Cell Culture And Effect Of MeJA On Important Secondary Metabolites And Its Molecular Mechanism In Rosmarinus Officinalis L.

Rosmarinus officinalis L.（rosemary）belongs to Labiatae.Rosemary is a medicinal plant with high nutritional and medicinal values.Its main functional components include flavonoids,terpenoids and phenols.Cell culture is a new way to produce secondary metabolites.In this study,the leaves of rosemary were used to induce callus and subculture.Establish rosemary cell suspension culture system by the selection of rosemary callus,optimization of the rosemary cell suspension culture system,In order to fully utilize the useful genetic resources of rosemary functional metabolites,rosemary suspension cells was used as a material for transcriptome analyses of different concentrations of MeJA.Based on the analysis of physiological and biochemical characteristics of rosemary suspension cells under the treatments of different concentrations of MeJA,high-throughput sequencing technologies were used to analyze the transcriptome of m RNAs and miRNAs in response to different concentrations of MeJA in rosemary suspension cells,and to verify some m RNAs,micro RNAs and target genes.Based on the study of transcriptome,the effects of MeJA and batch addition of sucrose on the culture of rosemary suspension cells and their functional metabolites in bioreactor were explored using q PCR.This study was aimed at exploring the mechanism of MeJA regulation on functional metabolites of rosemary suspension cells,and providing the scientific evidences for mining MeJA-responsive genes resources of rosemary suspension cells functional metabolites.The main results were as follows:1.Callus induction and establishment suspension culture system of rosemary cell and optimizationRosemary leaves were used as the initial material to induce rosemary callus.High yield callus was screened out by different hormone combinations during subculture and cell suspension culture,The results show that:MS+2,4-D 1mg/L,sucrose 30 g/L,4-D1mg/L and sucrose 30 g/L were the best medium for callus proliferation,and fresh weight per bottle was 7.03.Suspension cell culture system was established for the light yellow callus with fine particle,loose and fragile appearance.When establishing suspension culture system of rosemary cells,the cell growth showed a typical"s"curve,and the optimal culture cycle was 8-9 days.The sucrose content and inoculation amount of suspension culture system were optimized.The results showed that:the optimum medium for suspension culture was MS+2,4-D1mg/L,sucrose 30 g/L,inoculation amount 40 g/L,temperature 25℃,rotation speed120 rpm,rosemary cell fresh weight 35.72 g/100ml,dry weight 0.76 g/100ml rosmarinic acid content 3.15 mg/g·DW.In conclusion,establishment the suitable cell suspension culture system of rosemary.2.Analysis of secondary metabolite content and physiological and biochemical changes in rosemary suspension cells under different concentrations of MeJAThe suspension culture system of rosemary cells.were treated with MeJA solution on the 6th day in suspension culture of rosemary cells.The concentration of MeJA solution was 0,10,50,100,150 and 200μmol/L MeJA,respectively.Optimization of the optimal concentration of MeJA on the accumulation of secondary metabolites in the suspension of Rosmary cells.The results showed that compared with the culture without MeJA,the addition of 10-200μmol/L MeJA on the 6th day of culture promoted the flavonoids,rosmarinic acid and carnosic acid in rosemary suspension cells in varying degrees.When the concentration of MeJA was 100μmol/L,the rosmarinic acid content in suspension cells reached the highest value of 3.29 mg/g,which was 1.66times of the control group,200μmol/L The content of carnosic acid and flavonoid reached the highest value at MeJA,which were 2.71 mg/g and 21.38 mg/g,respectively,which were 1.84 and 1.44 times of the control group;the proliferation rate of suspension cells had no significant change,but the cell viability decreased with the increase of concentration,and the cell viability was the lowest at 200μmol/L MeJA.The optimization of MeJA adding time in the process of culture showed that MeJA was added on the 0,2nd（delay period）,4th（initial stage of logarithmic