Isolation and Characterization of a Gene Coding for Chitinase in Developing Winged Bean Seed (Psophocarpus Tetragonolubus )
Chitinase, which catalyse the hydrolysis of the β-1,4-N-acetyl-D-glucosamine linkages of the fungal cell wall polymer chitin, is involved in inducible defences of plants. The aim of this research is to isolate a chitinase genes from seed of winged bean. In order to isolate genes encoding for chit...
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| Format: | Thesis |
| Language: | English English |
| Published: |
2000
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| Subjects: | |
| Online Access: | http://psasir.upm.edu.my/id/eprint/8421/1/FSMB_2000_11_IR.pdf |
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| Summary: | Chitinase, which catalyse the hydrolysis of the β-1,4-N-acetyl-D-glucosamine
linkages of the fungal cell wall polymer chitin, is involved in inducible defences
of plants. The aim of this research is to isolate a chitinase genes from seed of
winged bean. In order to isolate genes encoding for chitinases from a cDNA
library of winged bean seed, two sets of degenerate primers were designed which
corresponded to conserved regions of chitinases class I and class IV. These were
then reverse transcribed and subsequently amplified using polymerase chain
reactions (RT -PCR) using 4 weeks old seeds cDNA as template. A 1.1 kb
fragment was cloned, and subjected to terminal sequence analysis to verify the
presence of sequences encoding for chitinases. Nucleotide sequence analysis showed that the fragment appears to encode for a class I chitinase, and this
fragment was then used as a probe to screen for a full length gene from the
winged bean seed cDNA library.
After library screening one clone, CHRZP was isolated and found to encode a
chitinase gene. The complete nucleotide sequence of the winged bean chitinase
(1324 bp) encoded a polypeptide of 289 amino acids that encodes for a basic
chitinase with cysteine-rich domain at the N-terminal. A Comparison of amino
acid sequence showed 88% similarity to a chitinase sequence isolated from Oryza
sativa. RNA blot hybridisation revealed that mRNA that corresponded to CHRZP
accumulates to high levels in leaves compared to seed, tuber and pod with a
transcript size of 1.0 kb. Southern hybridisation analysis indicated that this gene
was present as a single copy gene in the winged bean genome. |
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