Regulation Mechanism Of Methyl Jasmonate On Secondary Metabolism Of Isodon Rubescens

Isodon rubescens(Hemsl.)Hara,which is used as medicine from dry aerial parts,belongs to the genus Isodon of Labiatae.The main active components are diterpenoid,such as oridonin,ponicidin,and rosmarinic acid,which belongs to a phenolic acid.Methyl jasmonate(Me JA),as a plant stress adaptation hormone,regulates plant growth,development and secondary metabolism.In this thesis,I.rubescens was treated with gradient Me JA spray by nutrient solution culture method.It were analyzed to explore the effects of jasmonate signal on glandular trichome development and active components of I.rubescens that the morphological structure,glandular trichome frequency,secondary metabolite concentration,plant hormone changes,key enzyme gene expression changes and transcriptome of I.rubescens treated with Me JA.The regulation mechanism of oridonin can provide theoretical and experimental basis for the cultivation of excellent germplasm and the optimization of management measures of oridonin cultivation.The main results are as follows.(1)The distribution of oridonin in glandular trichomes was qualitatively analyzed by histochemical staining and desorption electrospray ionization-mass spectrometry(DESI-MS)method.The main accumulation of oridonin in glandular trichome was analyzed quantitatively by step extraction.Only the glandular trichomes containing terpenoids had fluorescence reaction after concentrated hydrochloric acid vanillin staining,but the non-secretory epidermis,leaf epidermis cells and mesophyll cells of I.rubescens had no fluorescence reaction.The fresh samples were scanned by DESIMS,and the signals were mainly distributed on the leaf surface.The concentration of oridonin in glandular trichome extract was 77.44%,and the concentration of oridonin in mesophyll extract was about 10.20%.The extraction rate was 87.64%.(2)The structure,shape and size of glandular trichome were observed by transparent leaf observation,scanning electron microscope and stereomicroscope,and the frequency of glandular trichome was counted.The results showed that Me JA treatment had an effect on the development of glandular trichome of I.rubescens,and50 μM concentration of Me JA promoted the formation of glandular trichome.The results showed that the leaf surface of I.rubescens had epidermis and glandular trichomes,and the glandular trichomes had head like glandular trichomes and shield like glandular trichomes.However,the frequency of glandular trichomes of I.rubescens was regulated by jasmonic acid signalling.The glandular trichomes increased at low concentration and decreased at high concentration.The effect of 50 μM concentration of Me JA on the formation of glandular trichomes was the best.(3)Through gradient screening pre-experiment(concentration of Me JA: 0 μM,50μM,100 μM,200 μM,400 μM)and 0-100 μM Me JA experiment,the morphological characters,vegetative growth,secondary metabolism and key enzyme gene expression of I.rubescens were determined,and the results showed that Me JA could regulate the growth,development and secondary metabolism of I.rubescens.50 μM Me JA promoted the growth of I.rubescens and the accumulation of oridonin.The results showed that the net photosynthetic rate,number of branches,biomass,oridonin concentration,oridonin accumulation and gene expression of key enzymes in related pathways of the plants were significantly increased at the appropriate concentration of Me JA(50 μM)compared with the control.Me JA had little effect on rosmarinic acid concentration,but the accumulation of rosmarinic acid increased with the increase of plant biomass.Me JA promoted the synthesis of oridonin and rosmarinic acid,but the promotion of diterpenoids was more obvious.(4)Exogenous application of Me JA could affect the hormone balance of I.rubescens.Treatment with 50 μM Me JA increased the concentration of endogenous jasmonates.Me JA treatment increased the concentrations of free auxin and cytokinin,increased the concentrations of indoleacetic acid glutamate,dihydrozeatin nucleoside and N6 isopentene adenine,and decreased the concentrations of bound auxin and cytokinin.Me JA reduced the concentration of gibberellin,abscisic acid and salicylic acid,but did not affect the concentration of ethylene.In summary,50 μM Me JA increased the concentration of free auxin and cytokinin to promote the growth of I.rubescens,and promoted the concentration of jasmonate in vivo to enhance the secondary metabolism of I.rubescens.(5)Through the analysis of transcriptome sequencing,354 differentially expressed genes were obtained after 50 μM methyl jasmonate treatment of I.rubescens,including195 up-regulated genes and 159 down-regulated genes,which were mainly enriched in secondary metabolite biosynthesis,material metabolism,plant MAPK signaling pathway,plant pathogenic microorganism interaction,plant hormone signaling pathway and so on.The regulation of Me JA on I.rubescens can promote plant growth,affect plant secondary metabolism and promote the formation of diterpenoids.In this project,oridonin is mainly distributed in glandular trichomes,it provided important experimental data and theoretical reference for the regulation mechanism of Me JA on the metabolism of I.rubescens,and lays a foundation for further study on the regulation mechanism of jasmonate hormones on the metabolism and active ingredient synthesis of I.rubescens.The reasearch provided theoretical basis and guidance suggestions for the optimization of cultivation and management measures of I.rubescens,and has great economic value for the effective development and rational utilization of I.rubescens.