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  • Title
  • DECLARATION
  • CERTIFICATE
  • ACKNOWLEDGEMENT
  • CONTENTS
  • ABBREVIATIONS
  • 1. General Introduction
  • 1.1. Natural Rubber - An Overview
  • 1.2. Para Rubber Tree - Havea brasiliensis
  • 1.3. Latex and Laticifers
  • 1.4. Rubber Biosynthesis
  • Fig. 1. General pathway of rubber biosynthesis
  • 1.5. Significance of Molecular Interventions for Hevea Crop Improvement
  • 1.6. Objectives
  • 2. Molecular Cloning and Characterization of the Genomic Sequence Coding for the Rubber Elongation Factor Protein from Hevea brasiliensis
  • 2.1. Introduction
  • 2.2. Materials and Methods
  • 2.2.1. Plant Material
  • 2.2.2. PCR Amplification of REF Gene
  • 2.2.3. Cloning of PCR Amplified REF Gene into Plasmid Vector
  • 2.2.4. Nucleotide Sequencing and Analysis
  • 2.2.5. Southern Blot Analysis
  • 2.3. Results
  • 2.3.1. Genomic DNA Isolation and PCR Amplification of REF Gene
  • 2.3.2. Cloning of REF Gene
  • 2.3.3. Sequence Characterisation of REF Gene
  • 2.3.4. DNA Blot Analysis
  • 2.4. Discussion
  • 2.4.1. PCR Amplification and Cloning of REF Gene
  • 2.4.2. Sequence Characterization of REF Gene
  • 2.4.3. Southern Blot Analysis
  • 3. Cloning and Characterization of the cDNA Encoding REF Protein from Hevea brasiliensis and its Expression in E coli and in Transgenic Tobacco (Nicotiana tabacum)
  • 3.1. Introduction
  • 3.2. Materials and Methods
  • 3.2.1. Cloning of REF cDNA by RT-PCR
  • 3.2.2. Heterologous Expression of Recombinant H. Brasiliensis REF Protein in E. Coli
  • 3.2.3. Genetic Transformation of Tobacco
  • 3.3. Results
  • 3.3.1. Cloning and Characterization of REF cDNA
  • 3.3.2. Heterologous Expression of REF Fusion Protein in E. Coli
  • 3.3.3. Genetic Transformation of Tobacco
  • 3.4. Discussion
  • 3.4.1. Cloning and Sequence Characterisation of REF cDNA
  • 3.4.2. Heterologous Expression of Recombinant REF Protein in E. coli
  • 3.4.3. Genetic Transformation of Tobacco
  • 4. Differential Expression Studies of Rubber Elongation Factor Gene in Hevea brasiliensis
  • 4.1 Introduction
  • 4.2. Materials arid Methods
  • 4.2.1. Plant Material
  • 4.2.2. RNA Extraction
  • 4.2.3. RNA Gel Blot Analysis
  • 4.2.4. REF cDNA Synthesis by RT- FCR
  • 4.2.5. REF Probe Synthesis
  • 4.2.6. Washing and Hybridization
  • 4.3. Results
  • 4.3.1. Differential Expression of REF Gene in Various Tissues of Hevea
  • 4.3.2. Effect of Tapping on REF Gent Expression
  • 4.3.3. REF Gene Expression in High Yielding and Low Yielding Clones
  • 4.3.4. Effect of Exogenous Ethephon Application on REF Transcript Level
  • 4.4. Discussion
  • 4.4.1. Differential Expression of REF Gene in Various Tissues of Hevea
  • 4.4.2. Effect of Tapping on REF Gene Expression
  • 4.4.3. REF Gene Expression in High Yielding and Low Yielding Hevea Clones
  • 4.4.4. Effect of Exogenous Ethephon Stimulation on REF Gene Expression
  • 5. Cloning and Functional Characterization of the Promoter Region of REF Gene from Hevea brasiliensis
  • 5.1. Introduction
  • 5.2. Materials and Methods
  • 5.2.1. Plant Materials
  • 5.2.2. PCR Amplification of Promoter Region of REF Gene
  • 5.2.3. Cloning and Sequencing of REF Gene Promoter
  • 5.2.4. Construction of REF Promoter / GUS Fusion
  • 5.2.5. Genetic Transformation of Tobacco
  • 5.3. Results
  • 5.3.1. PCR Amplification of Promoter Region of REF Gene
  • 5.3.2. Sequence Analysis of the Promoter Region of REF Gene
  • 5.3.3. Putative Regulatory Elements in the REF Promoter
  • 5.3.4. Construction of REF Promoter / GUS Fusion
  • 5.3.5. Transformation of Tobacco
  • 5.4. Discussion
  • 5.4.1. Amplification and Cloning of the 5 Upstream Region of REF Gene
  • 5.4.2. Sequence Characterization of REF Promoter Fragment
  • 5.4.3. Functional Characterization of REF Promoter in Transgenic Tobacco
  • Summary and Conclusion
  • References