Cloning And Functional Analysis Of Two Panax Notoginseng Antimicrobial Peptides Genes

Panax notoginseng?Burk?F.H.Chen is mainly produced in Wenshan Prefecture of Yunnan Province,and belongs to Panax in the Araliaceae.It is a traditional Chinese medicinal herb with various biological activities,such as reducing swelling,relieving pain,promoting blood circulation and alleviating blood stasis.Some fungal diseases including root rot and black spot severely affect the production and quality of the P.notoginseng medicinal materials.The root rot,which is mainly caused by Fusarium solani and F.oxysporum,is the most serious disease in P.notoginseng.The pretreatment of P.notoginseng with MeJA significantly increased its resistance to F.solani in our previous study.Therefore,high-throughput sequencing technology was used to perform transcriptome sequencing and analyze the gene expression profiling of MeJA pretreated P.notoginseng during F.solani infection.The transcriptome data analysis revealed that two P.notoginseng antimicrobial peptide genes were induced by exogenous MeJA treatment and responded to F.solani infection.To deeply understand the functions of two antimicrobial peptides in the interaction between P.notoginseng and F.solani,the following studies were carried out and the results are as follows:1.The full-length cDNA sequence of a defensin-like protein gene?PnDEFL1?was isolated from P.notoginseng by RACE.The full-length cDNA sequence of PnDEFL1 is 702 bp,and the ORF encodes a protein with 77 amino acid residues.During the infection of F.solani,exogenous MeJA pretreatment of P.notoginseng significantly increased the transcript accumulation level of PnDEFL1 compared with the sterile water treatment.In addition,the accumulation levels of PnDEFL1 in P.notoginseng root were induced in different degrees by MeJA,ETH,H₂O₂ and SA.The subcellular localization vector pBIN m-gfp5-ER-PnDEFL1 was constructed and transformed into Agrobacterium tumefaciens EHA105,which was used for transient expression of PnDEFL1-GFP fusion protein in onion?Allium cepa?epidermal cells.After two days of culture,the localization of PnDEFL1-GFP fusion protein was detected by a laser confocal microscopy.The green fluorescence of onion cells harboring the pBIN m-gfp5-ER-PnDEFL1 vector was specifically distributed in the cell wall,indicating that PnDEFL1 is an extracellular protein that localized in the plant cell wall.The PnDEFL1 protein with the signal peptide was expressed in E.coli BL21?DE3?,and the PnDEFL1 prokaryotic recombinant protein was approximately25 kDa.Then the PnDEFL1 protein was purified by nickel affinity chromatography.The antifungal assay showed that the PnDEFL1 recombinant protein inhibited the mycelial growth of F.solani,Botryosphaeria dothidea,F.oxysporum and Sclerotinia sclerotiorum in vitro,and the protein concentration is positively correlated with its anti-fungal activity.Meanwhile,the prokaryotic recombinant protein of PnDEFL1inhibited the germination of F.solani spores.Compared with the control?PDA liquid medium?,the spore germination rate of F.solani adding the recombinant protein of PnDEFL1 was about 33%.The over-expression vector pCAMBIA2300S-PnDEFL1was constructed and transformed into tobacco,and the T2 generation of transgenic tobacco plants were obtained.The results of qRT-PCR analysis showed that PnDEFL1was steadily expressed in T2 transgenic tobacco.The tobacco leaves and roots were inoculated with F.solani,and the resistance of PnDEFL1 transgenic tobacco lines to F.solani was greatly enhanced compared with wild type tobacco.In addition,the RNAi vector pHellsgate 2-PnDEFL1 was constructed and transformed into P.notoginseng leaves by A.tumefaciens.After 24 hours of A.tumefaciens transforming,the F.solani was inoculated.After 72 h of F.solani inoculation,the area of lesions produced in the P.notoginseng leaves expressing PnDEFL1 RNAi construct were significantly larger than control?transformed RNAi empty vector?.The above experimental results indicate that PnDEFL1 is an important disease resistance gene in P.notoginseng.2.The full-length cDNA sequence of a snakin antimicrobial peptide gene?PnSN1?was cloned from P.notoginseng by RACE.The full-length cDNA sequence of PnSN1 is 677 bp,and the ORF encodes a protein with 104 amino acid residues.The accumulation levels of PnDEFL1 in P.notoginseng roots were induced in different degrees by MeJA,ETH,H₂O₂ and SA.Compared with sterile water pre-treatment of P.notoginseng,the MeJA pre-treatment of P.notoginseng significantly increased the transcription levels of PnSN1 during the F.solani infection.Subcellular localization results that the green fluorescence of onion cells harboring the pBIN m-gfp5-ER-PnSN1 vector was specifically distributed in the cell wall,indicating that PnSN1 is an extracellular protein that localized in the plant cell wall.The PnSN1 protein with the signal peptide was expressed in E.coli BL21?DE3?,and the PnSN1 prokaryotic recombinant protein was approximately 28 kDa.The antifungal assay showed that the PnSN1 recombinant protein inhibited the mycelial growth of F.solani,F.oxysporum,F.verticillioide,and B.dothidea in vitro,and the protein concentration is positively correlated with its anti-fungal activity.Meanwhile,the prokaryotic recombinant protein of PnSN1 inhibited the germination of F.solani spores.Compared with the control,the spore germination rate of the recombinant protein of PnSN1 was about 32%.The plant overexpression vector pCAMBIA2300S-PnSN1 was constructed and transfected into tobacco by A.tumefaciens-mediated.The tobacco leaves and roots were inoculated with F.solani,and the resistance of PnSN1 transgenic tobacco lines to F.solani was greatly enhanced compared with wild type tobacco.In addition,the RNAi vector pHellsgate2-PnSN1 was constructed and transduced into P.notoginseng leaves by A.tumefaciens.After 72 h of F.solani inoculation,the area of lesions produced by P.notoginseng expressing PnSN1 RNAi construct was significantly larger than transformed RNAi empty vector.The above results indicate that PnSN1 is an important disease resistance gene in response to F.solani infection in P.notoginseng.The cloning of P.notoginseng snakin and defensin antibacterial peptide genes and functional analysis of PnSN1 and PnDEFL1 revealed that snakin and defensin antimicrobial peptide genes are involved in the molecular interaction between P.notoginseng and F.solani.This study help in further exploration of the molecular mechanism of disease resistance and defense in P.notoginseng,and it also provides candidate genes for plant disease resistance genetic engineering.