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Bioscience Methods 2014, Vol.6, No.1, 1-13
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8
The sequences of certain
StOSM
members, such as
StOSM-3B, -3F
and
-
5A, share greater than 95%
identity, which makes distinguishing each member
precisely by RT-PCR difficult. A visible distinction of
these
StOSMs
on the gel displaying the RT-PCR
products confirmed the specificity and reliability of
the eleven primer pairs to distinguish each gene by
RT-PCR. Therefore, these primers were used to
quantify the expression of the eleven
StOSM
mRNAs
in leaves
.
1.2.3 Real-time quantitative reverse transcription-PCR
(qRT-PCR) for
StOSM
expression analysis
Real-time qRT-PCR was performed for all
StOSM
members to determine the leaf RNA accumulation
patterns in response to decreasing water availability.
The mRNA accumulation of eleven
StOSMs
in the
leaf induced by 10% WCM (also DLP) as the
treatment and by 70% WCM as the control was
quantitatively assayed (Figure 10). Similar to the
qualitative assay
results
,
eight of eleven
StOSMs
under the 10% DLP condition displayed significant
increases in RNA accumulation compared with the
control condition.
The changes in the eleven
StOSMs
induced by drought
varied from gene to gene (Figure 4). Leaf
StOSM
mRNA accumulation for the genes upregulated by
DLP was 4-49-fold higher than in the control. The
peak
StOSM-
8E mRNA accumulation was greater than
49-fold higher than in the control.
StOSM
-297 mRNA
accumulation decreased 9-fold in response to drought
compared with the control.
StOSM
-251 showed exceptional leaf mRNA
accumulation. No significant difference was observed
in the relative levels of transcripts between the
treatment and control leaves (Figure 10). This finding
was not in agreement with the FPKM value and
qualitative assay results.
This comparison clearly revealed that the result of the
qualitative assay is in agreement with the mRNA
quantification and FPKM value results from the
RNA-Seq analysis (Table 2). This observation
indicates that drought does induce the regulation of
the expression of the eleven
StOSMs.
With eight
members of
StOSM
being upregulated and three being
downregulated,
StOSM
s play a positive role in
enhancing plant tolerance to stress.
Table 2 Comparison of the qualitative and quantitative
OSM
mRNA accumulation under water deficiency
TF
OSM
182
297
251
8E
1G
5A
3B
3F
3C
2D
306
FPKM
＋
－
－
＋
＋
＋
＋
＋
＋
＋
－
RT-PCR
＋
－
－
＋
＋
＋
＋
＋
＋
＋
－
qRT-PCR
＋
－
－
＋
＋
＋
＋
＋
＋
＋
－
Note:
－
stands for down-regulated;
＋
for up regulated
Therefore, osmotins can be considered to be drought
responsive molecules involved in withstanding water
deficits. Recent reports provide direct evidences to
support osmotin as a drought responsive molecule.
Expression of a osmotin-like SnOLP from
Solanum
nigrum
, 93.0% identity on aminoacid sequence with
OSM-3F (PGSC0003DMG400003044) in
S. tuberosum
confers drought tolerance in transgenic soybean
(Weber et al., 2014). Aslo, overexpression of OSM-3F
gene confers tolerance to salt and drought stresses in
transgenic tomato (
Solanum lycopersicum L.
) (Goel et
al., 2010).
Drought stress is an important environment factor
limiting crop production in some areas. Up to date, a
litter is known on how to enhance crop tolerance to
drought stress. This study not only provides a method
on how to screen isoforms of a gene from a genome
database, but also disclose response of osmotin in
drought tolerance. This is a practical option to dig a
genome for member identification of a gene family.
2 Discussion
From a metabolic point of view, the plant response to
water stress involves the generation of intercellular
components concerned with the regulation of water
potential. Osmotin is a component that accumulates
substantially when induced by water stress. Previous
studies revealed that the enhancement of osmotin
expression can increase plant tolerance to fungi and
abiotic stress. However, little is known regarding the
mechanism of osmotin in the response to stress.