Identification Of VQ Motif-Containing Family Gene In Wheat And Function Analysis Of TaVQ4-D In Plant Responsed To Drought And Salt Stress

Wheat(Triticum aestivum L.)is one of the world’s important food crops,accounting for 1/5 of the world’s crop planting area,and more than 40%of the world’s population on wheat as a staple food.However,grain yield and quality are severely reduced when wheat plants are subjected to environmental stresses such as drought.Therefore,maintaining stable yields under adverse conditions is an important objective of current wheat breeding programs.Some studies have shown that VQ genes are involved in various abiotic stress responses in plants,but the role of VQ proteins in wheat is poorly understood.In this study,VQ family genes of wheat were identified based on genomic information.And then,the function of TaVQ4-D gene was evaluated,and the mechanism of action of TaVQ4-D was studied.The main research results are as follows:1.Identification of VQ family genes in wheatIn this study,113 TaVQ genes(40 homoeologous groups)were identified in the wheat genome.Multi-sequence alignment of TaVQ proteins showed diversification of amino acid sequences in these VQ4 proteins in regions other than the highly conserved VQ motif structure FxxhVQxhTG.According to TaVQ protein sequence,TaVQ proteins can be divided into 8 subgroups(I-VIII).Gene structure analysis showed that 91.15%of TaVQ genes did not contain introns.Segmental duplication is the main way of gene expansion in wheat VQ family.The cis-elements analysis of the promoter region demonstrated that stress responses,hormone responses,growth and development,and WRKY binding elements are all widely distributed in TaVQ gene promoter.Gene expression analysis based on public RNA-seq data demonstrated that the expression pattern of TaVQ genes varies in different tissues.The results of RT-qPCR found that TaVQ genes displayed different expression levels under different abiotic and phytohormones stresses.2.Analysia function of drought and salt tolerance of TaVQ4-D gene in transgenic plants(1)Gene expression analysis showed that TaVQ4-D gene was induced by drought stress,and the expression level of TaVQ4-D gene was higher in wheat leaves,glume,and 5 days after pollination.The analysis of TaVQ4-D gene promoter activity showed that drought and salt stress could enhance the promoter activity of TaVQ4-D gene.Subcellular localization revealed TaVQ4-D protein localization in the nucleus the cytoplasm.(2)TaVQ4-D gene enhanced the tolerance of Arabidopsis and wheat to drought and salt stress.Under PEG and NaCl stress,the germination rate of Arabidopsis seeds with TaVQ4-D gene was significantly higher than WT.The survival rate,proline content,catalase(CAT)and superoxide dismutase(SOD)activities of Arabidopsis transgenic with TaVQ4-D gene significantly increased,while malondialdehyde(MDA)and O2-content decreased after drought and salt stress on adult Arabidopsis.After PEG and NaCl treatment,the leaves of the control plants curled and wilted,while the transgenic plants showed relatively good growth status,and the survival rate and fresh weight of TaVQ4-D transgenic wheat were significantly higher than those of the control.Drought and salt stress were applied to wheat seedlings growing in soil.The results showed that the wilt degree of transgenic wheat leaves was significantly less than that of the control,and the content of proline and SOD activity were significantly higher than control,while the content of O2-and MDA in the leaves were significantly reduced.Meanwhile,the expressions of stress-related genes TaRD29B,TaLEA7,TaDREBl,TaDREB3,TaP5CRS,TaCAT3,TaSOD1,TaGPx1-D and TaPOD1 were significantly up-regulated in transgenic TaVQ4-D wheat.BSMV-mediated silencing of TaVQ4-D expression decreased drought resistance in wheat.The content of proline and SOD activity in leaves of TaVQ4-D silenced wheat plants were significantly lower than WT,and the accumulation of O2-and MDA was higher.At the same time,the expression of stress-related genes was also down-regulated.3.Preliminary analysis of the mechanism of action of TaVQ4-D.TaVQ4-D was used as a bait protein to screen the wheat cDNA library.Two mitogen activated protein kinase(MAPK)genes TaMPK3 and TaMPK6 interacting with TaVQ4-D were obtained through sequencing and alignment.The interaction of TaVQ4-D with TaMPK3 and TaMPK6 in vitro and in vivo was verified by yeast two-hybrid,GST Pull-down and bimolecular fluorescence complementation assay.RT-qPCR analysis showed that TaMPK3 and TaMPK6 were induced by drought and salt stress.MAPK can transmit signals through phosphorylation of downstream substrates and play a role in plant growth and development and various stresses.TaVQ4-D protein was phosphorylated by TaMPK3 and TaMPK6 in vitro.LC-MS was used to detect the amino acid residues of phosphorylation modification of TaVQ4-D by TaMPK3 and TaMPK6.The results showed that a total of 13 and 5 possible phosphorylation sites were detected,respectively.Mutations at sites S145,S207,and T226 to the non-phosphorylated alanine(A)showed no significant change in the migration rate of phosphorylated TaVQ4-D,indicating that S145,S207,and T226 may not be the main phosphorylated sites of TaVQ4-D.Taken all together,this study analyzed the TaVQ family genes of wheat and identified the functions of TaVQ4-D,indicating that TaVQ4-D is involved in plant response to drought and salt stress by interacting with and as substrates for TaMPK3 and TaMPK6.These results provide candidate genes and theoretical basis for breeding new wheat varieties with drought resistance and salt tolerance.