Isolation And Functional Analysis Of A Mitogen-activated Protein Kinase Gene GhMPK11 From Cotton

Mitogen-activated protein kinase(MAPKs)that encode dual specificity threonine and tyrosine protein kinases are ubiquitous found in eukaryotes.MAPKs at the downstream of MAPK cascades can interact with target genes,which in turn cause various physiological and biochemical changes in the body.MAPK cascades are involved in plant growth and development,response to abiotic stresses such as drought,salinity,low temperature and biotic stresses includes diseases and insect pests,so studies the function of MAPKs on adversity stress play significant influence on plant in adaptation to environmental stress.However,researches of MAPKs are mainly focused on the model plant and limited on crop.Moreover in plants,researches on MAPKs are mainly focused on MPK3,MPK4 and MPK6 and other genes are relatively few studies.Cotton(Gossypium hirsutum)is one of the most important economic crops,the studies of MAPKs in cotton may help cotton adapt to adversity stress better.In this study,G.hirsutum is chosen as the experiment material and a novel gene GhMPK11 is isolated and characterized.The main results are as follows:(1)The full length cDNA of GhMPK11 is 1274 bp,containing a 95 bp 5'UTR,40bp3'UTR and 1098 bp open reading frame(ORF)that encodes a 365 amino acid protein with the predicted molecular weight 42 kDa and pI 6.44.The genomic structure analysis,multiply alignments and phylogenetic analysis demonstrates that the gene GhMPK11 belongs to group B family,in addition the subcelluar localization of GhMPK11 is detected by biolistic transformation system and Agrobacterium-mediated transient transformation,and all results reveal GhMPK11 localizes to nucleus.(2)The expression pattern of GhMPK11 is detected and the results show that hydrogen Peroxide(H2O2),abscissic acid(ABA),gibberellic acid(GA),Ralstonia solanacearum and Rhizoctonia solani can induce the expression level of GhMPK11 at different degree.So we hypothesize that GhMPK11 involves in signaling pathway and plant responses to pathogens.(3)GhMPK11 promoter sequence analysis indicates that there have some defense-responsive elements,development-related elements as well as GA-response element;GUS staining shows that ProGhMPK11::GUS transgenic Arabidopsis thaliana plants exhibit enhanced GUS activity after GA3 treatment,which further indicate that GhMPK11 involved in the GA signal pathway.(4)Using Agrobacterium-mediated transformation method,GhMPK11-overexpressing(OE)plants and empty vector(Vec)plants(control plants)are obtained and confirmed by PCR.Observe the growth of transgenic plants and find compared with Vec plants,OE plants exhibited a weak green intensity of the leaves.In addition,GhMPK11 transgenic plants contain higher GA3 content and higher expression level of GA-related genes,which suggest that GhMPK11 can regulate the expression of GA-related genes to alter GA3 content in plants.(5)The responses of GhMPK11 overexpression plants to pathogens are investigated.Results show that overexpression GhMPK11 can reduce the resistance of OE plants to R.solanacearum and R.solani treatment and also enhance the accumulation of reactive oxygen species(ROS),which suggest that GhMPK11 is involved in regulating cellular ROS homeostasis and biotic stress responses.(6)Genes related to GA,salicylic acid(SA)and jasmonic acid(JA)are analyzed before and after pathogen treatment.Results show that there are significant differences in the expression of GA-related genes after pathogen injection which preliminary conclude that that GhMPK11 regulate disease responses through GA signaling pathway.(7)The detached leaves are injected with R.solani which simultaneously treated with exogenous GA3,and results show that exogenous GA3 can counteract the influence of endogenous GA3 on disease responses and enhance plant susceptibility to pathogen injection.Combined with other results,it can be suggested that the GhMPK11 can regulate pathogen resistance through GA signaling pathway.(8)The expression level of ROS-related genes is detected after pathogen injection,as well as the activity of antioxidant enzymes.Compared with Vec plants,OE plants exhibited higher expression level of respiratory burst oxidase homolog gene(RbohB),lower level of antioxidant genes and lower activity of antioxidant enzymes.These results indicate that GhMPK11 can regulate cellular ROS homeostasis by regulating the expression ofROS-related genes.