*4.5. Total RNA Extraction and qRT-PCR Analysis*

The total RNA was extracted for Chinese cabbage tissues using TRIzol, while yeast RNA was extracted using the M5 EASYspin yeast RNA rapid extraction kit, MF158-01 (Mei5 Biotechnology, Co., Ltd. Beijing China). For yeast RNA extraction, the cells were grown until the OD600 value reached 0.3 at 28 ◦C, and then treated with 1 M NaCl for 12 h before the total RNA was harvested [45]. The first-stand cDNA was synthesized using a PrimeScript and RT reagent kit with gDNA Eraser (TAKARA). The SYBR Premix Ex-Taq Kit (TAKARA) was used for quantitative real-time PCR. All experiments were performed with three independent biological replications. The transcript levels were calculated using the 2ΔΔ-CT method. The TMP values of Chinese cabbage tissues were obtained from the Chinese cabbage database (http://brassicadb.cn (accessed on 22 March 2022)) for each S1fa gene. The primers used for qRT-PCR are presented in Supplementary Table S1.

#### *4.6. Yeast Constructs*

To construct the yeast (*Saccharomyces cerevisiae*) overexpression vectors, the coding sequences of Chinese cabbage genes, *Bra034084*, *Bra003132*, *Bra029784* and *Bra006994*, were cloned separately into the pRS-416-GFP vector. The coding sequences of the S1fa genes were amplified from Chinese cabbage cDNA with specific primers (Supplementary Table S1) and then inserted into the SPE1 site on pRS-416-GFP using the infusion cloning kit (Catalog no. 011614; Clontech) [46]. The sequence insertions were confirmed through SANGER sequencing and then used for the investigation of abiotic stress tolerance in yeast. To determine the subcellular localization of the S1fa proteins, the S1fa genes were inserted into pRS-416-GFP. The subcellular localization of the fusion proteins was observed under a Zeiss Axiophot fluorescence microscope as described previously [45].
