关键词: 小麦, GRAS转录因子, 热胁迫, 基因沉默, 蛋白互作

Abstract:

【Objective】The GRAS family constitutes a unique class of plant-specific transcription factors that play a pivotal role in plant development and stress response. To elucidate the function of GRAS family genes in wheat heat tolerance，which can provide genetic resources and theoretical foundation for wheat heat-resistant breeding.【Method】A potential heat stress-responsive transcription factor gene, TaGRAS34-5A, was identified through transcriptome analysis of TAM107 and Chinese spring wheat seedlings under high-temperature conditions. Subsequently, a bioinformatics analysis was performed on TaGRAS34-5A, and a phylogenetic tree was constructed to elucidate its molecular characteristics. The expression pattern of TaGRAS34-5A under various stresses, including high temperature, abscisic acid (ABA), ethylene (ETH), and salicylic acid (SA) treatments, were examined using real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) method. The subcellular localization of the TaGRAS34-5A protein was determined using wheat protoplast transient expression technique. Furthermore, the heat tolerance function of TaGRAS34-5A was validated using the heterologous expression system of Saccharomyces cerevisiae and the BSMV:VIGS (Barley stripe mosaic virus: Virus-Induced Gene Silencing) silencing technique. potential interacting proteins of TaGRAS34-5A were screened using yeast two-hybrid technology, and the heat tolerance function was verified, providing preliminary insights into its heat tolerance mechanism.【Result】TaGRAS34-5A, equipped with a characteristic GRAS domain and belongs to the GRAS transcription factor family, is localized to both the cell nucleus and cytoplasm. Bioinformatics analysis indicates that the TaGRAS34-5A promoter contains a large number of hormone response elements and light response elements, and it is most closely related to TaSCL14, OsGRAS23, and AtSCL14 in terms of phylogenetic relationships, suggesting its potential function in responding to oxidative stress. Its expression is upregulated under high-temperature, ethylene (ETH), abscisic acid (ABA), and salicylic acid (SA) treatments, peaking at 4, 6, 0.5, and 12 hours post-treatment, respectively, with the most significant induction observed under heat stress and SA. Functional assays in yeast demonstrated that heterologous expression of TaGRAS34-5A enhances the heat tolerance of the yeast. The results of BSMV:VIGS transient silencing experiment showed that after the 42 ℃ high-temperature treatment, TaGRAS34-5A silenced plants exhibited decreased chlorophyll content, reduced POD enzyme activity, increased cellular peroxidation, and decreased heat tolerance compared to the control. Preliminary studies on the heat tolerance mechanism suggest that TaGRAS34-5A exhibits strong transcriptional self-activation activity.it may modulate wheat heat tolerance by interacting with proteins such as the bZIP family transcription factor HBP-1b and the E3 ubiquitin ligase hel2, thereby regulating cellular redox homeostasis and detoxification processes, positively influencing the heat tolerance of wheat.【Conclusion】TaGRAS34-5A is induced by heat, ABA, ETH, and SA, and its encoded protein is located in the nucleus and cytoplasm. It exhibits transcriptional activation activity. Heterologous overexpression of TaGRAS34-5A enhances the heat tolerance of Saccharomyces cerevisiae. Silencing TaGRAS34-5A in wheat plants increases cellular peroxidation, decreases chlorophyll content, and reduces heat tolerance. TaGRAS34-5A may regulate the heat tolerance of wheat by modulating cellular redox state and detoxification processes.

Key words: wheat, GRAS transcription factor, heat stress, gene silencing, protein interaction