Functional Study Of FaMYB44.1 Transcription Factor In Regulating The Accumulation Of Anthocyanins In Strawberry Fruits

Cultivated Strawberries(Fragaria × ananassa Duch.)It is a perennial herbaceous plant of the Fragaria genus in the Rosaceae family.Its fruit is small and has high economic value,and it has nutritional,medical,and health functions.It is an important horticultural crop in the world,with a particularly rich Vc content and is known as the "fruit queen".Due to its significant commercial value and certain health benefits,it is loved by producers and consumers around the world.Fruits of Strawberry have been shown to have high antioxidant activity,which is positively correlated with the content of phenolic compounds,especially anthocyanins,mainly influenced by flavonoid synthesis pathways.Anthocyanins belong to flavonoid compounds and are natural water-soluble pigments.They are the main pigment components that present colors in fruits and vegetables.As important secondary metabolites,they play a crucial role in various processes of plant growth and development.In this experiment,bioinformatics methods were used to analyze the gene family of MYB transcription factors in cultivated strawberry,and combined with the expression profiles of fruits of different varieties of strawberry("Benihoppe","Snow White"and "Tokun")and"Tokun" fruits at different stages,Identify a series of candidate genes related to fruit quality.Through literature research,the target genes FaMYB44.1 and FaMYB44.3 of this study were further identified,and their molecular cloning,transcriptional activation,VIGS transient transformation into "Benihoppe",prokaryotic expression and other related experiments were analyzed.It was clear that FaMYB44.1 was a key gene for regulating anthocyanin synthesis in fruits of strawberry,and the regulatory network of FaMYB44.1 mediated anthocyanin synthesis in fruits of strawberry was further improved through EMSA and Dual-Luciferase Reporter experiment,The aim is to reveal the role of FaMYB44.1 transcription factor in the anthocyanin synthesis pathway of strawberries,as well as its regulatory function on strawberry metabolism pathway and fruit quality at the transcriptional level.This provides a theoretical basis for the regulation of anthocyanin synthesis in strawberries,and is of great significance for improving strawberry fruit quality and genetic improvement of other horticultural crops.We used fruits of strawberry as research materials to conduct functional identification of the FaMYB44.1 and FaMYB44.3.The main research findings are as follows:(1)The gene family of FaMYB was analyzed to screen key MYB candidate genes.A total of 407 MYB genes(FaMYBs)were identified within the entire genome of F.ananassa and named based on subgenomic locations.According to phylogenetic analysis,407 FaMYBs were divided into 36 subgroups.According to homology analysis,whole genome and segmental duplication are the main reasons for the expansion of the FaMYBs family.A total of 101 FaMYB loci with 1-6 alleles were identified from homologous genomes on homologous chromosomes.The differential expression maps of three varieties("Benihoppe","Snow White"and "Tokun")with different fruit qualities and ripening processes provide 8 candidate loci involved in fruit quality regulation.In this experiment,7/5/4 FaMYBs were selected as candidate genes involved in anthocyanin biosynthesis,sugar and organic acid synthesis,and fruit aroma,respectively.These results provide key FaMYBs for further analysis of gene regulation of strawberry fruit quality.Two MYB loci,mRNA26289 and mRNA24027,were selected as candidate genes for this study.The gene sequence homology rate of the same locus was very high,which showed high expression abundance in transcriptome analysis of different varieties of strawberry and "Tokun" at different stages.These two genes were named FaMYB44.1 and FaMYB44.3 respectively after BLAST and combining previous studies.(2)Cloning and expression analysis of genes.A phylogenetic evolutionary tree was constructed based on the amino acid sequences of MYB44 in FaMYB44.1,FaMYB44.3,and other species.The results showed that FaMYB44.1 had high homology with PbMYB44 in Pyrus bretschneideri,FaMYB44.3 had high homology with RcMYB44 in Rosa chinensis.Structural functional domain analysis shows that FaMYB44.1 and FaMYB44.3 have typical MYB binding domains.The CDS full-length coding sequence of FaMYB44.1 was successfully cloned.The analysis of tissue specific results shows that FaMYB44.1 and FaMYB44.3 have higher gene expression abundance in the early stage of fruit development,but lower expression levels in the later stage of fruit maturity,which may be negatively correlated with the accumulation process of anthocyanins in the fruit.The results of subcellular localization and self-activation experiments indicate that both are nuclear localization transcription factors with self activation activity,and suitable 3-AT inhibitory concentrations were further screened.(3)FaMYB44.1 participates in the synthesis and accumulation of strawberry anthocyanins.Using the VIGS virus vector to instantly silence the expression of FaMYB44.1 and FaMYB44.3 in strawberry fruit,the results showed that silencing the expression of FaMYB44.1 accelerated strawberry fruit coloring and increased anthocyanin accumulation,while silencing the expression of FaMYB44.3 did not show significant changes in strawberry fruit coloring.The results showed that FaMYB44.1 affected the accumulation of anthocyanins in strawberry fruits,while FaMYB44.3 had no significant effect on the accumulation of anthocyanins in fruits of strawberry.(4)EMSA,Dual-Luciferase Reporter and co injection of strawberry fruit experiments confirmed that FaMYB44.1 transcription factor specifically binds to the FaF3H promoter,transcriptionally inhibits its expression,and ultimately affects the synthesis and accumulation of anthocyanins in fruits of strawberry.EMSA showed that FaMYB44.1 specifically binds to the MBS CAACTG motif element on the FaF3H gene promoter,while FaMYB44.3 does not bind to the screened structural genes.Dual-Luciferase Reporter experiment confirmed that the transcription of FaMYB44.1 inhibited the expression of FaF3H gene;The co injection experiment of strawberry fruit confirmed that the FaMYB44.1 transcription factor ultimately affects the biosynthesis of anthocyanins in strawberry fruit by transcriptional inhibition of downstream anthocyanin key gene FaF3H expression.