陆地棉基因GhLDOX和Gh4CL3的功能解析
发布时间：2021-05-09 02:16
花青素是一类参与多种生物过程的植物黄酮类色素。LDOX是花青素生物合成途径中的一个下游基因,它能够编码白花青素并将其转化为花青素。迄今为止,还没有研究阐明LDOX同源基因在棉花花青素合成及生长发育中的作用。本研究通过CRISPR-Cas9系统对棉花GhLDOX基因进行精准编辑创造靶向突变体,并通过超表达技术提高GhLDOX基因的表达量,从而对棉花GhLDOX基因进行功能鉴定。结果表明,无论是在营养组织中还是在花中,CRldox基因突变体植株都完全不含花青素,而OELDOX基因高表达植株则表现出总花青素的增加。且花青素含量的变化与GhLDOX基因表达量呈成正相关。此外,为了揭示花青素在棉花毛状体发育中的作用,我们对转GhLDOX基因的棉花植株进行了叶片表皮毛和种子纤维的检测。发现花色素苷的产生与叶片表皮毛及纤维的起始和伸长呈显著正相关。CRldox突变体中花青素的缺失叶片表皮毛和种子纤维的起始和伸长的显著,而OELDOX高表达株系花青素含量增加,叶片和纤维长度增加。通过对GhLDOX基因敲除的植株叶片进行转录组测序,结果表明参与黄酮类化合物生物合成途径的基因表达差异明显,其中大部分基因在...
【文章来源】：华中农业大学湖北省 211工程院校 教育部直属院校
【文章页数】：167 页
【学位级别】：博士
【文章目录】：
List of abbreviations
Chapter 1 The leucoanthocyanidins dioxygenase (GhLDOX) gene regulates the formation & elongation of cotton trichomes and fiber via modulating auxin signalingpathway
摘要
ABSTRACT
1.1 INTRODUCTION AND LITERATURE REVIEW
1.1.1 Cotton as an Economical Crop
1.1.2 Plant Trichomes are Unique Differentiated Cells
1.1.2.1 Transcriptional Regulation Underlying Leaf-Trichomes Development
1.1.2.2 Associated Hormonal Mechanisms Underlying Trichome Development in Arabidopsis
1.1.3 Cotton Fiber Development
1.1.3.1 Cotton Fiber Transcriptional Regulation
1.1.3.2 Role of Hormones in Regulating Cotton Fiber Development
1.1.4 Flavonoids
1.1.4.1 The importance of flavonoids to plants
1.1.4.2 Flavonoid’s defensive role in cotton
1.1.4.3 Flavonoids are Responsible of Cotton Leaf Reddening
1.1.4.4 Flavonoids Function in Cotton Fiber Development
1.1.5 Anthocyanin Synthesis
1.1.5.1 Ldox: A Late Gene in Anthocyanin Biosynthetic Pathway
1.1.5.2 Role of Anthocyanins Coupled With Phytohormones in Cotton Fiber Development
1.1.5.3 Anthocyanins and Trichomes
1.1.5.4 Sugar/Light- Mediated Anthocyanin Accumulation
1.1.5.5 Anthocyanin Is a Buffer Zone to Protect Chlorophyll
1.1.6 CRISPR-Cas9: a promising tool for plant functional genomics
1.1.7 Aim of Study
1.2 Materials and methods
1.2.1 Construction of phylogenetic tree and digital expression profiling for Gh LDOX
1.2.2 Specific spacer sequences selection and sg RNA designing
1.2.3 Construction of RNA-guided CRISPR-Cas9 genome-editing vector
1.2.4 Construction of Gh LDOX overexpression vector
1.2.5 Plant material for Agrobacterium-mediated transformation in cotton
1.2.6 Initiation, Maintenance of Embryogenic Calli and Plant Regeneration
1.2.7 Plant Material and Growth Conditions
1.2.8 DNA Extraction
1.2.9 Hi- Tom Screening and Detection of On-Target Mutations Induced by CRISPR/Cas9 System
1.2.10 RNA Extraction, RT-PCR and q RT-PCR
1.2.11 Subcellular Localization of The GhLDOX Gene
1.2.12 RNA-Seq Analysis and Identification of DEGs
1.2.13 Total Anthocyanin Extraction and Quantitation
1.2.14 Fiber Quality Measurement of Transgenic Plants
1.2.15 In Vitro Ovule Culture Assay
1.2.16 Scanning Electron Microscopy (SEM)
1.2.17 Chemical Quantification of Plant Hormones
1.2.18 Measurement of Total Chlorophyll
1.2.19 Assays of total soluble sugars: glucose, fructose & sucrose
1.2.20 Gossypol Extraction and Quantification
1.2.21 Insect Bioassay
1.3 RESULTS
1.3.1 Characterization of Gh LDOX gene
1.3.1.1 Phylogenetic analysis of cotton LDOX genes
1.3.1.2 Expression profile analysis of Gh LDOX gene in cotton
1.3.2 Generation of Gh LDOX transgenic plants
1.3.3 Subcellular localization of Gh LDOX gene
1.3.4 Suppression of Gh LDOX leads to free anthocyanin plants
1.3.5 Gh LDOX affects plant trichomes development in cotton
1.3.6 Gene knock out of Gh LDOX significantly disrupted cotton fiber initiation and elongation
1.3.7 Transcriptome sequencing analysis
