| 博士学位论文评阅人、答辩委员会签名表 | 第3-6页 |
| 摘要 | 第6-7页 |
| Abstract | 第7-8页 |
| Abbreviations | 第13-14页 |
| CHAPTER 1 Literature review | 第14-30页 |
| 1.1 Importance of Soybean | 第14-15页 |
| 1.1.1 Classification and History of soybean | 第14页 |
| 1.1.2 Utilization and value/benefit of Soybean | 第14-15页 |
| 1.2 Impact of environmental abiotic stresses in soybean | 第15-17页 |
| 1.3 Small RNA involvement in post transcriptional regulation of gene expression inplant | 第17-19页 |
| 1.3.1 Micro RNAs and Si RNAs are new players in gene regulation | 第17页 |
| 1.3.2 Biogenesis and mechanism of mi RNAs pathway | 第17-18页 |
| 1.3.3 Biogenesis and mechanism of si RNAs pathway | 第18-19页 |
| 1.4 Micro RNAs involved in abiotic stress responses | 第19-24页 |
| 1.4.1 Micro RNA in drought stress | 第20页 |
| 1.4.2 Micro RNA in salt stress | 第20-21页 |
| 1.4.3 Micro RNA in cold stress | 第21-22页 |
| 1.4.4 Micro RNA in oxidative stress | 第22页 |
| 1.4.5 Micro RNA in nutrient deprivation | 第22-24页 |
| 1.5 Genetic marker and their applications in soybean | 第24-28页 |
| 1.5.1 Genetic improvement in soybean | 第24-25页 |
| 1.5.2 Basic molecular markers | 第25-26页 |
| 1.5.3 Advanced molecular markers | 第26页 |
| 1.5.4 Molecular marker application in soybean | 第26-28页 |
| 1.6 The significant of mi RNA and the objective of the studies | 第28-30页 |
| CHAPTER 2 Confirmation of stress response conserved mi RNA expression in soybeanseedlings | 第30-37页 |
| Abstract | 第30页 |
| 2.1 Introduction | 第30-31页 |
| 2.2 Materials and Methods | 第31-34页 |
| 2.2.1 Plant materials and treatments | 第31-32页 |
| 2.2.2 Total RNA extraction | 第32页 |
| 2.2.3 Qualified and quantified data of total RNA in 15% polyacrylamide gel | 第32-33页 |
| 2.2.4 Small RNA hybridization | 第33-34页 |
| 2.3. Results and Discussions | 第34-37页 |
| 2.3.1 Accumulation of conserved mi RNAs in soybean seedlings under stressescondition | 第34-37页 |
| CHAPTER 3 Prediction, experimentally verification and characterization for the targetgenes of gma-mi R166a | 第37-52页 |
| Abstract | 第37页 |
| 3.1 Introduction | 第37-39页 |
| 3.2 Materials and methods | 第39-43页 |
| 3.2.1 Plant materials and treatments | 第39页 |
| 3.2.2 Target genes prediction of gma-mi R166a | 第39页 |
| 3.2.3 Construction of c DNA library using six diverse genotypes of soybean seedlings | 第39-41页 |
| 3.2.4 Validation of target genes by 5'RLM RACE | 第41-42页 |
| 3.2.5 Bioinformatics data and data analysis | 第42-43页 |
| 3.3 Results and Discussions | 第43-52页 |
| 3.3.1 Target prediction of gma-mi R166a | 第43-46页 |
| 3.3.2 Verification of target cleavage sites by RACE | 第46-48页 |
| 3.3.3 Gm PHB of Glyma09g02750 is homologous to Arabidopsis PHABULOSA | 第48-50页 |
| 3.3.4 Phylogenectic analysis of Class III HD zip gene family | 第50-52页 |
| CHAPTER 4 Expression of gma-mi R166a and its target gene, Gm PHB, under variousenvironmental stresses | 第52-66页 |
| Abstract | 第52页 |
| 4.1 Introduction | 第52-53页 |
| 4.2 Materials and methods | 第53-55页 |
| 4.2.1 Plant materials and treatments | 第53页 |
| 4.2.2 Add Poly (A) to the 3’-terminal of mi RNA and synthesize the first-strand c DNA | 第53-54页 |
| 4.2.3 mi RNA quantification by real time Poly (A) based q RT-PCR | 第54页 |
| 4.2.4 First-strand c DNA synthesis for m RNA analysis of target gene | 第54-55页 |
