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Title
DECLARATION
CERTIFICATE
ACKNOWLEDGEMENT
CONTENTS
1. Introduction
Objectives of the Study
2. Review Of Literature
2.1. Defense Mechanisms in Plants - An Overview
2.1.1. Pre-existing defense structures
2.1.2. Induced biochemical defense
2.1.3. Recognition of the pathogen
2.1.4. The gene-for-gene hypothesis
2.1.5. Signal transduction and the hyper sensitive response
1 Diagrammatic representation of induced plant defense mechanisms
2.1.6. Pathogenesis related proteins
2.2. The Abnormal Leaf Fall Disease
2.2.1 Phytophthora spp.- The Unique Class
2.2.2. Attacks on Hevea
2.2.3. Symptoms of the disease
2 Occurrence of Abnormal Leaf Fall disease and its symptoms
2.2.4 Clonal susceptibility
2.2.5. Control measures
2.3. Plant β -1, 3-Glucanases and their Importance in Resistance
2.3.1. Nomenclature
2.3.2. Structural classes
2.3.3. Biological functions of β -1, 3 glucanases - Role in developmental processes
2.3.4. Defense related functions of β -1, 3 glucanases
2.3.4.1. Over-expression in response to pathogen attack
2.3.4.2. In vitro Studies
2.3.4.3. Release of fungal elicitors
2.3.4.4. Enhanced disease resistance by transgene expression
2.4. Molecular approaches in Hevea
3. Materials and Methods
3.1. Isolation, Cloning and Characterisation of Genomic and cDNA Sequences Coding for β -Glu Gene
3.1.1. Genomic DNA isolation
3.1.1. a. DNA quantification
3.1.2. Isolation of RNA from latex
3.1.3. First strand cDNA synthesis
3.1.4. Design of gene specific primers
3.1.5. PCR amplification of the gene from genomic and cDNA
3.1.6. Cloning of the PCR products
3.1.6.a. Elution of amplified products from agarose gels
3.1.6.b. Ligation
3.1.6.c. Transformation protocols for E. coli
3.1.7. Confirmation of cloning
3.1.7.a. Through PCR
3.1.7.b. Alkaline loess procedure for isolation of plasmids in large scale
3.1.7.c. Restriction analysis of the plasmid DNA
3.1.8. Sequencing and sequence analysis
3.1.8.a. PEG purification of plasmid DNA
3.1.8.b. Sequencing
3.1.9. Southern hybridisation
3.1.9.a. Restriction digestion of genomic DNA
3.1.9.b. Blotting
3.1.9.c. Preparation of labeled probes
3.1.9.d. Hybridisation
3.1.9.e. Washing of the blot and autoradiography
3.1.10. Isolation of promoter elements of the gene
3.2. Gene Expression Studies through Northern Hybridisation and RT-PCR Analysis
3.2.1. Plant Materials used
3.2.2. Fungal culture and inoculation
3.2.3. Isolation of RNA from leaf samples
3.2.4. Amplification of sequences for using as internal controls
3.2.4.a. Amplification of 18S ribosomal RNA gene
3.2.4.b. Amplification of β -actin gene
3.2.5. Northern hybridisation protocols
3.2.5.a. Electrophoresis of RNA
3.2.5.b. RNA blotting
3.2.5.c. Hybridisation and washing
3.2.5.d. Stripping of the blot
3.2.6. RT-PCR assay for differential expression
3.2.7. Analysis of auto radiograms and RT-PCR products
3.2.8. β -1, 3-Glucanase enzyme assays
3.2.9. Tissue specific expression
3.3. Construction of a Functional cDNA Clone and Purification of Recombinant Protein
3.3.1. Design of primers with restriction sites and PCR amplification
3.3.2. Cloning in expression vectors
3.3.3. Sequencing
3.3.4. Expression of the target gene
3.3.5. Purification of the target protein
3.3.6. Confirmation of the induced target proteins
3.3.6.a. Western blotting
3.3.6.b. Thrombin cleavage
3.3.7. Anti-fungal assay of purified recombinant proteins
4. Results
4.1.Isolation, Cloning and Characterisation of Genomic and cDNA Sequences Coding for β -Glu
