Molecular Mechanism Of Dirigent Genes Involved In The Interaction Of Panax Notoginseng-Fusarium Solani

Panax notoginseng（Burk）F.H.Chen is a traditional Chinese medicine,which is mainly cultivated in Wenshan,Yunnan province.The unique growth environment is prone to induce plant diseases and insect pests,especially fungal diseases.Root rot disease,caused by Fusarium solani and several pathogenic fungi,is one of important diseases of P.notoginseng which seriously reduces the yield and quality of medical materials of P.notoginseng.The research on genetics and breeding of resistance of P.notoginseng was rarely reported,so it is urgent to study the molecular interaction mechanism between P.notoginseng and root rot disease.It will provide a theoretical basis for breeding for disease resistance of P.notoginseng to identify and clone the defense related genes.The protein encoded by dirigent（DIR）gene can catalyze lignin monomer coupling and participate in the process of lignin biosynthesis,and they are an importent part of plant defense system.In this study,the expression sequence tags（EST）encoding the DIRs were obtained from the transcriptome sequencing of P.notoginseng infected by F.solani.Seven full-length cDNA sequences of DIR genes were cloned by rapid amplification of cDNA ends（RACE）.Bioinformatics analysis was used to analyze the characteristics of these seven genes and the deduced proteins.The expression patterns of PnDIRs were detected by quantitative real-time reverse transcriptase PCR（qRT-PCR）analyses.In addition,the prokaryotic expression vector of PnDIR1 was constructed,and the recombinant protein was induced and purified,then the antifungal activity was identified with in vitro plate antifungal experiments.Moreover,subcellular localization vector of PnDIR1 was constructed,and the location of PnDIR1 in plant cells was detected by laser scanning confocal microscopy.Furthermore,the plant overexpression vector of PnDIR1 was constructedand transformed into Nicotiana tabacum using Agrobacterium tumefaciens-mediated genetic transformation.The expression levels of some related genes were analyzed.The accumulation of lignin in transgenic tobacco was determined by Syros method,and the resistance of transgenic tobacco was analyzed.The main results are indicated as follows:1.Seven full-length cDNA sequences of DIR genes were isolated by RACE from P.notoginseng.The bioinformatics analysis demonstrated that those genes are typical DIRs.Phylogenetic analysis displayed that PnDIR1 belongs to DIR-a subfamily,but the other PnDIRs belong to DIR-b/d subfamily.The gene expression profile analysis indicated that seven genes can response to infection of F.solani,and the expression level of PnDIRs were up-regulated.Except for the PnDIR5,all the other PnDIRs answer to treatments with salicylic acid（SA）,Ethylene（ETH）,Methyl Jasmonate（MeJA）and H₂O₂.The PnDIR5 gene expression did not obviously respond to SA treatment,but PnDIR5 can answer to treatment of ETH,MeJA and H₂O₂ in the transcription level.PnDIR1 and PnDIR3 respond to all the molecule treatments,and their expression levels were up-regulated.2.The prokaryotic expression vector of pET-32a-PnDIR1 was constructedand transformed into Escherichia coli BL21（DE3）,and the recombinant protein was induced under 1 mM IPTG at 28℃,200rpm for 7 h.The molecular weight of PnDIR1recombinant protein is about 38kDa,and the PnDIR1 protein was purified by nickel column.The results of in vitro plate antifungal experiments indicated that the recombinant protein showed obvious inhibitory effect to mycelial growth of Gibberella moniliformis,Sclerotinia sclerotiorum,F.solani and F.oxysporum,and the antifungal activity can enhance with the increase of protein concentration.3.The subcellular localization vector of pBIN m-gfp5-ER-PnDIR1 was constructed and transformed into A.tumefaciens EHA105,which was used to infect onion（Allium cepa L.）epidermal cells.After two days of co-culture,the location of PnDIR1-GFP fusion protein was detected by laser scanning confocal microscopy.As a result,green fluorescence was specifically distributed on the cell wall,suggesting that the PnDIR1 protein was located at the plant cell wall.4.The plant overexpression vector pCAMBIA2300S-PnDIR1 was constructed and transformed into A.tumefaciens LBA4404.The transgenic tobacco was produced by leaf dish transformation,and the T2 generation transgenic tobacco was developed by by self-pollination.The results of qRT-PCR analysis showed that PnDIR1 was steadily expressed in transgenic lines,and the expression level some genes involved in the biosynthesis of lignin and lignan were increased compare with wild-type（WT）tobacco.Especially,the expression level of NtSAD2 in line D7 is 26.4 times higher than that in WT,and the expression level of NtP450 in line D13 is 37 times higher than WT.Meanwhile,the accumulation of lignin in transgenic tobacco was increased compare with WT.The resistance of PnDIR1 transgenic tobacco was significantly higher than WT after inoculation with F.solani,which revealed that overexpression of PnDIR1 enhance the resistance of transgenic tobacco to F.solani.In conclusion,P.notoginseng DIR gene family was involved in P.notoginseng defense response to F.solani,and they are important genes in P.notoginseng defense system,which can respond the infection by F.solani and treatment of signal molecules related to adversity stress.PnDIR1 is located at the cell wall,and the recombinant protein showed obvious inhibitory effect to growth of G.moniliformis,S.sclerotiorum,F.solani and F.oxysporum.The overexpression of PnDIR1 in tobacco induced some genes involved in biosynthesis of lignin and lignan,increased the accumulation of lignin,and enhanced the resistance of tobacco to F.solani.In short,isolation of P.notoginseng DIR gene family and function analysis of Pn DIR1 revealed that DIR gene family is involved in the molecular interaction between P.notoginseng and F.solani.This study laid the foundation for further exploring the mechanism of disease resistance in P.notoginseng and provided candidate genes for plant disease resistance gene engineering.