陸地棉基因GhLDOX和Gh4CL3的功能解析
發(fā)布時間：2021-05-09 02:16
花青素是一類參與多種生物過程的植物黃酮類色素。LDOX是花青素生物合成途徑中的一個下游基因,它能夠編碼白花青素并將其轉(zhuǎn)化為花青素。迄今為止,還沒有研究闡明LDOX同源基因在棉花花青素合成及生長發(fā)育中的作用。本研究通過CRISPR-Cas9系統(tǒng)對棉花GhLDOX基因進(jìn)行精準(zhǔn)編輯創(chuàng)造靶向突變體,并通過超表達(dá)技術(shù)提高GhLDOX基因的表達(dá)量,從而對棉花GhLDOX基因進(jìn)行功能鑒定。結(jié)果表明,無論是在營養(yǎng)組織中還是在花中,CRldox基因突變體植株都完全不含花青素,而OELDOX基因高表達(dá)植株則表現(xiàn)出總花青素的增加。且花青素含量的變化與GhLDOX基因表達(dá)量呈成正相關(guān)。此外,為了揭示花青素在棉花毛狀體發(fā)育中的作用,我們對轉(zhuǎn)GhLDOX基因的棉花植株進(jìn)行了葉片表皮毛和種子纖維的檢測。發(fā)現(xiàn)花色素苷的產(chǎn)生與葉片表皮毛及纖維的起始和伸長呈顯著正相關(guān)。CRldox突變體中花青素的缺失葉片表皮毛和種子纖維的起始和伸長的顯著,而OELDOX高表達(dá)株系花青素含量增加,葉片和纖維長度增加。通過對GhLDOX基因敲除的植株葉片進(jìn)行轉(zhuǎn)錄組測序,結(jié)果表明參與黃酮類化合物生物合成途徑的基因表達(dá)差異明顯,其中大部分基因在...
【文章來源】：華中農(nóng)業(yè)大學(xué)湖北省 211工程院校 教育部直屬院校
【文章頁數(shù)】：167 頁
【學(xué)位級別】：博士
【文章目錄】：
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
本文編號：3176419
【文章來源】：華中農(nóng)業(yè)大學(xué)湖北省 211工程院校 教育部直屬院校
【文章頁數(shù)】：167 頁
【學(xué)位級別】：博士
【文章目錄】：
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
本文編號：3176419
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