Isolation of drought related MYB transcription factor gene from brassica carinata
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Date
2006
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Publisher
CCSHAU
Abstract
The present study was conducted to isolate the drought
related MYB transcription factor gene from Brassica carinata
using RT-PCR approach. Primers were designed using AtMyb2
gene sequence of Arabidopsis thaliana, a well established drought induced
Myb gene. No similar sequence was found in Brassica species when gene
sequence was used in BLASTN search using NCBI website
www.ncbi.nlm.nih.gov. The primers were based on conserved DNA binding
domain of Myb gene.
Brassica carinata seeds were grown on MS medium for 14 days,
seedlings were uprooted and then exposed to air for 1hr/2hrs.dehydration
treatment. Total RNA was isolated using Trizol Reagent method. Total RNA
yield varied from 2250-2262.5 μg/g fresh wt. and two distinct ribosomal
bands (28S and 18S) were observed when total RNA was analysed on
formaldehyde and agarose gels. Isolation of mRNA was done using
Genelute mRNA miniprep kit and its concentration ranged from
148.8-211.7μg/g fresh wt. i. e. one-tenth of the total RNA.
RT-PCR was carried out using Qiagen one step RT-PCR kit with
Q-solution and total RNA as well as mRNA as template. All the three sets
of primers specific to Myb gene showed amplification. Set III (GhMYB)
amplified a product of around 200 bp. A common band of approx. 500 bp
was observed with Set I and Set II with mRNA as template.
The cDNA was eluted from gel and further amplified. The amplified
fragment was transformed using pPCR ScriptTM Amp SK(+) Cloning
Vector (Stratagene) and pDrive cloning Vector (Qiagen) in XL-Blue strain of
E. coli using blue-white selection. Transformation frequency in the range of
10.00 to 21.05 per cent was observed.
The transformed clones were characterized by carrying out plasmid
DNA isolation, PCR amplification of plasmid DNA and restriction enzyme
digestion of plasmid DNA. The isolated plasmid DNA from transformed
(white) colonies showed higher vector size when analyzed on agarose gel
suggesting that DNA fragment was inserted into vector. Further, the
plasmid DNA restriction pattern confirmed the cloning event.