Transcriptomic-Metabolomics Jointly Analysis Of Anthocyanin Metabolism For Variegated Testa In Peanut And Its MiRNA Interaction

Peanut is an important oil crop with economical interest.Peanut testa color showed significant differences in color,including white,red,purple,pink and variegated colors.In this study,the variegated testa peanut VG-01 was used as the material to study the differential genes in the pigment metabolism pathway of the variegated testa,laying a foundation for revealing the molecular regulation mechanism of anthocyanin synthesis in the variegated peanut,and promoting the functional genome of the variegated peanut.Scientific research is of great significance.The results of the molecular regulation mechanism of anthocyanin synthesis in peanut variegated testa are as follows:1.The RNA-Seq technology was used to analyze the RNA in the non-pigmented area B1,B2 and the pigmented area F1,F2 of at 30 days and 45 days after the needle was lowered.The Illumina sequencing platform was used for sequencing,and the DEGseq software was used to analyze the four groups The number of differentially expressed genes between samples and their up-and down-regulation.Differentially expressed genes（DEGs）in the flavonoid metabolism pathway in pigmented areas indicated that there were 27 DEGs highly related to the synthesis of variegated testa color among 1,050 DEGs.Of these 27,13 were up-regulated and 14 were down-regulated,including 3 PALs,1 C4H,2 CHSs,1 F3H,1 F3’H,2 DFRs,2 LARs,2 IAAs,4 bHLHs,and 9 MYBs.GO（Gene Ontology）analysis indicated that DEGs were similarly enriched in 3 branches.a total of 214 differential genes in F1-B1（F1 is the control group and B1 is the comparison group）were identified,of which 53 were up-regulated and 161 were down-regulated.There were 348 differential genes identified in F2-B2（F2 is the control group and B2 is the comparison group）,including 97 up-regulated and 251 down-regulated.There was a total of 152 differential genes in B1-B2（B1 is the control group and B2 is the comparison group）,including 82 up-regulated and 70 down-regulated genes,and 213 differential genes in F1-F2（F1 is the control group and F2 is the comparison group）,with 169 up-regulated and 44 down-regulated.2.Adopted the Blast comparison tool to compare the sequencing results with the reference genomes of diploid wild and tetraploid cultivated peanuts,and perform functional annotations.Adopted GO function to analyze the main biological functions of differentially expressed genes,adopted pathway to analyze the biochemical metabolic pathways and signal transduction pathways involved in differentially expressed genes,and analyzed the metabolic pathways of differentially expressed genes based on the KEGG database.GO（Gene Ontology）functional analysis showed that differential genes were significantly enriched in metabolic process and molecular functional components.The enriched GO items included:catalytic activity（GO:0003824）,organic metabolic process（GO:0071704）,molecular function（GO:0003674）,binding（GO:0005488）.KEGG（Kyoto Encyclopedia of Genes and Genomes）enrichment analysis screened out 6 metabolic pathways related to anthocyanin biosynthesis,including Phenylalanine metabolism,Phenylpropanoid biosynthesis,Flavone and flavonol biosynthesis,Flavonoid biosynthesis,Plant hormone signal transduction and Circadian rhythm-plant.3.The qualitative and quantitative detection of different parts of the variegated testa was carried out by liquid chromatography tandem mass spectrometry（LC-MS/MS）.The metabolites in the colored and non-stained areas were measured and analyzed.The results showed that 12 different products were detected in the pigmented and non-pigmented areas.The pigmented（F1 and F2）and non-pigmented（B1 and B2）areas were compared at DAF30 and DAF45.In the comparison of F1-B1,the total content of the 10 different metabolites measured in the pigmented areas was 7.24 times higher than that in the non-pigmented areas.The content of procyanidin A1,A2,B2,B3,delphinidin,cyanidin,cyanidin 3-O-galactoside,and rosinidin O-hexoside in the pigmented areas increased by 185.40-895.58%,while the relative content of petunidin 3-O-glucoside and cyanidin O-syringic acid in the pigmented areas were lower than in the non-pigmented areas,by 62.70-76.92%.In the comparison of F2-B2,the total content of the 11 different metabolites measured in the pigmented areas was 9.95 times higher than that in the non-pigmented areas.The relative content of procyanidin A1,A2,B2,B3,delphinidin 3-O glucoside,delphinidin,cyanidin,cyanidin 3-O-glueoside,rosinidin O-hexoside in the pigmented areas increased by 106.54-1,759.77%.However,compared with the pigmented areas,the relative content of cyanidin 3-O-galactoside and cyanidin O-syringic acid in the non-pigmented areas was increased by 35.25-80.35%.The relative content of