背景： 細(xì)菌在遇到環(huán)境變化,尤其是在應(yīng)激條件下,其非編碼RNA在蛋白表達(dá)重編程過程中起到關(guān)鍵作用。這些非編碼RNA參與細(xì)菌的各種胞內(nèi)過程,包括耐酸、鐵代謝、群體感應(yīng)、鎂和鈣離子運(yùn)輸、毒力、ABC轉(zhuǎn)運(yùn)系統(tǒng),外膜蛋白合成的抑制及轉(zhuǎn)錄因子的表達(dá)調(diào)控。在已知的非編碼RNA中,對(duì)反式編碼RNA的研究最為廣泛。在細(xì)菌基因組內(nèi),雖然順式編碼的反式轉(zhuǎn)錄已被證明是廣泛存在的,但它并未引起較多關(guān)注。這些反義RNA的合成、生理作用和作用機(jī)制至今仍是未知的。 目的： 細(xì)菌的復(fù)制體是由大量的酶組成的,這些酶相互間的協(xié)調(diào)作用得以完成染色體復(fù)制。通過對(duì)傷寒沙門菌轉(zhuǎn)錄組的高通量測(cè)序分析,發(fā)現(xiàn)一系列非編碼RNA,其基因位于與細(xì)菌復(fù)制過程有關(guān)基因的互補(bǔ)鏈。本論文旨在研究這些反義RNA在細(xì)菌復(fù)制過程中的調(diào)節(jié)作用。 方法： 1.反義RNA轉(zhuǎn)錄起始及終止位點(diǎn)的確定：利用5’和3’RACE (末端快速擴(kuò)增技術(shù))確定各個(gè)反義RNA的轉(zhuǎn)錄起始及終止位點(diǎn)。RACE分析技術(shù)和Northern blot用以確定反義RNA的全長(zhǎng)。 2.菌株構(gòu)建：構(gòu)建了相關(guān)反義RNA的高表達(dá)菌株,其帶有可被阿拉伯糖誘導(dǎo)的啟動(dòng)子。將反義RNA的基因全長(zhǎng)片段通過...

【文章頁(yè)數(shù)】：105 頁(yè)

【學(xué)位級(jí)別】：博士

【文章目錄】：
ABSTRACT
摘要
ABBREVIATIONS
CHAPTER 1 Introduction
    1.1 Background
    1.2 Salmonella
    1.3 Bacterial non-coding RNAs
    1.4 Bacterial antisense RNAs
        1.4.1 Characteristics of cis-encoded antisense RNAs
        1.4.2 Mechanisms of action of cis-encoded antisense RNAs in bacteria
        1.4.3 Methods to find asRNAs
    1.5 DNA Replication in Bacteria
        1.5.1 Replication origin architecture
        1.5.2 Overview of bacterial replication initiation
        1.5.3 Elongation of DNA
        1.5.4 Termination of DNA replication
    1.6 Objectives
    1.7 Relevance of the study
    1.8 Experimental design
    1.9 References
CHAPTER 2 An antisense RNA AsdA increases the mRNA stability of the gene encoding the replicationinitiation protein of S. Typhi
    2.1 Introduction
    2.2 Materials and Methods
        2.2.1 Bacterial strains,plasmids,and growth conditions
        2.2.2 Construction of strains overexpressing the asRNAs
        2.2.3 Construction of strains overexpressing the asRNAs in RNase Ⅲ and RNase E mutants
        2.2.4 Construction of fur mutant
        2.2.5 5'-RACE
        2.2.6 3'-RACE
        2.2.7 Growth kinetic analysis
        2.2.8 Expression analysis of asRNA under stress conditions
        2.2.9 Overexpression analysis
        2.2.10 RNA extraction
        2.2.11 Quantitative RT-PCR
        2.2.12 Northern blot analysis
        2.2.13 Growth curves
    2.3 Results
        2.3.1 Identification and mapping of the 5' and 3' ends of AsdA
        2.3.2 Analysis of AsdA expression under different growth conditions
        2.3.3 Strain constructions
        2.3.4 Effect of overexpression of asdA on dnaA mRNA level
        2.3.5 Effect of overexpression of asdA on the growth of S. Typhi
    2.4 Discussion
    2.5 References
CHAPTER 3 Identification and characterization of a cis antisense RNA of parC gene encoding DNAtopoisomerase Ⅳ of S. Typhi
    3.1 Introduction
    3.2 Materials and Methods
        3.2.1 Bacterial strains and culture conditions
        3.2.2 Strain and plasmid construction
        3.2.3 5'-and 3'-RACE
        3.2.4 RNA extraction
        3.2.5 Northern blot analysis
        3.2.6 Quantitative RT-PCR
        3.2.7 Growth curves
        3.2.8 Motility assay
    3.3 Results
        3.3.1 Identification of antisense RNA complementary to parC mRNA
        3.3.2 Expression of AspC under different growth conditions
        3.3.3 Strain constructions
        3.3.4 Effect of overexpression of AspC on parC mRNA level
        3.3.5 Effect of overexpression of AspC on the growth of S. Typhi
    3.4 Discussion
    3.5 References
GENERAL CONCLUSIONS
ACKNOWLEDGEMENTS
LIST OF PUBLICATIONS
APPENDICES



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