Study On The Molecular Mechanism Of WRKY28 Regulating ROS-and SA-Mediated Leaf Senescence In Oilseed Rape (Brassica Napus L.)

Leaf senescence,a precisely regulated process in plants,plays a vital role in nutrient remobilization.It also responds to changes from internal and external environments.Salicylic acid(SA)and reactive oxygen species(Birkenbihl et al.),being important hormone or signal molecule in plants can accelerate senescence.WRKY transcription factor(TF)gene family is one of the largest transcription factor gene families widely found in plants,which plays crucial roles in plant growth and development and in response to diverse biotic and abiotic stresses.There are few reports about WRKY gene in oilseed rape(Brassica napus L.)and the functions of many members have not been elucidated.Based on preliminary work in our lab,this study conducted further exploration on the biologic function and molecular regulatory mechanism of WRKY28 gene in oilseed rape.First,we constructed phosphor-mimicking form of WRKY28 gene through overlap PCR.When wild-type and phosphor-mimicking forms BnaWRKY28 gene were overexpressed individually in wild-type(WT)Arabidopsis thaliana,it was found that overexpression of the phosphor-mimicking form BnaWRKY28 ca can accelerate leaf senescence accompanied by accumulation of ROS.On this basis,we explored the molecular mechanism of BnaWRKY28 gene regulating leaf senescence using reverse transcription real-time fluorescent quantitative PCR(q RT-PCR),dual luciferase reporter assay(Dual-Luc),electrophoretic mobility shift assay(EMSA),bimolecular fluorescence complementation assay(Bi FC),co-immunoprecipitation assay(Co-IP),pull-down assay and chromatin immunoprecipitation assay(Ch IP)and other techniques.The main results are as follow.1.Overexpression of BnaWRKY28 ca gene in Arabidopsis thaliana can accelerate leaf senescence.The results of absolute quantitative RT-PCR showed BnaWRKY28 gene was expressed specifically in senescent leaves,which suggests phosphorus mimicking form of BnaWRKY28 gene can accelerate leaf senescence.2.BnaWRKY28 was localized in nuclei and regulated the expression of reporter gene under the control of W-box cis-element,indicating it acts as a transcriptional activator.Meanwhile,Bi FC assay,Co-IP assay and pull-down assay verified that BnaWRKY28 can interact with BnaCPK5,BnaCPK6 and BnaCPK11 in vivo and in vitro.Phosphorylation test proved BnaCPK5,BnaCPK6 and BnaCPK11 phosphorylated BnaWRKY28 in vitro.Hence,we speculated BnaCPK5,BnaCPK6 and BnaCPK11 phosphorylated BnaWRKY28 and then promoted leaf senescence by controlling the expression of downstream genes.3.To further explore the mechanism of BnaWRKY28 regulating leaf senescence,we detected the transcriptional level of ROS synthesis-related genes,SA synthesis-related genes and senescence-assocated marker genes in different transgenic lines compared to WT and GFP controls,through q RT-PCR.It was found that SA synthesis-related genes of ICS1(Isochorismate Synthase 1)and PBS3(avr Pph B susceptible 3),ROS synthesis-related gene of Rboh D(Respiratory Burst Oxidase Homolog D)and aging marker genes of SAG14(Senescence-Associated Gene 14)and SEN4(Senescence 4)were obviously up-regulated.EMSA and Ch IP-q PCR assays showed BnaWRKY28 ca can bind to W-box-containing fragments in the promoters of those genes directly,and dual-Luc assay indicated BnaWRKY28 ca can regulate the expression of those genes.These data suggest that BnaWRKY28 ca gene is involved in the process of leaf senescence via accumulation of SA and ROS.At the same time,it may promote leaf senescence by controlling the expression of senescence marker genes.Taken together,this study dissects the function and regulatory mechanism of BnaWRKY28 gene,which provides a theoretical basis to improve yield and resistance of oilseed rape by utilizing key WRKY transcription factor genes.