growth period）and6th（middle and late stage of logarithmic growth period）days of suspension culture,and the treatment concentration was 100 The results showed that the contents of rosmarinic acid,carnosic acid and flavonoids in the suspension cells added with MeJA on the 4th and 6th day were significantly higher than those on the 0 and 2nd day;the proliferation rate of suspension cells added with MeJA on the 0 and 2nd day was significantly lower than that of the control group,and the cell viability of suspension cells increased with the delay of adding time and was lower than that of the control group.The results showed that MeJA could promote the accumulation of rosmarinic acid,carnosic acid and flavonoid in rosemary suspension cells,and had no significant inhibitory effect on the growth of rosemary suspension cells during logarithmic growth period.The suspension culture of rosemary cells under different concentrations of MeJA,the enzyme activities of PAL,SOD,POD,CAT and PPO increased significantly with the increase of MeJA concentration in the range of 0-100μmol/L;the contents of MDA,H₂O₂ and Pro decreased significantly with the increase of MeJA concentration,The results showed that 10-200μmol/L MeJA could activate the enzyme antioxidant system of rosemary suspension cells,and the activities of SOD,CAT or POD were increased,which could eliminate the potentially dangerous O^2-and H₂O₂ in plants;the contents of endogenous hormones GA,IAA and JA increased significantly with the increase of MeJA concentration from 0 to 150μmol/L,and began to decrease at 200μmol/L,The content of ABA and SA decreased significantly with the increase of MeJA concentration in the range of 0-200μmol/L,The results showed that different concentrations of MeJA could coordinate or antagonize different endogenous hormones in rosemary suspension cells.3.Transcriptomic analysis of secondary metabolite accumulation in rosemary suspension cells under different concentrations of MeJAThe m RNAs of rosemary suspension cells under different treatments（CK,M10,M10 and M100）were sequenced using high-throughput sequencing,and the sequencing analysis and validation were conducted.A total of 7836,6797and 8310genes were differentially expressed（DEGs）in the CK vs M10,CK vs M50 and CK vs M100 combinations,.Among them,the number of up-regulated genes was3596,3025and 4528,and the number of down-regulated genes was 4240,3772 and 3782.The results of GO and KEGG enrichment analysis for differentially expressed genes indicated that some basic pathways were high lyenriched in MeJA response process,such as cell wall biogenesis,galactose metabolism,phenylpropanoid biosynthesis,flavonoid biosynthesis and Linoleic acid metabolism etc;Mapman analysis of the primary metabolism of differentially expressed genes showed that most of the differentially expressed genes were mainly distributed in cells,lipids,sucrose,starch,amino acids,tricarboxylic acid cycle（TCA）,ascorbic acid and glutathione pathways;Mapman analysis of secondary metabolism showed that most of the differential genes were mainly distributed in non mevalonate pathway,shikimic acid pathway,mevalonate pathway,phenylpropanoid compounds,lignin and lignans,flavonoids,carotenoids and alkaloids pathways.Based on the analysis of the expression heat map of JA biosynthesis and signal transduction pathway,phenylpropane biosynthesis,flavonoid biosynthesis,terpenoid biosynthesis,plant hormone signal transduction pathway and differentially expressed genes,it was found that most of the differentially expressed genes were up-regulated by MeJA,and different concentrations of MeJA had different effects on gene induction,such as 100μmol/L MeJA was higer than the 10 and 50μmol/L MeJA,for example JA biosynthesis and signaling pathway,the expression of LOX,AOS,JAZ and MYC2.MYB,AP2-EREBP,b HLH and other transcription factors were significantly affected by MeJA in rosemary suspension cells.The q PCR verification results indicated that the relative expression of the different concentrations of MeJA signaling network genes was consistent with the m RNAs sequencing results.4.miRNAs omics analysis of functional metabolite accumulation in rosemary suspension cells under different concentrations of MeJAThe miRNAs of rosemary suspension