Transcriptome Analysis Related To Isoflavone Biosynthesis And Identification Of R2R3-MYB Gene Family In Red Clover

Isoflavones,a class of secondary metabolites widely distributed in legume species,play a crucial role in plant-environment interactions and confer benefits to both human and animal health.However,animals are incapable of synthesizing isoflavones themselves and must rely on plants for ingestion of these metabolites.On the other hand,meeting the drug production and clinical demands with the isoflavone content in plants remains a significant challenge.Therefore,it is imperative to explore the biosynthesis mechanism of plant isoflavones for identifying excellent gene resources,enhancing forage and crop quality,and increasing the content of isoflavones.Red clover(Trifolium pratense L.)is a nutrient-rich and widely cultivated perennial legume.It is also one of the legumes with the greatest known isoflavone concentration,making it an ideal candidate for investigating the mechanism behind the biosynthesis of isoflavones.However,the molecular mechanisms that regulate the production and accumulation of isoflavones in this species remain poorly understood.Therefore,in this thesis,T.pratense cv.Minshan grown in Minxian,Gansu Province and T.pratense cv.Brilliant introduced from abroad were used as research materials to compare the contents of total isoflavones and their main components in aboveground tissues(flowers,stems and leaves)at different growing stages.Based on this comparison,key candidate genes were screened and transcriptome data from different varieties were analyzed to explore the molecular regulatory network of isoflavone biosynthesis in red clover.Finally,the R2R3-MYB gene family in red clover was systematically identified and analyzed at the genome-wide level using bioinformatics methods,and the candidate members that may regulate the isoflavone biosynthesis were predicted.The main results are presented below:1.The contents of total isoflavones in the leaves of two red clover varieties were compared at different growth stages,revealing that Minshan exhibited significantly or extremely significantly higher levels than Brilliant at three stages(vegetative stage,early flowering stage and full flowering stage)except for the budding stage.The concentrations of formononetin,biochanin A,and daidzein in the leaves of Minshan were higher than those of Brilliant across all growth stages,according to a further analysis of the isoflavone components between two red clover varieties,with the exception of genistein.2.A total of 6896 differentially expressed gene(DEGs)were screened from the transcriptome in the leaves of two red clover varieties at the budding stage and full flowering stage,of which 42 DEGs were found to be involved in isoflavone synthesis.At the budding stage,the key enzyme genes Tp38044(Tp HID),G14457(Tp IF7 Ma T)and Tp14926(Tp IFS),which are directly involved in isoflavone synthesis,showed higher expression levels in Minshan compared to Brilliant,while the expression levels of the key enzyme genes Tp2040(Tp F3’H)and Tp23355(Tp FLS)involved in flavonoid biosynthesis in Minshan were lower than those in Brilliant.At full flowering stage,4 DEGs were up-regulated in Minshan compared to Brilliant,and 3 of them(G13328 “Tp IF7 Ma T”、Tp36713 “Tp IF7GT”和 Tp36710“Tp IF7GT”)were involved in isoflavone biosynthesis.3.Analysis of transcription factor binding sites in isoflavone synthesis structural genes suggested that the MYB and b HLH families’ members are likely to be the primary transcription factors involved in regulating the biosynthesis of isoflavones.Additionally,weighted correlation network analysis(WGCNA)identified two significant modules(coral2 and darkslateblue).Based on the co-expression network constructed from related genes in two modules,three MYB transcription factor genes(Tp8814,Tp24919 and G11569)and one b HLH transcription factor gene(Tp30158)were associated with multiple isoflavone synthesis structural genes,indicating that they might play the important regulatory roles as hub genes in the biosynthesis of isoflavones of red clover.4.A total of 138 TpR2R3-MYB genes were identified in the genome-wide analysis of red clover.The collinearity analysis among species revealed that red clover and soybean(Glycine max L.)had the closest genetic distance.Phylogenetic analysis classified Tp MYBs into 30 subgroups,and the R2R3-MYBs in red clover were highly conserved during evolution.In addition,15 Tp MYB genes were related to the phenylpropanoid pathway in the G1,G3,G4,G6,G7 and G10 subgroups,and these 15 genes contained motif3,motif2,motif7 and motif1,suggesting that these four conserved motifs might also be associated with the phenylpropanoid pathway.5.The expression pattern analysis showed that the expression levels of Tp MYB79 and Tp MYB53 in Minshan were higher than those in Brilliant at full flowering stage,which was consistent with the results obtained from total isoflavone content determination.It was speculated that Tp MYB79 and Tp MYB53 were most probably involved in isoflavone biosynthesis.The above results laid a theoretical foundation for systematically elucidating the molecular basis of isoflavone biosynthesis in red clover and developing new germplasms of forages and crops with high content of isoflavones.