| Panax ginseng is a perennial herb of the genus Panax in the family Panax ginseng.It is the first of the three treasures in Northeast China and is known as the "King of the Hundred Grasses" for its high medicinal value.In China,ginseng is mainly grown in Jilin,Heilongjiang and Liaoning provinces in Northeast China.Because ginseng has a long growth cycle and strict requirements on growing environment,it is easily infected by a variety of diseases,which has a great impact on its quality and yield.At present,there is a lack of disease-resistant strains of ginseng,and the control of diseases is mainly through artificial application of chemical agents,which can play a role in the prevention and control in the short term,but long-term application will enhance the resistance of pathogens to pesticides,aggravate the damage to the soil environment,and speed up the spread of diseases.Therefore,with the development of genomics and transcriptome technology,it is necessary to search for disease-resistant genes in ginseng and breed disease-resistant strains to avoid further infection and transmission of the disease.In this paper,through the isolation and identification of ginseng root rot bacteria,screening of disease resistance materials,molecular marker-assisted mining of ginseng root rot resistance genes,and using molecular biology,transcriptomics technology to carry out the study of ginseng root rot resistance mechanism.The main research results are as follows:1.Isolation and identification of root rot fungus of ginsengEight pathogenic strains were isolated and purified from disinfected ginseng plants.The morphological characteristics of pathogenic bacteria and conidia were observed on PDA medium,and the species of pathogenic bacteria were identified by r DNA-ITS sequence analysis.The results showed that 6of the isolates were Fusarium solani,and the r DNA-ITS sequence was 99% similar to that of the Fusarium solani in Gen Bank.Two strains were Fusarium oxysporum,and the r DNA-ITS sequence was 99% similar to that of Fusarium oxysporum in Gen Bank.The pathogenicity of the two pathogens showed that the pathogenicity of Fusarium solanum was stronger than that of Fusarium oxysporum.The results of this study have a certain guiding effect on the study of disease resistance breeding and pathogenic mechanism of ginseng.2.Screening of disease-resistant materials of ginsengThe results showed that the incidence of inoculation by the two methods was basically the same,which indicated that in vitro resistance screening could reflect the resistance difference between the resistant materials,and in vitro leaf screening had the advantages of simple operation and easy observation compared with root block screening.The positive and negative sides of ginseng leaves were inoculated by the two inoculation methods of fungus block and fungus liquid respectively,and the results showed that the positive inoculation with fungus block had better disease effect.The feasibility of the screening method for resistance in isolated leaves was further proved by ginseng whisker tie-back test.By inoculation of different strains of ginseng,it was found that the disease resistance of FS was the highest and that of FX2 was the lowest.A total of 1424 strains of Panax ginseng were screened by leaf inoculation in vitro.Finally,50 strains of highly resistant materials and some highly susceptible materials were screened.3.Screening of core SSR molecular markers for disease resistance of ginsengFor screening SSR markers associated with ginseng disease resistance properties,strain with resistance to root rot and root rot of genomic DNA as a template,using agarose gel electrophoresis and polyacrylamide gel electrophoresis,two methods of the early stage of the laboratory screening 18 of SSR molecular marker polymorphism primers for the ginseng root rot disease resistance of the filter,the results show that the Primer P29 can distinguish root rot resistance and susceptible strains of ginseng,indicating that this marker may be related to root rot resistance of ginseng,which is expected to be used as a molecular marker to identify root rot resistance of ginseng and be used for early screening of ginseng root rot.4.Mining SNP Loci of Root Rot Resistance Gene in Panax ginsengBy PCR amplification of 21 genes related to disease resistance,we sequenced the sequence that could expand the band between root rot resistance and susceptible material of ginseng.Through sequence alignment,only one SNP site was found in the resistant and susceptible strains of PR5 gene,and the site changed from G mutation to a mutation in susceptible strains.The above SNP sites were further verified by using the DNA of 6 root rot resistant and 6 root rot susceptible ginseng lines screened in the previous stage as templates.Sequence alignment results showed that the above SNP sites did not appear in the resistant strains,so the SNP sites may have nothing to do with disease resistance.Further real-time quantitative PCR analysis of the gene showed that the expression level of the gene in susceptible strains was higher than that in resistant strains,indicating that the infection of pathogenic bacteria may induce the overexpression of the gene.5.Transcriptomics study on the anti-root rot mechanism of ginsengTranscriptome analysis of ginseng root rot showed that there were significant differences between resistant and susceptible strains,and metabolic and cellular processes played a dominant role in biological processes.Among the cellular components,the cellular components and membrane components were significantly enriched,while in the molecular function category,the catalytic activity and binding genes were the most numerous.To KEGG metabolic pathway enrichment analysis,plant pathogen interactions and MAPK signal pathways in the key genes are expressed in the resistant strains were significantly raised,such as coding calcium dependent protein kinase,LRR serine/threonine protein kinase and cysteine protease gene,these disease-resistant gene expression enhanced resistance to root rot in ginseng. |
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