本研究采用一個(gè)棉花種間雜種BC₂F₂的回交自交系群體,進(jìn)行干旱脅迫處理以及基因型測(cè)序（GBS）和分型,定位了耐旱的數(shù)量性狀位點(diǎn)（QTLs）。在穩(wěn)定的QTL區(qū)域內(nèi)進(jìn)一步挖掘基因,在干旱條件下評(píng)價(jià)基因的功能。參考陸地棉[（AD）1]、亞洲棉（A2）和雷蒙德氏棉（D5）基因組測(cè)序數(shù)據(jù),對(duì)NLP、LEA、TH、CDK、CYP450和Alba等基因家族進(jìn)行了全基因組鑒定分析;對(duì)各個(gè)基因家族中的部分新基因進(jìn)行了克隆,轉(zhuǎn)化到擬南芥或陸地棉中進(jìn)行了干旱和鹽脅迫或缺氮條件下性狀鑒定,通過(guò)正向和反向遺傳學(xué)手段分析了它們的功能。1.BC₂F₂群體的耐旱QTL定位采用自陸地棉和毛棉種間雜交的BC₂F₂群體的188個(gè)系,構(gòu)建了一個(gè)含有10888個(gè)SNP標(biāo)記的遺傳圖譜。圖譜全長(zhǎng)4191.3 c M,平均標(biāo)記距離0.1047 c M,覆蓋At和Dt兩個(gè)亞基因組各約51%和49%。該圖譜包括30個(gè)穩(wěn)定的耐旱QTLs,其中有3個(gè)成簇分布（簇1、4和10）,在每條染色體含有1個(gè)位點(diǎn)以上的簇被... 

【文章來(lái)源】：中國(guó)農(nóng)業(yè)科學(xué)院北京市

【文章頁(yè)數(shù)】：302 頁(yè)

【學(xué)位級(jí)別】：博士

【文章目錄】：
摘要
abstract
LIST OF ABBREVIATIONS
DEDICATIONS
CHAPTER ONE INTRODUCTION
    1.1 Cotton and Challenges in Cotton Production
        1.1.1 Drought and Salt Stress Coping Strategies in Cotton
        1.1.2 Plant’s Transcription Factors and Gene Functions in Plants Under Drought and Salt Stress Conditions
    1.2.Morpho‐physiological Mechanism of Cotton Plants in Responses to Drought Stress
    1.3.Biochemical and Molecular Mechanism of Drought Tolerance in Cotton
    1.4.Drought Effect and Adaptive Response by Cotton Plants
    1.5.Strategies to Induce Abiotic Stress Tolerance in Cotton
        1.5.1.Marker‐Assisted Selection(MAS)Based on Drought‐Related QTLs/Transcription Factors
        1.5.2.Genotyping by Sequencing and QTL Mapping of Drought Tolerant Traits
        1.5.3.Mapping of the BC_2F_2 Progenies and Mining of Drought Stress Responsive Genes
        1.5.4.The NODULE-INCEPTION-like proteins(NLPs)role in cotton
        1.5.5.Characterization of the Late Embryogenesis Abundant(LEA)Proteins Family in Cotton
        1.5.6.Functional Characterization of Cyclin Dependent Kinase Gene Family in Cotton
        1.5.7.Trihelix Transcription Factors and Overexpression of Gh_A05G2067(GT-2)in Cotton
        1.5.8.Identification and Functional Characterization of the CYP450 Genes in Cotton
        1.5.9.The Alba Genes and their Putative Role in Cotton under Abiotic Stress Conditions
    1.6.The Objective of this Research
CHAPTER TWO MATERIALS AND METHODS
    2.1 Plant Materials and Treatments
        2.1.1 Mapping Population and Drought Treatment
        2.1.2.Materials for Stomata Examination and RT-qPCR Analysis of the Five Genes under Drought and/or Salt Treatments
        2.1.3.Materials Used For RNA Extraction for Examining Expression Patterns of NLP Genes under Nitrogen Deficiency Treatments
        2.1.4.Materials Used For Transformation or VIGS Researches of Six Functional Gene Families
    2.2 Data Collection of Various Traits on the BC_2F_2 Populations under Drought stress Condition
        2.2.1 Soil Moisture,Soil Temperature,and Soil Electrical Conductivity
        2.2.2.Determination of the Morphological Characteristics of Plants
        2.2.3 Cell Membrane Stability(CMS)
        2.2.4 Relative Leaf Water Content(RLWC)
        2.2.5 Excised Leaf Water Loss(ELWL)
    2.3.Sample Collection,GBS library Preparation,Sequencing,and SNP Genotyping
        2.3.1.Extraction,Quantification and Quality Determination of DNA
        2.3.2.The GBS Library Preparation,Sequencing and SNP Genotyping
        2.3.3.Genotyping by Sequencing(GBS)Protocol
    2.4.Data Analysis,Linkage Map Construction,QTL Mapping,and Gene Mining
        2.4.1.Data Analysis
        2.4.2.Linkage Map Construction and QTL Mapping
        2.4.3.Gene Mining,Functional Annotation,Phylogenetic tree,and Gene Structure Analysis
        2.4.4.RNA Sequence Analysis of the Mined Genes
        2.4.5.The miRNA Target and Promoter Analysis
