Cloning And Functional Analysis Of Two Panax Notoginseng Pathogenesis-related Proteins

Panax notoginseng?Burk?F.H.Chen is a traditional Chinese herbal medicine of the genus Panax in Araliaceae.Studies have shown that P.notoginseng saponins have many biological activities such as anti-tumor,anti-virus,cholesterol lowering and immunity,and are important drugs for preventing cardiovascular and cerebrovascular diseases.Its unique growth environment is easy to induce the occurrence of pests and diseases,especially fungal diseases.Among them,the problem of root rot has become a serious obstacle to the development of the planting industry in P.notoginseng,which has greatly affected the quality and yield of P.notoginseng.The main pathogen causing root rot is Fusarium solani.Therefore,through the study of the molecular interaction mechanism between P.notoginseng and root rot fungi,we found some disease resistance and defense genes of P.notoginseng,which provided a theoretical basis for the genetic breeding of P.notoginseng resistance.Our previous study found that pretreatment of P.notoginseng with methyl jasmonate?MeJA?increased the resistance to P.notoginseng to F.solani,and was inoculated with F.solani by pretreatment with methyl jasmonate.The transcriptome sequencing of P.notoginseng revealed that some pathogenesis-related proteins?PRs?genes can simultaneously respond to the treatment of exogenous methyl jasmonate and F.solani.In this paper,two PRs PnPR10-3 and PnPR like in response to MeJA were cloned by RACE technology,and their expression characteristics and functions were further analyzed.1.A PR10 gene PnPR10-3 was cloned from P.notoginseng by RACE.The full-length cDNA of the PnPR10-3 gene gene is 836 bp,wherein the open reading frame?ORF?encodes a protein of 158 amino acids and the molecular weight is 16.95kDa.Quantitative reverse transcriptase PCR?qRT-PCR?analysis showed that PnPR10-3 gene responded to F.solani infection,Moreover,the treatments of MeJA,ethylene?ETH?,hydrogen peroxide?H₂O₂?and salicylic acid?SA?induced the expression of PnPR10-3 gene to different degrees.After treatment with MeJA for 72 h,the expression of PnPR10-3 was about 3 fold higher than that of control.For the ETH treatment at 48 h,the expression of PnPR10-3 was about 4 fold higher than that of control.After treatment with H₂O₂ for 12 h,the expression of PnPR10-3 was about 6fold higher than that of control;after treatment with for 24 h,the expression of PnPR10-3 was about 4.3 fold higher than that of control.To further detect the activity of PnPR10-3,a prokaryotic expression vector pET-32a-PnPR10-3 was constructed to induce recombinant protein.The recombinant protein was improved significantly for the growth of F.oxysporum,F.solani,Colletotrichum gloeosporioides and F.verticillioides and the higher the protein concentration,the more obvious the inhibitory effect.The ribonuclease activity assay showed that the PnPR10-3recombinant protein has an in vitro RNase activity of approximately 350 U mg^-1.The subcellular localization vector pBIN m-gfp5-ER-PnPR10-3 was constructed to infect the onion epidermal cells,and the results indicated that PnPR10-3 was localized to the cytoplasm.At the same time,the over-expression vector pCAMBIA2300S-PnPR10-3was constructed and transformed into tobacco,and the T2 generation of transgenic tobacco plants was obtained.QRT-PCR analysis showed that PnPR10-3 was stably expressed in T2 tobacco,and the resistance of transgenic tobacco to F.solani was significantly stronger than that of Wild type?WT?.Further,the RNA interference?RNAi?vector pHellsgate2-PnPR10-3 was constructed and transferred into A.tumefaciens.After transient expression in P.notoginseng leaves,F.solani was inoculated.Compared with the control,the disease symptoms leaves of the experimental group were more severe.It indicated that the transient expression of pHellsgate2-PnPR10-3 in P.notoginseng leaves could reduce the resistance of P.notoginseng to F.solani.Moreover,qRT-PCR revealed that the transient expression of RNAi vector in P.notoginseng leaves significantly reduced the transcription level of PnPR10-3 gene in P.notoginseng leaves.2.A PR gene PnPR like was cloned from P.notoginseng using RACE technology.The full-length cDNA of the PnPR like gene gene is 976 bp,wherein the ORF encodes a protein of 238 amino acids and the molecular weight is 26.64 kDa.QRT-PCR analysis showed that PnPR like gene can respond to F.solani infection,Moreover,the induction of salicylic acid SA,ETH,MeJA and H₂O₂ can induce the expression of PnPR like gene to different degrees.After treatment with MeJA for 24 h,the expression of PnPR like was about 5.2 fold higher than that of control.After treatment with SA,the expression of PnPR like was about 5 fold higher than that of control.After treatment with H₂O₂ for 12 h,the expression of PnPR like was about 3.2fold higher than that of control.After treatment with ETH for 12 h,the expression of PnPR like was about 2.4 fold higher than that of control.To further detect the activity of PnPR like,the prokaryotic expression vector pET-32a-PnPR like was constructed to induce recombinant protein.Plate antibacterial experiments showed that the recombinant protein was significantly increased for the growth of Gibberella moniliformis,C.gloeosporioides and F.solani and the higher the protein concentration,the more obvious the inhibitory effect.The RNase activity assay showed that the PnPR like recombinant protein has an in vitro RNase activity of approximately 325 Umg^-1.Subcellular localization analysis indicated that PnPR like was localized to the cytoplasm.At the same time,the over-expression vector pCAMBIA2300S-PnPR like was constructed and transformed into tobacco,and the T2 generation of transgenic tobacco plants was obtained.QRT-PCR analysis showed that PnPR like was stably expressed in T2 tobacco,and the resistance of transgenic tobacco to F.solani was significantly stronger than that of WT.Further,the RNAi vector pHellsgate2-PnPR like was constructed and transferred into A.tumefaciens.After transient expression in P.notoginseng leaves,F.solani was inoculated.Compared with the control,the disease symptoms leaves of the experimental group were more severe.It indicated that the transient expression of pHellsgate2-PnPR like in P.notoginseng leaves could reduce the resistance of P.notoginseng to F.solani.Moreover,qRT-PCR revealed that the transient expression of RNAi vector in P.notoginseng leaves significantly reduced the transcription level of PnPR like gene in P.notoginseng leaves.In summary,PnPR10-3 and PnPR like respond to the infection of F.solani,and the prokaryotic recombinant proteins have obvious inhibitory activity against several pathogenic fungi such as F.solani.Overexpression of two P.notoginseng PRs in tobacco enhanced resistance to F.solani,indicating that these two genes play an important role in the resistance of P.notoginseng to F.solani.