| Ginseng(Panax ginseng C.A.Mey.),a perennial herb belonging to the Araliaceae ginseng genus,is one of the traditional luxury Chinese medical herbs.Ginseng is vulnerable to infection by various pathogens,which may cause a variety of diseases during its long period of growth.Currently,prevention and control of ginseng diseases is mainly based on chemical control.Although chemical control can effectively limit the occurrence of diseases,the residue of pesticides endangers the human body and hampers ecological development.With the development of genetic technologies,mining of endogenous disease resistance genes and cultivating disease-resistant varieties may provide novel methods to prevent ginseng diseases.Additionally,using molecular detection for early diagnosis of the pathogen may contribute to the prevention and control of ginseng diseases.In the present study,we focused on the research of ginseng disease resistance.First,NBS disease resistance genes in the transcriptomes of ginseng were studied,which may provide the theoretical basis for the development and utilization of these resistance genes.Second,the disease resistance mechanism of ginseng induced by Alternaria panax Whetz infection as well as molecular detection methods for the pathogen were also studied.These may provide a scientific basis for disease resistance breeding,prevention and treatment of black spot disease in ginseng.The main findings of this article are as follows:1.Ginseng NBS(PgNBS)resistance genes were identified from transcriptomes of 14 tissues of Jilin ginseng.The structures,evolution and expression profiles of these PgNBS genes were systematically analyzed,and these results laid the foundation for functional research and disease resistant breeding in ginseng.Four hundred and twelve PgNBS gene transcripts,derived from 284 gene models,were identified from the transcriptomes of 14 ginseng tissues.These genes were classified into eight types: TNL,TN,CNL,CN,NL,N,RPW8-NL and RPW8-N.Seven conserved motifs were identified in both the Toll/interleukin-1 receptor(TIR)and coiled-coil(CC)groups,whereas six were identified in the RPW8 group.Phylogenetic analysis showed that the PgNBS gene family is ancient,with the vast majority of its genes originating before ginseng as a species.In spite of their family grouping,the PgNBS genes have differentiated dramatically and may be sorted into numerous functional categories.The expression profile differed across tissues,ages of roots and the roots of different genotypes.However,the genes are coordinated in expression,forming a single co-expression network2.A total of 21 samples of ginseng leaves pretreated with Alternaria panax Whetz were collected,and their transcriptomes were sequenced using high-throughput sequencing technology.A series of differentially expressed genes were identified and analyzed using bioinformatics methods in order to reveal the molecular mechanisms of the interaction between ginseng and Alternaria panax Whetz.This information may be used for mining anti-disease germplasm resources and disease resistance breeding.A total of 102.42 Gb of sequence information was obtained from transcriptome sequencing of 21 samples.Analysis of differentially expressed genes showed that 1472,1841,703,1310,2591,2941 genes were up-regulated at 1,6,12,24,48 and 72 hours after pathogen treatment,respectively;there were 1089,1460,1095,1612,7000 and 2723 genes were down-regulated,respectively.Gene Ontology(GO)annotation showed that these differentially expressed genes fell into three categories: cellular components,molecular function and biological processes.Differentially expressed genes annotated in biological process were significantly enriched in response to stimulus(GO: 0050896),response to stress(GO: 0006950),etc.Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway analysis showed that differentially expressed genes were significantly enriched in plant hormone single transduction(ko04075),carbon metabolism(ko01200),and protein processing in endoplasmic reticulum(ko04141),etc.Plant-pathogen interaction pathway analysis revealed that CDPK,CALM,WRKY25 and RIN4 protein-encoding genes were varied from 1.2-5.3 times after pathogen treatment.3.The expression patterns of PgNBS genes in the leaves of ginseng treated with Alternaria panax Whetz were analyzed.We found some PgNBS genes that respond to Alternaria panax Whetz.Thus,these genes are potentially associated with defense against Alternaria panax Whetz colonization in ginseng.To investigate the expression patterns of PgNBS genes induced by pathogen growth,the sequences of 284 PgNBS genes were used as a query and aligned to the sequences obtained from the transcriptomes of ginseng leaves treated with Alternaria panax Whetz.The gene expression dates were isolated and analyzed.The results showed that 44 PgNBS genes were specifically expressed in the leaves of ginseng.These genes showed different expression levels and expression profiles when treated with Alternaria panax Whetz,among which PgNBS010,PgNBS023,PgNBS42,PgNBS62,PgNBS91 were significantly up-regulated under the treatment of Alternaria panax Whetz,we presumed that these genes are more important for ginseng resistant to Alternaria panax Whetz.4.A rapid and accurate molecular detection technology was established for identification of an important ginseng pathogen: Alternaria panax Whetz.This laid a theoretical foundation for early diagnosis and dynamic monitoring of pathogenic bacteria in the field and provided a new paradigm for the prevention and treatment of ginseng diseases.In this work,a single-tube nested PCR-lateral flow biosensor assay(STNPCR-LFBA),which combined single tube nested PCR and lateral flow biosensor assays,was developed for rapid identification of Alternaria panax Whetz in soil samples.STNPCR-LFBA retained the sensitivity of traditional two-step nested PCR while simplifying the test procedure and reducing the risk of cross-contamination.The PCR product was checked by a lateral flow biosensor assay,which provided the basis for migration of the test to a point-of-care(POC)microfluidic format.STNPCR-LFBA was specific to Alternaria panax Whetz,and no cross-reactions were observed in other non-target samples;the limit of detection was up to 0.01 pg of Alternaria panax Whetz genomic DNA.STNPCR-LFBA is fast,accurate and simple to operate,and it can be used for the detection of Alternaria panax Whetz in soil. |
