Expression analysis of tae-miR399 and TaUBC24 at freezing stress in Dn1
Tillering node is an important organ to winter wheat overwintering (K. K. Peng et al., 2021). To investigate the changes in tae-miR399 andTaUBC24 expression levels in fields under naturally decreasing temperature, the qRT-PCR was performed in Dn1 tillering nodes. The expression of tae-miR399 decreased at 0 ℃ and -10℃ (reached the minimum at 0 ℃), and then it increased significantly at -25℃ (induced more than 9 folds) (Fig. 2A). The expression of TaUBC24 was opposite to that of miR399 in tillering nodes, reached the maximum at 0℃ (induced more than 8 folds), then decreased and peaked the minimum at -25℃ (Fig. 2B).
To eliminate the influence of spatial-temporal differences and other factors in the field on Dn1 tae-miR399, we conducted qPCR experiments on the tillering nodes of Dn1 seedlings cultured in the incubator with short-term freezing treatment. The results showed that the expression pattern of tae-miR399 was similar to that of the field. During cold acclimation at 4℃, the expression of miR399 decreased, and then increased significantly at -10℃ (induced more than 15 folds) (Fig. 2C). The expression change of its target gene TaUBC24 was still opposite to the expression change of tae-miR399. It increased significantly and peaked at 4°C, and then decreased significantly at -10°C, reaching the lowest peak (Fig. 2D). Based on these results, we deduced that tae-miR399 played a positive role in achieving plant tolerance to freezing stress. Meanwhile, from the expression pattern assays, we speculated that tae-miR399 might regulate TaUBC24expression.