Study On Methyl Jasmonate Regulating The Release Of Secondary Metabolites From Broccoli Hairy Roots

The broccoli（Brassica oleracea L var.italica Planch）is a Brassica oleracea species belonging to the Brassicaceae family,which is dominated by the consumption of flower balls consisting of countless pedicels and flower buds,has attracted much attention because of the abundance of bioactive compounds including glucosinolates（GLS）,carotenoids,vitamins,mineral elements,phenolics,and so on.Methyl jasmonate（Me JA）treatment increased the level of plant secondary metabolites and caused their release into liquid medium,including Glucoraphanin（GRA）and sulforaphane（SF）,which are the main active components of broccoli;GRA and SF in broccoli hairy roots under Me JA treatment would be largely released into liquid medium,but the release mechanism has not been reported.In this study,hairy roots of broccoli were induced by the exogenous addition of Me JA,and the response surface experiments were designed using Design-expert8.0.6 software on the basis of single factor experiments with inoculum volume,p H,and culture temperature as independent variables and the total production of GRA and SF in the broccoli hairy root culture system as the response value;Using Illumina sequencing technology,we investigated the molecular mechanism of exogenous inducer Me JA in regulating the in vitro release of GRA and SF from broccoli hairy roots at different times,and the following conclusions were obtained:（1）When the inoculation volume of hairy roots was 0.15 g,the rotation speed was 110 r/min,the temperature was 25℃,the GRA production in hairy roots was significantly higher than that in other treatments;at this time,the MYR activity was maximal and the SF production in the medium was significantly higher than that in other treatments.Therefore,under these conditions,it is helpful for the synthesis of GRA and the release of SF into the medium.（2）When p H was 6.0,the yield of GRA in hairy roots was 23.43 times higher than that in medium.At when p H was 5.5,SF yield in medium was significantly higher than that in other treatments;MYR activity is maximal at this time,it can be concluded that when the p H is 5.5,the release of SF into the medium reaches the maximum.（3）When the medium volume was 100 m L,the yield of GRA in hairy roots and medium was significantly higher than that in other treatments.When the volume of the medium was 110 m L,MYR activity reached the maximum,at this time,the yield of SF in the medium was also significantly higher than other treatments.Therefore,a control medium volume of 100 m L facilitated the biosynthesis of GRA and SF in hairy roots,while a control medium volume of 110 m L facilitated SF release into the medium.（4）On the basis of the single factor experiments,the mathematical model was established by using box Behnken design in response surface analysis,the model significance test P<0.05 showed that the model was significant;the P value of mismatch was 0.3701>0.05,the mismatch was not significant.The correction coefficient R²_adj of the model is 0.8629,which can explain about 86.29%of the change of the response value.The determination coefficient R² is 0.9091,which has a good fitting degree and small experimental error.the regression equation is used to determine the optimal culture conditions:hairy root quantity of 0.15 g,culture temperature is 25.44℃,p H 5.56,under the condition of the verification experiment,broccoli hairy root of GRA and SF,total output of 2080μg/flask,theory of regression model to predict the output of 2440μg/flask,verify the value is lower than forecast,the difference is 360.（5）10 mmol/L Me JA treatment of broccoli hairy roots for 0,3,6,9,12 h,the GRA yield in hairy roots was significantly higher than the GRA yield in the medium,and both reached the maximum at 9 h;while hairy roots The yield of SF in the roots reached the maximum at 12 h;the activity of MYR in hairy roots decreased with the extension of Me JA treatment time,and the difference was significant;at 0 h,the activity of MYR was the largest,and at 12 h,the activity of MYR was the smallest.（6）A total of 4733 differential genes were involved in regulation at 0,3,6,9,and12 hours after Me JA treatment,of which 1024 genes were up-regulated and 3709genes were down-regulated.The KEGG pathway enrichment analysis of the common up-regulated differential genes revealed that the“SNARE interaction in vacuolar transport”pathway（ko04130）was enriched in the first place;the ABC transporter related genes ABCB19,ABCG6,ABCG36,ABCB9 were up-regulated at 0,3,6,9 h,respectively.The above results indicate that broccoli hairy root culture conditions are the main factor restricting the synthesis of GRA and SF,and the release of SF is regulated by multiple genes and multiple pathways.Although this study initially uses the transcriptome to study the molecular mechanism of SF release,which can provide theoretical support for the extraction of GRA and SF from fermentation broth in industrial production,it is necessary to fully reveal the molecular mechanism and signal regulation network of GRA and SF release Through the joint analysis of transcriptome,proteome and metabolome.