4.1.1. Isolation of nucleic acids
4.1.2. PCR amplification of β -1, 3-glucanase gene
3 DNA isolated from leaf tissues
4 RNA isolated from latex and leaf tissues
5 PCR amplification of β-glu gene
6 Amplification of the first strand cDNA
4.1.3. Cloning of the PCR products
7 Schematic diagram of cloning of PCR products in T-vectors
8 PCR with the white / blue colonies to confirm the presence of the insert
9 pGEM vector diagram
10 Confirmation of cloning through restriction analysis
4.1.4. Nucleotide sequencing and sequence analysis
11 Genomic sequences coding for Hevea β-1, 3- glucanase
12 Amino acid sequence of β-glu deduced from the cDNA sequence
13 Amino acid sequence comparison of Hevea mature β-glu with three reported sequences
14 Prediction of the N-terminal signal peptide using SignalP? Protein analysis software.
15 Glycosylation site prediction by NetNGlyc protein analysis tool.
4.1.5. Southern hybridisation
16 Genomic DNA digested with restriction enzymes for Southern blotting
17 Southern hybridisation of genomic DNA with β- glu probe
18 Southern hybridisation with 14 genotypes of Hevea
4.1.6. Isolation of the promoter elements of β -glu gene through RAGE
19 Diagrammatic representation of the RAGE technique
20 Adapter ligated genomic DNA fragments amplified with adapter specific and gene specific primers
21 Nested PCR with the products of the first PCR as template
22 Cloning of the RAGE products in pGEM vector 23 The promoter sequence of rubber β-glu
4.2. Gene expression studies through northern hybridisation and RT-PCR analysis
4.2.1. Fungal infection to the plant material
4.2.2. Isolation of RNA from infected leaves
4.2.3. Amplification of sequences for using as internal controls
24 Phytophthora Sporangial development
25 Zoospore liberation prior to inoculation
26 Plants inoculated with Phytophthora and maintained under favourable conditions for disease development
27 RNA isolated from infected leaf samples
28 PCR amplification of a part of the 18S RNA gene
29 Amplification of a part of the actin gene
30 Sequence of 18 S RNA gene in Hevea
31 Partial sequence of 13-actin gene in Hevea.
4.2.4. Northern hybridisation
32 Northern blot hybridisation analysis and corresponding graphics plot showing differential expression of p-1, 3- glucanase gene
4.2.5. RT-PCR assay for differential expression
33 Relative RT-PCR analysis of control and infected leaf tissues
34 Induction of β- glu transcripts in the infected leaf tissues at different time intervals
35 Comparison of the induction pattern of β-glu in tolerant and susceptible clone in response to pathogen infection
36 Graphic representation of the comparison of β-glu induction in tolerant and susceptible clones
4.2.6. Measurement of enzyme activity
37 Graphic plot on the induction of β-glu expression relative to actin in control and infected leaves of tolerant clone RRII 105.
38 Graphic plot on the induction of β-glu expression relative to actin in control and infected leaves of susceptible clone RRIM 600
39 Comparison of Β-glu expression determined by northern hybridisation and relative RT-PCR
40 Enzyme activity in the infected tissues of tolerant and susceptible clones
41 Northern blot showing constitutive expression of β-glu in the latex
42 Expression levels of β-glu in the latex of different clones
4.2.7. Tissue specific expression
4.3. Construction of functional cDNA clone and purification of recombinant proteins
4.3.1. PCR amplification with restriction site attached primers
4.3.2. Cloning in expression vectors
43 PCR amplification of mature β-1, 3-glucanase gene
44 pGEX and pET vectors isolated for cloning of the gene
45 Diagram of pET 32a+ and pGEX-2T expression vectors
46 Expression vectors double digested with EcoR I and BamH I.
47 Cloning in pGEX and pET vectors
48 PCR analysis of the potential recombinant colonies
49 Confirmation of cloning in pGEX vector through restriction analysis
50 Confirmation of cloning in pET vector through restriction analysis
51 Sequence of the recombinant pGEX vector, showing the reading frame.
52 Sequence of the recombinant pET vector showing the reading frame
53 Recombinant protein expression in pGEX vector
4.3.3. Expression of the target gene
4.3.4. Purification of the target protein
54 Recombinant protein expression with pET vector
55 Column purification of the recombinant fusion protein expressed by the pGEX vector
4.3.5. Confirmation of the induced proteins
56 Purification of pET recombinant protein
57 Purification of pET control protein
58 Western blot showing the detection of target proteins
59 Cleavage of N-terminal fusion tag of the purified recombinant protein
60 Anti-fungal activity of purified Hevea recombinant β-1, 3-glucanase against Phytophthora meadii.
61 Inhibition of fungal growth by purified enzyme
4.3.6. Anti-fungal assay of recombinant proteins
5. Discussion
5.1. Characterisation of β -1, 3-glucanase gene in rubber tree
5.2. Differential expression of Hevea β- glu during pathogenesis
5.3. Recombinant Heveao β -glu and its anti-fungal properties
Prospects of the study
6. Summary and Conclusion
References
List of Figures
ABBREVIATIONS