The Regulation Effect Of Hormones On The Growth And Anthocyanin Synthesis Of The Suspension Culture Red Cyclocarya Paliurus Cells

Anthocyanins are secondary metabolites of flavonoids,which are widely distributed in various plants and play an important role in physiological activities such as reproduction and stress resistance.In recent years,anthocyanins have been widely used in foods,cosmetics and medicines,because of their good color,safety and health characteristics.At present,anthocyanins are mainly extracted from plant materials.However,due to the long growth cycle and susceptibility to environmental,climatic,pest,and other factors,raw materials will become a constraint factor for developing and utilizing anthocyanins.However,due to the long growth cycle and susceptibility to environmental,climatic,pest,and other factors,raw materials will become a constraint factor for developing and utilizing anthocyanins.Plant cell culture has multiple outstanding advantages such as a short growth cycle and independence in natural environments,and therefore will become an important way to produce anthocyanins.Unfortunately,owing to the lack of good cell lines,instability of the production process and relatively high cost,this potential method has not yet been widely used.Although the concentration is extremely low,hormone plays an important role in the growth and secondary metabolism of plants.In this thesis,based on the effect of hormones on the growth of the suspension-cultured Cyclocarya paliurus（C.paliurus）red cells,were screened and optimized.On this basis,the regulation of hormones on cells anthocyanin synthesis were studied.Meanwhile,the molecular regulation mechanism of hormone on anthocyanin synthesis was explored by multi-omics integration analysis.The main results and conclusions were as follows:（1）Hormones were screened and optimized,taking cells growth as an indicator.The“1+1”model（i.e.,one auxin plus one cytokinin）was used for hormone screening and optimization.Selected one auxin from 1-naphthylacetic acid（NAA）,2,4-dichlorophenoxy acetic acid（2,4-D）,3-Indoleacetic acid（IAA）or Indole-3-butytric acid（IBA）and selected one cytokinin from Kinetin（KT）,6-benzylaminopurine（6-BA）or Zeatin（ZT）to screen and optimize based on the effect on cells growth.The results showed that the seven hormones affected the growth of cells in varying degrees.NAA was the best growth-promoting hormone in auxin,and the biomass increased by 20%compared with the control group at 0.5 mg/L.Cytokinin KT had the best effect,and the biomass increased by 28%at 0.5 mg/L.The results of the hormone optimization test showed that when the medium hormones were 0.5 mg/L KT+0.3 mg/L NAA,the cell state was good and grew rapidly.The biomass of C.paliurus cells could reach 19.167±0.425 g/L（dry weight）.（2）The regulatory effect of hormones on the synthesis of anthocyanin in cells was systematically studied,and the regulatory parameters of the optimized hormones were optimized.The“2+1”model was adopted,that is,on the basis of the above two optimized hormones for growth,another hormone was added.The effect of abscisic acid（ABA）,ethephon（ETH）,Methyl Jasmonate（Me JA）,Gibberellins A₃（GA₃）,IAA,2.4-D,24-epibrassinolide（24-EB）and Thidiazuron（TDZ）on anthocyanin synthesis in suspension culture of C.paliurus cells were studied.The results showed that GA₃,IAA,ETH,2,4-D,ABA and TDZ promoted anthocyanin synthesis,especially GA₃had the best promotion effect,while Me JA and 24-EB inhibited anthocyanin synthesis.The optimized GA₃control parameters were as follows:1.0 mg/L GA₃,added on the 4th day and harvested at 72h after addition.Under the optimized conditions,the total content and yield of anthocyanins in the cells reached 2.92 and 2.83 times of the control group,respectively.（3）The dynamic changes in signaling molecules under the regulation of GA₃were explored.The concentrations of NO,H₂O₂,SA and JA signaling molecules in suspension culture of C.paliurus cells showed temporal changes after GA₃treatment.H₂O₂appeared at three concentration peaks at 1,4 and 24h after GA₃addition.SA appeared at a higher peak at 4 and 24h,and JA appeared at a peak at 12h.The change in NO was relatively small,but the concentration of GA₃was always higher than that of the control group.At0.5h after the addition of GA₃,the four signal molecules all responded,and the concentration began to increase.By comparison,the variation of SA was larger,followed by H₂O₂,JA and NO.