*2.7. Prediction of miRNAs Targeting the S1fa Genes in Chinese Cabbage*

miRNAs are a class of non-coding single strand RNA molecules of approximately 22 nucleotides, which are encoded by endogenous genes and may be involved in the activation of genes in response to abiotic stresses. A total of 39 miRNAs targeting the S1fa genes in Chinese cabbage are presented in Table 2, including 5 miRNAs targeting *Bra003132* (*ath-miR5661*, *ptc-miR397c*, *mtr-miR2641*, *hvu-miR6214* and *hme-miR-278*), 11 miRNAs targeting *Bra006994* (ptc-*miR397c*, *zma*-*miR399e-5p*, *aly-miR160c*-*3p*, *ath-miR5661*, *osa-miR160a-3p*, *osa-miR160b-3p*, *zma-miR160b-3p*, *zma-miR160g-3p*, *bdi-miR160b-3p, bdimiR160c-3p* and *ata-miR160c-3p*), 11 miRNAs targeting *Bra029784* (*aly-miR838-3p*, *zmamiR399e-5p*, *ath-miR838*, *osa-miR3982-3p*, *bdi-miR398b, osa-miR2095-3p*, *aly-miR4248a*, *alymiR4248b*, *aly-miR4248c*, *gma-miR4363* and *bdi-miR7757-3p.1*) and 12 miRNAs targeting *Bra034084* (*aly-miR838-3p*, *zma-miR399e-5p*, *ath-miR838*, *mtr-miR2673a*, *mtr-miR2673b*, *gmamiR4363*, *bdi-miR398b*, *osa-miR2055*, *osa-miR3982-3p*, *cca-miR6116-3p*, *stu-miR8050-3p* and *gma-miR9752*).

*Antioxidants* **2022**, *11*, 1782


**Table 2.** Prediction of miRNAs targeting the S1fa genes of Chinese cabbage.

#### *2.8. S1fa Overexpression in Response to Abiotic Stresses in Yeast*

To elucidate the function of the S1fa genes in abiotic stress tolerance, we generated an overexpression model of yeast using the pRS416 vector. The yeast cells with S1fa overexpression were exposed to abiotic stresses (75 μM-Cd, 75 mM-Hg, 100 mM-Al, 50 mM-Cu, 100 mM-Co, 1M-NaCl, 2M-Mannitol, and cold and heat stresses (24 h stress followed by two days with the normal temperature)) (Figure 7(A,B)). The overexpression of *Bra034084*, *Bra003132*, *Bra029784* and *Bra006994* and EV (empty vector) showed no effects on Al, Mannitol, Co and cold stresses, However, the cells were sensitive to NaCl, Hg and Cd stresses, compared with EV (Figure 7(C,F,H)). Under NaCl stress, *Bra006994* and *Bra029784* were highly sensitive, when compared to EV (Figure 7(H)). Likewise, under heat stress (38 ◦C) and cold stress (4 ◦C), all cells with the overexpression of Chinese cabbage S1fa genes grew slightly slower than EV (Figure 7(A,B)). Cells overexpressed with *Bra006994* and *Bra029784* were more sensitive to these stresses (Figure 7(A,B)). These results indicate that the *Bra006994* and *Bra029784* genes play an important role in response to salt stresses.

(**a**)

**Figure 7.** *Cont*.
