声明
List of abbreviations
CHAPTERⅠ-INTRODUCTION
1.1 General Introduction
1.2 Auxin and shoot branching regulation
1.3 The auxin signaling pathway
1.4 SL signaling and shoot branching regulation
1.4.1 Discovery of SL
1.4.2 Chemistry of SL molecule
1.4.3 Biosynthesis of SL
1.4.4 SL Transport
1.4.5 SL perception and signaling
1.4.6 Traits governed by SL signaling pathway
1.5 Objectives
CHAPTERⅡ-IDENTIFICATION, GENE CLONING AND FUNCTIONAL ANALYSIS OF THE hd103 MUTANT
2.1 Background
2.2 MATERIALSAND METHODS
2.2.1 Plant material and growth condition
2.2.2 Phenotypic analysis
2.2.3 Genetic analysis
2.2.4 Map-based cloning
2.2.5 Confirmation of the candidate mutation
2.2.6 Gene expression constructs and transformation
2.2.7Plant transformation
2.2.8 Construction of over expression vector
2.2.9Construction of RNAi vectors
2.2.10 Construction of CRISPR/CAS9 genome-edited lines
2.2.11Determination of expression pattern of OsIAA16
2.2.12 Construction of promoter-GUS vector
2.2.13Determination of GUS activity
2.2.14 Assessment of early auxin-responsive genes
2.2.15 Gene structure and phylogenetic analysis
2.2.16 Auxin effect on plant growth and development
2.2.17 Yeast two-hybrid analysis
2.2.18GST Pull-down assay
2.2.19 Split luciferase assay for in vivo protein-protein interaction
2.2.20 Protocol for SDS PAGE preparation and Western blotting
2.2.21 Protein stability experiment
2.2.22 Gene expression of SL pathway genes
2.2.23 Phyto-hormone analysis
2.2.24ExternalSLapplication and tiller development assay
2.2.25SL/auxin-mediated degradation of OsD53 protein
2.2.26 Construction of knockout vectors using CRISPR-CAS9 to edit the putative interacting ARFs
2.2.27 Construction of complementation lines with WT/deleted AuxRE in SL pathway genes
2.3 RESULTS
2.3.1 Altered plant architecture in the hd103 mutant
2.3.2 Genetic analysis of the hd103 mutant
2.3.3 Map-based cloning of thehd103 locus
2.3.4OsIAA16protein structure and domain analysis
2.3.5 Phylogenetic analysis of OsIAA16
2.3.6 Over-expression of OsIAA16 in WT background mimics the hd103 mutant phenotype
2.3.7 Knock-down or knock-out of OsIAA16 in hd103 suppresses the high tillering dwarf mutant phenotype
2.3.8 Expression pattern of the OsIAA16 gene
2.3.9 Determination of auxin levels in the WT and hd103 mutant
2.3.10 Effect of C185T substitution on the OsIAA16 protein stability
2.3.11 Effect of C185T substitution on the auxin-related phenotypes
2.3.12 Effect of C185T on auxin responsiveness
2.3.13 Effect of C185T substitution on the early auxin-responsive gene expression
2.3.14 Effect of C185T substitution on its binding with auxin receptor TIR1
2.3.15 Identification of the OsIAA16 interacting Auxin response Factors (ARFs)
2.3.16 Auxin-strigolactone cross-talk in shoot branching regulation
2.4 DISCUSSION
2.4.1 The altered phenotype in hd103 mutant is the result of defective auxin signaling
2.4.2 Auxin modulates SL pathway
2.5 CONCLUSION
CHAPTER Ⅲ-SUCROSE ALLEVIATES THE STRIGOLACTONE MEDIATED TILLER BUD SUPPRESSION THROUGH THE F-BOX PROTEIN D3
3.1 INTRODUCTION
3.2 MATERIALSAND METHODS
3.2.1 Plant material and growth conditions
3.2.2 D53 quantification assay using callus tissues
3.2.3 Gene expression analysis
3.2.4In vivo luciferase activity assay
3.3 RESULTS
3.3.1 The hd103 mutant is hyper-responsive to sucrose like other SL pathway mutants
3.3.2 Effect of sucrose on SL-mediated tiller bud suppression
3.3.3 Effect of sucrose on the SL-induced degradation of D53 protein
3.3.4 Effect of sucrose on SL signaling genes
3.3.5 Impact of sucrose on the transcriptional regulation of SL signaling genes
3.3.6 The d3 mutant is hyper-responsive to sucrose
3.3.7 Involvement of sucrose and SL antagonism in other developmental processes
3.4 DISCUSSION
3.4.1 Sucrose inhibits SL signaling and alleviates the SL inhibition of tillering
3.4.2 Sucrose inhibits SL perception by suppressing D3
3.4.3 D3 acts partly independently of SL to inhibit bud outgrowth
3.4.4 Involvement of sucrose and SL antagonism in other developmental processes
3.5 CONCLUSION
CHAPTERⅣ-GENERAL CONCLUSION
参考文献
致谢
CURRICULUM VITAE
中国农业科学院;
