ISOLATION AND EXPRESSION OF rafpl AND rafp2 ENCODING ANTIMICROBIAL PEPTIDES FROM A LOCAL CULTIVAR OF Raphanus sativus
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Date
2007-08-30
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University of Agricultural Sciences GKVK, Banglore
Abstract
Pathogens are known to develop varying levels of resistance to the
applied chemicals, resistant varieties and biological molecules.
Antimicrobial peptides that act mainly through disintegrating the cell
membrane of the pathogen offer a new option for creating resistance to the
pathogens.
In this study, a working database with reported AMPs was
constructed, which could be queried and updated. 350 bp and 300 bp DNA
fragment corresponding to rafpl and rafp2 genes were amplified using
specific primer pairs, designed based on their published nucleotide
sequence in Raphanus sativus. The rafpl and rafp2 from a local cultivar of
R. sativus were cloned in pTZ57R/T and transferred to E. coli DH5a. The
presence of the inserts was confirmed by sequence analysis and nucleotidenucleotide
BLASTn search. The sequence comparison showed that the
cloned rafpl and rafp2 had 99 per cent homology with respective, published
sequences of R. sativus. The defensin genes rafpl and rafp2 were subcloned
in a prokaryotic expression vector pET28a(+). The SDS-PAGE analysis of
total protein from recombinant clones pKKK166A and pKKK166B in E. coli
BL21pLysS showed the expression of 12.6 kDa and 10.6 kDa protein,
respectively. The crude protein extract from transformed E. coli reduced the
growth of Sclerotium rolfsii, Rhizoctonia bataticola and Fusarium solani.
The rafpl and rafp2 inserts were further subcloned into a plant
transformation vector pHSlOO and mobilized into Agrobacterium
tumefaciens LBA4404 by triparental mating. The A. tumefaciens with
recombinant clone pKKK207B containing rafp2 was used to transform
tobacco. The putative transformants were subjected to PGR analysis and
four plants were found to be PGR positive.
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