Study On The Accumulation Of Secondary Metabolites Of Mulberry Suspended Cells By Inductor

Terpenoids,phenols and alkaloids produced by mulberry secondary metabolism have various physiological activities.In recent years,the method of plant cell culture to produce secondary metabolites has the characteristics of being free from environmental restrictions and promoting cell proliferation,and has been used in the industrial production of plant secondary metabolites.In the process of plant cell suspension culture,enzyme activity in cell metabolism can be activated by adding inducers,which can increase the yield of secondary metabolites and sometimes can induce new compounds.Therefore,based on the establishment of the mulberry suspension cell culture system,different concentrations elicitors were added to the mulberry suspension cell to promote the secondary metabolites accumulation in the mulberry suspension cells.The main research results are as follows:The leaves of the mulberry Yu-711 were used as explants to induce callus and initially establisht suspension cell lines.Mulberry suspension cell growth curve showed "S" type that add 1.5 mg/L 6-BA + 0.5 mg/L 2,4-D + 0.1 mg/L NAA in MS liquid medium.The highest peak of living cells growed the 16 th day in suspension culture.After 16 days of cultivation,the nutrient consumption in the medium and the toxic metabolites accumulation result in the growth slow.Therefore,in order to maintain the exuberant cell division ability and cell activity,the subculture time of mulberry suspension cell culture is about 16 days.Different concentrations of coronatine(COR),β-CD,and chitosan were used as inducers to induce mulberry suspension cells,and influence of their secondary metabolites 1-deoxynojirimycin(DNJ),total phenol,total flavonoids accumulation on mulberry suspension cells were studied.The concentration of total DNJ in the mulberry suspension cell line treated with 1.0 μmol/L COR induced an increase of264% compared with the control group(P<0.05).The content of total flavonoids in the 1.0 mmol/L cyclodextrin-treated group was higher than control group,increase of102%(P<0.05);the total phenolic concentration increased by 44.10 % compared with the control group(P<0.05);the total DNJ content of the 100.0 mg/L chitosan treatment group increased significantly(P<0.05),an increase of 216% compared with the control group.It was shown that COR,β-CD and chitosan could inducesuspension cells to increase secondary metabolism production as an inducers.The induction of mulberry suspension cells by single factor 1.0 μmol/L COR,1.0mmol/L β-CD,10.0 mmol/L β-CD and induction of mulberry suspension cells by complex inducer showed that in the treatment with 1.0 μmol/L COR and 1.0 mmol/Lβ-CD,compared with the control group,the content of flavonoids,DNJ and total phenolic in the secondary metabolites of the β-CD-induced treatment group were increased by 113.62%,306%,and 23.34%,respectively.It was shown that treatment with 1.0 μmol/L COR and 1.0 mmol/L β-CD complexes of mulberry suspension cells significantly increased the secondary metabolites flavonoids,DNJ,total phenolic content,and antioxidant activity.The effect was better than the single factor induction of COR and β-CD.After the induction of mulberry suspension cells for the first time,transcriptome analysis was performed to screen and identify differential expression of genes related to flavonoid metabolism.By analyzing the transcriptomes of mulberry suspension cells induced by COR,a total of 741 genes were up-regulated and 593 genes were down-regulated.Among these differentially expressed genes,23 genes associated with flavonoid secondary metabolites were identified through Blast homology alignment.The relationship between differential gene expression and the metabolic yield of flavonoids can be used to provide more adequate evidence for improving the synthesis and molecular regulation of flavonoids in secondary metabolites in mulberry suspension cells.In conclusion,this study successfully established a mulberry suspension cell system and found that three suitable inducers(corondin,β-CD,chitosan)could effectively improve the secondary metabolite yield of mulberry suspension cells.The optimal concentration and compound of the induction effect were optimized and screened.The identification and analysis of genes related to flavonoids synthesis of the secondary metabolites of Mulberry suspension cells provided a high reference value for the study of Mulberry suspension cells.