cyanidin and delphinidin are highest in colored metabolites in the comparison F1-Bl,which is same as F2-B2.In the comparison of B1-B2,at DAF45,the total content of the seven different metabolites measured was 1.33 times higher than that at DAF30 in the non-pigmented areas.The relative content of petunidin 3-O-glucoside,delphinidin,cyanidin 3-O-galactoside at DAF45 increased by 43.73-81.62%,while the relative content of cyanidin O-syringic acid,cyanidin 3-O-glucoside,procyanidin A1,rosinidin O-hexoside at DAF45 decreased by 152.39-313.82%compared with DAF30.The metabolites between F1 and F2 were almost identical.At DAF45,the total content of the four different metabolites measured was 3.35 times higher than that at DAF30 in the pigmented areas.The relative content of delphinidin 3-O-glucoside,petunidin 3-O-glucoside,and rosinidin O-hexoside at DAF45 was 48.44-92.25%higher than at DAF30,while the relative content of cyanidin O-syringic acid at DAF45 was lower by 386.24%compared to DAF30.The comparative analysis of the determination results showed that cyanidin and delphinidin were the decisive pigments that caused the peanut seed coat to form mottling.4.The combined analysis of metabolome and transcriptome showed that the differential genes in the flavonoid biosynthetic pathway are directly related to the synthesis of delphinidin and cyanidin.The Fl-B1 correlation results showed a higher delphinidin and cyanidin content in the pigmented areas compared with the non-pigmented areas.Concomitantly,FPKM（Fragments Per Kilobase of exon model per Million mapped fragments）of 2 CHSs and 1 C4H increased in the pigmented areas.The F2-B2 correlation results showed that the FPKM of 2 DFRs,1 F3’H,1 F3H,and 2 LARs in non-pigmented areas increased,and different metabolites resulted in the highest procyanidin content in the non-pigmented areas.5.Adopting TRIZOL method to extract total RNA,synthesize the corresponding cDNA,designed fluorescent real-time quantitative PCR（Quantitative Real-time,qRT-PCR）primers to form the color of the testa in the transcriptome sequencing results according to the representative sequence corresponding to the sequence library required for sequencing.The difference genes were verified.Fluorescence quantitative qPCR verification was performed on 20 genes related to anthocyanin metabolism.The results showed that 11 genes were verified in F1-B1 and F2-B2,12 genes were verified in B1-B2,and 10 genes were verified in F1-F2,consistent with transcriptome results.The 20 selected differential genes in the four comparison groups showed similar qPCR expression trends to the transcriptome detection results.6.The study showed that the metabolic pathways related to the difference in the color synthesis of the variegated testa included phenylpropane biosynthesis,flavonoid biosynthesis,isoflavone biosynthesis and rhythm-plant.From miRNA sequencing results,86 differentially expressed miRNAs were screened out.20 miRNAs were related to the synthesis of peanut testa color anthocyanins,including miR₈,miR₅0,miR₅1 and miR₂39-x that simultaneously target anthocyanin,anthocyanidin and IFS target genes;Five structural genes that regulate anthocyanin biosynthesis:miR₃98-x that regulates CHS target genes,miR₄82 that regulates 4CL target genes,miR₂66 and miR₁82 that regulate F3’H target genes and miR₅ that regulate anthocyanin 3-O-glucoside target gene;MiR858-y targets the regulatory genes in anthocyanin biosynthesis which regulates MYB2 and MYB3;MiR₁0,miR₁5,miR₆1,miR₇2,miR₁02,miR₁16,miR-123,miR₁93,miR₂56 and miR₈62-z targeting CYP450 target genes.The combination of miRNA sequencing and transcriptome analysis of KEGG enrichment indicated that flavonoid biosynthesis is the most direct metabolic pathway for the synthesis of testa variegation.This study is of great significance for comprehensively revealing the molecular mechanism of anthocyanin synthesis in variegated testa peanut.In short,the flavonoid biosynthetic pathway was the most direct metabolic pathway for the formation of variegated testa peanut.The different content of delphinidin and cyanidin were the main metabolites in the formation of variegated testa peanut.During the development of variegated testa peanut,14 main structural enzyme genes and 21 regulatory genes in the pigmented and non-pigmented areas affected the formation of variegated peanut testa,including phenylalaninase and cinnamic acid-4-hydroxylase,Chalcone synthase,dihydroflavonol 3-hydroxylase,dihydroflavonol-3’-hydrogenase,dioxyflavonol-4-reductase and colorless reductase and 7 bHLH,14 MYB regulatory genes.It layed the foundation for revealing the molecular regulation mechanism of anthocyanin synthesis in peanut,and provided candidate genes for genetic improvement of peanut.At the same time,it was of great significance to promote the research on functional genomics of peanut.