cells under different treatments（CK,M10,M10 and M100）were sequenced using high-throughput sequencing,and the sequencing results were verified and analyzed.A total of 69 known miRNAs of 22miRNA families and 181 novel miRNAs were obtained;86 differentially expressed miRNAs,including 32 known miRNAs and 54 novel miRNAs,were screened out in CKvs M10,CKvs M50 and CKvs M100 comparison groups;4,7 and 4 known miRNAs and 6,23 and 6 novel miRNAs were specifically expressed in CKvs M10,CKvs M50and CKvs M100 comparison groups,respectively.KEGG enrichment analysis of miRNA target genes showed that MeJA had a variety of corresponding pathways on secondary metabolites of rosemary suspension cells,including plant hormone signal transduction,linoleic acid metabolism,α-linolenic acid metabolism,flavone and flavonol biosynthesis,anthocyanin biosynthesis,etc.;analysis of miRNA target genes by Cytoscape showed that miR156a-5p,miR160,miR160a-5p,miR167d＿1、miR396a-3p＿4、miR396a-3p＿5、miR396b、miR6300、novel＿mir151、novel＿mir176 and novel＿mir179 were most likely involved in the effect of MeJA on rosemary suspension cells.Mapman and the expression heat map analysis of different expressed miRNAs showed that different concentrations of MeJA significantly regulated cell wall,amino acids,starch and sucrose,tricarboxylic acid cycle,flavonoid metabolism,proteolytic enzymes and signal transduction,among which miRNAs and their target genes involved in plant hormone signal transduction:miR160a-5p and its target genes ARF,miR171b-3p,miR171b-3p＿3 and miR171d＿1 and its target genes DELLA,miR171b-3p and its target genes ETR,miR156a,miR156a-5p and novel＿mir4 and its target gene BRI1 interact with each other to regulate the accumulation of metabolites in rosemary suspension cells;peroxidase,a phenylpropane biosynthesis pathway gene,gene 3AT is targeted by miR167d＿1 of anthocyanin biosynthesis pathway,gene IF7MAT of flavone and flavonol biosynthesis pathway and isoflavone biosynthesis pathway were targeted by novel＿mir176、novel＿mir11 and novel＿mir179 can be targeted and affect the metabolism and synthesis of anthocyanins and flavonoids.The q PCR results showed that there was a complex regulatory relationship between miRNAs and target genes in rosemary suspension cells,including both positive and negative regulation.In response to different concentrations of MeJA in rosemary suspension cells,some miRNAs can negatively regulate their target genes at specific concentrations of MeJA.5.Optimization of large-scale culture conditions of rosemary suspension cells in5L bioreactoThis study obtained cultivation regularities of rosemary cells in the bioreactor.In this study,the culture conditions of Rosmarinus embryogenic suspensions in bioreactor were initially optimized to achieve the purpose of increasing the production of rosemary cells and functional metabolites.Results showed that the optimal ventilation of suspension cells cultured in bioreactor was 100 ccm,and agitation speed was 100 rpm,Culture period was 14 days.Based on the established and optimized suspension culture system of rosemary suspension cells,cell growth,flavonoid and rosmarinic acid contents,cell vitality,substrate consumption and so on were firstly studied in 100μmol/L MeJA treatments.Results found that the dry weight of the cells treated was no significant change,,the expression of JA signaling pathway transcription factor MYC2 and structural gene PAL、CHS were analyzed by q PCR were significantly up-regulated,the flavonoid contents increased by 4.62 mg/g and the rosmarinic acid contents increased by 0.49 mg/g compared to those without MeJA after 14 days culture.Based on the established and optimized cells suspension culture system of rosemary,cell growth,flavonoid and rosmarinic acid contents,cell vitality,substrate consumption and so on were firstly studied in batch addition of carbon source treatments.Results:the dry weight of the cells treated increased by 0.3 g/L,t he rosmarinic acid contents increased by 0.19 mg/g and the flavonoid contents increased by 0.89 mg/g by sucrose solution was added on the sixth day after 14 days culture.The results showed that the accumulation of flavonoids and rosmarinic acid in rosemary suspension cells could be improved by adding sugar sources in batches.