## 4 Materials and Methods
### TrhSFB2 Gene Isolation from White Clover and Bioinformatics Analysis  
Initially, the homologous sequences of _TrHSFB2a_ were obtained through the BLAST program of NCBI \"[[http://www.ncbi.nlm.nih.gov/Blast](http://www.ncbi.nlm.nih.gov/Blast)]([http://www.ncbi.nlm.nih.gov/Blast](http://www.ncbi.nlm.nih.gov/Blast)) (accessed on 12 April 2020)\" [51] by searching and identifying conserved sequences region of _TrHSFB2a_ with closely related species and then aligning these sequences with partially published white clover genomes on the NCBI database [[https://www.ncbi.nlm.nih.gov/genome/13404](https://www.ncbi.nlm.nih.gov/genome/13404)]([https://www.ncbi.nlm.nih.gov/genome/13404](https://www.ncbi.nlm.nih.gov/genome/13404)) (accessed on 12 April 2020) [52] to obtain gene sequence information for the designing primers. The premier primer-5 software was used to design primers for _TrHSFB2a_ amplification, and thereby, obtained a full-length CDS sequence of _TrHSFB2a_ from the cDNA library by using the primer pair _TrHSFB2-F_ CTCGCGAACCTTAGAACTCTCA and _TrHSFB2-R_ TCCCTAATCCATCCAACATCAGGTGTA through the touch-down PCR method and Phanta Max Master Mix (see the procedure detail in Supplementary Methods and Supplementary Table S1). The full-length amplified PCR fragments were excised and cloned into the pMD19 simple vector for sequencing; after confirming the _TrHSFB2_ sequence from three sequenced clones through a homology search on the NCBI database, an open reading frame of 924 bp was cloned into the pBI21 overexpression vector (detail below). The molecular weight and pI values of primary _TrHSFB2_ protein sequences were determined using the ExPASy software [[http://web.expasy.org/protparam](http://web.expasy.org/protparam)]([http://web.expasy.org/protparam](http://web.expasy.org/protparam)) (accessed on 1 March 2022) [53]. Clustal Omega \"[[https://www.ebi.ac.uk/Tools/msa/clustalo/](https://www.ebi.ac.uk/Tools/msa/clustalo/)]([https://www.ebi.ac.uk/Tools/msa/clustalo/](https://www.ebi.ac.uk/Tools/msa/clustalo/)) (accessed on 1 March 2022)\" [54] and PSORT \"[[https://www.genscript.com/psort.html](https://www.genscript.com/psort.html)]([https://www.genscript.com/psort.html](https://www.genscript.com/psort.html)) (accessed on 10 December 2020)\" [55] were used for multiple sequence alignment and subcellular protein localization, respectively. The motif and conserved domains were identified using \"[[https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi](https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi)]([https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi](https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi)) (accessed on 1 March 2022)\" [56], and open reading frames (ORFs) were predicted using the NCBI program [[https://www.ncbi.nlm.nih.gov/orfinder](https://www.ncbi.nlm.nih.gov/orfinder)]([https://www.ncbi.nlm.nih.gov/orfinder](https://www.ncbi.nlm.nih.gov/orfinder)) (accessed on 10 May 2020) [57]. The construction of the phylogenetic tree was carried out through the MEGA 11 bioinformatic tool. Plasmid Construction, Genetic Transformation and Developing Homozygous Overexpression Lines of TrHSFB2a  
pSUPER1300-GFP and pBI121 vectors were used for subcellular localization and for constructing the overexpression of _Arabidopsis_, respectively. The complete coding region (without stop codon) of _TrHSFB2a_ was amplified from a previously constructed pMD19 simple vector by the PCR reaction using Prime STAR(r) Max DNA-Polymerase (Takara-Biology Technology (Beijing) Co., Beijing, China) by following manual instructions, and inserted into the restriction sites of _Xbal_ and _BamHI_ of the linearized pBI121 (containing CaMV 35S promoter) and _Xbal_ and _Kpn1_ site of the linearized pSuper1300-GFP using the EasyGeno Assembly Cloning kit (TIANGEN Biotech (Beijing) Co., Ltd., Beijing, China) to generate _TrHSFB2::GFP_ in-frame fused protein according to manual's instruction. The primers for amplifying the coding region and flanking vector sequences for both constructs are enlisted in Supplementary Table S1.