Verification of the function of tae-miR399 in the freezing
tolerance in transgenic Arabidopsis thaliana
To describe the function of tae-miR399 in tolerance against freezing
stress, we constructed it into an expression vector under the control of
the cauliflower mosaic virus 35S promoter (OEmiR399). After producing
Arabidopsis transgenic plants through several cycles of kanamycin
resistance selection, PCR analysis of genomic DNA with the primers
specific to tae-miR399 and 35S corroborated 9 independent transgenic
lines (Fig. S3A). The total RNA of these 9 Arabidopsis lines was
extracted for their miR399 expression level by PCR semi-quantitative
detection, and the wild type Arabidopsis plant (WT) was used as a
control. The results showed that miR399 was expressed in all OEmiR399
transgenic Arabidopsis plants. Although WT also has miR399 endogenously,
the expression of miR399 in OEmiR399 transgenic Arabidopsis plants was
significantly higher than that in WT. Two lines (OEmiR399-4 and
OEmiR399-6) were selected and self-crossed to obtain stable homozygous
lines.
To investigate the effects of tae-miR399 on tolerance to freezing stress
in Arabidopsis, one-month-old Arabidopsis plants (WT,
OEmiR399-4 and OEmiR399-6) were
transferred to the 4°C for 3 d for cold acclimation and then the
temperature dropped to -10°C for 2 h. Before treatment, there was no
obvious difference in the morphological parameters including synchronous
growth, leaf size and color among these Arabidopsis lines (Fig. 4A), and
the primary roots of OEmiR399 lines were significantly longer than WT
(Fig. 4B).After the cold treatment, the leaves of all Arabidopsis lines
curled as the temperature decreased. Obviously, the leaf color of WT is
darker than that of OEmiR399 lines. Moreover, the tae-miR399
overexpressing plants showed higher survival rates than the rest after
recovering culture (Fig. 4C). To verify whether tae-miR399 improved the
freezing tolerance of Arabidopsis, physiological indicators of all
Arabidopsis lines were determined. The results showed that there was no
significant difference in electrical conductivity and MDA content among
all lines at 24°C (Fig. 4D and E). With the decrease of temperature, the
electrical conductivity and MDA content of all lines increased. At 4°C
and -10°C, the MDA content of OEmiR399 lines were lower than that of WT.
The electrical conductivity of OEmiR399 lines were lower than that of WT
at -10°C (Fig. 4D and E). Generally, the OEmiR399 lines showed better
physiology performance under freezing treatment as compared to other
lines.
qRT-PCR analysis of miR399 and AtUBC24 in all Arabidopsis lines
showed that the expression of miR399 in OEmiR399 transgenic Arabidopsis
plants was always significantly higher than that of WT, and the
expression of its target gene AtUBC24 was always significantly
lower than that of WT (Fig. 5A and B). Moreover, at -10°C, the
expression of miR399 in WT significantly increased and the expression ofAtUBC24 in WT decreased obviously. At 4°C, the expression of
miR399 in OEmiR399 transgenic Arabidopsis plants decreased and the
expression of AtUBC24 in OEmiR399 transgenic Arabidopsis plants
increased (Fig. 5A and B).