| 4.2.5 Target gene quantification by real time q RT- PCR | 第55页 |
| 4.2.6 Bioinformatic and phylogenectic tree analysis | 第55页 |
| 4.3 Results and Discussions | 第55-66页 |
| 4.3.1 Analysis of mature and precursor of mi R166 in different species | 第55-57页 |
| 4.3.2 Expression of gma-mi R166a in various stresses | 第57-59页 |
| 4.3.3 Gm PHB is a strong candidate target gene of gma-mi R166a under stresses | 第59-61页 |
| 4.3.4 Variety specific expression of gma-mi R166a in different tissue | 第61页 |
| 4.3.5 Stem loop gma-mi R166a considerably conserved in diverse genotype of soybeanseedlings | 第61-63页 |
| 4.3.6 Cis-acting element and promoter analysis of gma-MIR166a | 第63-66页 |
| CHAPTER 5 Construct overexpression of gma-MIRNAs and functional analysis of gma-MIR166a | 第66-80页 |
| Abstract | 第66页 |
| 5.1 Introduction | 第66-68页 |
| 5.2 Material and methods | 第68-74页 |
| 5.2.1 Construction of Ca MV35s::gma-MIRNAs cassettes in binary vectorp CAMBIA3300 | 第68-72页 |
| 5.2.2 Transformation of precursor of gma-MIRNAs to Arabidopsis by using floral dipmethod | 第72-73页 |
| 5.2.3 Screening of transgenic plants | 第73页 |
| 5.2.4 Expression analysis of gma-mi R166a and Gm PHB in transgenic Arabidopsis.. 605.2.5 Salt stress treatment in transgenic Arabidopsis | 第73页 |
| 5.2.5 Salt stress treatment in transgenic Arabidopsis | 第73-74页 |
| 5.3 Results and Discussions | 第74-80页 |
| 5.3.1 Overexpression of gma-MIR166a in Arabidopsis has two distinct phenotypes | 第74-75页 |
| 5.3.2 Phenotypic characters between wild type & gm-MIR166a in Arabidopsis | 第75-76页 |
| 5.3.3 Repression of target genes PHB by overexpression of gma-MIR166a inArabipdosis by real time q RT-PCR | 第76-77页 |
| 5.3.4 Mild overexpression of gma-MIR166a confers salt tolerance in Arabidopsis | 第77-80页 |
| CHAPTER 6 Allelic diversity within the genus Glycine by gma-mi R1511 In Del markerand characterization of its function | 第80-98页 |
| Abstract | 第80页 |
| 6.1 Introduction | 第80-82页 |
| 6.2 Materials and Methods | 第82-84页 |
| 6.2.1 Plant Materials for allelic diversity analysis | 第82页 |
| 6.2.2 Bioinformatics data and data analysis | 第82-83页 |
| 6.2.3 Genomic DNA extraction and sequencing | 第83页 |
| 6.2.4 Stress treatment in three different types of allelic variation in annual wildsoybean | 第83-84页 |
| 6.2.5 Gene expression analysis of three different types of allelic variation of gma-mi R1511/mi R1511* and its target Gm RPL4a | 第84页 |
| 6.3 Results and Discussions | 第84-98页 |
| 6.3.1 Functional marker gma-mi R1511 In Del as an exemplar for allelic variationanalysis | 第84-85页 |
| 6.3.2 Sequence analysis of different allelic variation in genus Glycine | 第85-87页 |
| 6.3.3 Phylogenetic analysis of mi R1511 | 第87-89页 |
| 6.3.4 Distribution of allelic diversity by gma-mi R1511 In Del marker in China andneighborhood | 第89-93页 |
| 6.3.5 Confirm present or absent of gma-mi R1511 in different accessions of annualwild soybean | 第93-95页 |
| 6.3.6 Expression of target gene (Gm RPL4a) of gma-mi R1511 under stresses | 第95-96页 |
| 6.3.7 Anti expression of gma-mi R1511/mi R1511* and its target gene Gm RPL4a understresses condition | 第96-98页 |
| CHAPTER 7 Summary | 第98-102页 |
| REFERENCES | 第102-123页 |
| APPENDIX | 第123-143页 |
| ACKNOWLEDGEMENTS | 第143-145页 |
| AUTHOR BIOGRAPHY | 第145页 |
