| Drought and salt stresses are the main environmental factors that could cause the reduction of crop yield,and restrict the crop cultivation.As an important crop,wheat has developed the complicated regulatory mechanism in response to various abiotic stresses during its evolution.The in-depth study of wheat abiotic stress response mechanism is of great significance for breeding new wheat cultivars with drought and salt tolerances.Plants respond to environmental stresses by regulating the expressions of related genes through a complex network of transcription factors.Trihelix transcription factor family members extensively participate in regulation of plant growth,development and in responses to abiotic stresses.However,there is no systematic identification of Trihelix gene family in wheat,and few functional studies on Trihelix genes in wheat have been reported.In the present study,all the Trihelix gene family members were identified in hexaploid wheat and its close relatives through multiple bioinformatics methods,and their gene structures and evolutionary relationships were also analyzed.Moreover,the expression profiles of wheat Trihelix genes were analyzed through systematic biology approaches.On this basis,Trihelix gene Ta GT-75 was cloned from the model wheat cv.Chinese Spring,and then Ta GT-75-overexpression transgenic Arabidopsis and wheat were generated to further investigate its function in response to drought and salt stresses.The main results are as follows:(1)We identified a total of 94 Trihelix genes in Triticum aestivum,and further identified22 Trihelix genes in T.urartu,29 in Aegilops tauschii and 31 in Brachypodium distachyon.We analyzed the chromosomal locations and orthology relations of the identified Trihelix genes,and no Trihelix gene was found located on chromosomes 7A,7B,and 7D of wheat,reflecting the uneven distributions of wheat Trihelix genes.According to the phylogenetic analysis,the 186 identified Trihelix proteins in wheat,rice,B.distachyon and Arabidopsis were clustered into 5 major clades,named as GT-1,GT-2,SIP1,SH4,and GT?.Based on conservative motif and gene structure analysis,Trihelix genes belonging to the same clades usually shared similar motif compositions and exon/intron structural patterns.Gene duplication is the main driving force for the Trihelix gene family expansion in the speciation and evolution of wheat.Five and three pairs of wheat Trihelix genes were identified as tandem duplication genes and segmental duplication genes,respectively,validating that there were more intrachromosomal gene duplication events in wheat genome comparing to other grass species.(2)The tissue-specific and differential expression profiling of Trihelix gene family under cold and drought stresses were analyzed by using the public RNA-seq data,and it was found that homologous genes in three subgenomes usually shared similar expression profiles.Ten wheat Trihelix genes were selected from those with differential expressions under abiotic stresses according to the RNA-seq data,and the q RT-PCR method was used to confirm their expression profiles under multiple abiotic stress treatments.The results showed that these ten selected Trihelix genes generally responded to abiotic stresses including drought,salt and cold conditions.Weighted gene co-expression network analysis was conducted to research on the co-expression network of related genes in wheat under drought and other stresses.GO enrichment analysis of genes highly correlated with Ta GT-75 revealed that these genes were mainly involved in biological processes including stress response,transcriptional regulation,and RNA metabolism,and had the abilities of binding to proteins and DNA,localized in the membranous organelles and nuclei.(3)The Ta GT-75 was isolated from T.aestivum L.cv.Chinese Spring.Agrobacterium tumefaciens-mediated transient expression of the fused protein in tobacco leaves showed that Ta GT-75-GFP was localized in the nucleus.Transactivation activity analysis showed that Ta GT-75 had no transactivation activity.While the binding activity analysis confirmed that Ta GT-75 could bind to the GT elements.(4)Phenotypic analysis of wild type and Ta GT-75-overexpression transgenic Arabidopsis plants showed that Ta GT-75 enhanced plant tolerances to drought and salt stresses.Under osmotic stress,Ta GT-75-overexpression Arabidopsis plants had smaller stomatal aperture in leaves than wild type plants.Under the treatments of Na Cl and mannitol,the root length was longer and the fresh weight was higher in Ta GT-75-overexpression Arabidopsis plants compared to wild type plants.Furthermore,Ta GT-75 was found to enhance the stress tolerance of transgenic Arabidopsis through regulating the expressions of genes associated with proline and soluble sugar biosynthesis,and the expressions of some LEA gene family members in response to drought and salt stresses.Besides,Ta GT-75increased the antioxidant enzyme activities and reduced the oxidative damages in transgenic Arabidopsis plants by modulating the expression levels of genes encoding antioxidant enzymes in response to oxidative stress.To further study the biological functions of Ta GT-75 in wheat,Ta GT-75 gene was transformed into wheat cv.Chinese Spring through particle bombardment,and six Ta GT-75-overexpressing transgenic wheat lines in T2 generation were finally obtained.The phenotypic analyses of wild type and Ta GT-75-overexpressing transgenic wheat plants indicated that Ta GT-75 could increase tolerances to drought and high salt stresses in transgenic wheat.In conclusion,all Trihelix genes were systematically identified in wheat and its close relatives.Their evolutionary relationships and expression profiles under abiotic stresses were also analyzed.Ta GT-75 was cloned from hexaploid wheat,and overexpression of Ta GT-75 could enhance the drought and salt tolerances of transgenic Arabidopsis and wheat.The above results laid a foundation for the subsequent functional research of Ta GT-75,and provided a candidate gene for the wheat genetic improvement. |
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