Anthocyanin Accumulation Mechanism Of Allium Polyphyllum And Functional Verification Of AwMYB114 Gene Based On Transcriptome And Metabolome Analysi

Himalayan onion(Allium wallichii Kunth),a species of Allium genus in Amaryllidaceae family,is a bulbous perennial herb,and integrates ornamental,medicinal and edible value,so it has a broad prospect of development and utilization.At present,research on A.wallichii mainly focuses on the identification of medicinal active components,chromosome karyotype analysis and pollination ecology,but its molecular mechanism of anthocyanin synthesis and regulation have not been reported,which seriously limits the ornamental value and the application potential in landscaping of the A.wallichii.Therefore,from the perspective of anthocyanin synthesis and accumulation,this study firstly carried out metabolomic and transcriptome analysis with the petals of purple flower(P)and white flower(W)of A.wallichii as materials,screened the differential metabolites and differential expression genes(DEGs)related to anthocyanin synthesis,and then excavated the key genes that play a role in anthocyanin synthesis and accumulation of A.wallichii,and finally verified the function of key genes by transforming petunia hybrida W115.This study not only reveals the mechanism of the difference in color between purple and white flowers of A.wallichii,but also lays a theoretical foundation for the development,utilization,and innovation of germplasm resources of A.wallichii and other Allium plants.The main research results of this study are as follows:(1)The UPLC-MS/MS technology was used to detect the flavonoid metabolomic of the petals of purple and white flowers of the A.wallichii during their blooming period(S5),and combined with the database MWDB,a total of 352 metabolites were detected,with flavonol and flavone metabolites being the most abundant.Then there are 120 differential metabolites were obtained through differential metabolites screening,with white flowers as the control,of which 54 are up-regulated and 66 are down-regulated in purple flowers.KEGG pathway enrichment analysis of differential metabolites showed that 5 differential metabolites were significantly enriched in the anthocyanin biosynthesis pathway,and compared to white flowers,of which 4 are up-regulated and accumulated in purple flowers,including pelargonidin-3,5-O-diglucoside,pelargonidin-3-O-rutinoside,peonidin-3-O-glucoside,and pelargonidin-3-O-glucoside.One is down-regulated and accumulated in purple flowers,which is petunidin-3-O-glucoside.Pelargonidins are the main anthocyanin of A.wallichii,with the highest up-regulation of pelargonidin-3,5-O-diglucoside in the purple flowers,reaching 118 folds.This result indicated that pelargonidin-3,5-O-diglucoside plays an important role in the accumulation of anthocyanins in A.wallichii.(2)The RNA-seq technology was used to sequence the transcriptome of petals of three flower developmental stages(S1,S3 and S5)of purple flower and white flower in A.wallichii.In P＿S1 vs W＿S1,1324 DEGs were screened.In P＿S3 vs W＿S3,5098 DEGs were screened.In P＿S5 vs W＿S5,373 DEGs were screened.Venn plot analysis shows that there are a total of 6494 DEGs between purple and white flowers during the S1,S3,and S5 stages,of which 5 genes are differentially expressed at all three stages of the two flower colors.GO enrichment and KEGG pathway enrichment analysis found that DEGs were mainly enriched in molecular function and biological processes,and there are DEGs present in pathways related to anthocyanin synthesis,such as “phenylpropanoid biosynthesis”,“flavonoid biosynthesis” and“anthocyanin biosynthesis”.Due to the lack of reference genes used in q RT-PCR,this study screened the reference genes under 6 different conditions.Firstly,the full-length sequences of 8 reference genes were cloned and quantitative primers were designed for q RT-PCR.Then,the expression stability of 8 reference genes was compared by Delta Ct,ge Norm,Norm Finder,Best Keeper and Ref Finder,and the expression stability of the selected ideal reference genes was verified by DFR gene.The results showed that TUB2 and GAPC were the most stable reference genes,while ACT1 was the most unstable reference gene.Then,25 full-length sequences of genes related to anthocyanin accumulation were cloned,and 6 DEGs related to anthocyanin accumulation and 9 DEGs in other biosynthetic pathways were selected for q RT-PCR with TUB2 as an internal reference.The results showed that the transcriptome data were reliable.It was also further known that Aw ANS,Aw DFR and AwMYB114 genes were differentially expressed at different flower development stages of A.wallichii.It was speculated that Aw ANS,Aw DFR and AwMYB114 genes played a key role in the accumulation of anthocyanin in A.wallichii.(3)The key transcription factor AwMYB114 for anthocyanin synthesis regulation was identified from A.wallichii.AwMYB114 has the closest relationship with the members of the sixth and seventh subgroups of Arabidopsis R2R3-MYB family.This study firstly cloned the CDS sequence of the AwMYB114 gene,and then constructed an overexpression vector of the gene to transform the white flower line W115 of Petunia hybrida.Through phenotype observation,it was found that compared to the wild-type,the callus,leaves,petals,and even plants of the AwMYB114 transgenic petunia lines showed varying degrees of color deepening,appearing as light pink,deep pink,or purple.The expression levels of the AwMYB114 gene were detected by q RT PCR in different lines of transgenic Petunia hybrida.It was found that as the expression level of the gene increased,the petal color of the transgenic Petunia hybrida line changed from light pink to purple,and the highest expression level line,even the entire plant,showed a purple color.The above indicates that the AwMYB114 gene plays a regulatory role in the accumulation of anthocyanins in A.wallichii.