Real - Time pcr assay for #-1, 3-glucanase gene expression in black pepper(piper nigrum L)
By: Dukare Kiran shankar.
Contributor(s): Nazeem P A (Guide).
Material type:
BookPublisher: Vellanikkara Centre for Plant Biotechnology and Molecular Biology, College of Horticulture 2009Description: 120.DDC classification: 660.6 Online resources: Click here to access online Dissertation note: MSc Abstract: Cultivated black pepper (Piper nigrum) varieties are susceptible to Phytophthora rot, caused by Phytophthora capsici, resulting in yield losses upto 50 per cent. Since conventional breeding programmes for this perennial spice crop are complex and time consuming, an attempt was made to unravel the mechanism for disease tolerance at the molecular level.
-1,3-glucanases are the pathogenesis-related proteins reported in black pepper. The present study was carried out to confirm the role of -1,3-glucanases in the defence mechanism and to unravel the mode of expression in comparison with the resistant genotype, Piper colubrinum.
Real-time PCR (polymerase chain reaction) assay and northern blotting were performed to determine the expression pattern of the gene encoding -1,3-glucanases in both susceptible and resistant genotypes after infection with the fungus Phytophthora capsici. The data was normalized with a stably expressing housekeeping gene. Southern hybridization was carried along with real-time PCR to determine the gene copy number in both P. nigrum and P. colubrinum.
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was identified as the reference gene (endogenous control), since it was more stable than 18S rRNA and actin genes studied. The real-time PCR assay indicated marked difference in -1,3-glucanase at transcript level in the genotypes studied, when the data was normalized with endogenous control. The rate of increase in transcript level after infection with the pathogen was low and gradual in P. nigrum while it was much higher and faster in P. colubrinum. The glucanase enzyme activity at healthy stage in P. colubrinum was 88.00 unit/mg protein/10 min, while it was only 26.29 unit/mg protein/ 10 min in P. nigrum. Upon inoculation with the fungus, the gene expression was elevated over four times within two hours after inoculation in P. colubrinum while it was much lower in P. nigrum. The northern blot analysis also indicated differential expression of the gene. Southern hybridization indicated that the gene copy number varied in two species, while real-time PCR assay confirmed that it was almost double in P. colubrinum than in P. nigrum.
The results clearly indicate the positive role of -1,3-glucanases in the defence mechanism and highlight the differential gene expression in susceptible and resistant genotypes.
| Item type | Current location | Call number | Status | Date due | Barcode |
|---|---|---|---|---|---|
Theses
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KAU Central Library, Thrissur Theses | 660.6 DUK/RE PG (Browse shelf) | Available | 172934 |
MSc
Cultivated black pepper (Piper nigrum) varieties are susceptible to Phytophthora rot, caused by Phytophthora capsici, resulting in yield losses upto 50 per cent. Since conventional breeding programmes for this perennial spice crop are complex and time consuming, an attempt was made to unravel the mechanism for disease tolerance at the molecular level.
-1,3-glucanases are the pathogenesis-related proteins reported in black pepper. The present study was carried out to confirm the role of -1,3-glucanases in the defence mechanism and to unravel the mode of expression in comparison with the resistant genotype, Piper colubrinum.
Real-time PCR (polymerase chain reaction) assay and northern blotting were performed to determine the expression pattern of the gene encoding -1,3-glucanases in both susceptible and resistant genotypes after infection with the fungus Phytophthora capsici. The data was normalized with a stably expressing housekeeping gene. Southern hybridization was carried along with real-time PCR to determine the gene copy number in both P. nigrum and P. colubrinum.
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was identified as the reference gene (endogenous control), since it was more stable than 18S rRNA and actin genes studied. The real-time PCR assay indicated marked difference in -1,3-glucanase at transcript level in the genotypes studied, when the data was normalized with endogenous control. The rate of increase in transcript level after infection with the pathogen was low and gradual in P. nigrum while it was much higher and faster in P. colubrinum. The glucanase enzyme activity at healthy stage in P. colubrinum was 88.00 unit/mg protein/10 min, while it was only 26.29 unit/mg protein/ 10 min in P. nigrum. Upon inoculation with the fungus, the gene expression was elevated over four times within two hours after inoculation in P. colubrinum while it was much lower in P. nigrum. The northern blot analysis also indicated differential expression of the gene. Southern hybridization indicated that the gene copy number varied in two species, while real-time PCR assay confirmed that it was almost double in P. colubrinum than in P. nigrum.
The results clearly indicate the positive role of -1,3-glucanases in the defence mechanism and highlight the differential gene expression in susceptible and resistant genotypes.
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