托品烷生物堿（tropane alkaloids,TAs）是醫(yī)學上最古老的傳統(tǒng)藥物之一,已經有近3000年的使用歷史。TAs主要包括莨菪堿和東莨菪堿,這兩種生物堿均可以作為抗膽堿藥物在臨床上廣泛應用于止痛、麻醉、戒毒脫癮、抗暈動和治療帕金森病等。藥用TAs的生產完全依賴于與從茄科特定的藥源植物中提取獲得,這些藥源植物主要有:顛茄（Atropa belladonna）、曼陀羅（Datura stramonium）和澳洲毒茄雜交種（Duboisia hybrid）等。在TAs藥源植物中,托品烷生物堿含量極低,無法滿足市場需求。如在顛茄中,莨菪堿僅為葉片干重0.02%-0.17%,東莨菪堿僅為葉片干重0.01%-0.08%。通過生物技術手段提高藥源植物中TAs的含量,是目前解決TAs供求矛盾最為有效的手段,也是相關行業(yè)一致追求的目標。開展TAs生物合成途徑的分子生物學研究是采用生物技術手段改造TAs生物合成途徑的前提。顛茄是《中國藥典》收錄的TAs藥源植物。近年來,隨著對托品烷生物堿生物合成途徑研究的不斷深入,對顛茄等TAs藥源植物中TAs生物合成途徑中的許多相關基因進行了分離和鑒定,使通過... 

【文章來源】：西南大學重慶市 211工程院校 教育部直屬院校

【文章頁數(shù)】：85 頁

【學位級別】：博士

【文章目錄】：
摘要
Abstract
CHAPTER 1 LITERATURE REVIEW
    1.1 Atropa belladonna
    1.2 Alkaloids
    1.3 Tropane alkaloids
    1.4 Biosynthetic pathway network of TAs and related genes
        1.4.1 TAs and related metabolites: biosynthetic pathway
        1.4.2 Enzymes and genes in the TA biosynthetic pathway
CHAPTER 2 INTRODUCTION
    2.1 Research purpose and significance
    2.2 Scope and contents of this research
    2.3 Technical route
CHAPTER 3 MATERIALS AND METHODS
    3.1 Experimental materials and reagent consumables
        3.1.1 Plant materials
        3.1.2 Strains and plasmids
        3.1.3 Instruments and equipment
        3.1.4 Kits and reagents
        3.1.5 Preparation of conventional reagents and culture medium
        3.1.6 Antibiotic preparation and storage method
    3.2. General method and procedures
        3.2.1 RNA extraction and determination
        3.2.2 Reverse transcription of RNA
        3.2.3 Agarose gel electrophoresis of DNA products
        3.2.4 Recovery of the target strip
        3.2.5 Ligation
        3.2.6 Preparation of DH5a competent cells
        3.2.7 Transformation of products or recombinant plasmids into DH5a competent cells
        3.2.8 Transformation of recombinant plasmids into Agrobacterium rhizobium C58C1 competent cells
        3.2.9 Extraction of plasmids
        3.2.10 TPS extraction of A. belladonna genomic DNA (g DNA)
    3.3 Cloning and analysis of Ab MPO gene
        3.3.1 Extraction and detection of total RNA of A. belladonna
        3.3.2 Synthesis of the first c DNA
        3.3.3 Cloning of target gene
        3.3.4 Acquisition of the Ab MPO gene by 5'RACE
        3.3.5 Cloning of the full-length c DNA of the Ab MPO gene
    3.4 Bioinformatics analysis of Ab MPO gene
    3.5 Construction of RNAi silencing vector of A. belladonna and obtaining engineered bacteria
    3.6 Establishment of root culture system
        3.6.1 Activation of engineered bacteria
        3.6.2 Transformation of A. belladonna explants by engineered Agrobacterium strains
        3.6.3 PCR identification of transgenic A. belladonna roots
        3.6.4 Expanded culture of transgenic A. belladonna root
        3.6.5 Induction of hairy roots of A. belladonna by A. rhizobium C58C1
    3.7 Gene expression analysis
        3.7.1 Fluorescence quantitative PCR
        3.7.2 Analysis of Ab MPO gene expression in transgenic hairy roots
    3.8 Determination of alkaloid content in A. belladonna roots
Chapter 4 RESULTS
    4.1 Cloning of the Ab MPO genes from A. belladonna
    4.2 Bioinformatics analysis of the Ab MPO gene
        4.2.1 Multiple alignment of Ab MPO1 and Ab MPO2 amino acid sequences
        4.2.2 Reconstruction of the Ab MPO molecular phylogenetic tree
    4.3 Tissue profiles of Ab MPO1 and Ab MPO2
    4.4 Construction of interference vectors p BIN19-Ab MPO1-Ri and p BIN19-Ab MPO2-Ri and transfer into C58C1
        4.4.1 Construction of interference vector p BIN19-Ab MPO1-Ri and p BIN19-Ab MPO2-Ri
        4.4.2 Obtaining engineered bacteria
    4.5 Suppression of Ab MPO1/Ab MPO2 in root cultures of A. belladonna
        4.5.1 Fluorescence q PCR detection of transgenic hair roots
        4.5.2 Determination of alkaloids in hairy roots of transgenic A. belladonna
Chapter 5 DISCUSSION
Supplementary
REFERENCES
Acknowledgements


【參考文獻】：
期刊論文
[1]轉NtPMT和HnH6H轉變莨菪堿型顛茄為東莨菪堿型顛茄[J]. 權紅,夏科,曾俊嵐,陳敏,蘭小中,廖志華.  藥學學報. 2016(12)
[2]顛茄托品烷生物堿合成途徑基因表達分析與生物堿積累研究[J]. 強瑋,王亞雄,張巧卓,李金弟,夏科,吳能表,廖志華.  中國中藥雜志. 2014(01)



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