颠茄N-甲基腐胺氧化酶基因的克隆与功能研究
发布时间：2021-08-04 12:19
托品烷生物碱（tropane alkaloids,TAs）是医学上最古老的传统药物之一,已经有近3000年的使用历史。TAs主要包括莨菪碱和东莨菪碱,这两种生物碱均可以作为抗胆碱药物在临床上广泛应用于止痛、麻醉、戒毒脱瘾、抗晕动和治疗帕金森病等。药用TAs的生产完全依赖于与从茄科特定的药源植物中提取获得,这些药源植物主要有:颠茄（Atropa belladonna）、曼陀罗（Datura stramonium）和澳洲毒茄杂交种（Duboisia hybrid）等。在TAs药源植物中,托品烷生物碱含量极低,无法满足市场需求。如在颠茄中,莨菪碱仅为叶片干重0.02%-0.17%,东莨菪碱仅为叶片干重0.01%-0.08%。通过生物技术手段提高药源植物中TAs的含量,是目前解决TAs供求矛盾最为有效的手段,也是相关行业一致追求的目标。开展TAs生物合成途径的分子生物学研究是采用生物技术手段改造TAs生物合成途径的前提。颠茄是《中国药典》收录的TAs药源植物。近年来,随着对托品烷生物碱生物合成途径研究的不断深入,对颠茄等TAs药源植物中TAs生物合成途径中的许多相关基因进行了分离和鉴定,使通过...
【文章来源】：西南大学重庆市 211工程院校 教育部直属院校
【文章页数】：85 页
【学位级别】：博士
【文章目录】：
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
【参考文献】：
期刊论文
[1]转NtPMT和HnH6H转变莨菪碱型颠茄为东莨菪碱型颠茄[J]. 权红,夏科,曾俊岚,陈敏,兰小中,廖志华. 药学学报. 2016(12)
[2]颠茄托品烷生物碱合成途径基因表达分析与生物碱积累研究[J]. 强玮,王亚雄,张巧卓,李金弟,夏科,吴能表,廖志华. 中国中药杂志. 2014(01)
本文编号：3321685
【文章来源】：西南大学重庆市 211工程院校 教育部直属院校
【文章页数】：85 页
【学位级别】：博士
【文章目录】：
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
【参考文献】：
期刊论文
[1]转NtPMT和HnH6H转变莨菪碱型颠茄为东莨菪碱型颠茄[J]. 权红,夏科,曾俊岚,陈敏,兰小中,廖志华. 药学学报. 2016(12)
[2]颠茄托品烷生物碱合成途径基因表达分析与生物碱积累研究[J]. 强玮,王亚雄,张巧卓,李金弟,夏科,吴能表,廖志华. 中国中药杂志. 2014(01)
本文编号：3321685
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