Moleculor Mechanism Of Cotton GhWRKY21 Transcription Factor Regulating Plant Drought And High Temperature Resistance

As the main raw material of cotton textile industry,cotton is one of the important economic crops in China.A variety of abiotic stresses(such as drought and high temperature)have seriously affected the production of cotton in China.WRKY transcription factor is a transcription factor specific to higher plants,which is widely involved in the regulation of plant stress responses to environment stresses.Because of the complexity of functions,the role of WRKY transcription factor in cotton is still unclear,and the molecular mechanism of WRKY transcription factor in regulating plant resistance needs to be further clarified.Here,we isolated and characterized the cotton group IId WRKY transcription factor gene GhWRKY21.The important role of GhWRKY21 in the regulation of abiotic stress resistance of cotton was clarified by using bioinformatics,molecular biology,biochemistry and other techniques,and the molecular mechanism of GhWRKY21 in the regulation of drought resistance of cotton was analyzed.The main results are as follows:(1)The full length sequence of GhWRKY21 is 1494 bp,containing a 173 bp5’-untranslated region(5′-UTR),a 324 bp 3′-untranslated region(3′-UTR)and a 996 bp open reading frame(ORF).The ORF encoded a protein composed of 331 amino acid residues with a predicted molecular mass and isoelectric point of 36.8 kDa and 9.51,respectively.Analysis of the amino acid sequence alignment and a phylogenetic tree showed that GhWRKY21 is a member of group Ⅱd WRKY proteins.(2)Subcellular localization indicated that the GhWRKY21 protein specifically localized in the nucleus.The expression patterns of GhWRKY21 in different tissues and under abiotic stresses were analyzed.The results showed that GhWRKY21 was found in cotton roots,stems and leaves.The expression level of GhWRKY21 could be induced by abiotic stress such as drought and high temperature,and also induced by abscisic acid(ABA)salicylic acid(SA),methyl jasmonate(JA)and other signal molecules.(3)GhWRKY21-silenced cotton lines were generated using VIGS(virus-induced gene silencing)technology.Functional analysis showed that silencing GhWRKY21 significantly improved the tolerance to drought and high temperature stress of cotton,and significantly reduced the damage degree of cotton leaves.(4)GhWRKY21-overexpressed transgenic tobacco was obtained by agrobacterium tumefaciens-mediated leaf disc transformation.By measuring the germination rate,survival rate,water loss rate,stomatal opening and other indicators of transgenic tobacco under drought stress and phenotypic analysis,it was found that the overexpression of GhWRKY21 reduced the drought resistance of transgenic plants.In addition,GhWRKY21-overexpressing plants were sensitive to drought stress,and the sensitive phenomena of seed germination stage and root growth stage disappeared after ABA synthesis inhibitor was added.Further analysis of guard cells revealed that GhWRKY21 was involved in ABA-mediated stomatal closure.These results suggest that the sensitivity of GhWRKY21 transgenic plants to drought stress is ABA related.(5)By analyzing the promoter elements of all type A PP2 C protein phosphatase in ABA signaling pathway,possible candidate genes for GhWRKY21 regulation were obtained.The results of Y1 H analysis and EMSA test showed that GhWRKY21 binds to the promoter of GhHAB.In addition,overexpression of GhWRKY21 in protoplasts of cotton increased the expression level of GhHAB,and silencing of GhWRKY21 in cotton also reduced the expression level of GhHAB.The above results indicated that GhWRKY21 negatively regulated ABA-mediated drought resistance by regulating GhHAB expression.(6)To analysis the transgenic tobacco treated with high temperature in the germination stage and seedling stage,it was demonstrated that GhWRKY21 overexpression in Nicotiana benthamiana enhanced plant sensitivity to heat stress.Combined with the changes of heat-related genes in plants,it was suggested that GhWRKY21-overexpressed plants could regulate the heat resistance by influence the expression levels of heat-related genes.