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Molecular Plant Breeding Provisional Publishing
Molecular Plant Breeding 2012, Vol.3, No.4, 37
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49
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47
Figure 9
p35SAcS
vector containing the pat selection marker
gene under CaMV 35S promoter and 35S terminator
Note: This vector was used in combination with both type of
vectors conferring constitutive and inducible expression of
HarChit
and
HarCho
3.3 Molecular analysis of putative transgenics
The first step of transgenic analysis is to prove them at
DNA level and then at RNA and lastly at protein level.
Therefore all the BASTA selected plants were first
checked by PCR and Southern analysis and then
Northern Blot/rt PCR analysis was done. Lastly the
activity of the overexpressed proteins was seen using
pathological analysis with pathological fungi (
Erysiphe
graminis
f.sp.
tritici
).
3.4 DNA isolation and Southern Blot analysis
Genomic DNA was isolated by the protocols described
by Palotta et al., 2000. Quality and quantity of isolated
DNA was seen by using the
spectrophotometer from
Eppendorf, Germany and running DNA on agarose gel.
DNA of each sample was divided into three portions;
one was digested with endonuclease that cuts only once
in the cassette, other was digested with endonuclease (s)
cutting the expression cassette out of the plasmid, third
portion was uncut. 25µg from each portion was run on
0.8% agarose gel along with non transgenic wild type
and plasmid DNA as negative and positive controls
respectively. DNAwas transferred onto Nylon membrane
Hybobond NX (Ammersham Braunschweig Germany)
by capillary method (Sambrook et al., 1989) and cross
linked at 1250 J using Stratalinker TM 1800UV
Crosslinker
(Stratagene, La Jolla, USA). The membrane
transferred DNA was hybridized to the DIG labelled
gene specific probes amplified by primer pairs given in
table 2 and detected by CSPD substrate using the
recommended protocols.
3.5 RNA isolation and Northern Blot/RT-PCR
Freshly isolated total RNA was run on the 1 percent
degeneration agarose gel in 1X MEN buffer. RNA
was transferred to Hybond
TM
N+ Nylon membrane by
capillary transfer using 10 X SSC solutions and fixed
to membrane using the method mentioned above for
Southern Blot analysis. For the detection hybridization
positive single stranded gene specific DNA probes
were radioactively labelled with P
32
according to
protocols given by manufacturers of DNA labelling kit
(MBI Fermentas st. Leon-Rot, Germany). Detections
were done on the X-ray films (Amersham, USA).
Northern blot analysis was done for transgenics where
the genes (
HarChit
and
HarCho
) were constitutively
expressed. The trangenics where the genes were under
inducible promoter, the induction was done by rubbing
the leaf surface with sea sand and total RNA was
isolated eight hours afterwards (Leckband and Loerz,
1998 and Serazetdinova et al., 2005) cDNA was
synthesized from the total RNA using 18-mer
oligonucleotide (Oligo(dT)
18
) primer, dNTPs, RNAse
inhibitor and the Moloney murine leukaemia virus
reverse transcriptase (M-MuLV) as recommended by
the reagents manufacturer (Fermentas Life Science, St.
Leon Germany). Gene specific primers were used to
amplify the transcripts of
HarChit
and
HarCho
out of
cDNA from respective transgenic plants and lines.
3.6 Inoculation with
Erysiphe graminis
f sp.
tritici
Inoculation studies with
Erysiphe graminis
f sp.
tritici
was performed on detached leaf segments using the
method given below. All the pathological assays were
revised at least thrice.
3.7 Detached leaves infections
In these experiments second leaf of two weeks old
seedlings of T
1
transgenic (Both over expression and
knock down lines) and control lines (wild type Florida
as well as transgenic line containing
bar
gene only)
were cut into 3 cm long segments. These segments
were cultured on an anti-senescence media containing
0.4% agar, 10 ppm (parts per million) benzamidazole
and 1ppm silver nitrate. The protocols have already
been used by Oldach et al., 2001 and Girgi et al., 2006.
Inoculations were done in the settling tower by blowing
the freshly harvested powdery fungal material mixed
with equal volume of baby talcum powder onto the
cultured leaf segments and at least one hour was given
to settle the fungus down onto the plates. It was made
sure that at least 300
-
400 conidia should be available
per cm². Conidia were counted by putting a scale in the