Isolation And Functional Anaysis Of Cytosolic Acetoacetyl-CoA Thiolase Gene TaAACT1 In Wheat

Sharp eyespot and Fusarium stem rot（FCR）are the main diseases affecting wheat production in China.Breeding and promoting disease-resistant wheat cultivars is an effective way to prevent and control Sharp eyespot and Fusarium stem rot in wheat,but no wheat material that is completely immune to sharp eyespot or FCR has been found so far.Therefore,exploring the resistance genes of wheat related to sharp eyespot and FCR has significant theoretical and practical value.In this study,a wheat cytosolic acetoacetyl-CoA thiolase gene TaAACT1 related to resistance to sharp eyespot was screened from wheat by transcriptome data and RT-qPCR analysis.The gene functional analysis was analyzed by virus-induced gene silencing（VIGS）technology,aiming to provide reference for wheat molecular breeding for disease resistance.The results are as follows:（1）Cloning of TaAACT1 gene and functional analysis of its resistance to wheat sharp eyespot.The results of RNA-Seq and RT-qPCR analysis showed that the relative transcription level of TaAACT1 gene in wheat cv.CI12633 with resistance to sharp eyespot was significantly higher than that of susceptible cultivars;In the resistant wheat cv.CI12633,TaAACT1 quickly responded to the stress of Fusarium graminearum,and its expression level was significantly upregulated,indicating that TaAACT1 was involved in the early defense response of wheat to sharp eyespot;The results of protein sequence alignment and phylogenetic analysis indicate that TaAACT1,along with rice AACT（Oryza saliva AACT（OsAACT））and corn AACT（Zea mays AACT（ZmAACT））,is clustered in the same branch and belongs to the cytosolic acetoacetyl-CoA thiolase（AACT/ACAT）protein.The TaAACT1 is composed of 418 amino acids,with a 100%similarity in amino acid sequence to the corresponding protein（XP₀201631811）of Aegilops tauschii,indicating that TaAACT1 in wheat may originate from Aegilops tauschii.VIGS technology was used to silence-TaAACT1 for reducing the resistance of wheat to sharp eyespot.The TaAACT1-GFP protein was transiently expressed in wheat protoplasts using PEG mediated method,and subcellular localization analysis was performed.The results showed that the TaAACT1 protein was localized within the cytoplasm.（2）Functional analysis of TaAACT1 gene for resistance to FCR.The results of SNP analysis combined with wheat variant omics analysis showed that 13 SNPs related to the resistance of wheat to Fusarium graminearum were found in the TaAACT1 gene sequence.The results of RT-qPCR analysis showed that the relative transcription level of TaAACT1 gene in wheat cv.CI12633,which is resistant to FCR,and significantly higher than that of susceptible cultivars.The RT-qPCR analysis results showed that TaAACT1 rapidly responded to Fusarium graminearum stress and significantly upregulated gene expression in the wheat cv.CI12633 resistant to FCR.TaAACT1 gene expression is induced by the defense related hormone jasmonic acid（JA）.The results of VIGS technology,RT-qPCR,and analysis of infection types showed that the expression level of TaAACT1 gene in wheat plants after TaAACT1-silencing was significantly reduced,and TaAACT1-silencing significantly weakened the resistance of wheat to FCR disease.The results of RT-qPCR analysis showed that the relative transcriptional levels of defense genes TaChitinase2,TaChitinase3,TaChitinase4,and TaDefansin were significantly reduced in wheat plants with TaAACT1-silencing.The above results indicate that TaAACT1 can positively regulate the expression levels of TaChitinase2,TaChitinase3,TaChitinase4,and TaDefansin,thereby regulating the resistance of wheat to FCR.