Cloning and Characterization of Two Novel Genes "BeSNAC1" an
发布时间：2020-12-20 21:54
NAC和WRKY转录因子(TFs)在环境胁迫中起着关键的作用,然而关于NAC和WRKY转录因子在竹子中的研究报道较少。慈竹是重要的经济竹种,其具有很强的生长能力,可以在各种生境中生长,在恶劣的环境条件下也能存活。本研究的主要目的是鉴定让慈竹抵抗非生物胁迫环境相关的转录因子,并将其转化到小麦中,研究其功能。本研究成功的从慈竹中克隆得到两个新的胁迫相关的转录因子BeSNAC1”和“BeWRKY2”,并对其进行生物信息学、组织表达、酵母单杂交和亚细胞定位分析。利用基因枪法对小麦“DN7742”的幼胚愈伤组织进行遗传转化,并获得转基因植株,为BeSNAC1”和“BeWRKY2”功能的深入研究奠定基础,也为抗性小麦品种改良提供理论依据。主要研究结果如下:1.通过使用生物信息学方法和来自相应植物的相应数据库的信息来分离基因。使用来自水稻的应激响应性SNAC1的氨基酸序列,通过同源克隆法克隆NAC TF。尽管为了分离WRKYTF,利用来自Phyllostachys杂环基因组数据库和Bambusa Emeiensis转录组数据库的信息。分离的TF分别命名为“BeSNAC1”和“BeWRKY2”。和“B...
【文章来源】：西南科技大学四川省
【文章页数】：146 页
【学位级别】：硕士
【文章目录】：
摘要
ABSTRACT
List of abbreviations
1 Introduction
1.1 Research significance
1.1.1 Wheat as a chief source of food
1.1.2 Wheat production and demand
1.2 Abiotic stresses
1.2.1 Drought
1.2.2 Salinity
1.3 Research Background
1.3.1 Transcription factors （TFs）
1.3.2 NAC Transcription factors
1.3.3 WRKY Transcription factors
1.4 Research objectives
1.5 Work layout
2 Cloning and bioinformatics analysis of BeSNACl and BeWRKY2 genesfrom Bambusa emeiensis
2.1 Testing materials,main instruments and reagents
2.1.1 Testing materials
2.1.2 Main instruments for testing
2.1.3 Main reagents and other materials for testing
2.1.4 Reagents and medium preparation
2.2 Testing Methods
2.2.1 Selection and cloning of genes
2.2.2 Total RNA extraction
2.2.3 cDNA synthesis via RT-PCR
2.2.4 PCR amplification of coding sequence of BeSNAC1 &BeWRKY2
2.2.5 Validation of amplicons by Gel electrophoresis
2.2.6 Purification of validated PCR amplicons
2.2.7 Restriction digestion
2.2.8 Vector and genes purification
2.2.9 Ligation of purified amplicons to pMD19-T vector
2.2.10 Transformation of recombinant plasmids intoEscherichia coli （DH5a）
2.2.11 Blue-white screening
2.2.12 PCR confirmation of target genes ligation
2.2.13 Sequencing of genes
2.2.14 Culture expansion and plasmids extraction
2.2.15 Enzymatic validation of successful ligation
2.3 Bioinformatics analysis of BeSNACl and BeWRKY2 genes
2.3.1 The conserved motif analysis of BeSNACl and BeWRKY2
2.3.2 Multiple protein sequence alignment of BeSNAC1 andBeWRKY2
2.3.3 The phylogenetic analysis of BeSNAC1 and BeWRKY2
2.3.4 The domain structure analysis of BeSNAC1 and BeWRKY2
2.4 Results and Analysis
2.4.1 Total RNA extraction
2.4.2 PCR Amplification of BeSNACl and BeWRKY2
2.4.3 The enzymatic validation of BeSNAC1 and BeWRKY2
2.4.4 Sequencing results analysis
2.4.5 Domain structure analysis of the BeSNAC1
2.4.6 Domain structure analysis of the BeWRKY2
2.4.7 Multiple protein Sequence alignment of BeSNAC1
2.4.8 Multiple protein Sequence alignment of BeWRKY2
2.4.9 Phylogenetic analysis of BeSNAC1
2.4.10 Phylogenetic analysis of BeWRKY2
2.4.11 The conserved domain structure of BeSNACl
2.4.12 The conserved domain structure of BeWRKY2
3 Yeast one-hybrid screening of BeSNAC1 and BeWRKY2
3.1 Testing materials, main instruments and reagents
3.1.1 Materials for testing
3.1.2 Main instruments for testing
3.1.3 Reagents and other materials
3.1.4 Main reagents preparation
3.2 Testing methods
3.2.1 PCR amplification of BeSNACl and BeWRKY2
3.2.2 Gel electrophoresis validation of PCR amplicons
3.2.3 Purification of validated PCR amplicons
3.2.4 Restriction digestion
3.2.5 Vector and genes purification
3.2.6 Ligation of genes with pEG202
3.2.7 Transformation of recombinant plasmids into Escherichiacoli （DH5α）
3.2.8 Escherichia coli （DH5a） culture
3.2.9 PCR confirmation of target gene ligation
