Cloning And Functional Verification Of MYB Transcription Factors Related To Flavonol Synthesis In Juglans Sigillata Dode

MYB transcription factor(TF)can regulate the expression of enzyme genes related to flavonol synthesis.Juglans sigillata Dode ’Qianhe 7’ is widely planted in southwest China,and its fruit is rich in flavonols,which has high health value.In this study,the MYB gene family of Juglans sigillata was studied,and its bioinformatics analysis was carried out to screen out members that regulated flavonols synthesis.Candidate MYB genes were cloned and their functions were verified by overexpressing in callus and silencing in green husks.The binding sites of candidate MYB genes regulating flavonol synthesis were further identified by DAP-seq.The results are as follows:(1)A total of 222 MYB TFs were screened from Juglans sigillata.JsMYB121,JsMYB141,JsMYB148 and JsMYB170 were clustered with At MYB11,At MYB12 and At MYB111.The FPKM values of JsMYB141 and JsMYB170 were well correlated with these important structural genes.They may be the key genes to regulate flavonols synthesis in walnut fruit.(2)Overexpressing JsMYB141 in callus showed stronger orange fluorescence than wild type,and the transcription levels of Js PAL,Js CHS,Js F3’5’H and Js FLS increased significantly,as well as the contents of myricetin,rutin,catechin and epicatechin.After JsMYB141 was silenced,the browning of green husks accelerated,and the expression levels of Js PAL,Js C4 H,Js4CL,Js CHI,Js F3’H,Js F3’5’H and Js FLS decreased significantly,as well as the contents of rutin,catechin and epicatechin.(3)Overexpressing JsMYB170 in callus showed stronger orange fluorescence than wild type,and the transcription levels of Js4 CL,Js CHS,Js CHI,Js FLS and Js DFR increased significantly,as well as the contents of quercetin,rutin,catechin and epicatechin.After JsMYB170 was silenced,the browning of green husks accelerated,the expression levels of Js C4 H,Js CHS,Js CHI,Js F3’H,Js FLS and Js DFR decreased significantly,as well as the contents of myricetin,kaempferol,rutin,catechin and epicatechin.(4)DAP-seq screened 15 target genes related to flavonol synthesis of JsMYB141,including 4 PAL,7 4CL,1 C4 H and 3 FLS.25 target genes related to flavonol synthesis of JsMYB170 were screened,including 6 PAL,10 4CL,3 CHS,1CHI,1 Js F3’5’H,2 FLS and 2 C4 H.AC-element is an important binding element for JsMYB141 and JsMYB170 to regulate flavonols synthesis.