Transcriptome Analysis Of The Molecular Mechanism Of Methyl Jasmonate Regulating The Synthesis Of Secondary Metabolites In Broccoli Hairy Roots

Broccoli,as a “Star” species in Brassica,has been studied by many scholars because it is rich in anticancer material sulforaphane(SF).SF is one of the strongest anti-cancer and prve-cancer natural active ingredients recognized in vegetables so far.At present,SF was mainly obtained from the seeds or seedlings of broccoli,which had low yield and high cost.It has been reported that the use of hairy roots could improve the content of secondary metabolites synthesis.The culture system of hairy roots of broccoli has been successfully established in our laboratory.The content of SF in hairy roots of broccoli is higher than that in leaves and young roots of sterile seedlings.However,the study about nutrient consumption and micro-environmental changes in the culture system during the growth of hairy roots of broccoli,as well as the molecular mechanism of SF synthesis regulated by exogenous methyl jasmonate(MeJA)was not relatedly reported.Thus,the nutrient consumption and micro-environment changes in the broccoli hairy root multiplication cycle culture system during 30 days were first studied.Then based on the dynamic changes of hairy root growth,as well as the dynamic changes of GRA and SF synthesis,the molecular mechanism of MeJA regulating GRA and SF synthesis in the hairy roots was analyzed by transcriptomics.The conclusions are following:1.Nutrient consumption and micro-environmental changes in culture system during hairy root growth of broccoli.(1)In the growth process of hairy roots of broccoli for 30 days,the consumption of ammonium nitrogen was less than that of nitrate nitrogen.On 24 th day,the lowest ammonium nitrogen content was 47.64 mmol/L,which was 59.06% of the initial amount.On 30 th day,the lowest ammonium nitrogen content was 18.21 mmol/L,which was 33.69% of the initial amount.During hairy roots culture of broccoli,the consumption ratio of ammonium nitrogen(minimum)and nitrate nitrogen was 1:1.09.(2)During the growth of hairy roots of broccoli for 30 days,the carbon source was consumed.On 30 th day,the sucrose content in the medium was 40.75 mmol/ L,and the sucrose consumption rate(consumption rate = consumption quantity / initial quantity)was 53.50%.It could be concluded that more sucrose was consumed in the proliferation period(9-18 days).(3)During the growth of hairy roots in broccoli for 30 days,the conductivity of the culture system was 6.16 ms/cm on 24 th day,and the decrease rate of conductivity was 5.01%(decrease rate = decrease quantity/initial quantity);on 30 th day,the conductivity of the culture system was 5.225 ms/cm,and the decrease rate of conductivity was 19.43%.During the hairy root decay period(24th-30 th day)of broccoli,the electrical conductivity of the culture system decreased significantly.(4)The pH of the culture system changed during the growth of hairy roots of broccoli.The initial pH of the culture system was 5.921,4.318 on 18 th day and 4.651 on 30 th day.The pH of the culture system decreased first and then increased.2.Effects of MeJA on growth of the hairy roots and secondary metabolite synthesis in hairy roots of broccoli(1)Studies on the dynamic changes of broccoli hairy root proliferation showed that the growth of hairy roots of broccoli could be divided into five periods: lag period(0-6 days),differentiation period(6-9 days),proliferation period(9-18 days),static period(18-21 days)and decay period(after 21 days).(2)The 21 day,the proliferation amount of hairy root of broccoli is 18.25 times than the initial amount,and the change of proliferative multiple was large in the proliferative period.(3)Studies on the dynamics of the synthesis of GRA and SF in hairy roots of broccoli.The results showed that a large number of GRA and SF in the hairy roots of broccoli were synthesized at rest period,and the production of GRA in hairy roots was more than that in medium,but the production of SF in medium was more than that in hairy roots.(4)The GRA yield of hairy root was 5049.27 μg/flask under the treatment of 10 mmol/L MeJA concentration,which was 2.79 times than that of the control group.Under the same conditions,the SF yield was the largest,2549.34 μg/flask,which was 2.22 times than that of the control.3.Transcriptome analysis of secondary metabolite synthesis related genes by MeJA induced in hairy roots of broccoli(1)The hairy roots of broccoli were treated respectively by 10 mmol/L MeJA for 0,3,6,9,12 hours.Compared with 0 h,the yield of GRA and SF increased under other treatments,and the overall trend was increased at first and then decreased.The yield of GRA and SF reached the highest at 9 h,which was 2.22 times and 1.74 times of 0 h,respectively.(2)1195 differentially expressed genes were observed in hairy roots treated by 10 mmol/L MeJA for 0,3,9 h.Among them,304 genes were up-regulated expression and 763 genes were down-regulated expression.(3)By KEGG pathway enrichmentanalysis for common up-regulated expression genes at 0,3th,9th h,we found that the enrichment of "glucosinolate biosynthesis" pathway(KO00966)ranked 20 th.(4)BCAT2,MAM1,CYP79B1,CYP83B1,UGT74B1 and FMOGS-OX5,the six key genes regulating GRA synthesis,were up-regulated expression at 0,3th h and down-regulated expression at 9th h.(5)The pathway map of the biosynthesis of GRA and GRA converted to SF in hairy roots of broccoli induced by MeJA was simulated(see the result analysis).