托品烷生物堿（tropane alkaloids,TAs）是一類從茄科特定的植物中提取的含氮小分子有機(jī)物,主要包括為莨菪堿、山莨菪堿、東莨菪堿等,其中,莨菪堿和東莨菪堿在在臨床上作為抗膽堿藥物廣泛用于鎮(zhèn)痛,麻醉、戒毒脫癮和帕金森病的治療。由于野生TAs藥源植物中,托品烷生物堿含量極低,TAs的市場(chǎng)供求矛盾日益突出。因此,利用分子生物技術(shù),通過(guò)引入TAs生物合成途徑關(guān)鍵酶基因或調(diào)節(jié)蛋白基因來(lái)打破特定的限速步驟,從而顯著增加靶向產(chǎn)物莨菪堿和東莨菪堿的含量,是目前TAs代謝工程領(lǐng)域的主要研究方法,而開(kāi)展對(duì)TAs生物合成途徑基因的功能鑒定是這一切的前提。顛茄是《中國(guó)藥典》收錄的重要的TAs商業(yè)藥源植物.目前對(duì)以顛茄主要研究對(duì)象的TAs生物合成途徑的解析取得了一定的進(jìn)展,然而,有一些步驟依然,尚未明確。目前的研究表明,TAs的生物合成起始于鳥(niǎo)氨酸和精氨酸的脫羧反應(yīng)。目前對(duì)腐胺生物合成的研究多集中于對(duì)ADC和ODC作用的評(píng)價(jià),而對(duì)于精氨酸酶的在TAs生物合成的的研究鮮見(jiàn)報(bào)道,這嚴(yán)重限制了我們對(duì)TAs生物合成機(jī)制的理解和對(duì)相關(guān)基因的利用,因此本文以顛茄為材料對(duì)精氨酸酶基因展現(xiàn)了相關(guān)的研究。相關(guān)研究結(jié)... 

【文章來(lái)源】：西南大學(xué)重慶市 211工程院校 教育部直屬院校

【文章頁(yè)數(shù)】：84 頁(yè)

【學(xué)位級(jí)別】：博士

【文章目錄】：
摘要
Abstract
CHAPTER 1 LITERATURE REVIEW
    1.1 Background
    1.2 Atropa belladonna
    1.3 Tropane alkaloids
    1.4 TA biosynthesis pathway in Atropa belladonna
    1.5 Genes and enzymes in the biosynthetic pathway of the TAs
    1.6 Plant transgenic technology
CHAPTER 2 INTRODUCTION
    2.1 Research purpose and significance
    2.2 Scope and content of research
    2.3 Outline of Methodology
CHAPTER 3 Gene Cloning, Purification, Enzymatic assay of Arginase
    3.1 Introduction
    3.2 Materials and Methods
        3.2.1 Plant material
        3.2.2 Strains and Plasmids
        3.2.3 Instruments and equipment
        3.2.4 Kits and reagents
        3.2.5 Antibiotic preparation and storage method
    3.3 General Methods and Procedures
        3.3.1 Extraction and Detection of Total RNA
        3.3.2 Agarose gel electrophoresis of DNA products
        3.3.3 Recovery of PCR Target Bands
        3.3.4 Reverse transcription of RNA
        3.3.5 connection reaction
        3.3.6 Preparation of E.coli competent cells
        3.3.7 Transformation of competent cells of E.coli
        3.3.8 Identification and Sequencing of Recombinant
        3.3.9 Extraction of Recombinant Plasmid
        3.3.10 Cloning and Analysis of ARG Gene from Atropa belladonna
        3.3.11 Bioinformatics Analysis of AbARG Gene
        3.3.12 Tissue profile analysis of AbARG
        3.3.13 Construction of Prokaryotic Expression Vector of AbARG Gene
        3.3.14 Time Gradient Induction of AbARG Recombinant Protein
        3.3.15 Induction of AbARG Recombinant Protein
        3.3.16 Purification of AbARG Recombinant Protein
            3.3.16.1 Ni column regeneration
            3.3.16.2 Disposal of dialysis bags and desalination of protein dialysis
            3.3.16.3 Protein dialysis desalination
        3.3.17 Functional Identification and Enzymatic Kinetics Analysis of AbARG Recombinant Protease Activity
    3.4 Results and Analysis
        3.4.1 Obtaining and Analysis of Full-length cDNA of AbARG Gene
        3.4.2 Multiple Alignment of Amino Acid Sequences of AbARG
        3.4.3 Phylogenetic Analysis of AbARG
        3.4.4 AbARG tissue expression profiles
        3.4.5 Construction of AbARG Gene Prokaryotic Expression Vectors
        3.4.6 A small amount of induced expression of recombinant AbARG protein
        3.4.7 Purification of Recombinant AbARG Proteins
        3.4.8 Analysis of Ornithine producing by TLC
        3.4.9 Enzymatic Kinetics Analysis of AbARG Recombinant Protein
CHAPTER 4 Study on the role of Arginase in the TAs Pathway of A.belladonna
    4.1 Introduction
    4.2 Material and methods
        4.2.1 Plant material
        4.2.2 Strains and plasmids
        4.2.3 Instruments and equipment
        4.2.4 Kits and reagents
        4.2.5 Antibiotic preparation and storage method
        4.2.6 Transformation of recombinant plasmid into Agrobacterium rhizogenes C58C1 competent cells
    4.3 Construction of RNAi silencing vector of A.belladonna and engineering bacteria obtaining
    4.4 AbARG gene overexpression vector construction
    4.5 Analysis the suppression of AbARG-RNAi expression patterns
    4.6 Analysis of overexpression AbARG expression patterns
    4.7 Agrobacterium rhizogenes C58C1 transforms A.belladonna with leaf disk method
    4.8 The establishment of A.belladonna root culture system
        4.8.1 Activation of engineering bacteria C58C1-pHELLSGATE-AbARG
        4.8.2 Activation of engineering bacteria C58C1-pBI121-AbARG
        4.8.3 PCR identification of transgenic belladonna root
        4.8.4 Extended culture of transgenic belladonna root
    4.9 Extraction and detection of tropane alkaloids
        4.9.1 Extraction of alkaloids from the root
        4.9.2 Extraction of alkaloids from medium
        4.9.3 HPLC detection of atropine alkaloids
    4.10 Result and analysis
        4.10.1 Construction of the interference vector pHELLSGATE12-AbARG-Ri
        4.10.2 Construction of the overexpression vector pBI121-AbARG
        4.10.3 Collection and analysis of hairy roots of transgenic AbARG
        4.10.4 Collection and analysis of hairy roots of transgenic lines
        4.10.5 Fluorescence Quantitative PCR Detection of Transgenic Hair Roots
        4.10.6 Determination of alkaloids in hairy roots of transgenic belladonna
Chapter 5 Discussion and Conclution
    5.1 Outlook
Supplementary
REFERANCE
ACKNOWLEDGEMENTS


【參考文獻(xiàn)】：
期刊論文
[1]對(duì)《中國(guó)藥典》2005年版一部含牡丹皮制劑質(zhì)量控制的探討[J]. 田友清,丁平,燕憲海,胡文江.  中國(guó)中藥雜志. 2008(03)



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