Cloning and characterization of gene from mustard (Brassica napus)
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Date
2007
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UAS, Dharwad
Abstract
The overexpression of non-expressor of PR proteins (NPR1) 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, NPR1 gene
from mustard (Brassica napus) was cloned by using gene specific primers. The gene
encoding NPR1 protein was amplified and cloned in pTZ57R/T vector, sequenced and
analyzed in silico. Cloned NPR1 gene had 98 per cent homology with reported NPR1 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 NPR1 protein of mustard has an ankyrin repeat
and a BTB/POZ (Broad-complex, Tramtrack, and Bric-a-brac/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 evolutionary relationship between
these proteins.
To facilitate transformation of crop plants, NPR1 gene was subcloned into a plant
transformation vector pHS100 at XbaI and BamHI 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 PCR checked with
NPR1 gene specific primers, 5 were positive. These positive plants need to be analyzed
further for the expression of NPR1 gene.