| Panax notoginseng(Burk)F.H.Chen,also known as Tiansanqi,has the effects of promoting blood circulation,removing blood stasis,reducing swelling and pain,stopping bleeding,replenishing blood,and improving the body’s immunity with high medicinal value.In recent years,fungal diseases such as root rot,black spot and round spot have seriously affected the quality of P.notoginseng and the sustainable development of P.notoginseng industry,among which the root rot of P.notoginseng mainly caused by Fusarium solani is most serious.In the previous study,our research group found that the jasmonic acid signal is an important signal pathway for P.notoginseng to respond to F.solani,and a series of jasmonic acid-responsive resistancerelated genes(PnDEFL1,PnSN1,PnPR10-3,PnPR10-3,PnDEFL1,PnSN1,PnPR10-3,PnChI1,PnGlu1,PnPGIP)were isolated from P.notoginseng.WRKY transcription factors play an important role in regulating transcription reprogramming of plant defense against pathogen infection.It has received extensive research attention in recent years,but the research on WRKY transcription factor of P.notoginseng is rarely reported,and it is unclear whether WRKY transcription factor is involved in regulating defense response against pathogen in P.notoginseng.Therefore,this paper isolated the WRKY gene from P.notoginseng,which cooperate with jasmonic acid signal to participates in defense response to F.solani infection.Its expression profile in P.notoginseng roots were analyzed after signal molecule treatment and during the infection of F.solani.In addition,the PnWRKY9 gene which is responsive to jasmonic acid and has a high expression level was selected for structural and subcellular localization analysis,functional verification and study on transcriptional regulation characteristics of P.notoginseng defensin gene PnDEFL1.This paper helps to further understand the mechanism of P.notoginseng WRKY transcription factor cooperating with jasmonic acid signal in defense response against F.solani and the molecular interaction mechanism between P.notoginseng and root rot pathogen.The main research results of this article are summarized as follows:1.The P.notoginseng WRKY gene family was obtained by transcriptome sequencing technology,and 30 P.notoginseng WRKY genes with complete ORF were cloned,which were named PnWRKY1-PnWRKY30.The conserved structure of these genes,phylogenetic tree and the expression pattern oduring F.solani infection after treatment with methyl jasmonate were analyzed.The results showed that the WRKY gene family of P.notoginseng had a complete conserved domain with highly homologous in the evolutionary process.Ten WRKY genes including PnWRKY5/6/9/11/15/21/22/25/28/30,responded to methyl jasmonate treatment,and their transcription levels were increased during the infection of F.solani.2.The PnWRKY9 with high expression level was selected for in-depth functional research.Sequence analysis showed that the gene contained a WRKY domain and a CX4-5-CX22-23-H-X1-H zinc finger domain.The fusion expression vector of PnWRKY9 and GFP was constructed and transformed into Agrobacterium tumefaciens EHA105 capable cells,which was expressed instantaneously in onion epidermal cells.The subcellular localization of PnWRKY9 protein was observed in the nucleus under a laser confocal microscope.Plant overexpression vector p CAMBIA2300S-PnWRKY9 was constructed and transformed into WT tobacco for stable expression,and the resistance analysis results showed that the PnWRKY9 transgenic tobacco showed enhanced resistance to F.solanum.Subsequently,the RNAi expression vector of PnWRKY9 was constructed and transformed into P.notoginseng leaves for transient expression,and then was inoculated with F.solani.The results showed that the expression of RNAi vector in P.notoginseng leaves enhanced the sensitivity to F.solani.Functional verification analysis showed that PnWRKY9 is a positive regulator in P.notoginseng modulating the defense response to F.solani.3.To verify that PnWRKY9 is a transcription factor regulating the resistance genes in P.notoginseng the promoter of P.notoginseng defensin gene PnDEFL1(PPnDEFL1)was cloned,which contains a W-box and then was used in the electrophoretic mobility shift assay(EMSA)and yeast one hybrid(Y1H)to analyze the binding of PnWRKY9 to PPnDEFL1 and transcriptional activation.The prokaryotic expression vector of PnWRKY9 was constructed,the recombinant protein was induced by isopropyl β-Dthiogalactoside(IPTG),and the PnWRKY9 recombinant protein was purified by NiNTA column affinity chromatography for EMSA experiment,The results showed that PnWRKY9 protein can specifically bind to the PPnDEFL1 fragment containing W-box.The PnWRKY9 prey vector and the promoter PPnDEFL1 bait vector were constructed and transformed into yeast competent cells for Y1 H experiment.The results showed that the PnWRKY transcription factor showed transcriptional activation of PPnDEFL1.At the same time,the fusion expression vector of promoter PPnDEFL1 and GUS gene was constructed and transformed into PnWRKY9 transgenic tobacco and WT tobacco,respectively,and the GUS activity of co-expressed transgenic tobacco was determined.The results showed that the GUS activity of PPnDEFL1 and PnWRKY9 co-expressed tobacco was significantly stronger than that of PPnDEFL1 sexpressed transgenic tobacco.It can be seen that the PnWRKY9 transcription factor can transcriptionally activate the promoter of the PnDEFL1 gene and positively regulate the expression level of PnDEFL1.The WRKYs of P.notoginseng are important transcription factor genes that cooperate with the jasmonic acid signal pathway to participate in the defense response to F.solani.There are 10 WRKY genes responding to the treatment of methyl jasmonate,and their transcription levels increase during the infection of F.solani.Among them,the PnWRKY9 is a nuclear localization protein that specifically binds to the promoter sequence of the root rot disease resistance gene PnDEFL1,and PnWRKY9 positively regulates the transcription level of PnDEFL1.Combination with the jasmonic acid signal,the PnWRKY9 transcription factor positively regulates the expression of defensin gene,and plays an important regulatory role in the defense response of P.notoginseng to F.solani. |
