The Study On The Mechanism Of Lilium Regale Wilson Defensin Antimicrobial Peptide Gene LrDef1 Responding To Fusarium Oxysporum

Lilium regale Wilson is a unique wild species in China,it has strong resistance to Fusarium oxysporum,the pathogen of Fusarium wilt.It is an excellent germplasm resource for lily disease resistance breeding.In this study,a defensin antimicrobial peptide gene（LrDef1）responsed to F.oxysporum induction was isolated based on the analysis of the transcriptome sequencing data of L.regale at the previous stage.The full-length c DNA sequence of LrDef1 was obtained by RT-PCR amplification and its function was analyzed.The expression profile of LrDef1 was analyzed by q RT-PCR;The expression site of LrDef1 was analyzed;LrDef1 recombinant protein was induced and purified to investigate its antifungal activity in vitro;LrDef1 was overexpressed into the model plant tobacco（Nicotiana tabacum）to analyze the resistance of transgenic tobacco to F.oxysporum.Based on the positive regulatory factor Lr WRKY3 in L.regale resistance against Fusarium wilt obtained from a previous study,and investigated the transcriptional regulation of LrDef1 by Lr WRKY3.To provide insight into the regulatory mechanism of the defense response in L.regale and to reveal the molecular mechanism of L.regale response to F.oxysporum invasion.The main research work and main results of this paper are as follows:1.The cDNA sequence of a defensin antimicrobial peptide gene of L.regale was obtained by RT-PCR and named as LrDef1.The full-length c DNA of LrDef1 was 501bp,with a 225-bp coding region,and was predicted to encode a protein containing 74amino acid residues.In the q RT-PCR analysis,LrDef1 transcripts were detected in L.regale roots,stems,leaves,flowers,and scales,and its expression was induced by F.oxysporum infection and four adversity-associated signaling molecules,methyl jasmonate（Me JA）,salicylic acid（SA）,hydrogen peroxide,and ethephon.2.The p BIN m-gfp5-ER-LrDef1 subcellular localization vector was constructed,and transient expressed in onion epidermal cells.LrDef1 located in the plant cell wall was observed under laser scanning microscope.The p ET32a-LrDef1 prokaryotic expression vector was constructed and LrDef1 recombinant protein was obtained by heterologous expression in Escherichia coli BL21（DE3）.Plate inhibition assays showed that LrDef1 recombinant protein had an inhibitory effect on the growth of Alternaria alternata and F.solani,F.oxysporum.The overexpression vector p CAMBIA2300s-LrDef1 was constructed and overexpressed in tobacco to obtained the T₂ generation LrDef1 transgenic tobacco.q RT-PCR analysis showed that LrDef1 was stably expressed in T₂ generation transgenic tobacco,and LrDef1 enhanced the resistance of tobacco to F.oxysporum.3.The promoter fragment of LrDef1（p LrDef1）was obtained by genome-walking technique.Bioinformatics analysis showed that the p LrDef1 contained W-box element that could specifically bind to WRKY transcription factors.Electrophoretic mobility shift assay results revealed that Lr WRKY3-His recombinant protein could specifically bind p LrDef1 fragment containing W-box sequence.The recombinant bait vector p Ab Ai-p LrDef1 and the recombinant prey vector p GADT7-Lr WRKY3 were constructed to carry out yeast single hybridization experiments,the results indicated that Lr WRKY3could bind p LrDef1 and activate its transcription in yeast.The plant vector（p BI121-p LrDef1-GUS）ofβ-glucuronidase（GUS）reporter gene driven by p LrDef1 was constructed and transformed into Lr WRKY3 transgenic tobacco and wild-type tobacco,respectively.GUS activity analysis showed that LrDef1 promoter responded to treatment with multiple hormones（abscisic acid,SA and Me JA）and pathogen fungal infection（F.oxysporum,Phoma tracheiphila and A.alternata）.The GUS activity of p LrDef1-GUS/Lr WRKY3 co-expressing transgenic tobacco was significantly higher than that of p LrDef1-GUS transgenic tobacco,suggesting that Lr WRKY3 activated the expression of downstream reporter gene of LrDef1 promoter,thereby enhancing GUS activity.4.Through transcriptional sequencing analysis of Lr WRKY3 transgenic tobacco,KEGG pathway enrichment results of differentially expressed genes showed that a lot of SA/JA signaling pathways,MAPK signaling pathways,flavonoid biosynthesis and plant-pathogen interaction related gene expressions were significantly up-regulated in Lr WRKY3 transgenic tobacco,and including two tobacco defensin antimicrobial peptide genes.The results of LC-MS showed that the expression of Lr WRKY3 in tobacco increased the contents of endogenous Me JA and Me SA,indicating that Lr WRKY3positively regulates the molecular interaction between L.regale and F.oxysporum by activating SA/JA signal transduction pathway and regulating the expression of disease resistance genes such as defensin.In conclusion,LrDef1 is a F.oxysporum resistance gene,and Lr WRKY3 regulates LrDef1 expression by specifically binding to the W-box in the LrDef1 promoter and activating its transcription to participate in the molecular interaction between L.regale and F.oxysporum.