        2.4.6.RT-qPCR Validation of the Key Functional Genes
    2.5.Functional Characterization of Six Stress Responsive Gene Families in Cotton
        2.5.1.Identification of Stress Responsive Gene Families in Cotton
        2.5.2.Chromosomal Locations and Syntenic Analysis of Abiotic Stress-Responsive Gene Families
        2.5.3.Phylogenetic and Gene Structure Analysis of the Abiotic Stress Responsive Genes
        2.5.4.Prediction of miRNA’s Targeting the Various Stress Responsive Genes in Cotton
        2.5.5.Cis-regulatory Element Analysis and Gene Ontology(GO)Annotation
        2.5.6.RNA Isolation and RT-qPCR Verification
    2.6.Functional Analysis of the Stress Responsive Gene Families in Cotton
        2.6.1.Transformation of the Novel Genes,LEA2,CDK and TH in A.thaliana
        2.6.2.Profiling of Abiotic stress Responsive Genes in Transformed Arabidopsis plants
        2.6.3.Biochemical Assays in Transgenic and WT Arabidopsis Lines Under Abiotic Stress
        2.6.4.Stress Conditions and Physiological Measurements on the Overexpressed and the Wild Types
        2.6.5.Determination of the Subcellular Location of Gh_D12G2017(CDKF4)Protein
        2.6.6.Virus Induced Gene Silencing of TH,CYP450,Alba and NLP Genes in Cotton
        2.6.7.Profiling of Stress-Responsive Genes on the VIGS and WT Cotton Plants
        2.6.8.Physiological and Biochemical Evaluations of the VIGS Plants and WT Cotton Plants
        2.6.9 Root Evaluation of the VIGS and WT Cotton Plants
CHAPTER THREE RESULTS
    3.1.QTL Mapping and Functional Characterization of Key Genes
        3.1.1.Phenotype Variation between Parental Lines and their BC2F1 Generation
        3.1.2 Correlation Analysis
        3.1.3.Microscopic Examination of the Parents and Their BC2F1 Generation
        3.1.4.SNP detection,InDel Detection,Annotation,Genetic Markers,and Marker Coding
        3.1.5.Genetic Map Construction
        3.1.6.High-density Genetic Linkage Map Construction
        3.1.7.Evaluation of Reorganization Relationship and Collinearity of Genetic Maps and Genomes
        3.1.8.Gene Mining Within the GBS Marker Regions of the Mapping Population
        3.1.9.Chromosome Mapping of the Genes Mined for the Dominant Domain,Pkinase
        3.1.10.RNA Sequence Data of the Genes of the Pkinase Domain
        3.1.11.The miRNA Target Analysis of the271 Dominant Genes
        3.1.12.Cis-regulatory Element Analysis
        3.1.13.Gene Ontology(GO)Analysis of the Mined Genes
        3.1.14.RT-qPCR Validation of the Candidate Genes
        3.1.15.Identification of Consistent and Clustering QTLs for Physio-Morphological Traits
        3.1.16.Gene Mining within the3 Major QTLs Clusters Regions
        3.1.17.Bioinformatics Analysis of the Mined Genes within the Major QTL Clusters
            3.1.17.1 Physiochemical-Subcellular Localization of the Proteins Encoded by the Mind Genes
            3.1.17.2 Gene Ontological Annotation of the Mind Genes
        3.1.18.Phylogenetic Tree Analysis of the Protein Encoded by the Mined Genes
        3.1.19.The miRNA Target and Cis-regulatory Element Analysis of the Mined Genes
            3.1.19.1 Determination of the various miRNA targeting the Mined Genes
            3.1.19.2 Determination of the Various Cis-regulatory Element Associated with the Mined Genes
        3.1.20.RNA Seq.Expression Analysis of the Mined Genes
        3.1.21.RT-qPCR Validation of the Key Genes
    3.2 A Novel NLP Genes Identified within the QTL Region Enhances N deficiency Tolerance
        3.2.1 Identification and Sequence Analysis of the Cotton NLP Proteins
        3.2.2 Phylogenetic Tree Analysis