1.3.7.1 Identification and functional analysis of DEGs
1.3.8 Altering the expression of GhLDOX gene modulated the expression of auxin genes pathway
1.3.9 Suppression of GhLDOX Downregulates Genes Involved in Chlorophyll Metabolic Pathway
1.3.10 The impact of the molecular and biochemical basis of Gh LDOX expression on soluble sugars accumulation in cotton plants
1.3.11 Differential Gene Expression of Key Genes in Flavonoids Biosynthetic Pathway of GhLDOXTransgenic Plants
1.3.12 Insect bioassay for GhLDOX transgenic lines
1.4 DISCUSSION
1.4.1 Anthocyanin Regulates Cotton Trichomes Initiation and Elongation Through Modulating AuxinPathway
1.4.2 GhLDOX Anthocyaninless Mutant Inversely Modulates Nitrate and Carbon Metabolism
1.4.3 Anthocyanin and Secondary Metabolites Toxicity Enhanced Resistance to Cotton Bollworm
Chapter 2 Characterization and functional analysis of the 4-coumarate:coenzyme A ligase 3 (Gh4CL3)improved cotton resistance against biotic stresses through lignin-mediated enhanced-defense mechanism
摘要
ABSTRACT
2.1 Introduction and Literature Review
2.1.1 Cotton (Gossypium hirsutum) pathogens and pests
2.1.1.1 Plant pathogens
2.1.1.2 Plant feeding-pests in cotton
2.1.2 Plants Defense Mechanisms
2.1.2.1 Constitutive (pre-existing) Defense Mechanisms
2.1.2.2 Induced defense mechanisms
2.1.3 Primary vs secondary metabolites related to plant defense responses
2.1.3.1 Lignin and phenylpropanoid biosynthetic pathways
2.1.3.2 Chemical structure of lignin: as a primary compound
2.1.3.3 Role of lignification in plant defense
2.1.3.4 Role of Flavonoids in plant defense
2.1.3.5 Effect of phenolic content and lignin modifications on resistance to pathogens
2.1.4 Plant 4-coumarate: coenzyme A ligase (4CL)
2.1.4.1 Roles of 4CL in plant development and response to environmental stresses
2.1.5 Study objectives
2.2 Materials and methods
2.2.1 Isolation and sequence retrieval of 4CL gene family in four cotton species
2.2.2 Gene Structure and Conserved Motifs Analyses
2.2.3 Construction of phylogenetic tree and digital expression profiling for Gh4CL3
2.2.4 Specific spacer sequences selection and sg RNA designing
2.2.5 Construction of RNA-guided CRISPR-Cas9 genome-editing vector
2.2.6 Construction of Gh4CL3 overexpression vector
2.2.7 Plant material for Agrobacterium-mediated transformation in cotton
2.2.8 Initiation, maintenance of embryogenic calli and plant regeneration
2.2.9 Plant Material and Growth Conditions
2.2.10 DNA extraction
2.2.11 Hi- Tom Screening and Detection of On-Target Mutations Induced by CRISPR/Cas9 System
2.2.12 RNA extraction, RT-PCR, and qRT-PCR
2.2.13 Subcellular localization of the Gh4CL3 gene
2.2.14 Verticillium dahliae inoculation
2.2.15 Lignin histochemical test
2.2.16 Determination of total lignin content
2.2.17 Insect bioassay
2.2.18 Chemical quantification of plant hormones
2.3 RESULTS
2.3.1 Characterization of 4CL gene family in cotton
2.3.1.1 Identification of 4CL Gene Family Based on Wide Genome Analysis in The GenusGossypium Spp
2.3.1.2 Gene structure and conserved motifs analyses of Gh4CL
2.3.1.3 Phylogenetic Analysis of cotton 4CL genes
2.3.2 Generation of Gh4CL3 transgenic cotton lines
2.3.3 Subcellular localization of Gh4CL3 gene
2.3.4 Overexpression or down-regulation of Gh4CL3 altered plant resistance to V.dahliae
2.3.5 Altering Gh4CL3 gene expression in cotton plants modulated lignin content
2.3.6 Suppression of Gh4CL3 gene conferred susceptibility to cotton bollworm
2.3.7 Lignin reduction triggers salicylic acid accumulation in CR4CL3 plants
