Discovery And Analysis Of WRKY Transcription Factor Gene Family In Lilium Regale Wilson

Lilium spp.is a world-famous fresh-cut flower and occupies an important position in the domestic and foreign flower industry.During the propagation of bulbs and the production of fresh-cut flowers,lilies are susceptible to various pathogens such as fungi,viruses,and bacteria.At present,there are dozens of lily diseases,among which Fusarium spp.is the most serious disease in lily production.The WRKY transcription factor is a plant-specific supergene family that plays an important role in regulating plant development,stress responses,and transduction of hormonal signals.In this study,42 WRKY genes were discovered from Lilium regale Wilson,a wild lily with high resistance to Fusarium wilt.According to phylogenetic analysis,these Lr WRKYs were classified and their genome structure was analyzed.Quantitative reverse transcription PCR(q RT-PCR)was used to analyze the expression pattern of Lr WRKYs in L.regale.In addition,the resistance-related gene Lr WRKY2was cloned and its function was analyzed.In conclusion,this study provides valuable information about the structural and functional characteristics of the Lr WRKYs,which not only deepens our understanding of the molecular mechanism of L.regale resistance to Fusarium oxysporum f.sp.lilii,but also lays a foundation for the following study of WRKY genes cloning,functional analysis and transcriptional regulation mechanism in the stress resistance of L.regale.The research work of this paper and the main results obtained are as follows:1.Forty two full-length cDNA sequences of WRKY were isolated by reverse transcription PCR(RT-PCR)from L.regale.There are three conserved motifs in WRKY transcription factors,which are conserved Motif 1,2 and 3.The results of cluster analysis showed that WRKY members of L.regale were divided into three classes:I,II and III,most of which belonged to class II.According to the evolution of WRKY DNA binding domain,class II are further divided into five subclasses,namely IIa,IIb,IIc,IId,and IIe,most of which are class IIc.The results of gene structure analysis showed that except Lr WRKY3,the number of exons and introns of other WRKY genes were between 2?4 and 1?3 respectively.2.The qRT-PCR analysis showed that under normal growth conditions,most WRKY members of L.regale had higher expression in roots and lower expression in leaves,stems and flowers.Salicylic acid(SA),methyl jasmonate(Me JA),ethylene(ET)and hydrogen peroxide(H₂O₂)treatment inhibited the expression of Lr WRKY27,28,36,and 40,but up-regulated the expression of 14 genes Lr WRKY1,2,3,and so on.After F.oxysporum infection,the transcription levels of WRKY genes were different,but the expression levels of 7 genes such as Lr WRKY2 were significantly induced by F.oxysporum infection.3.The subcellular localization recombinant vector of Lr WRKY2 gene was constructed and transferred into Agrobacterium tumefaciens EHA105.Agrobacterium-mediated infection of onion epidermal cells,after dark culture,green fluorescent protein was observed under laser confocal microscope.The green fluorescent protein expressed by Lr WRKY2-GFP fusion gene was specifically distributed in the nucleus of onion epidermal cells,indicates that Lr WRKY2 is a protein located in the nucleus.4.The specificity of LrWRKY2 protein binding to PLr PR10-5 was analyzed in vitro by electrophoretic mobility shift assay(EMSA).The recombinant p ET-32a-Lr WRKY2 plasmid was transformed into the prokaryotic expression strain BL21(DE3),and induced by isopropyl-1-thio-?-D-gac-topyranoside(IPTG,1 m M in the end),at a temperature of 28°C for 6 h.The target protein of the inclusion body was obtained and the inclusion body protein was denatured.After purification,renaturation and concentration,the target protein can be obtained which meets the requirements of EMSA.Biotinylated probes were also synthesized.Recombinant protein and probe binding experiments showed that Lr WRKY2 protein specifically binds to W-box.5.The yeast one-hybrid system was used to analyze the transcriptional activation activity of L.regale transcription factor Lr WRKY2.The PLr PR10-5 and Lr WRKY2gene sequences were inserted into the p Bait-Ab Ai bait plasmid and p GADT7 prey vector,respectively.The prey vector p GADT7-Lr WRKY2 was transformed into a yeast cell Y1HGold containing p Ab Ai-PLr PR10-5 recombinant bait plasmid by a PEG/Li Ac conversion method,and cultured in a selective medium SD/-Leu medium(200 ng/m L Ab A).The results showed that Lr WRKY2 could specifically bind to the promoter sequence of the PLr PR10-5 gene and activate the transcription of PLr PR10-5.6.LrWRKY2 was overexpressed in tobacco,and positive plants with stable expression were successfully obtained.The q RT-PCR results indicate that Lr WRKY2is stably overexpressed in transgenic tobacco,while Lr WRKY2 overexpression increased the expression of some defense-related genes in transgenic tobacco.Four strains of Lr WRKY2 transgenic tobacco were selected for F.oxysporum in vivo bacteriostasis experiments.The results showed that the resistance of transgenic tobacco to F.oxysporum was much higher than that of wild type tobacco.In conclusion,the WRKY gene family(Lr WRKY1?Lr WRKY42)were successfully obtained in this study.The q RT-PCR analysis showed that the expression level of WRKY genes in the normal growth state of L.regale had root tissue advantages.In addition,WRKY gene family responded to the treatment of several signaling molecules and responded to the infection of F.oxysporum.Among them,Lr WRKY2 is induced and regulated by SA,Me JA,ET and H₂O₂ signal molecules,and can be expressed by F.oxysporum.Subcellular localization analysis showed that Lr WRKY2is a protein located in the nucleus.Prokaryotic expression of Lr WRKY2 recombinant protein has the property of specifically binding to W-box.In addition,yeast one-hybrid analysis showed that Lr WRKY2 could specifically bind to the promoter sequence PLr PR10-5 and activate the transcription of PLr PR10-5.Finally,Lr WRKY2overexpression in tobacco increased expression of some defense-related genes in transgenic tobacco and enhanced tobacco resistance to F.oxysporum.