CLONING AND CHARACTERIZATION OF NPRl GENE FROM MUSTARD (Brassica naous)
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Date
2007-08-30
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University of Agricultural Sciences GKVK, Banglore
Abstract
The overexpression of non-expressor of PR proteins [NPRl] gene
has proved effective in providing resistance to a broad spectrum of
pathogens in different plant species, indicating its functionality across a
wide taxonomic range. In the present study. NPRl gene from mustard
[Brassica napus) was cloned by using gene specific primers. The gene
encoding NPRl protein was amplified and cloned in pTZ57R/T vector,
sequenced and analyzed in sUico. Cloned NPRl gene had 98 per cent
homology with reported NPRl gene of mustard at both nucleotide and
protein level. It has four exons with three stretches of internal introns
(642-851.1588-1801,1970-2055). The NPRl protein of mustard has an
ankyrin repeat and a BTB/POZ (Broad-complex, Tramtrack, and Bric-abrac/
pox virus and zinc finger) domain and codes for 579 amino acids.
Limited homology is also detected to the mammalian transcription factor
inhibitor IkBa, This implies a close evolutionaiy relationship between
these proteins.
To facilitate transformation of crop plants, NPRl gene was
subcloned into a plant transformation vector pHSlOO at Xbal and BarhHl
restriction sites. The recombinant vector pHSAM was mobilized into
Agrobacterium tumefaciens LBA4404 by tri-parental mating.
A. tumefaciens with recombinant clone pHSAM was used for transforming
tobacco. A number of kanamycin resistant plants were generated. Of the
15 plants PGR checked with NPRl gene specific primers, 5 were positive.
These positive plants need to be analyzed further for the expression of
NPRl gene.
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