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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