Cloning And Functional Characterization Of TaHsfC2aL In Common Wheat

Wheat is the second largest food crop in China.In the scenario of climate change,the occurrence of extreme weather events severely restricted wheat production.Developing thermotolerant wheat varieties is the most economical and effective approach to resolve the problem and ensure wheat production,while revealing the molecular mechanism of wheat thermotolerance and exploring elite heat-tolerant gene resources are the premise and cornerstone for the improvement of thermotolerant wheat varieties.In this study,a wheat HSF coding gene Ta Hsf C2 a L was cloned in Hanxuan10,a variety with prominent tolerances to drought and heat stresses.Its expression patterns and subcellular localization were analyzed,and its interacting protein was identified.Furthermore,its function was characterized by transforming rice,and the underline the molecular mechanism were revealed.The nucleotide sequence polymorphisms of the Ta Hsf C2 a L were also analyzed,the functional marker was developed accordingly,and the elite haplotype was identified.The detailed results are as follows:1.A wheat heat shock transcription factor gene Ta Hsf C2 a L was cloned,locating on chromosome 4B in common wheat.The size of its genomic DNA was 1150 bp,and the c DNA was 1041 bp,encoding a putative protein with 273 amino acid resides.Ta Hsf C2 a L contained a specific HSF-domain of HSFs.Multiple sequence alignments showed that the HSF domain was highly conserved among the orthologs from various plant species.Subcellular localization revealed that Ta Hsf C2 a L was specifically localized in the nucleus.2.Various cis-acting elements were identified in the promoter region of TaHsfC2 aL,including hormone responsive elements,as well as elements related to the responses to low temperature and dehydration stresses.Tissue specific expression assays showed that Ta Hsf C2 a L was highly expressed in young spike at jointing,peduncle at heading and flag leaf at flowering stage.Furtheromore,it was induced by hormone and stress.3.Phenotyping results showed that overexpression of TaHsfC2 aL conferred pouncedenhanced tolerance to high temperature in rice,which was supported by the physiological index,including remarkably reduced malondialdehyde contents and significantly increased cell membrane stability in the transgenic lines relative to WT controls after a four-day heat stress.Further gene expression assays revealed heteroexpression of Ta Hsf C2 a L resulted in significant transcriptional augment of downstream heat shock protein genes.Therefore,it was speculated that the improved thermotolerance might be attributed to the upregulation of small molecule HSP coding genes.4.Multiple candidate interactors were obtained by screening the wheat c DNA library in yeast cells,one of which was heat association protein 32(HSA32).The interactions between Ta Hsf C2 a L and Ta HSA32 were validated by yeast two-hybridization,BIFC and LCI experiments,and the interaction only occurred in the nucleus.5.Subcellular localization indicated that Ta HSA32 was present in the cytoplasm,nucleus and cell membrane,partially overlapping with that of Ta Hsf C2 a L,hinting the potential of their interaction in wheat.Gene expression assays showed that Ta HSA32 was highly expressed in young spike at jointing,peduncle at flowering,and flag leaf at grain-filling stage.Ta HSA32 was also induced by various abiotic stresses.The overlapped spatiotemporal expressions of Ta Hsf C2 a L and Ta HSA32 indicated that there was a possibility of their interactions under the abovementioned conditions.6.Transcritonal activation assays showed Ta HsfC2 a L was a transcriptional activator.It could interact with Ta HSA32 and bind to the promoter of heat protection genes,and thus cotrolling their expressions.7.Association analysis showed that Hap-1 was a major haplotype in lanrace population with higher plant height,longer peduncle and penultimate internode,while Hap-2 and Hap-4 were major haplotypes in the population of morden varietie with shorter plant height,peduncle and penultimate internode.Geographical distribution of haplotypes showed that the Hap-2 and Hap-4 were positively selected in wheat breeding.