## 2 Results  
### TrHSFB2a Was Predicated as One of the Class B HSF Proteins  
The sequence of the isolated _TrHSFB2a_ gene comprises an open reading frame (ORF) of 924 bp (Figure 1a), which constitutes a protein of 307 amino acids (Figure 1c). Interpreting scan results showed that this protein belonged to the heat shock transcription family and was predicated as the heat stress transcription factor B-2a (HSFB2-a). It contained a heat shock factor-type, DNA-binding domain (HSF-DNA-bd) of 93 amino acids (20-113), commonly associated with DNA-binding transcription factor activity, sequence-specific DNA binding and regulation of DNA-dependent cellular transcription. The N-terminal region contained a helix-turn-helix motif (H2-T-H3) in the center of the DNA binding domain (DND) (Figure 1c). Based on the homology of amino acids, _TrHSFB2a_ showed an overall 80.46%, 70.61% and 73.74% homology with _TrHSFB2b_, _MhHSFB2b_ and _CarHSFB2a_, respectively (Figure 1b). Additionally, amplification and sequencing of _TrHSFB2a_ from white clover genomes showed it has one intron of 107 bp, similar to other HSF genes of plants. Based on conserved domains of HSF genes, these are divided into Class A, B and C members. Phylogenetic tree constructed using 27 HSF proteins showed _TrHSFB2a_ clustered with class B HSF proteins of closely related species and _Arabidopsis_, indicating that it is one of class B HSF proteins (Figure 1d).  
Figure 1: The characteristics of _TrHSFB2a_ gene and protein sequence, and the phylogenetic analysis. (**a**) shows amplified DNA fragment of _TrHSFB2a_ gene from white clover cDNA separated on 1.5% agarose gel, (**b**) sequence similarity of the predicted protein of _TrHSFB2a_ gene with other species, (**c**) comparison of amino acids sequences of _TrHAFB2a_ with related HSF proteins—highly homolog amino acids residue are shaded and conserved domains are boxed. (**d**) Phylogenetic relationship of the protein of _TrHSFB2a_ gene with other plant species and other members of HSF family of the _Arabidopsis thaliana_. Triangles size in branches represents bootstrap values. Phylogenetic tree analysis was based on minimum evolution using Mega (version 11).  
Bioinformatics tools were used to understand physiochemical and structural characteristics of TrHSFB2a and results are presented in Supplementary Figure S1. Using pro-param predication [31], it was found that the molecular formula of TrHSFB2a was C1499H2383N4250A75511, molecular weight was 34,311.63, theoretical isoelectric point was 8.7, instability index was 49.92, and it was an unstable protein. DeepTMHMM [32] predicted that it was a globular protein and had no transmembrane structure (Supplementary Figure S1a). Hydrophobicity analysis using ProtScale revealed that it was a hy-drophobic protein (Supplementary Figure S1b), with an average hydrophitability \(-0.566\). The secondary structure of the encoded protein predicted by SOPMA [33] showed that the secondary structure was composed of 33. 88% \(\alpha\) helix, 2.93% \(\beta\)-turn, 55.70% random coil and 7.49% extended strand (Supplementary Figure S1d). The phosphorylation sites of protein sequence determined by Netphos 3.1 server [34] showed TrHSFB2a protein had 58 phosphorylation sites, including 32 serine sites, 19 threonine sites and 7 tyrosine sites (Supplementary Figure S1c). Protein signal peptide analysis using Signalp 4.1 server [35] showed that the protein had no signal peptide (Supplementary Figure S1e). The spatial protein structure predicted by SWISS-Mod. server [36] revealed that the three-dimensional structure had a monomer structure (Supplementary Figure S1f).