        3.2.3 Physiochemical Properties of the Cotton Proteins Encoded by the NLP Genes
        3.2.4 Gene Structure Analysis and Motif Identification of the Cotton NLP Proteins
        3.2.5 Cis-regulatory Element Analysis of the Cotton NLP Genes
        3.2.6 The miRNA Target Prediction of the Cotton NLP Genes
        3.2.7 RT-qPCR Validation of the Selected GhNLP Genes
        3.2.8 Silencing of Gh_A05G3286(NLP5)and Evaluation of VIGS and WT plants
        3.2.9 Stress Responsive Genes profiling and biochemical analysis on NLP knocked plants
    3.3.Genomewide Analysis and Functional Characterization of LEA Gene Family
        3.3.1.Identification of the LEA Genes in Cotton
        3.3.2.Phylogenetic Analyses,Gene Structure and Protein Motifs of LEA Genes in Upland Cotton
        3.3.3.Phylogenetic Analyses of the LEA Proteins in Cotton with Other Plants
        3.3.4.Chromosomal Distribution of Cotton LEA Genes
        3.3.5.Gene Duplication and Syntenic Analysis
        3.3.6.Prediction of LEA Genes(mRNA)Targeted by miRNAs in Upland Cotton
        3.3.7.Gene Ontology(GO)Annotation
        3.3.8.Cis-regulatory Element Analysis
        3.3.9.Upland Cotton LEA Genes Expression Analysis under Drought Stress
    3.4.Comprehensive Analysis of Cotton LEA2 Genes
        3.4.1.LEA2 Proteins Identification in Cotton and Other Plants
        3.4.2.Phylogenetic Analyses of LEA2 Proteins in G.hirsutum,G.arboreum,and G.raimondii
        3.4.3.Physiochemical Analysis and Subcellular Localization of LEA2 Genes in Upland Cotton
        3.4.4.Genomic Organization and Motif Detection of the LEA2 Proteins in Cotton
        3.4.5.Chromosomal Location and Duplication events of the LEA2 Genes in Cotton
        3.4.6.Cis-regulatory Element Prediction in LEA2 Proteins
        3.4.7.Gene Ontology(GO)Annotation
        3.4.8.Prediction of LEA2 Genes Targeted by miRNAs
        3.4.9.Analysis of Tertiary Protein Structure of Upland Cotton LEA2s
        3.4.10.LEA2 Gene Interactional Dynamics under Drought Stress
        3.4.11.RNA Seq.Expression Patterns of LEA2 Genes in Different Tissues of Upland Cotton
        3.4.12.Expression Profiles of LEA2 Genes in Leaf,Stem,and Roots of Upland Cotton
        3.4.13.Expression Profiles of LEA2 Genes under Drought Stress
        3.4.14.RT-qPCR Analysis of the Transformed Gene in Upland Cotton Tissues
        3.4.15.CotAD_24498 Overexpressed Plants have Increased Root Growth and improved Tolerance to Drought Stress
        3.4.16.Transcripts Investigation of Drought Stress-Responsive Genes
        3.4.17.Oxidants and Antioxidant Enzyme Determination in the Transgenic Lines
    3.5.Functional Characterization of the CDK Genes under Drought and Salt Stress Condition
        3.5.1.Identification and Sequence Analysis of CDK Proteins in Cotton Genome
        3.5.2.Gene Structure and Amino Acid Motif Analysis of the CDK Genes in Cotton
        3.5.3.Phylogenetic Analyses and Protein Alignments of the CDKs Proteins
        3.5.4.Chromosomal Distribution of Cotton CDK Genes
        3.5.5.Gene Duplication,Orthologs,Paralogs and Selection Type of the CDK Genes
        3.5.6.Promoter(Cis-regulatory Element)Analysis
        3.5.7.RNA Seq.Analysis of the CDK Genes under Drought and Salt Stress Condition
        3.5.8.RT-qPCR Analysis of the Cotton CDK Genes under Drought and Salt Stress
        3.5.9.RT-qPCR Validation of the Highly Upregulated CDK Genes
        3.5.10.Determination of the Subcellular Localization of Gh_D12G2017(CDKF4)Protein
        3.5.11.RT-qPCR Analysis of Gh_D12G2017(CDKF4)Gene in Upland Cotton Tissues
        3.5.12.The Response of the Overexpressed and WT Plants under Drought and salt Stress Conditions..