3.2.10 Culture expansion and plasmids extraction
3.2.11 Enzymatic validation
3.2.12 Yeast EGY-48 Competent cells Preparation and Yeasttransformation
3.2.13 PCR validation of target genes
3.3 Results and Analysis
3.3.1 The PCR amplification of BeSNAC1 and Be WRKY2
3.3.2 Restriction digestion and ligation
3.3.3 Enzymatic validation
3.3.4 RCR validation of target genes in yeast
3.3.5 Transcriptional activity analysis of BeSNAC1 and BeWRKY2
4 Sub-Cellular localization analysis BeSNAC1 and BeWRKY2
4.1 Testing materials, main instruments and reagents
4.1.1 Materials for testing
4.1.2 Main instruments for testing
4.1.3 Main reagents and other materials
4.1.4 Main reagents and medium preparation
4.2 Testing methods
4.2.1 PCR amplification of BeSNAC1 and BeWRKY2
4.2.2 Gel electrophoresis validation of PCR amplicons
4.2.3 Purification of validated PCR amplicons
4.2.4 Restriction digestion
4.2.5 Vector and genes purification
4.2.6 Ligation of genes with pTEX-GFP
4.2.7 Insertion of the vector into Escherichia coli （DH5α）
4.2.8 Escherichia coli （DH5α） culture
4.2.9 PCR confirmation of target genes ligation
4.2.10 Culture expansion and plasmids extraction
4.2.11 Enzymatic validation
4.2.12 The onion epidermal cells preparation
4.2.13 Gold preparation
4.2.14 Coating plasmids with gold
4.2.15 Transformation using particles delivery system
4.2.16 Microscopic analysis
4.3 Results and Analysis
4.3.1 The gel electrophoresis validation of PCR amplicons
4.3.2 Restriction digestion and ligation
4.3.3 Enzymatic validation
4.3.4 Subcellular localization of BeSNACl and BeWRKY2
5 The expression pattern analysis of BeSNAC1 and BeWRKY2 of Bambusaemeiensis
5.1 Testing materials, main instruments and reagents
5.1.1 Testing materials
5.1.2 Main instruments for testing
5.1.3 Main reagents and other materials for testing
5.1.4 Reagents preparation
5.2 Testing methods
5.2.1 Plant materials and stress treatments
5.2.2 Total RNA extraction
5.2.3 cDNA synthesis via RT-PCR
5.2.4 The qRT-PCR primers
5.2.5 Real-time PCR Primers specificity detection
5.2.6 Expression pattern analysis of BeSNACl and BeWRKY2 byQuantitative Real-time PCR （qRT-PCR）
5.2.7 Statistical analysis
5.3 Results and Analysis
5.3.1 Gel electrophoresis analysis of Total RNA
5.3.2 The qRT-PCR primers specificity detection
5.3.3 Expression pattern analysis of BeSNACl via quantitativeReal-time PCR （qRT-PCR）
5.3.4 Expression pattern analysis of BeWRKY2 via quantitativeReal-time PCR （qRT-PCR）
6 Construction of overexpression vector, wheat transformation andvalidation of overexpressed plants
6.1 Testing materials, main instruments and reagents
6.1.1 Testing materials
6.1.2 Main instruments for testing
6.1.3 Main reagents and other materials for testing
6.1.4 Reagents and mediums preparation
6.2 Testing methods
6.2.1 Overexpression vector construction
6.2.2 Ex-plant preparation and Callus induction
6.2.3 Gold preparation
6.2.4 Coating overexpression vectors with gold
6.2.5 Osmotic treatment and transformation
6.2.6 Validation and overexpression analysis of target genes intransformed plants
6.3 Results and Analysis
6.3.1 The gel electrophoresis validation of PCR amplicons
6.3.2 Restriction digestion and ligation
6.3.3 Enzymatic validation
6.3.4 Callus induction and regeneration of seedlings bombardedwith BeSNACl
6.3.5 Callus induction and regeneration of seedlings bombardedwith BeWRKY2
6.3.6 Validation and overexpression analysis of BeSNACl intransformed plants
