颠茄精氨酸酶基因的克隆与功能鉴定
发布时间：2021-03-05 21:12
托品烷生物碱（tropane alkaloids,TAs）是一类从茄科特定的植物中提取的含氮小分子有机物,主要包括为莨菪碱、山莨菪碱、东莨菪碱等,其中,莨菪碱和东莨菪碱在在临床上作为抗胆碱药物广泛用于镇痛,麻醉、戒毒脱瘾和帕金森病的治疗。由于野生TAs药源植物中,托品烷生物碱含量极低,TAs的市场供求矛盾日益突出。因此,利用分子生物技术,通过引入TAs生物合成途径关键酶基因或调节蛋白基因来打破特定的限速步骤,从而显著增加靶向产物莨菪碱和东莨菪碱的含量,是目前TAs代谢工程领域的主要研究方法,而开展对TAs生物合成途径基因的功能鉴定是这一切的前提。颠茄是《中国药典》收录的重要的TAs商业药源植物.目前对以颠茄主要研究对象的TAs生物合成途径的解析取得了一定的进展,然而,有一些步骤依然,尚未明确。目前的研究表明,TAs的生物合成起始于鸟氨酸和精氨酸的脱羧反应。目前对腐胺生物合成的研究多集中于对ADC和ODC作用的评价,而对于精氨酸酶的在TAs生物合成的的研究鲜见报道,这严重限制了我们对TAs生物合成机制的理解和对相关基因的利用,因此本文以颠茄为材料对精氨酸酶基因展现了相关的研究。相关研究结...
【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】：84 页
【学位级别】：博士
【文章目录】：
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
【参考文献】：
期刊论文
[1]对《中国药典》2005年版一部含牡丹皮制剂质量控制的探讨[J]. 田友清,丁平,燕宪海,胡文江. 中国中药杂志. 2008(03)
本文编号：3065872
【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】：84 页
【学位级别】：博士
【文章目录】：
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
【参考文献】：
期刊论文
[1]对《中国药典》2005年版一部含牡丹皮制剂质量控制的探讨[J]. 田友清,丁平,燕宪海,胡文江. 中国中药杂志. 2008(03)
本文编号：3065872
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