| Chlorogenic acid is one of the main effective functional components of antibacterial,anti-virus,anti-cancer,lowering blood lipid and lowering blood sugar,and anti-oxidation effects of traditional Chinese herbal medicines-Lonicera japonica Thunb.It has been widely used in medicine,food,cosmetics and other fields.The efficient biosynthesis of chlorogenic acid and has gained increasingly attention in recent years.Many experts have concentrated on the exploration of the key enzyme genes for the chlorogenic acid synthesis pathway.In this paper,Lonicera japonica Thunb.suspension cell culture,established by our research group in the previous experiments,was used as the study material.Firstly,the elicitors of high-yield chlorogenic acid was selected and the induction conditions were optimized;secondly,Illumina Hiseq X Ten platform was used to analyze the differentially expressed genes(DEGs)in Phenylpropanoid biosynthesis under the induction of Me JA,key enzyme genes for the production of chlorogenic acid were screened;lastly,the consumption rules of nutrients in Lonicera japonica Thunb.suspension cells solution which after induction and normal growth were studied.Below are key research findings:(1)The study on induction technology of high-yield chlorogenic acids in Lonicera japonica Thunb.suspension cells and its products accumulation rules: The main secondary metabolites are chlorogenic acid,isochlorogenic acid A,isochlorogenic acid B,and isochlorogenic acid C.Three efficient elicitors were selected: methyl jasmonate(Me JA),salicylic acid(SA),ultraviolet irradiation(UV).The best condition followed: the final concentration of Me JA at 200μmol/L,the final concentration of SA at 50μmol/L and the irradiation time of UV at 2h,under this condition the total content of chlorogenic acids reached up to 9.68% in dry cells.The concentration of isochlorogenic acid A was the highest,followed by chlorogenic acid,among all the products.The added elicitors affected the growth of suspension cells slightly,with the wet weight and dry weight of cells decreased by 22.54%、7.25% respectively.(2)The consumption rules of nutrients in Lonicera japonica cell suspension culture solution: It displayed the tendency declining rapidly at the beginning then slowly,they all recovered slightly in the later period.The consumption rate of the induced group was slower than the control group;the total sugar consumption was slower than that of the reducing sugar.Ammonium nitrogen consumption was faster than nitrate nitrogen,and the amount of nitrogen source had picked up in the later period,which are similar to other plant suspension cells.The amount of these substances can be adjusted or added halfway in large-scale culture of cells.(3)The transcriptome sequencing and analysis Lonicera japonica Thunb.suspension cells induced by Me JA: the transcriptome sequencing of four suspension cell samples(0h group without Me JA,Me JA induced 4h,10 h and 20 h)were carried out respectively using Illumina Hiseq X Ten platform.A total of 46,858 Unigenes were obtained,Average Length,N50,and N90 were 810.85 bp,1304 bp and 326 bp,respectively;the resulting Unigenes were compared with databases of KOG,NR,Swiss-Prot,Tr EMBL,KEGG,GO,and there were 33,286(71.04%)was successfully annotated,3827 genes were annotated in all databases,and a large amount of annotation information was obtained.12641,8134,and4862 DEGs between 4h and 0h,10 h and 0h,and 20 h and 0h after induction;screened 20 Unigenes related the CGA biosynthesis:TRINITY＿DN15699＿c4＿g2(C4H),T R I N I T Y＿D N 1 5 9 9 0＿c 3＿g 3(PA L),T R I N I T Y＿D N 1 4 3 3 3＿c 0＿g 1(H C T),T R I N I T Y＿D N 1 4 0 1 3＿c 1＿g 9(C 3 ’ H),T R I N I T Y＿D N 1 3 4 11＿c 1＿g 1(C A D),a n d TRINITY＿DN14179＿c1＿g1(4CL).Speculated the possible synthesis pathway of CGA in Lonicera japonica suspension cells to be hydroxylation of C3’H to CGA.Provide a reference for further research of the induction and regulation biosynthesis mechanisms of CGA and utilizing genetic engineering enhance accumulation chlorogenic acids. |
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