Transcriptional Activation Assay of LbSAD2 in Yeast Cells and in situ Hybridization of LbSAD2 in L. bicolor

The ORF of LbSAD2 was introduced into the vector pGBKT7/BD using Ndel digestion sites according to the instruction of an In-Fusion HD Cloning Kit (Clontech Laboratories, Inc.). The vectors pGBKT7/BD (empty control), pGBKT7-LbSAD2 (experimental group), pGBKT7-lam (negative control), and pGBKT7-LbTTG1 (positive control) were separately transformed into Y2H Gold yeast (Saccharomyces cerevisiae) cells using the Yeastmaker Yeast Transformation System 2 (TaKaRa). After an initial 3-day culture on SD/–Trp medium, the transcriptional activity of the yeast was evaluated according to their growth on SD/–Trp medium for 2 days at 30°C (Guo et al., 2013). β-Galactosidase activity was measured based on the growth on SD/–Trp/X-α-gal plates (Han et al., 2019).

In order to verify whether the location of LbSAD2 was related to the salt gland, in situ hybridization of LbSAD2 was carried using the developing leaves (the first leaves after germination for 5–8 days) of L. bicolor according to Leng et al. (2021). In brief, after fixed with 4% paraformaldehyde and the leaves were embedded in paraffin following gradient alcohol dehydration, 8-μm thin sections were treated with proteinase K, and then hybridized with 6 ng/μl hybridization solution at 37°C overnight. Localization of LbSAD2 digoxin-labeled probe (5′-DIG-GCGAAGACAGAAUCAACACGAACUGGGAGC-3′, purified by HPLC) was detected as blue-violet.

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