Gene Regulation Study On Biosynthesis Of Phenolic Acids Compounds And Their Derivatives By Suspension Culture Cells Of Bletilla Striata

Objective:1.To establish and optimize the efficient biosynthesis of phenolic acid compounds and their derivatives of cell suspension culture system of Bletilla striata(B.striata).2.To screen the key genes which regulating the biosynthesis of phenolic acids compounds and their derivatives,and analyze and verify the mechanism of gene interaction.3.To explore the regulatory mechanism of ARF gene on biosynthesis of phenolic acid compounds and their derivatives in B.striata.Methods:1.Based on the existing research,the amount of phenolic acid compounds and their derivatives was taken as the main monitoring index,and the orthogonal experimental design method was used to optimize the parameters of various key factors,and the induction rate of suspension cells was calculated after 40 days of culture.Loose,bright yellow callus were then selected and inoculated on the same proliferation medium.The callus proliferation rate was calculated by weighing at the second and fourth subcultures.After 60 days of subculture,phenolic acid compounds and their derivatives were highly efficient produced from 15 kinds of optimized proliferation media.After 30 days of high efficiency culture,the contents of p-Hydroxybenzyl Alcohol(HBA)、Dactylorhin A、Militarine and Coelonin were determined by HPLC.2.The suspension cell material in method 1 was used to conduct transcriptome sequencing analysis using the Illumina technology and PacBio SMRT technology,in which the cell material was pooled by the cells of the efficient biosynthesis of phenolic acid compounds and their derivatives at 10 time points.3.According to the transcriptome sequencing results in method 2,a series of bioinformatics softwares were used to perform functional annotation,lncRNA,SSR analysis,and the major metabolic pathways affecting the synthesis of phenolic acid compounds and their derivatives were selected according to KEGG pathway.4.According to the results in method 3,we compared each two developmental stages of suspension culture cells in B.striata,and analyzed the DEGs among 8 time points(3 Dpi,18 Dpi,27 Dpi,30 Dpi,33 Dpi,36 Dpi,39 Dpi and 42 Dpi).R language was applied to screen out the key regulated genes related to the biosynthesis of phenolic acid compounds and their derivatives.qPCR was conducted to analyze the changes in gene expression levels at different time points,and the relationship between phenolic acid compounds and their derivatives was analyzed.5.Bioinformatics arrays were employed to explore ARF genes from the resultes of method 3.Phylogenetic analysis was conducted on ARF proteins in Arabidopsis thaliana and Dendrobium candidum.R language was used to perform Pearson correlation analysis on the expression of BsARF genes and genes related to the synthesis of phenolic acid compounds and their derivatives.Results:1.Culture system construction of suspension cells for efficient biosynthesis of phenolic acid compounds and their derivatives.Using the seeds as explants,the optimal culture medium for cell suspension culture was MS+1.0 mg/L 6-BA+2.0 mg/L 2,4-D+0.5 mg/L NAA+30g/L sucrose.The optimal medium for subculture was 1/2MS+1.0 mg/L 6-BA+3.0 mg/L 2,4-D+0.5 mg/L NAA+30 g/L sucrose.After adding different precursors and organics,the results showed that the contents of HBA and Coelonin in suspension culture cells cultured in 1/2MS+1.0 mg/L 6-BA+3.0 mg/L 2,4-D+30 g/L sucrose+50 mg/L phenylalanine were higher than those in other groups(p<0.05).The contents of DactylorhinA and Militarine in suspension culture cells cultured in MS+1.0 mg/L6-BA+3.0 mg/L2,4-D+30 g/L sucrose+20mg/L phenylalanine were higher than those in other treatment groups(p<0.05).2.Full-length transcriptome sequencing and bioinformatics analysis.Full-length transcriptome databases in B.striata were constructed,and functional annotation and related bioinformatics analysis of the obtained unigenes were performed.A total of 100,276 high-quality full-length transcripts were obtained,with an average length of 2,530 bp and an N50 of 3,302 bp.About 52%of total sequences were annotated against the Gene Ontology,53,316 unigenes were hit by KOG annotations and divided into 26 functional categories,80,020 unigenes were mapped by KEGG annotations