2.3.8 Altering The Expression of Gh4CL3 Gene Modulates Gene Expression of Key Genes inFlavonoids Biosynthetic Pathway
2.4 Discussion
Reference
SUPPLEMENTARY TABLE 1
Appendix 1: Protein sequence of 4CL genes used for the phylogenetic tree
Appendix 2: List of publications
ACKNOWLEDGEMENT
本文编号：3176419
【文章来源】：华中农业大学湖北省 211工程院校 教育部直属院校
【文章页数】：167 页
【学位级别】：博士
【文章目录】：
List of abbreviations
Chapter 1 The leucoanthocyanidins dioxygenase (GhLDOX) gene regulates the formation & elongation of cotton trichomes and fiber via modulating auxin signalingpathway
摘要
ABSTRACT
1.1 INTRODUCTION AND LITERATURE REVIEW
1.1.1 Cotton as an Economical Crop
1.1.2 Plant Trichomes are Unique Differentiated Cells
1.1.2.1 Transcriptional Regulation Underlying Leaf-Trichomes Development
1.1.2.2 Associated Hormonal Mechanisms Underlying Trichome Development in Arabidopsis
1.1.3 Cotton Fiber Development
1.1.3.1 Cotton Fiber Transcriptional Regulation
1.1.3.2 Role of Hormones in Regulating Cotton Fiber Development
1.1.4 Flavonoids
1.1.4.1 The importance of flavonoids to plants
1.1.4.2 Flavonoid’s defensive role in cotton
1.1.4.3 Flavonoids are Responsible of Cotton Leaf Reddening
1.1.4.4 Flavonoids Function in Cotton Fiber Development
1.1.5 Anthocyanin Synthesis
1.1.5.1 Ldox: A Late Gene in Anthocyanin Biosynthetic Pathway
1.1.5.2 Role of Anthocyanins Coupled With Phytohormones in Cotton Fiber Development
1.1.5.3 Anthocyanins and Trichomes
1.1.5.4 Sugar/Light- Mediated Anthocyanin Accumulation
1.1.5.5 Anthocyanin Is a Buffer Zone to Protect Chlorophyll
1.1.6 CRISPR-Cas9: a promising tool for plant functional genomics
1.1.7 Aim of Study
1.2 Materials and methods
1.2.1 Construction of phylogenetic tree and digital expression profiling for Gh LDOX
1.2.2 Specific spacer sequences selection and sg RNA designing
1.2.3 Construction of RNA-guided CRISPR-Cas9 genome-editing vector
1.2.4 Construction of Gh LDOX overexpression vector
1.2.5 Plant material for Agrobacterium-mediated transformation in cotton
1.2.6 Initiation, Maintenance of Embryogenic Calli and Plant Regeneration
1.2.7 Plant Material and Growth Conditions
1.2.8 DNA Extraction
1.2.9 Hi- Tom Screening and Detection of On-Target Mutations Induced by CRISPR/Cas9 System
1.2.10 RNA Extraction, RT-PCR and q RT-PCR
1.2.11 Subcellular Localization of The GhLDOX Gene
1.2.12 RNA-Seq Analysis and Identification of DEGs
1.2.13 Total Anthocyanin Extraction and Quantitation
1.2.14 Fiber Quality Measurement of Transgenic Plants
1.2.15 In Vitro Ovule Culture Assay
1.2.16 Scanning Electron Microscopy (SEM)
1.2.17 Chemical Quantification of Plant Hormones
1.2.18 Measurement of Total Chlorophyll
1.2.19 Assays of total soluble sugars: glucose, fructose & sucrose
1.2.20 Gossypol Extraction and Quantification
1.2.21 Insect Bioassay
1.3 RESULTS
1.3.1 Characterization of Gh LDOX gene
1.3.1.1 Phylogenetic analysis of cotton LDOX genes
1.3.1.2 Expression profile analysis of Gh LDOX gene in cotton
1.3.2 Generation of Gh LDOX transgenic plants
1.3.3 Subcellular localization of Gh LDOX gene
1.3.4 Suppression of Gh LDOX leads to free anthocyanin plants
1.3.5 Gh LDOX affects plant trichomes development in cotton
1.3.6 Gene knock out of Gh LDOX significantly disrupted cotton fiber initiation and elongation
1.3.7 Transcriptome sequencing analysis
1.3.7.1 Identification and functional analysis of DEGs
1.3.8 Altering the expression of GhLDOX gene modulated the expression of auxin genes pathway
1.3.9 Suppression of GhLDOX Downregulates Genes Involved in Chlorophyll Metabolic Pathway
1.3.10 The impact of the molecular and biochemical basis of Gh LDOX expression on soluble sugars accumulation in cotton plants
