Expression of myb gene in brassica tournefortii L.under drought stress
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Date
2009
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Publisher
CCSHAU
Abstract
Brassica tournefortii, a highly drought tolerant Brassica
species was used to study myb gene expression under drought stress.
Seeds of Brassica tournefortii were grown on MS medium.
Then 14 days seedlings were uprooted and given drought stress by
subjecting them to air drying, PEG (-2 to -8 bar) and mannitol (100 mM
to 400 mM) treatments. Total RNA isolation from stressed seedlings was carried out using Trizol reagent yielding 17.52-36.8 Fg/ml of total RNA.
One- step RT-PCR was carried out using total RNA as template with
BjMyb-1 primer designed from conserved domain of AtMyb2 and its
homologous. BjActin primers were used in RT-PCR which served as
control, as actin gene is constitutively expressed in all tissues. Exposure
to drought stress for 15 minutes and 30 minutes (air drying) gave no
amplification showing air drying upto 30 minutes does not induce any
myb expression. An amplified product of 250 bp was obtained on
exposure to drought stress for 60 minutes with air drying, PEG (-2 to -8
bar) and mannitol (100 mM to 400 mM) treatments. The transcript level
was found similar in all treatments irrespective of drought treatments.
cDNA was eluted out from the gel and purified cDNA was
transformed using pDrive cloning vector (Quigen) in XL-blue strain of
E.coli using blue- white selection. Transformed clones were characterized
by plasmid DNA isolation, PCR amplification of plasmid DNA with gene
specific primers. Plasmid DNA from transformed clones showed higher
molecular weight than untransformed plasmid DNA on agarose gel
electrophoresis confirmed the insertion of DNA fragment into the
plasmid. PCR amplification of plasmid DNA also confirmed the
successful cloning.
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Keywords
Fruits, Genotypes, dna, Acidity, Rapd, Limes, Genetics, Confectionery, Polymorphism, Developmental stages