Identification Of Pomegranate R2R3-MYB Family And Expression Analysis In The Lignin And Anthocyanin Synthesis

A common deciduous shrub plant,the pomegranate(Punica granatum L.)has used in medicine,food,ornamentation,and greenery.The molecular basis for the development of pomegranate seed traits has attracted a lot of attention in recent years.According to research,the amount of lignin and anthocyanin determines the color and hardness of pomegranate seeds.A variety of biological processes are regulated by the R2R3-MYB transcription factor,including the pathways for the synthesis of lignin and anthocyanins.In order to expolre PgMYB,which is associated to lignin and anthocyanin synthesis,we first identified members of the R2R3-MYB transcription factor family from the entire pomegranate genome.Then,using transcriptome analysis to examine how their expression is regulated in the pathways for the synthesis of lignin and anthocyanins.This article offers a theoretical framework for future investigation into the process by which pomegranate seed traits are formed.Three pomegranate cultivars were used for this study: ’Tunisia’(low lignin content of seed coat,red soft seed),’Hongyu’(high lignin content of seed coat,red hard seed),and’Baiyu’(high lignin content of seed coat,white hard seed).At 40 days,80 days,and 120 days following flowering,a total of 27 samples were obtained from seeds.To give information to support the investigation of PgMYB expression patterns,transcriptome sequencing was done on all samples.The main results obtained from the study were as follows:(1)The R2R3-MYB transcription factor family from pomegranates has been identified and analyzed.With amino acid lengths ranging from 99 to 1516 amino acids,Pomegranate found 186 members of the R2R3-MYB transcription factor,all of which are hydrophilic proteins with shared motif structures.PgMYBs were split into 34 subgroups,and each subgroup’s members share some of the same protein structural domains and gene structures.Screening of nine PgMYBs connected to lignin synthesis and eight PgMYBs connected to anthocyanin synthesis from pomegranate R2R3-MYB members based on homologous genes with similar activities.(2)The transcriptome of several pomegranate types and harvest times was assessed using a total of 36.01–49.3 million Raw reads,containing 5.822–8.026 Gb of nucleotide sequencing data.After quality control,the linkers and low-quality data from the sequencing raw data were removed to produce 12.02 million clean read pairs.178.31 Gb worth of bases were used in total.Bases larger than 5.33 Gb were found,and the average GC content across 27 samples ranged between 49.13% and 50.56%.The Q20 and Q30 values were also higher than 96% and 91%,respectively.This finding suggests that the transcriptome data are trustworthy and can enable further data analysis.(3)With a higher number of differential genes in the biosynthesis of secondary metabolites(Ko01110),phenol propane biosynthesis(Ko00940),and flavonoid biosynthesis(Ko00941),the differentially expressed genes(DEGs)of Tunisia and Hongyu were enriched to 106 KEGG pathways.The DEGs of "Hongyu" and "Baiyu" were enriched for a total of 30 KEGG pathways,with the metabolic pathway(Ko01100)and Ko01110 having the highest number of differential genes.At 40 days,80 days,and 120 days after flowering in "Tunisia," DEGs were enriched on 133 and 125 KEGG pathways,and DEGs were found on the Ko00940 and Ko00941 pathways,respectively.In conclusion,DEGs were discovered in several comparison groups and were enriched in various pathways.(4)Analysis of PgMYB’s lignin synthesis-related expression pattern: Eight of the nine PgMYBs showed considerably higher expression in the Tunisia and Hongyu comparative group.PgMYB79 expression peaked in both kinds at 40 days after blooming,fell steadily after that,and was consistently greater in the ’Hongyu’ variety than the ’Tunisia’ variety at all times.It was deduced that PgMYB79 would facilitate the synthesis of lignin because its expression was positively linked with lignin concentration.The expression of PgMYB115 and PgMYB2 peaked at 40 days after anthesis as well,and it was higher in Tunisia than in Hongyu at all times.It is likely that PgMYB115 and PgMYB2 are negatively regulating the production of lignin because their relationship to lignin content was negatively associated.The results of the Q-PCR assay on the above genes were consistent with the above results.(5)PgMYB expression pattern analysis in relation to anthocyanin synthesis In the comparative groups of "Hongyu" and "Baiyu," all 8 PgMYBs were significantly expressed.PgMYB105 and PgMYB70 expression both increased with time,peaking 120 days after blooming.Both genes’ expression was higher in "Hongyu" than "Baiyu" over all time periods,and it positively connected with the amount of anthocyanins,likely promoting the synthesis of anthocyanins.In both cultivars,PgMYB65 expression peaked 40 days after flowering and subsequently gradually decreased.At 120 days after flowering,PgMYB104 expression peaked.It had been growing steadily since flowering.In all eras,"Baiyu" had higher PgMYB65 and PgMYB104 expression than "Hongyu," In every phase,PgMYB65 and PgMYB104 expression was higher in "Baiyu" than "Hongyu," and they were inversely linked with anthocyanin concentration,suggesting that they may prevent the synthesis of anthocyanins.