1.3.11 Differential Gene Expression of Key Genes in Flavonoids Biosynthetic Pathway of GhLDOXTransgenic Plants
1.3.12 Insect bioassay for GhLDOX transgenic lines
1.4 DISCUSSION
1.4.1 Anthocyanin Regulates Cotton Trichomes Initiation and Elongation Through Modulating AuxinPathway
1.4.2 GhLDOX Anthocyaninless Mutant Inversely Modulates Nitrate and Carbon Metabolism
1.4.3 Anthocyanin and Secondary Metabolites Toxicity Enhanced Resistance to Cotton Bollworm
Chapter 2 Characterization and functional analysis of the 4-coumarate:coenzyme A ligase 3 (Gh4CL3)improved cotton resistance against biotic stresses through lignin-mediated enhanced-defense mechanism
摘要
ABSTRACT
2.1 Introduction and Literature Review
2.1.1 Cotton (Gossypium hirsutum) pathogens and pests
2.1.1.1 Plant pathogens
2.1.1.2 Plant feeding-pests in cotton
2.1.2 Plants Defense Mechanisms
2.1.2.1 Constitutive (pre-existing) Defense Mechanisms
2.1.2.2 Induced defense mechanisms
2.1.3 Primary vs secondary metabolites related to plant defense responses
2.1.3.1 Lignin and phenylpropanoid biosynthetic pathways
2.1.3.2 Chemical structure of lignin: as a primary compound
2.1.3.3 Role of lignification in plant defense
2.1.3.4 Role of Flavonoids in plant defense
2.1.3.5 Effect of phenolic content and lignin modifications on resistance to pathogens
2.1.4 Plant 4-coumarate: coenzyme A ligase (4CL)
2.1.4.1 Roles of 4CL in plant development and response to environmental stresses
2.1.5 Study objectives
2.2 Materials and methods
2.2.1 Isolation and sequence retrieval of 4CL gene family in four cotton species
2.2.2 Gene Structure and Conserved Motifs Analyses
2.2.3 Construction of phylogenetic tree and digital expression profiling for Gh4CL3
2.2.4 Specific spacer sequences selection and sg RNA designing
2.2.5 Construction of RNA-guided CRISPR-Cas9 genome-editing vector
2.2.6 Construction of Gh4CL3 overexpression vector
2.2.7 Plant material for Agrobacterium-mediated transformation in cotton
2.2.8 Initiation, maintenance of embryogenic calli and plant regeneration
2.2.9 Plant Material and Growth Conditions
2.2.10 DNA extraction
2.2.11 Hi- Tom Screening and Detection of On-Target Mutations Induced by CRISPR/Cas9 System
2.2.12 RNA extraction, RT-PCR, and qRT-PCR
2.2.13 Subcellular localization of the Gh4CL3 gene
2.2.14 Verticillium dahliae inoculation
2.2.15 Lignin histochemical test
2.2.16 Determination of total lignin content
2.2.17 Insect bioassay
2.2.18 Chemical quantification of plant hormones
2.3 RESULTS
2.3.1 Characterization of 4CL gene family in cotton
2.3.1.1 Identification of 4CL Gene Family Based on Wide Genome Analysis in The GenusGossypium Spp
2.3.1.2 Gene structure and conserved motifs analyses of Gh4CL
2.3.1.3 Phylogenetic Analysis of cotton 4CL genes
2.3.2 Generation of Gh4CL3 transgenic cotton lines
2.3.3 Subcellular localization of Gh4CL3 gene
2.3.4 Overexpression or down-regulation of Gh4CL3 altered plant resistance to V.dahliae
2.3.5 Altering Gh4CL3 gene expression in cotton plants modulated lignin content
2.3.6 Suppression of Gh4CL3 gene conferred susceptibility to cotton bollworm
2.3.7 Lignin reduction triggers salicylic acid accumulation in CR4CL3 plants
2.3.8 Altering The Expression of Gh4CL3 Gene Modulates Gene Expression of Key Genes inFlavonoids Biosynthetic Pathway
2.4 Discussion
Reference
SUPPLEMENTARY TABLE 1
Appendix 1: Protein sequence of 4CL genes used for the phylogenetic tree
Appendix 2: List of publications
ACKNOWLEDGEMENT
本文编号：3176419
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