| Tianma(Gastrodiae rhizome)is the dried tuber of Gastrodia elata Blume(Orchidaceae),which has the effects of relieving wind and relieving spasm,suppressing liver yang,and dispelling wind and removing obstruction in meridians.G.elata Bl.f.glauca S.Chow is a forma of G.elata.The commercial ones have high drying rate,and good quality.It is the main plant source of Gastrodiae rhizome.G.elata Bl.f.glauca S.Chow is distributed in the provinces from northeast to southwest and Changbai Mountain area is one of the important producing areas of G.elata Bl.f.glauca S.Chow.G.elata are often infected by pathogenic microorganisms during its cultivation,and disease prevention and control is a matter of great concern for the production of G.elata.According to incomplete statistics,the incidence of G.elata disease is 6% to 17%,and fungal diseases are the main ones,resulting in yield reduction of 10% to 30%,and even no harvest in severe areas.80% life cycle of G.elata grows underground.The vegetative growth of tubers,the source of Gastrodiae rhizome,is completely underground.The occurrence of diseases is difficult to observe,and it is inconvenient to carry out mechanical(physical)control.The use of chemical pesticides such as fungicides may damage the symbiotic bacteria of G.elata,which also caused a decrease in their production.In addition,G.elata may also be infected by pathogenic bacteria during its storage period,causing rot symptoms as well as economic losses.The selection and breeding of resistant varieties of G.elata is a sustainable strategy for disease prevention and control of G.elata.With the rapid development of biotechnology,research on molecular breeding based on omics in the field of medicinal plants is increasingly being carried out.The discovery of key genes for G.elata disease is the basis of G.elata molecular breeding.However,the molecular breeding of G.elata is still in its infancy.The related genes of G.elata disease have not been reported yet,and its disease defense mechanism remains to be clarified.Therefore,this study carried out the discovery and functional analysis of the genes related to the disease defense of G.elata in Changbai Mountain,and analyzed the mechanism of action of the candidate gene Ge JAZ1 for disease defense of G.elata on the basis of transcriptomics.Finally,the four agronomic traits of G.elata in Changbai Mountain has been evaluated for disease resistance,and the main research results are as follows:1.Screening of defense genes for Changbai Mountain G.elata disease.Through transcriptome sequencing and bioinformatics analysis,7540 differentially expressed Unigenes(DEGs)were identified.According to KEGG pathway,gene expression differential FC,DEGs expression level and related studies reported in the literature,10 disease defense candidate genes were screened,and Gen Bank ID of them were MZ298646,MZ298645,MZ298652,MZ298643,MZ298647,MZ298654,MZ298648,MZ298649,MZ298650,MZ298651.2.q RT-PCR verification of candidate genes and metabonomic analysis.Using q RT-PCR technique,the transcript levels of 10 candidate genes screened out by transcriptome were analyzed.LC-MS analysis of metabolite levels and correlation analysis with transcript levels were conducted.The results showed that expression trends of nine genes were consistent with the transcriptome results.Thirteen differential metabolites were significantly enriched in propanoate metabolism,oxidative phosphorylation,zeatin biosynthesis,purine metabolism,phenylalanine metabolism,tyrosine metabolism,pyruvate metabolism,nicotinate and nicotinamide metabolism,sulfur metabolism,phenylpropanoid biosynthesis pathways.They are consistent with the transcriptome DEGs enrichment results.3.Obtaining and sequence analysis of candidate genes for disease defense.Primers were designed according to the ORF sequence of the candidate genes,and 6 target genes were obtained by RT-PCR amplification.According to the results of Blast,the 6 genes were named Ge JAZ2,Ge WRKY33,Ge TGA1,Ge CML18,Ge ERF1,Ge JAZ1.Among them,Ge JAZ1 was further identified as a key candidate gene.4.Functional analysis of Ge JAZ1,a candidate disease defense gene of Changbai Mountain G.elata.(1)Transient transformation of Ge JAZ1 in Changbai Mountain G.elata.The transiently expressed vector p HB-Ge JAZ1-YFP was constructed by seamless cloning and homologous recombination technology,and the recombinant plasmid p HB-Ge JAZ1-YFP was transferred into the tuber tissue of Changbai Mountain G.elata by Agrobacterium-mediated method for transient transformation.PCR results showed that Ge JAZ1 gene was integrated into G.elata genome;q RTPCR results showed that Ge JAZ1 gene was transcribed at a high level on the second day after Agrobacterium infection;SDS-PAGE showed that Ge JAZ1-YFP fusion protein was expressed in tuber tissues of Changbai Mountain G.elata.(2)Determination of defense-related indexes and medicinal components of Ge JAZ1 transiently expressed G.elata.Microplate reader was used to determine the defense-related indicators,and HPLC was used to determine six medicinal components of G.elata.The results showed that,compared to the control group,the activity of superoxide dismutase(SOD),peroxidase(POD)and catalase(CAT)activities in Ge JAZ1 transiently expressed G.elata was higher,and the content of malondialdehyde(MDA)did not change significantly.There was no significant change for 6 medicinal components in Ge JAZ1 transiently expressed of G.elata.5.Defense mechanism analysis of Ge JAZ1.(1)Bioinformatics analysis of Ge JAZ1 gene sequence.The Ge JAZ1 gene open reading frame(ORF)is 516 bp in length,encoding 171 amino acids,with a protein molecular weight of18544.14 and an isoelectric point of 8.43.According to the conserved domains,7 G.elata JAZ proteins were retrieved from the Changbai Mountain G.elata transcriptome database.(2)Ge JAZ1 transcription regulation analysis.Transcriptome sequencing of Ge JAZ1 transiently expressed G.elata showed that differentially expressed transcription factors or enzyme genes appear in signal transduction pathways(e.g.MAPK signal transduction,phytohormone signal transduction,plant-pathogen interaction),biosynthetic pathways(e.g.steroids,ubiquinone,terpenoids,phenylpropanes,alkaloids,flavonoids)and sugar and lipid-related pathways.Transcriptome-transcriptome analysis shows that Ge JAZ1 is indirectly involved in the disease defense response of G.elata in Changbai Mountain.6.Evaluation of disease resistance of G.elata with different agronomic traits.Analysis of the expression levels of defense candidate genes for four agronomic traits and determination of defense-related indicators showed that the disease defense gene Ge TGA1 has the highest expression level in long-rachis and large-fruited G.elata;SOD activity is the highest in longrachis and mid-fruited G.elata.POD activity is highest in long-rachis and small-fruited G.elata,CAT activity is higher in long-rachis and large-fruited G.elata,and MDA content is higher in long-rachis and large-fruited G.elata.Analysis from the above defense-related indicators,the potential disease resistance of long-rachis G.elata is stronger. |
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