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Genomics and Applied Biology, 2011, Vol.2 No.2
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Figure 5 Phylogenetic tree for the upregulated and down regulated genes compared with ALY-family gene in
Nicotiana benthamiana
Note: A: Based on DNA nucleotide sequence; B: Based on deduced amino acid sequence
is relocalized in the presence of P19 from the nucleus
to the cytoplasm, whether the P19 protein similarly is
localized to the cytoplasm or nucleus, and whether the
P19 protein efficiently suppresses RNA silencing. The
RRM domain is conserved in all ALY proteins and has
been shown previously to be necessary and sufficient
for the P19
-
ALY interaction. Our findings indicate
that the interaction among the three genes (cytochrom
oxidase, ALY-family genes, and
P19
) attenuates the
TBSV infection in tomato. We therefore deduce that
these three genes could be considered as member
genes tomato’s innate plant defense system. We also
note that the expression of these genes are directly
proportional to infection time.
It remains to show whether the protein interactions
and RNA-binding capacities of the ALY RRM domain
are directly related to P19 siRNA binding and
silencing suppression activity, and whether the ALY
proteins affect P19 function primarily by altering its
sub-cellular location.
2 Materials And Methods
2.1 Propagation of the Egyptian isolate of TBSV
Purified
Tomato bushy stunt Virus
(TBSV) was
provided by Dr. Fayza Aref, Faculty of science,
Alexandria University, Egypt. The virus was propagated
in
Solanum lycopersicum
by mechanical inoculation
of leaves with purified virus particles. Plants used for
inoculation were 15~20 days old. Mock-inoculated
plants were manually inoculated with the inoculation
buffer (0.01 mol/L phosphate buffer pH 7.0, 0.5 g
Na
2
SO
3
and 1% celite). Plants were grown at 25~30
℃
for two weeks until the viral symptoms appear.
2.2 Resistant and susceptible Tomato cultivars and
symptoms of TBSV
Leaves from inoculated and healthy plants were
collected periodically from the three different varieties
(TY70/84 and TY70/70, resistant to TBSV, TY20,
susceptible to TBSV) and stored at
-
80
℃
. The plants
were maintained for two weeks post inoculation. This
period is enough for the appearance of well charac-
terized symptoms.
2.3 Isolation of total RNA from tomato plant samples
and cDNA synthesis
About 100 mg of the frozen tomato plant leaves
(infected and healthy) was subjected to RNA extraction
using mRNA extraction kit (QIAGene, Germany)
according to the manufacturer’s instructions. For cDNA
synthesis, 2.5 µL from RNAwas combined with 5 µL of a
2
×
reverse transcription mixture containing (50 mmol/L
Tris-HCl (pH 8.3), 50 mmol/L KCl, 4 mmol/L MgCl
2
,
20 mmol/L dithiothreitol), 2.5 µL dNTPs (4 mmol/L),
1 µL oligo dT primer (Promega), 13 µL of RNAs free
water and 1 µL (50 unit/µL) of
Murine Leukemia
Virus
(MLV) reverse transcriptase and incubated at
37
℃
for 1 hr, followed by one cycle for inactivating
the enzyme at 75
℃
for 10 minutes.