6.3.7 Validation and Overexpression analysis of BeWRKY2 intransformed plants
6.3.8 Ti generation of BeSNACl and BeWRKY2 overexpressedplants
7 Discussion and Conclusion
ACKNOWLEDGEMENT
DEDICATION
References
Academic papers and research findings
本文编号：2928615
【文章来源】：西南科技大学四川省
【文章页数】：146 页
【学位级别】：硕士
【文章目录】：
摘要
ABSTRACT
List of abbreviations
1 Introduction
1.1 Research significance
1.1.1 Wheat as a chief source of food
1.1.2 Wheat production and demand
1.2 Abiotic stresses
1.2.1 Drought
1.2.2 Salinity
1.3 Research Background
1.3.1 Transcription factors （TFs）
1.3.2 NAC Transcription factors
1.3.3 WRKY Transcription factors
1.4 Research objectives
1.5 Work layout
2 Cloning and bioinformatics analysis of BeSNACl and BeWRKY2 genesfrom Bambusa emeiensis
2.1 Testing materials,main instruments and reagents
2.1.1 Testing materials
2.1.2 Main instruments for testing
2.1.3 Main reagents and other materials for testing
2.1.4 Reagents and medium preparation
2.2 Testing Methods
2.2.1 Selection and cloning of genes
2.2.2 Total RNA extraction
2.2.3 cDNA synthesis via RT-PCR
2.2.4 PCR amplification of coding sequence of BeSNAC1 &BeWRKY2
2.2.5 Validation of amplicons by Gel electrophoresis
2.2.6 Purification of validated PCR amplicons
2.2.7 Restriction digestion
2.2.8 Vector and genes purification
2.2.9 Ligation of purified amplicons to pMD19-T vector
2.2.10 Transformation of recombinant plasmids intoEscherichia coli （DH5a）
2.2.11 Blue-white screening
2.2.12 PCR confirmation of target genes ligation
2.2.13 Sequencing of genes
2.2.14 Culture expansion and plasmids extraction
2.2.15 Enzymatic validation of successful ligation
2.3 Bioinformatics analysis of BeSNACl and BeWRKY2 genes
2.3.1 The conserved motif analysis of BeSNACl and BeWRKY2
2.3.2 Multiple protein sequence alignment of BeSNAC1 andBeWRKY2
2.3.3 The phylogenetic analysis of BeSNAC1 and BeWRKY2
2.3.4 The domain structure analysis of BeSNAC1 and BeWRKY2
2.4 Results and Analysis
2.4.1 Total RNA extraction
2.4.2 PCR Amplification of BeSNACl and BeWRKY2
2.4.3 The enzymatic validation of BeSNAC1 and BeWRKY2
2.4.4 Sequencing results analysis
2.4.5 Domain structure analysis of the BeSNAC1
2.4.6 Domain structure analysis of the BeWRKY2
2.4.7 Multiple protein Sequence alignment of BeSNAC1
2.4.8 Multiple protein Sequence alignment of BeWRKY2
2.4.9 Phylogenetic analysis of BeSNAC1
2.4.10 Phylogenetic analysis of BeWRKY2
2.4.11 The conserved domain structure of BeSNACl
2.4.12 The conserved domain structure of BeWRKY2
3 Yeast one-hybrid screening of BeSNAC1 and BeWRKY2
3.1 Testing materials, main instruments and reagents
3.1.1 Materials for testing
3.1.2 Main instruments for testing
3.1.3 Reagents and other materials
3.1.4 Main reagents preparation
3.2 Testing methods
3.2.1 PCR amplification of BeSNACl and BeWRKY2
3.2.2 Gel electrophoresis validation of PCR amplicons
3.2.3 Purification of validated PCR amplicons
3.2.4 Restriction digestion
3.2.5 Vector and genes purification
3.2.6 Ligation of genes with pEG202
3.2.7 Transformation of recombinant plasmids into Escherichiacoli （DH5α）
3.2.8 Escherichia coli （DH5a） culture
3.2.9 PCR confirmation of target gene ligation
3.2.10 Culture expansion and plasmids extraction
3.2.11 Enzymatic validation
3.2.12 Yeast EGY-48 Competent cells Preparation and Yeasttransformation
3.2.13 PCR validation of target genes
3.3 Results and Analysis
3.3.1 The PCR amplification of BeSNAC1 and Be WRKY2
3.3.2 Restriction digestion and ligation
