic tree analysis
3 Biochemical analysis of SiRe₀427
    3.1 Materials and Methods
        3.1.1 Strains, plasmids and culture conditions
        3.1.2 Isolation of S. islandicus genomic DNA
        3.1.3 Purification of DNA by gel extraction
        3.1.4 Cloning of the gene of SiRe₀427
        3.1.5 Construction of the plasmids for protein purification in E. coli
        3.1.6 Plasmid extraction by the Plasmid Mini Kit
        3.1.7 Expression of wild type SiRe₀427 and mutant proteins in E. coli
        3.1.8 SDS-PAGE and native PAGE assays
        3.1.9 Detection of his-tagged wild type and mutant proteins by western blotting
        3.1.10 Construction of overexpression and complementation strains
        3.1.11 DNA binding assay
        3.1.12 In vitro pull-down assay
        3.1.13 Chemical cross-linking assay
        3.1.14 Phosphoribohydrolase activity assay
        3.1.15 SisLOG activity as a function of concentration
        3.1.16 Determination of the optimal temperature
    3.2 Results and discussion
        3.2.1 Construction of expression plasmid and protein purification
        3.2.2 Determination of oligomeric status of SiRe₀427 by size exclusion chromatography
        3.2.3 Determination of the oligomeric status of SiRe₀427 by chemical cross-linking and nativePAGE analyses
        3.2.4 SiRe₀427 is not a DNA binding protein
        3.2.5 SiRe₀427 hypothetical protein is not DprA protein
        3.2.6 The hypothetical SiRe₀427 protein is a LOG homologue protein
        3.2.7 SiRe₀427 has no hydrolysis activity on cAMP
4 Genetic analysis of SiRe₀427
    4.1 Materials and methods
        4.1.1 SiRe₀427 gene knockout strategy
        4.1.2 Construction of gene knockout plasmid
        4.1.3 Electro-transformation and generation of pMID-SiRe₀427-T
        4.1.4 Genotype verification of SiRe₀427 mutant strain
        4.1.5 Construction of SiRe₀427 overexpression and complementation strains
        4.1.6 Determination of the growth curves
        4.1.7 Microscopy observation
        4.1.8 Sensitivity of △sire₀427 mutant strain to UV irradiation
    4.2 Results and discussion
        4.2.1 Analysis of S. islandicus pMID-SiRe₀427 gene knockout strain
        4.2.2 The sire₀427 gene is dispensable
        4.2.3 Phenotype analysis of the deletion mutant
        4.2.4 Cells morphology analysis
        4.2.5 Analysis of the sensitivity of the mutant strain△sire-0427to DNA damaging agent
5 Discussion and perspectives
    5.1 Evolution of cytokinins
    5.2 Lonely Guy(LOG) protein homologue in archaea
    5.3 Summary
Appendix Ⅰ-Reagents
Appendix Ⅱ-Instruments
Appendix Ⅲ-Media
Appendix Ⅳ- Solutions
Appendix Ⅵ- Systems for PCR, double enzyme digestion and ligation
Appendix Ⅶ SOE PCR
Appendix Ⅷ-Gel extraction kit for DNA from agarose gel
Appendix Ⅸ-Plasmid mini kit for plasmid extraction
Appendix Ⅹ-Transformation of Sulfolobus islondicus cells
Appendix Ⅺ-TABLE 1 Oligonucleotides used in this study
Appendix Ⅻ TABLE 2 Strains and plasmids used in this study
References
Acknowledgement
List of published papers
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