###### Abstract  
Heat shock transcription factors (HSF) are divided into classes A, B and C. Class A transcription factors are generally recognized as transcriptional activators, while functional characterization of class B and C heat shock transcription factors have not been fully developed in most plant species. We isolated and characterized a novel HSF transcription factor gene, _THSFB2a_ (a class B HSF) gene, from the drought stress-sensitive forage crop species, white clover (_Trifolium repns_). _THSFB2a_ was highly homologous to _MtHSFB2b_, _CarHSFB2a_, _AnHSFB2b_ and _HHSFB2a_. The expression of _THSFB2a_ was strongly induced by drought (_PEG6000_ 15% \(w/r\)), high temperature (35 \({}^{\circ}\)C) and salt stresses (200 mM L\({}^{-1}\) NaCl) in white clover, while subcellular localization analysis showed that it is a nuclear protein. Overexpression of the white clover gene _TrHSFB2a_ in _Arabidopsis_ significantly reduced fresh and dry weight, relative water contents (RWC), maximum photosynthesis efficiency (Fv/Fm) and performance index on the absorption basis (PIABS), while it promoted leaf senescence, relative electrical conductivity (REC) and the contents of malondialdehydedehyde (MDA) compared to a wild type under drought, heat and salt stress conditions of _Arabidopsis_ plants. The silencing of its native homolog (_AtHSFB2a_) by RNA interference in _Arabidopsis thaliana_ showed opposite trends by significantly increasing fresh and dry weights, RWC, maximum photosynthesis efficiency (Fv/Fm) and performance index on the absorption basis (PIABS) and reducing REC and MDA contents under drought, heat and salt stress conditions compared to wild type _Arabidopsis_ plants. These phenotypic and physiological indicators suggested that the _THSFB2a_ of white clover functions as a negative regulator of heat, salt and drought tolerance. The bioinformatics analysis showed that _THSFB2a_ contained the core B3 repression domain (BRD) that has been reported as a repressor activator domain in other plant species that might repress the activation of the heat shock-inducible genes required in the stress tolerance process in plants. The present study explores one of the potential causes of drought and heat sensitivity in white clover that can be overcome to some extent by silencing the _TrHSFB2a_ gene in white clover. _Trifolium repns; heat shock factor; abiotic stress; overexpression; gene silencing_4.0.10.390.1223.12769. and shows significant growth retardation under stress conditions [1], which restrict its development and production globally [2; 3]. Therefore, it is of great theoretical and practical significance to strengthen the research to improve drought tolerance in white clover and cultivate drought-tolerant varieties by identifying, studying and manipulating downstream genes and molecular mechanisms. Heat shock proteins (HSPs), as molecular chaperones, play significant roles in responding to environmental stresses and taking part in protein folding, assembly and stability, which are important threats under stressed conditions in plants [4; 5]. The expression of HSPs is controlled by heat shock transcription factors, containing conserved binding domains known as heat stress elements (HSE), mostly existing in promoter regions of the heat stress-inducible/responsive genes and are conserved almost in all eukaryotes. Structurally heat shock transcription factors (HSFs) comprise of a main DNA-binding domain (DBD) of a helix-turn-helix motif in the N-terminal region, which generally binds with the HSE present in the promoter region of heat stress-inducible genes [6]. Following the DBD domain, there is an oligomerization domain (OD), associated with hept hydrophobic repeat motifs, and a NLS (Nuclear localization signals) domain containing a basic amino acid residue motif and a leucine-rich motif in the HR-C region for export (NES). The C-terminal region is comparatively less conserved, comprising of hydrophobic, aromatic and acid amino acid (AHA) motifs, which are essential for transcriptional activity [6].