Phylogenetic and Expression Analysis of Soybean HXK Genes

The sequences of the GmHXKs proteins obtained from the G. max genome were aligned using DNAMAN 7.0 software to search for conserved domains by inspection using sites present in AtHXK1 as a reference. To compare evolutionary relationships, the putative HXKs from G. max, A. thaliana, Solanum lycopersicum, O. sativa and Nicotiana tabacum were used to construct the phylogenetic tree using MEGA-X with the neighbor-joining (NJ) method and 1,000 bootstrap replicates (Kumar et al., 2018 (link)). Expression data on GmHXK gene family members at different developmental stages and in different tissues under normal conditions were downloaded from the Soybase (https://www.soybase.org/). Data on differential expression for only 14 members were eventually obtained and used for subsequent analysis.
To analyze expression pattern of soybean seedlings under salt stress, soybean seedlings were grown in a growth chamber under greenhouse conditions of 28°C under a16-h light/8-h dark cycle. Three-week-old seedlings were treated with 0.5% NaCl (salt stress) or drought treatment (10%PEG 6000). The root samples of the seedlings were collected after treatment for 2-h, 8-h, 24-h, and 72-h. Then, different samples were frozen quickly in liquid nitrogen, and stored at −80°C for RNA extraction and analysis. Total RNA was isolated using the Plant RNA Kit (CWBIO, Beijing, China), and its concentration and purity were determined by Nanodrop2000 nucleic acid analyzer (Thermo, America). First-strand cDNA was synthesized from 0.5 µg of total RNA using the HiFi-MMLV cDNA Kit (CWBIO, Beijing, China), and then used as a template for qRT-PCR analysis using gene-specific primers (Supplementary Table S1). Data analysis of RT-qPCR was performed using 2^−ΔΔCT method (Livak and Schmittgen, 2001 (link)).