Discovery And Functional Verification Of Drought Tolerance Related Gene WRKY18 In Cotton

Cotton is an important cash crop in the world.With the warming of global climate,extreme environmental climate such as drought and high temperature threatens cotton production.Under the circumstance of decreasing cotton planting area year by year,it is particularly important to excavate drought-tolerant genes and improve cotton drought resistance.In this study,a WRKY18 gene related to drought tolerance in cotton was identified by intron functional markers and correlation analysis of drought tolerance traits.On this basis,the structure,expression and drought tolerance function of the WRKY18 gene were further analyzed.The main results are as follows:1.Discovery of drought-tolerance related genes:The genotype of 264 upland cotton populations was analyzed by intron functional markers developed in our laboratory.The association analysis between these polymorphic markers and drought-tolerance related phenotypic data traits of the upland cotton populations was further carried out,and five polymorphic markers were screened.The five markers were EPIC356,EPIC43,EPIC50,EPIC93 and EPIC309.WRKY18,a functional gene corresponding to EPIC356,was found to be significantly associated with relative SOD value and relative emergence rate,and was a candidate gene for drought tolerance in cotton.2.Structural expression characteristics of WRKY18:Through PCR amplification and sequencing analysis,the open reading frame(ORF)of WRKY18 was composed of 1551 bases,encoding 516 amino acids.It contains two WRKY domains.According to the expression of WRKY18 in tissues and organs,WRKY18 was predominantly expressed in roots,stems and leaves of cotton.The identification of drought-induced expression of WRKY18 showed that the expression of WRKY18 increased significantly at 72h,indicating that WRKY18 would respond to drought stress.Subcellular localization showed that WRKY18 functioned in the nucleus.3.Functional analysis of WRKY18:Through PCR amplification of the specific region of WRKY18,the VIGS vector was constructed,and the VIGS function of WRKY18 was verified in cotton.After silencing the target gene,the drought tolerance of WRKY1 8 in cotton was studied by observing the changes of physiological indices of watered 15%PEG plants and watered control plants.The results showed that there was no significant change in phenotype among the plants without water injection,no-load injection and silence of WRKY18,while the growth of the plants treated with 15%PEG was inhibited,suggesting that drought stress could inhibit the growth and development of the plants.Under drought stress,leaf abscission of plants silenced with WRKY18 gene was more serious than that of plants without or without WRKY18 gene injection.By comparing the changes of silent gene in drought treatment and water control with those of non-injected plants,it was found that the above-ground dry matter weight,POD,MDA,PRO and H2O2 values of plants silenced with WRKY18 gene did not change significantly,but the underground dry matter weight and relative SOD values showed significant changes.Very significant changes indicated that silence of WRKY18 would affect the growth of cotton roots and the changes of SOD value,which led to the decrease of drought tolerance of cotton plants.The overexpression vector of WRKY18 promoter was constructed.The heterologous expression of the 35S promoter was analyzed in Arabidopsis wild type Col-0 by flower dipping method.Five positive clones were obtained,named OE-1,OE-2,OE-3,OE-4 and OE-5,respectively.The drought tolerance of five positive clones was tested.Arabidopsis thaliana was treated with mannitol of 0 mM,100 mM,150 mM,200 mM and 250 mM to simulate drought and observe the growth of its root length.The results showed that the root length of OE-2,OE-4 and OE-5 overexpressed Arabidopsis thaliana under mannitol treatment was significantly or extremely significantly higher than that of WT.OE-2 was significantly increased under mannitol treatment at 200 mM and 250 mM,while OE-4 was significantly increased under mannitol treatment at 150 mM,200 mM and 250 mM,while OE-5 was significantly increased under mannitol treatment at 100 mM,150 mM,200 mM and 250 mM,while OE-5 was significantly increased under mannitol treatment at 100 mM,150 mM,200 mM and 250 mM.The results showed that overexpression of WRKY18 in Arabidopsis thaliana could promote the growth of root length and improve the drought tolerance of plants.