| Transcription factors（TFs）play a crucial role in plants in response to biotic and abiotic stresses.Many transcription factor,such as heat shock factor（HSF）,WRKY,MYB,NAC,bZIP,zinc finger protein,AP2/ERF,DREB,ERF,b HLH,have been found to be associated with plant tolerance to stress stress.However,the key TFs of rice response to heat stress were rarely identified,and the mechanism of TFs in rice response needs to be fully elucidated.At present,the large accumulation of biomolecular big data promotes the continuous maturity and application of bioinformatics technology.From omics big data,the identification of genes or proteins of rice in response to environmental stress has become an effective research strategy.Therefore in this study,the omics data of rice was downloaded and analyzed to identify key transcription factors of rice under high temperature stress,then modern molecular techniques were applied to validate the functions of the genes.The main study contents and results are presented as follows.Enrichment analysis of THS within 3 Kb of TSS using Homer software showed that 1652 THS were positively enriched and 156 THSs negatively enriched,and the differentially enriched THS represented7.07%of the total.1.Identification of key TFs in rice in response to heat stressFirstly,transcriptomic sequencing（RNA-seq）and epigenomic sequencing（ATAC-seq）data of rice under heat stress were downloaded and analyzed,and then the results of these two datasets were integrated.Analysis of the ATAC-seq raw data revealed that heat stress increased the distribution frequency of THS（Transposase Hypersensitive Site）from 0to 3 Kb of promoter regions in the rice genome,while decreased the distribution frequency of THS in other regions.Meanwhile,heat stress enhances the signal at the TSS（Transcription Start Sites）and makes the area near the TSS more accessible.Enrichment analysis of THS within 3Kb of TSS using Homer software showed that 1652 THS were positively enriched and 156 THSs negatively enriched,and the differentially enriched THS represented 7.07%of the total.Finally,BLAST tool was used to identify the heat stress differentially enriched THSs in the rice reference genome.As a result,five key transcription factors of rice in response to heat stress were obtaied.Secondly,we performed transcriptomic data analysis of rice under heat stress.We then screened the differentially expressed genes by using the criteria±2-fold difference and P-value<0.05.After obtaining the differentially expressed genes we identified the common genes between the differentially expressed and the genes with the differential enriched THSs.Finally we obtained three transcription factors which were differentially expressed in rice under heat stress,namely OsbZIP14,Os MYB2 and Os HSF7.2.Functional validation of key transcription factors in rice response to heat stressRT-PCR was applied to detect the expression levels of OsbZIP14,Os MYB30 and Os HSF7 in rice under heat stress,and the results were consistent with the above omics big data analysis results.Then,one of the key transcription factors,OsbZIP14,was analyzed for subcellular localization and transcription activity,which showed that OsbZIP14 is a typical nuclear TF with transcriptional activation capacity.Finally,overexpressing rice plants with OsbZIP14 were constructed to further verify the function of this gene in response to heat stress.Subsequently it was observed that the survival rate of OsbZIP14 overexpressing rice lines under heat stress was significantly higher than wild type.Additionally,3,3-diaminobenzidine（DAB）and trypan blue staining test results showed that H2O2accumulation and cell death levels in OsbZIP14 overexpression plants were significantly lower than those in wild type.The above results further verified that OsbZIP14 is a key transcription factor responding to heat stress in rice.3.The mechanism of OsbZIP14,a key transcription factor in rice response to heat stressFirst,a yeast two-hybrid library was used to verify the interacting proteins of OsbZIP14,and found OsbZIP14 and OsbZIP58 interaction.Additionally,the bimolecular fluorescence complementation（Bi FC）experiment further verified the above results.We then used protein interaction prediction tools to discover OsbZIP14 interactions with OsbZIP58 products through hydrogen bonds and salt bridges.However,OsbZIP58 is the key gene regulating the accumulation of rice grain storage material under heat stress,and the expression of OsbZIP58 gene has increased significantly in the Cas9 mutant of OsbZIP14 gene under high temperature stress.This indicates that OsbZIP14 gene negatively regulates OsbZIP58 gene expression and affects rice grain development during filling stage.In conclusion,this study identified three rice key TFs in response to heat stress,and through the dry and wet test,found that OsbZIP14 gene expression and OsbZIP58 gene expression through interaction,which affect the accumulation of storage material in rice grain,thus affect the tolerance of rice to heat stress.The results provide candidate genes for the subsequent genetic improvement of rice,and also lay a foundation for deeply exploring the molecular mechanism of heat stress tolerance in rice. |