（4）Transcriptome detection and analysis of suspension culture of C.paliurus cells regulated by GA₃:Illumina Hi Seq sequencing platform was used to perform transcriptome sequencing of 21 samples cells from 7 groups after GA₃treatment at 24,48 and 72h（named GA1＿24,GA1＿48,GA1＿72）and the corresponding cells without GA₃treatment at0,24,48 and 72h（named GA0＿0,GA0＿24,GA0＿48,GA0＿72）.The 84,568,598 Unigenes were obtained with an average length of 1052.97 bp;the obtained Unigenes were compared and annotated with the six public databases GO,KEGG,Pfam,egg NOG,Swissprot and NR.Further differential expression analysis showed that 318,734 and 1,332 genes were significantly different between GA0＿24 vs GA1＿24,GA0＿48 vs GA1＿48,and GA0＿72 vs GA1＿72,respectively.Sample PCC（Pearson correlation coefficient）and PCA（Principal component analysis）analysis showed that there was good repeatability in the 7 groups of samples,and there were significant differences between groups,indicating that GA₃treatment caused significant changes in intracellular gene expression levels.Through KEGG pathway enrichment analysis,GA20OX2,GAI,A2OX2,GA3OX1 and GIDIB five genes related to GA₃synthesis and transduction pathway,three PR-1 and three NPR1 genes related to SA signal transduction pathway,and four genes related to JA signaling pathway including jasmonic acid O-methyltransferase,LOX6,LOX15 and LOX2 were found.These signal transduction related genes were differentially expressed under the regulation of GA₃.In terms of anthocyanin synthesis pathway structural genes,the expression of 4CL,F3H,F3’H,DFR,LDOX and 3GT were significantly up-regulated,while the expression of F3’5’H and ANR were down-regulated.In terms of transcriptional regulatory genes,the expressions of MYB5,b HLH122,ERF003 and b ZIP44 were significantly up-regulated,while the expressions of MYB4,MYB44,MYC2 and b HLH137 were significantly down-regulated.These analyses results showed that GA₃regulation caused changes in the expression of signal transduction related genes,resulting in significant changes in the expression of related structural genes and regulatory genes,and ultimately promoted the accumulation of anthocyanins.（5）Metabolome detection and analysis of suspension culture of C.paliurus cells under the action of GA₃.UPLC-MS/MS technology was used to detect and analyze the targeted anthocyanins metabolites of two groups of cell samples（GA0＿72 and GA1＿72）with or without GA₃treatment.16 metabolites related to the anthocyanin synthesis pathway were identified,including 8 anthocyanins,2 procyanidins and 6 flavonoids.The content of cyanidin-3-O-galactoside in the cells treated with GA₃reached 53.01±5.82μg/m L,which was 3.22 times compared with the control group.（6）Detection and analysis of suspension culture of C.paliurus cells proteome after GA₃treatment（GA0＿72 vs GA1＿72）.28,311 peptides and 5,578 proteins were identified by TMT labeling quantitative proteome detection.The molecular weight of the protein is mainly distributed in the range of 10 k Da～60 k Da.The coverage of the protein is mainly less than 50%,and most of the proteins have corresponding peptides≥2.PCA analysis showed that proteins were significantly different between the GA₃treatment group and the control group.There were 192 differentially expressed proteins between the two groups,of which 90 were up-regulated and 102 were down-regulated.Through PPI（protein-protein interaction）network analysis,GA₃treatment regulated protein expression through various related pathways,in which the protein expression of anthocyanin 3-O-glucosyltransferase was significantly up-regulated and anthocyanin reductase was significantly down-regulated.The changes in these proteins promoted the significant accumulation of anthocyanins.（7）The mechanism of anthocyanin synthesis regulated by GA₃was explored by transcriptome,metabolome and proteome correlation analysis.Multi-omics integration analysis showed that GA₃regulation resulted in up-regulation the expression of 4CL,F3H,F3’H,DFR,LDOX and 3GT genes and their corresponding proteins,and down-regulation of ANR gene and its protein expression,which eventually led to a significant increase in anthocyanin synthesis.