Production Of Chlorogenic Acid And Its Derivatives By Gardenia Jasminoides Cell Culture And Elicitation Of Methyl Jasmonate

Gardenia Jasminoides Eill(G.Jasminoides)is a common plant with dual-purpose of drug and food,and there are variety of bioactive secondary metabolites in its fruit.Among them,chlorogenic acids(CGAs)is one of the most important bioactive secondary metabolites but has not attracted sufficient attention from researchers due to its low content.Our group has induced callus of G.Jasminoides for the production of CGAs but found that its content was still low in the callus.In the current paper,conditions of callus induction were studied,and the technical system of producing CGAs by G.Jasminoides suspension cell was established via screening the callus with excellent status.Meanwhile,the qualitation and quantitation of major secondary metabolites in the G.Jasminoides cell were performed.Methyl jasmonate(Me JA)was used to stimulate and significantly enhance the accumulation of CGAs in the G.Jasminoides.On this basis,We carried out the RNA-seq of G.Jasminoides suspension cell elicited by Me JA and investigated the molecular mechanism of Me JA elicitation on CGAs biosynthesis form the perspective of gene regulation.The main results are as follows:(1)Callus induction and suspension culture establishment of G.Jasminoides: four types callus were obtained by the induction of different hormone content combination.Result showed that MS media with 0.3 mg·L-1 KT and 0.5 mg·L-1 NAA was most suitable for GJE callus induction and produced a callus formation rate of 97.78 ± 1.54 %.After continuously screening,the callus with a high growth rate,compact texture,and granular surface was obtained and was suitable for establishment of suspension culture.Subsequently,we carried out the optimization of culturing condition during suspension culture,including inoculation quantity,liquid filling quantity,rotation rate,and photoperiod.The most optimal condition was 15 % inoculation quantity + 40 % liquid filling quantity + 120 rpm + continuous photoperiod,under which cell biomass reached the maximum of 23 ± 0.25 g·L-1.(2)Qualitative and quantitative analysis of CGAs in G.Jasminoides cell: Seven CADs compounds were identified via HPLC-TOF-MS/MS,namely,3-caffeoylquinic acid,4-caffeoylquinic acid,5-caffeoylquinic acid,3,5-dicaffeoylquinic acid,4,5-dicaffeoylquinic acid,3,5-dicaffeoyl-4-O-(3-hydroxyl-3-methyl)-glutaroylquinic acid,and malonyl-4,5-O-dicaffeoylquinic acid.This is the first identification of malonyl-4,5-O-dicaffeoylquinic acid from GJE.Methodological evaluation indicated the HPLC method could be applied to quantify the CGAs content,and the maximum total CGAs content and yield was 1.24 ± 0.07 mg·g-1 and 23.42 ± 0.9 mg·L-1,respectively.(3)Elicitation of CGAs in G.Jasminoides cell and the screening of elicitors: the optimal elicitor,which showed the most significant effect on CGAs accumulation,was screened among SA,Me JA,ANE,and SNP.Result indicated that all those four elicitators exhibited varying degrees of promoting effect on the CGAs biosynthesis in G.Jasminoides cell.Among them,Me JA showed the best promoting effect and its optimized condition was as follows: on the 5th day of culture,Cells were co-cultured with 200 ?M for 3 days and then reached the maximum total CADs content and yield,up to 20.98 ± 1.1 mg·g-1 and 232.32 ± 9.1 mg·L-1,which were 19.51 times and 14.14 times those of the control,respectively.(4)RNA-seq of G.Jasminoides suspension cell elicited by Me JA: the control(G0h),the cell eilicited by 200 ?M for 8 h(G8h),and the cell eilicited by 200 ?M for 20 h(G20h)were collected and submitted for RNA-seq on Illumina Hi Seq 2500 platform,which generated 57069 high-quality Unigenes with average and N50 length of 655 and 1051 bp.Then the total unigenes were aligned to public database for annotation,including NR,Pfam,String,Swissprot,GO,COG and KEGG,Among which 80.7 % Unigenes were annotated in at least one database.(5)Analysis of differentially expressed genes in cells under Me JA elicitation: two comprasion(G0h_vs_G8h and G0h_vs_G20h)generated 3611 and 3405 differentially expressed genes(DEGs),respectively.In the KEGG enrichment of DEGs,Several pathways were enriched,which were relevant to biosynthesis and signal transduction of JA and CGAs biosynthesis.14 EDGs associated with JA biosynthesis and signal transduction were up-regulated.2158 and 1978 Unigenes differentially expressed in TF family in two comparisons,and many of them were classified into the TFs family which could respond to JA elicitation(b HLH,MYB,ERF,WRKY,b ZIP,and AP2).17 MYB TFs were up-regulated,among which MYB111,MYB60,MYB85,and MYB15 were involved in regulation of “Phenylpropanoid biosynthesis”.In “Phenylpropanoid biosynthesis” pathway,11 Unigenes were significantly up-regulated and associated with CGAs biosynthesis.In addition,the RT-q PCR method was used to verify the expression of 9 up-regulation genes related to CGAs biosynthesis.The results showed that the expression of these 9 genes was consistent with the result from whole-transcriptome sequencingOverall,In the current paper,the technical system of producing CGAs by G.Jasminoides suspension cell was established and the maximum total yield of CGAs was obtained under Me JA elicitation.On this basis,the molecular mechanism of Me JA elicitation on CGAs biosynthesis was investigated,which provide reference for popularization and application of producing CGAs by Jasminoides suspension cell.