Molecular Mechanism On The GST1 Gene Encoding The Key Enzyme Of Glutathione Synthesis In Wheat Seedlings Response To Copper Stress

Wheat is one of the most important food crops,and is inevitably damaged by drought,heat,temperature and heavy metals during its growth and development.Ascorbic acid(ASA)-glutathione(GSH)play important roles in plant responses to abiotic stresses.In this study,we determined the expression of ASA-GSH cycle related enzymes and genes.The gene of glutathione S-transferase(TaGST1)was significantly induced and expressed in wheat response to Cu2+ stress,speculating that it could play an important role in the tolerance of Cu2+stress in wheat.Furthermore,the promoter sequence of TaGST1 gene was isolated;TaWRKY74,the upstream transcription factor regulating the expression of TaGST1 gene,was screened out by using yeast one hybrid technology.The tolerance of TaWRKY74 transcription factor to Cu2+ stress in wheat was verified by using BSMV-VIGS technology.The main results are as follows:(1)Under Cu2+ stress,the contents of ASA and GSH in wheat were increased,and the genes APX,DHAR,MDHAR,GR and GST1,which are related to ASA-GSH cycle,were induced significantly.Among them,TaGST1 was induced to express at the highest level under the condition of 1 mM Cu2+ concentration,which suggested that TaGST1 was the key gene of ASA-GSH synthesis in wheat response to Cu2+ stress tolerance.(2)After Y1H between the promoter of TaGST1 gene and the Cu2+-stressed cDNA library,TaWRKY74,the upstream transcription factor regulating the expression of TaGST1,was isolated by yeast one hybrid technology.By comparing the transcription factor sequence with wheat database,it was found that it was located on the 5D chromosome;the coding region contained 1056 bp nucleotides and 351 amino acids,including WRKYGQK and C2HC zinc finger domains;the subcellular was located on the nucleus and had transcriptional activation activity.(3)The binding of Ta WRKY74 to the TaGST1 promoter was mainly through the W-box element on the Ta GST1 promoter.(4)BSMV-VIGS technology was used to verify the function of TaWRKY74 gene under copper stress.First,the BSMV-VIGS-TaWRKY74 vector was constructed and inoculated on wheat leaves.After inoculation,the wheat plant phenotype was observed and qPCR was used to detect TaWRKY74 gene expression,and wheat silencing plants with significantly reduced TaWRKY74 gene expression were successfully obtained.TaGST1 expression in TaWRKY74 silenced plants was found to be consistent with TaWRKY74 expression,indicating that TaWRKY74 regulates TaGST1 expression.The TaWRKY74 silenced wheat plants were treated with Cu2+ stress for 10d,and it was found that the plant height,root length,dry weight and fresh weight of TaWRKY74 gene silenced wheat plants were significantly reduced by 9.38%,13.56%,22.35%and 21.02%respectively compared with the control;Cu2+ content,GST enzyme activity and GSH content were significantly reduced in leaves and roots by 11.90%,27.66%,12.63%and 9.76%,23.47%,7.01%respectively;malondialdehyde(MDA)and H2O2 content in leaves and roots significantly increased by 3.70%,2.58%and 14.29%,25.24%;the expression trends of TaWRKY74 and TaGST1 are consistent with their transcription levels.The results indicated that TaWRKY74 affects the content of GSH by regulating the expression of TaGST1 gene and participates in wheat Cu2+stress tolerance.