Cloning And Functional Study Of R2R3-MYBs Genes Related To Flavonol Synthesis In Petunia Axillaris

Petunia hybrida is an important ornamental plant.As a model plant for genetic research,Petunia hybrida has a long history and is a classic model plant for flower color and gene function research.Flavonol is one of the color sources of flowers and fruits,mainly white to light yellow,and can be used as a signal for information transmission between pollinators and other organisms,participating in plant hormone signal transmission,promoting pollen tube growth,and protecting plants from UV-B radiation.MYB transcription factor is an important regulator of flavonol synthesis.At present,the research on MYB transcription factor of Petunia hybrida mainly focuses on the synthesis and release of volatile aroma compounds and the color,texture and spots of corolla,while the research on the regulation of flavonol synthesis is only MYB-FL.In view of this,in order to identify more MYB transcription factors related to flavonol regulation,and determine the downstream target gene of MYB-FL and the effect of gene overexpression on the regulation of flavonol in corolla,we conducted this study,which will provide reference for the exploration of MYB protein and the regulation of flavonol synthesis in Petunia axillaris.The main results are as follows:1.Through evolutionary analysis and multiple sequence alignment analysis,three R2R3 MYB transcription factors(MYB-FL,Pa MYB40,and Pa MYB1)were selected from the P.axillaris genome,and their full length CDS was cloned.The open reading frame lengths of MYB-FL,Pa MYB1,and Pa MYB40 were 1056 bp,1218 bp,and 1065 bp,respectively,encoding 351,405,and 354 amino acids;Both contain the R2R3 MYB domain and the SG7 motif unique to flavonol regulated transcription factors,while the C-terminus of Pa MYB1 and Pa MYB40 contain the SG7-2 motif.2.The expression levels of MYB-FL,Pa MYB1 and Pa MYB40 in different tissues and at different development stages of corolla were detected by q RT-PCR.Taking the root as a reference,q RT-PCR detection showed that the transcription level of MYB-FL in the corolla was much higher than that of other tissues,reaching its peak in the S1 stage,which was significantly higher than other tissues and stages.Pa MYB1 is expressed in stems,leaves,and flowers,and is highly expressed in stems.The expression level of Pa MYB40 showed a small difference in all tissues of P.axillaris,which was 1-4 times higher than that in the root.The expression level in S5 was significantly higher than that in other tissues.3.The content of flavonol in transgenic plants and P.axillaris was detected by HPLC.The content of myricetin in the corolla of MYB-FL mutant mediated by CRISPR/Cas9 was significantly lower than that of P.axillaris.When MYB-FL was overexpressed,significantly increased expression level of Pa FLS,the myricetin content in petunia corolla was significantly higher than that in the control.After DPBA staining,the corolla showed a brighter yellow fluorescence.To sum up,MYB-FL regulates biosynthesis of flavonol in P.axillaris corolla.It is concluded that MYB-FL activates flavonol synthesis and inhibits anthocyanin synthesis.4.Yeast single hybrid(Y1H)test showed that p GADT7-MYB-FL and p Ab Ai-Pa FLS-Pro co-transformed yeast survived and grew normally on the selective medium containing SD/-Leu/250 ng/m L-Ab A,but the control did not grow,indicating that MYB-FL could interact with the Pa FLS promoter.Double fluorescein enzyme test showed that compared with the control,when p Green II-62-SK-MYB-FL and p Green II-0800-LUC-Pa FLS-Pro co-transformed,Tobacco leaves show bright fluorescent signals.This further indicates that MYB-FL can bind to the promoter of FLS and activate its expression.5.Pa MYB1 knockout and overexpression mutants were obtained through CRISPR/Cas9 technology and P.axillaris overexpression genetic transformation.There was no significant change in the phenotype of Pa MYB1-M and Pa MYB1-OE plants.After Pa MYB1 is knocked out,The content of myricetin in the corolla of P.axilaris significantly decreased,indicating that Pa MYB1 is involved in the regulation of flavonols in the corolla;The content changes in roots and leaves are relatively small,while the quercetin content in Pa MYB1-OE roots is significantly increased.Pa MYB1 can promote the biosynthesis of flavonols in P.axillaris roots.6.After Pa MYB40 was knocked out and overexpressed,there was no significant change in the plant phenotype.HPLC detection revealed that there was little change in the content of myricetin,quercetin,and kaempferol in the roots and leaves of Pa MYB40-M and Pa MYB40-OE.When Pa MYB40 was knocked out,the expression levels of Pa FLS and Pa F3 H were significantly lower than those of the control,but the content of myricetin and anthocyanins in the corolla increased.After overexpression of Pa MYB40,the content of myricetin and anthocyanins decreased.In summary,Pa MYB40 negatively regulates Biosynthesis of flavonols in the corolla of P.axillaris.7.The expression patterns of FLS in the single mutants of Pa MYB1,Pa MYB40 and MYB-FL are different,but hardly detected in the three mutants,which indicates that the three transcription factors may partially complement each other in the regulation of FLS expression,and the three genes may have functional redundancy in some tissues of P.axillaris.