Study On The Metabolism Of Fruit Flavonoids With The Regulation Of Strawberry FaMYB11 Gene

Strawberry(Fragaria × ananassa Duch)belongs to the genus Rosaceae,which has the characteristics of bright fruit color,tender meat,rich nutrition and rich flavonoids,and is very popular among consumers.The research on the regulation of strawberry flavonoid metabolism has always been the focus of strawberry research.Strawberry FaMYB11 gene can regulate the metabolism of strawberry flavonoids,but the mechanism is still unclear.In this study,FaMYB11 was studied using fluorescence quantification,over-expression,and yeast two-hybrid techniques using cultivated strawberry ‘red face’ as the material to explore the mechanism by which FaMYB11 regulates the metabolic pathway of flavonoids in strawberry fruits.The results obtained are as follows:1.Using the cultivated strawberry ’Hongyan’ as material,the FaMYB11 gene was cloned,and its CDS region was 927 bp after bioinformatics analysis,encoding a polypeptide containing 308 amino acids,and its protein molecular weight was predicted to be 34.9k D,isoelectric point It is 6.35,without signal peptide and transmembrane structure.Subcellular localization results show that FaMYB11 is localized in the nucleus.2.The FaMYB11 promoter was cloned to obtain a promoter of 2218 bp.Bioinformatics analysis revealed that the promoter contained multiple MYB and MYC binding sites,as well as multiple light-responsive elements,as well as fungal trigger response elements and low temperature.3.Using real-time fluorescence quantitative analysis of FaMYB11 gene expression in various tissues,the results show: FaMYB11 is expressed in all tissues of strawberry,the highest expression in functional leaves,and in the fruit development process,in the white fruit stage The expression level of FaMYB11 was the highest in the fruits,and the expression level of FaMYB11 in the fruits decreased after entering the color transformation period.4.Construction of FaMYB11 overexpression vector,transient over-expression in the ’Hongyan’ fruit of the white fruit stage,the results showed that the expression levels of anthocyanins and proanthocyanidins in the fruit were significantly increased;fluorescence quantitative analysis showed that the over-expression of FaMYB11 gene caused flavonoids The expression levels of structural genes F3’H,ANR and LAR in the metabolic pathways all increased significantly,and had no significant effect on the structural genes of flavonoid metabolism such as PAL,CHS,CHI,F3 H and OMT.5.To verify the interaction between FaMYB11 protein and transcription factors related to flavonoid metabolism,construct FaMYB11 yeast vector and transfer it to yeast for verification.Yeast two-hybrid results show that FaMYB11 protein can interact with FaEGL3 and FaGL3 proteins.6.Construction of FaMYB11-p GEX prokaryotic expression vector to induce fusion protein expression in E.coli.The results showed that the size of the target protein was about 62.5 k Da.SDS-PAGE found that most of the recombinant protein existed in the form of inclusion bodies.GST resin column was used.The supernatant was purified to obtain the purified FaMYB11 fusion protein.Based on the above results,this experiment found that FaMYB11 regulates the strawberry flavonoid metabolism pathway by regulating the expression of structural genes F3’H,ANR,LAR.At the same time,it was found that FaMYB11 protein interacts with FaEGL3 and FaGL3,which lays the foundation for further research on the function of FaMYB11 gene for the subsequent construction of stable transgenic plants,EMSA and other experiments to clarify its regulation mechanism of strawberry flavonoid metabolism.