        3.5.13.Evaluation of Stress Responsive Genes on the Tissues of Transgenic and WT Plants
    3.6.Genomewide Identification and Functional Analysis of Gh_A05G2067(GT‐2)
        3.6.1.Identification of Trihelix Proteins in Cotton
        3.6.2.Phylogenetic Analysis of Cotton Trihelix Proteins with Other Plants
        3.6.3.Genomic Organization and Chromosomal Distribution of the Cotton Trihelix Genes
        3.6.4.RNA Sequence Analysis Profiled under Abiotic Stress Conditions
        3.6.5.RT-qPCR Validation of the Selected Genes
        3.6.6.RT-qPCR and subcellular determination of the Transformed Gene,Gh_A05G2067(GT-2)
        3.6.7.Oxidant and Antioxidant Enzymes Assays in Gh_A05G2067(GT-2) -Overexpressed plants
        3.6.8.Physiological Traits Evaluation under Drought and Salt Stress Conditions
        3.6.9.Profiling of Abiotic Stress Responsive Genes in GT-2-Overexpressed Lines and WT
        3.6.10.Physiological Traits Evaluation in Gh_A05G2067(GT-2) -Silenced Plants
        3.6.11.Biochemical and RT-qPCR Analysis of the Abiotic Stress-Responsive Genes
    3.7.Reverse Genetics Reveals the Putative Role of CYP450 Gene Family in Cotton
        3.7.1.Identification of the Cotton Cytochrome P450(CYPs)Genes
        3.7.2.Subcellular Localization Analysis of the Cotton Cytochrome P
        3.7.3.Cis-regulatory Element Analysis of the Cotton Cytochrome P
        3.7.4.Chromosomal Mapping of the Upland G.hirsutum Cytochrome CYP450 Genes
        3.7.5.RNA Seq.Analysis and RT-qPCR Validation of the Upland Cotton CYP450 Genes
        3.7.6.Expression Analysis of Gh_D07G1197 and Gh_A13G2057 in VIGS and WT Plants
        3.7.7.Evaluation of Performance of the VIGS Plants and WT plants
        3.7.8.Stress-Responsive Gene Profiling on the Tissues of VIGS Plants and WT Plants
    3.8.Functional Characterization of Aba Genes via RNAi Method in Cotton
        3.8.1 Identification of the Alba Proteins in Cotton Species
        3.8.2 Phylogenetic Tree Analysis of the Cotton Alba Proteins
        3.8.3 Physiochemical Properties of the Cotton Alba Proteins
        3.8.4 Gene Structure and Motif Identification
        3.8.5 The miRNA Target on the Varies Alba Genes in Cotton
        3.8.6 Cis-regulatory Elements Analysis of the Cotton Alba Genes
        3.8.7 RNA Seq.Analysis and RT-qPCR Validation of the Various Alba Genes
        3.8.8 The Efficiency of Knockdown of the Two Alba Genes in Cotton
        3.8.9 Physiological and Root Assays of the VIGS Plants and the WT Plants
        3.8.10 Biochemical and Abiotic Stress Responsive Profiling on the VIGS and WT plants
CHAPTER FOUR DISCUSSION
    4.1.QTL Mapping and Functional Characterization of Key Genes
    4.2.GBS Mapping and Analysis of Conserved Genes that Respond to Drought Stress tolerance
    4.3.A Novel NLP Genes Identified Within the QTL Enhancing Nitrogen Deficiency Tolerance in Cotton
    4.4.Characterization of the LEA Proteins Family in Cotton under Drought Stress Condition
    4.5.Functional Characterization of the CDK Gene Family in Cotton
    4.6.Genomewide Identification and Functional Analysis of the Trihelix Transcription Factors
    4.7.Reverse Genetics Reveals the Putative Role of CYP450 Gene Family in Cotton
    4.8 Functional Analysis of the Alba Genes in Cotton
CONCLUSION
    5.1.QTL Mapping and Functional Characterization of Key Genes within the QTL regions
    5.2 Genomewide Identification of the Six Gene Families in Cotton
    5.3 RNA and RT-qPCR Validation of Protein Encoded by the Various Gene Families
    5.4 Functional Characterization of the Five Gene Families Under Drought and/or Salt Stress
REFERENCES
ACKNOWLEDGEMENT
CURRICULUM VITAE



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