3.3.3 Enzymatic validation
3.3.4 RCR validation of target genes in yeast
3.3.5 Transcriptional activity analysis of BeSNAC1 and BeWRKY2
4 Sub-Cellular localization analysis BeSNAC1 and BeWRKY2
4.1 Testing materials, main instruments and reagents
4.1.1 Materials for testing
4.1.2 Main instruments for testing
4.1.3 Main reagents and other materials
4.1.4 Main reagents and medium preparation
4.2 Testing methods
4.2.1 PCR amplification of BeSNAC1 and BeWRKY2
4.2.2 Gel electrophoresis validation of PCR amplicons
4.2.3 Purification of validated PCR amplicons
4.2.4 Restriction digestion
4.2.5 Vector and genes purification
4.2.6 Ligation of genes with pTEX-GFP
4.2.7 Insertion of the vector into Escherichia coli （DH5α）
4.2.8 Escherichia coli （DH5α） culture
4.2.9 PCR confirmation of target genes ligation
4.2.10 Culture expansion and plasmids extraction
4.2.11 Enzymatic validation
4.2.12 The onion epidermal cells preparation
4.2.13 Gold preparation
4.2.14 Coating plasmids with gold
4.2.15 Transformation using particles delivery system
4.2.16 Microscopic analysis
4.3 Results and Analysis
4.3.1 The gel electrophoresis validation of PCR amplicons
4.3.2 Restriction digestion and ligation
4.3.3 Enzymatic validation
4.3.4 Subcellular localization of BeSNACl and BeWRKY2
5 The expression pattern analysis of BeSNAC1 and BeWRKY2 of Bambusaemeiensis
5.1 Testing materials, main instruments and reagents
5.1.1 Testing materials
5.1.2 Main instruments for testing
5.1.3 Main reagents and other materials for testing
5.1.4 Reagents preparation
5.2 Testing methods
5.2.1 Plant materials and stress treatments
5.2.2 Total RNA extraction
5.2.3 cDNA synthesis via RT-PCR
5.2.4 The qRT-PCR primers
5.2.5 Real-time PCR Primers specificity detection
5.2.6 Expression pattern analysis of BeSNACl and BeWRKY2 byQuantitative Real-time PCR （qRT-PCR）
5.2.7 Statistical analysis
5.3 Results and Analysis
5.3.1 Gel electrophoresis analysis of Total RNA
5.3.2 The qRT-PCR primers specificity detection
5.3.3 Expression pattern analysis of BeSNACl via quantitativeReal-time PCR （qRT-PCR）
5.3.4 Expression pattern analysis of BeWRKY2 via quantitativeReal-time PCR （qRT-PCR）
6 Construction of overexpression vector, wheat transformation andvalidation of overexpressed plants
6.1 Testing materials, main instruments and reagents
6.1.1 Testing materials
6.1.2 Main instruments for testing
6.1.3 Main reagents and other materials for testing
6.1.4 Reagents and mediums preparation
6.2 Testing methods
6.2.1 Overexpression vector construction
6.2.2 Ex-plant preparation and Callus induction
6.2.3 Gold preparation
6.2.4 Coating overexpression vectors with gold
6.2.5 Osmotic treatment and transformation
6.2.6 Validation and overexpression analysis of target genes intransformed plants
6.3 Results and Analysis
6.3.1 The gel electrophoresis validation of PCR amplicons
6.3.2 Restriction digestion and ligation
6.3.3 Enzymatic validation
6.3.4 Callus induction and regeneration of seedlings bombardedwith BeSNACl
6.3.5 Callus induction and regeneration of seedlings bombardedwith BeWRKY2
6.3.6 Validation and overexpression analysis of BeSNACl intransformed plants
6.3.7 Validation and Overexpression analysis of BeWRKY2 intransformed plants
6.3.8 Ti generation of BeSNACl and BeWRKY2 overexpressedplants
7 Discussion and Conclusion
ACKNOWLEDGEMENT
DEDICATION
References
Academic papers and research findings
本文编号：2928615
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