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Molecular Plant Breeding Provisional Publishing
Molecular Plant Breeding 2012, Vol.3, No.4, 37-49
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41
from young spikes of the entire Southern Blot analysis
positive T
0
and selected T
1
progeny plants was run on
1% degeneration gel at the rate of 15 µg per probe/lane
(3A) and transferred to the Nylon membrane by capillary
method under clean environment and cross linked later.
This RNA was then hybridized with 400 bp long radio
actively labelled
HarChit
specific single stranded
DNA and were detected on X-Rays film. Here I.A
-
18,
I.A
-
5, I.A
-
2 and I.A
-
17 are transgenic lines. –ve= is
the RNA from control non transgenic plant. T
0
is the
RNA from primary transgenic plant and T
1
P
1
to T
1
P
4
is the RNA from four selected plants from progeny of
the primary transforments.
1.6 Induction of
HarChit
and
HarCho
genes under
the control of
Vst1
promoter
The expression of
HarChit
and
HarCho
genes was
induced by wounding with sea sand on the leaf
surfaces in the transgenics where they were under
Vst1
promoter. RNA was isolated eight hours after
induction as reported by Leckband, 1997 and cDNA
was synthesized. Later on PCR reactions were done
using c-DNA as template and gene specific primers
for both
HarChit
and
HarCho genes
. All the five
plants which were Southern Blot analysis positive for
pVst-HarChit
showed an expected band of 400 bp and
all the six plants positive for
pVst-HarCho
showed an
expression for
HarCho
gene. The PCR product from
all of them was compared with negative (non transgenic)
control, PCR (H
2
O was used as second negative to check
any contamination) and positive control (plasmid
DNA specific product). The PCR products were also
Figure 3 Northern analyses of T
0
and T
1
generation with
pUbi-HarChit
construct
Table 2 Primer sequences used for Southern, Northern and RT-PCR
Primer name
Primer sequence (5′-3′)
Length (bp)
Product (bp)
Iqrar-cho400for
GACAACCACAAATCGGGAACG
21
400
Iqrar-cho400rev
CGTGGCCGTTATCACCGCT
19
Iqrar-chit400for
GAGAAGAGAGCCAACGGATACGC
23
400
Iqrar-chit400rev
TCAATACCATCGAAACCCCAATC
23