Study On The Biosynthesis Mechanism Of Flavonoids In Different Growth Stages Of Ampelopsis Megalophylla

Ampelopsis megalophylla Diels et gilg is a medicinal plant belonging to genus Ampelopsis of family Vitaceae.The leaves are the main medicinal part,and could be used for the treatment of hypertension and so on.The previous study of our group showed that flavonoids were the main active components,including dihydromyricetin(ampelopsin),myricetin,myricetin and quercetin.The contents of these flavonoid secondary metabolites in the leaves of different periods were significantly different.In this study,two-dimensional electrophoresis was used for the proteomics study of A.megalophylla.The transcriptome sequencing on the leaves in different seasons was performed using the Illumina sequencing technology,and abundant gene function annotation information was obtained.At the same time,based on the differences of gene expression among the leaves in different seasons,the roles of related functional genes in the plant flavonoid biosynthesis were explored.The specific research contents and results were described as follows:1.The contents of ampelopsin and myricetin in the leaves of A.megalophylla collected in May,August and October were determined by HPLC.The results showed that: for ampelopsin: May >August >October;for myricetin: May > October > August.2.The proteins in the leaves of A.megalophylla collected in May,August and October were extracted by phenol extraction method,and the proteinswere analyzed by two-dimensional electrophoresis.Based on the Coomassie brilliant blue G-250 staining,the proteins in the leaves were well separated in both horizontal and vertical directions,and thus the map with clear background,high resolution and reproducibility were obtained.The IPG strips in the range of p H 4-7 were selected for subsequent experiments.Two parallel experiments were performed on each of the three samples,and the matching rate was 82.11%,73.07%,78.29%,respectively.The results showed that this test method had high resolution and matching rate,good stability and reproducibility.The isoelectric point and molecular weight of total proteins in the leaves of A.megalophylla were mainly in the range of 5.0-6.0 and 15-25 KDa,respectively.3.The protein spots on gel spectra were proceeded by matching software for data analysis and pairwise comparison.The protein spots with more than2-fold expression were taken as differential proteins,and 13 proteins were identified by MALDI-TOF/TOF mass spectrometer.According to the results,a positive correlation could be preliminarily deduced between the content of ampelopsin,the main active component in the leaves of A.megalophylla,and the expression of the two proteins F3 H and F3H-1.4.The transcriptome sequencing data of leaves collected in May,August and October were obtained using Illumina sequencing technology.The effective data of all samples were over 95% in Q20 and over 90% in Q30.83,886 unigenes were obtained by data assembly,with an average length of814 bp,a maximum length of 17,500 bp and a length of 1,648 bp in N50,indicating the good sequencing and assembly quality.Compared with data in GO,KEGGko,Pfam,Eggnog,Signal P and Tm HMM,59.67% of the sequences have high homology with reported genes in other species.5.A total of 22,788 unigenes were annotated into the GO database,which were divided into three main functional categories,and further divided into 52 subgroups,including 14 cell components(CC),14 molecular functions(MF)and 24 biological processes(BP).In KEGG database,we searched all the unigenes of A.megalophylla,and commented 15,149 unigenes in total.Transcriptome data showed that 1,458 transcripts encoded potential transcription factors,which could be divided into 47 families.Pearson product moment correlation coefficient analysis showed that the greater the seasonal difference,the smaller the correlation between the leaves and the greater the difference in gene expression;the closer the season,the greater the correlation between the leaves and the smaller the difference in gene expression.The co-genes satisfying the screening criteria FDR < 0.05 and ?log2Fold Change?> 1 were set as differential expression genes.There were 9,096 DEGs in May and August,including 3,905 increased in May and 5,191 decreased in August.There were 10,613 DEGs in May and October,of which 3,747 were increased in May and 6,866 were decreased in October.There were 393 DEGs in August and October,of which 64 were increased in August and 329 were decreased in October.A total of 14,446 DEGs are mapped to GO database,of which 4,063 are annotated in KEGG database.The differences were mainly reflected in the biosynthesis of flavonoid(ko00941)and the biosynthesis of flavone and flavonol(ko00944).In the flavonoid biosynthesis pathway(ko00941),the up-regulated enzymes include flavonoid-4-hydroxylase(F4H),dihydroflavonol-4-reductase(DFR),caffeinated coenzyme A-O-methyltransferase.In the biosynthesis of flavonoids and flavonols(ko00944),the up-regulated enzymes include flavonoid-3',5'-hydroxylase(F3'5'H),flavonoid3'-hydroxylase(F3'H),flavonoid 3-methyltransferase(F3MT),flavonoid 3',5'-methyltransferase(F3'5'MT).6.To study the biosynthesis pathway of flavonoids in A.megalophylla,it was observed that 31 unigenes encoding 15 key enzymes were related to the biosynthesis of flavonoids.Among them,18 DEGs,including CHS,PAL,F3 H,F3'5'H,C4 H,F3'H,ANS,F3'5'MT and F3 MT,were the highest in May,while8 DEGs,including LAR,HCT and DFR,were the highest in October.The glucosyltransferase and UDP-glycosyltransferase,which are related to flavonoid accumulation and biological modification,have the same expression level.Most genes are highly expressed in August or October,while the genes encoding mannosyltransferase and xylosyltransferase are highly expressed in May.Based on our analysis,five DEGs were annotated to be MYB,two DEGs were annotated to be b HLH,and three DEGs encoded WD40 repeat protein,78 DEGs were annotated as CYP450.7.Through the analysis on the KEGG metabolic pathway and the expression of genes related to flavonoids biosynthesis,five DEGs related to the biosynthesis of dihydromyricetin,myricetin,myricetin and quercetin were screened out,which were FLS,F3'5'H,F3 OM,DFR and LAR respectively.The correlation coefficient between RNA and q RT-PCR was r2=0.9222(r=0.9603),which verified the accuracy of transcriptome sequencing.The expression level of F3'5'H and F3 OM in the transcriptome sequencing was May > August > October,which was consistent with the content of dihydromyricetin.It was verified that F3'5'H in KEGG pathway can catalyze dihydroquercetin to dihydromyricetin.At the same time,FLS was sequenced and expressed in August > May > October,which was consistent with the content determination of myricetin.It was confirmed that FLS can catalyze the synthesis of myricetin from quercetin.8.We successfully cloned and isolated the full length of Am CHS Gene from A.megalophylla by q RT-PCR and RACE.The gene encoded a hydrophilic protein with a molecular weight of 42.8 KDa.It contains an open reading frame(ORF)with a length of 1,182 bp,encoding 393 amino acid residues.The average value of signal peptide encoded by Am CHS is 0.143,which is less than 0.5.The similarity between this protein and blasted CHS inthe family Vitaceae was 94%,indicating the accuracy of the sequence.Homology analysis showed that the similarity with Glycine max,Petuniax hybrida,Polygonum cuspidatum,Arabidopsis and Ginkgo biloba was 87.79%,82.26%,84.56%,84.21% and 82.70%,respectively.In phylogenetic tree,they are closely related to grapes,which is consistent with the traditional classification method,indicating that the close related species have similar genetic information of CHS Gene.