Abstract: 【Background】 Pepper is widely cultivated as a vegetable around the world. With the increasing frequency of extreme high temperature weather in recent years, heat stress has become one of the main environmental factors affecting pepper productivity, due to its feature of warm-prone but heat-sensitive. Therefore, it is very important for pepper production to clarify its mechanisms supporting heat-tolerance and then develop pepper varieties with heat tolerance. 【Objective】 Since the heat shock transcription factor HsfA2 plays important roles in plant heat tolerance, the upstream transcription factor of pepper CaHsfA2 was screened and functionally analyzed in heat-tolerance in this study, in order to provide theoretical basis for further understanding the mechanisms of heat tolerance of pepper.【Method】The 955bp promoter sequence upstream of start codon of CaHsfA2 was used as the bait, the yeast one-hybrid (Y1H) technology was applied to screen the upstream transcription factors of CaHsfA2, and their interaction were checked by Y1H point-to-point hybridization, dual-luciferase reporter system (Dual-Luciferase), and LUC assay (LCA). For the candidate upstream transcription factors of CaHsfA2, its dynamic expression under heat stress in pepper heat-tolerant line R9 were analyzed by qRT-PCR technology; its subcellular localization were fulfilled through the transient gene expression technology, and its functional analysis in heat tolerance were performed using the virus-induced gene silencing technology (VIGS). 【Results】 CaBES1 was identified as the candidate upstream transcription factor of CaHsfA2, and their interaction was confirmed. By the analysis of Dual-Luciferase system and CaHsfA2 expression in CaBES1-silenced pepper plants, it is suggested that CaBES1 negatively regulates the transcription of CaHsfA2. The result of subcellular localization showed that CaBES1 was expressed in both cell membrane and nucleus. After heat stress treatment, the fluorescence signal in the nucleus was enhanced, which was consistent with the property of CaBES1 transferring from cytoplasm to nucleus when it performs its biological functions. By dynamic expression pattern analysis, under heat stress, the expression level of CaBES1 decreased firstly and then increased, which also indicates that CaBES1 can respond to heat signal and lays a foundation for the further functional study in heat tolerance. After CaBES1 was silenced in pepper, by comparing the phenotype, relative electrical conductivity and chlorophyll content of silenced plants and control plants, it was inferred that silencing of CaBES1 increased the expression of CaHsfA2 and enhanced the heat tolerance of pepper. 【Conclusion】 CaBES1 inhibits pepper heat tolerance by negatively regulating the expression of CaHsfA2.

Key words: pepper, CaHsfA2, CaBES1, heat stress