| Panax notoginseng(Burk.)F.H.Chen is a pearl among Chinese herbal medicines,has high medicinal value and is widely planted in Wenshan of Yunnan Province.However,due to the long growth cycle of P.notoginseng and the stress of fungal,bacterial and viral diseases,the sustainable development of P.notoginseng industry has been seriously hindered.Therefore,it is urgent to deeply study the influence of viral diseases on the growth and development of P.notoginseng and the mechanism of P.notoginseng responding to viral diseases.In the previous reports on P.notoginseng viral diseases,only a few studies have identified several pathogens of P.notoginseng viral diseases by traditional means,such as morphological observation and serological analysis,and there is no research on the molecular mechanism of P.notoginseng against viral infection.Hence,the deep sequencing of small RNAs(s RNAs)was used to identify the viruses of diseased P.notoginseng with chlorosis,mosaic and pitting symptoms in this study.In addition,real-time fluorescent quantitative PCR(q PCR)technology is used to establish a rapid viral detection system to provide a faster and more accurate viral detection means.At the same time,the influence of viral diseases on the biosynthesis of P.notoginseng saponins(PNS)and photosynthesis was analyzed.Finally,the interaction mechanism between P.notoginseng and viruses was studied through transcriptome,proteome and metabolome analysis,which provided theoretical basis for the prevention and treatment of P.notoginseng viral diseases.The main results of this paper are as follows:1.Through deep sequencing of s RNA of diseased P.notoginseng with three symptoms,Panax notoginseng A(Pn VA)and Panax cyrptic virus 4(PCV4)were identified.The existence of the two viruses was verified by RT-PCR,and possible spherical viral particles were found by transmission electron microscopy(TEM).Based on the phylogenetic analysis of RNA-dependent RNA polymerase gene sequence of PCV4,it is speculated that PCV4 belong to alphapartitivirus.In addition,a q PCR detection method for Pn VA and PCV4 was established.Compared with RT-PCR detection method,this method has the advantages of more accuracy and sensitivity,and it also has high specificity and repeatability.2.In the aspect of saponin biosynthesis,the expression levels of key genes including 3-hydroxy-3-methylglutaryl-coa synthase(HMCAS)and squalene epoxidase(SE)in diseased P.notoginseng with chlorosis,mosaic and pitting symptoms were significantly down-regulated compared with the healthy P.notoginseng(control),and the content of several ginsenosides also decreased.Among them,the synthesis of saponins from diseased P.notoginseng with chlorosis symptoms is most inhibited by viruses,while that of diseased P.notoginseng with mosaic symptoms is least affected.In the aspect of photosynthesis,the expression levels of photosynthesis related genes including ribose diphosphate carboxylase(Rubisco)and fructose 1,6-diphosphate aldolase(FBA)in P.notoginseng with three symptoms were all down-regulated.In addition,the photosynthetic parameters including chlorophyll content as well as the enzymatic activity of Rubisco 、FBA and FBP are obviously decreased.3.In addition,the interaction mechanism between P.notoginseng and viruses was preliminarily studied based on transcriptome,proteome and metabolome differential analysis.The differentially expressed genes in RNA sequencing,are mainly enriched in several pathways including plant hormone signal transduction,plant-pathogen interaction and MAPK signal pathway.The differential proteins in proteome are mainly related to porphyrin and chlorophyll metabolism,photosynthesis and oxidative phosphorylation.The differential metabolites in metabolome are mainly involved in ABC transporter,glucosinolates biosynthesis and starch and sucrose metabolism.Multiomics analysis based on metabolic pathways showed that viral infection inhibited physiological pathways such as photosynthesis and sugar metabolism of P.notoginseng,and speculated that hypersensitive response(HR),oxidative phosphorylation,salicylic acid(SA)and jasmonic acid(JA)signal pathways were involved in the defense response of P.notoginseng against viruses.In a word,the results of this paper enrich the identification methods and detection methods of the pathogens of P.notoginseng viral diseases,and have a deeper understanding of the influence of viral diseases on the growth and development of P.notoginseng.Moreover,multi-omics joint analysis preliminarily reveals the molecular mechanism of the interaction between P.notoginseng and viruses,which is beneficial to the prevention and control of P.notoginseng viral diseases in the future. |
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