and clustered into 363 pathways.Furthermore,15,133 long-chain non-coding RNAs(lncRNAs)were detected.And 68,996 coding sequences were identified based on SSR analysis,among which 30 pairs of primers selected at random were amplified and obtained stable bands.3.Analysis of DEGs between key nodes of suspension culture cells in B.striata.Compared with high-throughput transcriptome sequencing data at different time points,it was found that the comparison group containing the most DEGs on 3 Dpi and 42 Dpi contained 7,006 DEGs,among which 3,894 genes were up-regulated and 3,112 genes were down-regulated.Metabolic pathways related to biosynthesis of phenolic acid compounds and their derivatives analysis showed that Phenylpropanoid biosynthesis,Starch and sucrose metabolism and Glycolysis/Gluconeogenesis pathways in each of the two time points is all have significant differences in the expression.It was showed that the differential accumulation of these four metabolic pathways at every two time points was related to the differential expression of key genes,which might be the primary reason for the different accumulation of major secondary metabolites in suspension culture cells of B.striata.4.Screening of target genes regulating the synthesis of phenolic acids and their derivatives and association analysis of their expression levels.The chemical structure and content analysis of phenolic acid compounds and their derivatives showed that from 33 Dpi,the content of HBA and Dactyl orhin A began to decrease,while the content of Militarine and Coelonin increased exponentially.It is speculated that HBA and Dactylorhin A may promote the accumulation of Militarine and Coelonin.The correlation analysis between DEGs and differentially accumulated metabolites indicated that metabolism-related genes might be one of the reasons for the difference in the content of secondary metabolites at different time points.In this study,14 candidate genes involved in the synthesis of Coelonin,18 involved in the synthesis of HBA,23 involved in the synthesis of DactylorhinA and 41 involved in Militarine were screened out.Subsequently,10 unigenes were randomly selected for qPCR verification,and the results showed that the expression patterns of the 10 unigenes at different time points were consistent with the results of transcriptome sequencing.5.Identification and analysis of ARF gene family in suspension culture cells of B.striata.Bioinformatics analysis software was used to predict and analyze the physicochemical properties,secondary structure,subcellular localization and conserved amino acid sequence of BsARFs protein and constructed phyletic tree.The expression level of BsARFs at different time points was also analyzed.The results showed that a total of 28 ARF genes were identified in B.striata.The length of the encoded proteins ranged from 230-860aa,the molecular weight of the proteins ranged from 25.81655 to 95.78813 kDa,and the isoelectric point ranged from 5.17 to 10.05.Subcellular localization was predicted to be in the nucleus.The conserved domain analysis showed that all the 27 members contained B3 and ARF domains,and some proteins contained Aux/IAA domains,with a total of 20 conserved motifs.The phylogenetic tree showed that the 28 BsARFs,37 AtARFs and 22 DcARFs could be divided into three large groups and five subfamilies.The results of gene expression analysis showed that the ARF gene was closely related to phenolic acid compounds and their derivatives related genes PAL,P450 and BGLU.At different time point,these genes showed strong synergism at expression level among ARF17 with P450,PAL1,PAL2,PAL3 genes,ARF1 with PAL2,PAL3,and ARF22,ARF26 with BGLU4.Conclusion:This study successfully built a liquid culture system to produce synthetic phenolic acid compounds and their derivatives,and further built a transcriptome database with full-length of 100,276 unigenes,then candidate genes related to phenolic acid compounds and their derivatives were screened,in which 14 unigenes related to Coelonin,and 18 unigenes related to HBA,23 unigenes related to DactylorhinA,41 unigenes related to Militarine.Finally,the growth of suspension cells during the whole culture period was analyzed.A total of 28 BsARF genes were identified and speculated to involve in the regulation of secondary metabolism of phenolic acids derivatives.