Identification And Functional Analysis Of MYB Transcription Factors Regulating The Flavonoid Biosynthesis In Ginkgo Biloba L

Ginkgo biloba is the only remaining member of Ginkgoaceae,which is an important economic tree species.Flavonoids are a kind of polyphenol compounds,which are the important secondary metabolites with diverse biological functions in G.biloba.The flavonoid biosynthesis is a complex process that influenced by a wide range of internal and external factors.With the deepening of research on flavonoids,it was found that MYB transcription factors play important roles in the regulation of flavonoid transcription.At present,a large of MYB transcription factors involved in the regulation of flavonoid synthesis have been identified in many species.However,most of research is limited to the cloning realted structural genes,little is known about the regulatory mechanism of MYB in G.biloba.Therefore,in the present study,we took G.biloba as the experimental material to screen and identify MYB related to the regulation of flavonoid synthesis by means of bioinformatics.And the function and molecular regulation mechanism of candidate MYB were studied by gene cloning,genetic transformation,transcriptome sequencing and other methods.The main results were as follows:(1)Based on the G.biloba reference genome and the transcriptome dadas measured in the previous period,94 members of MYB family were screened by bioinformatics.According to the characteristics of the amino acid sequence domain,the MYB family of G.biloba was divided into three catagories,including R2R3-MYB(69 members),1R-MYB(22 members)and 3R-MYB(3 members).The phylogenetic tree of R2R3-MYB of G.bloba and Arabidopsis thaliana was constructed,and the MYB which was closed realted to S4,S5,S6 and S7 subgroups of A.thaliana was obtained preliminarily.After the phylogenetic tree analysis of MYBs involved in the regulation of flavonoid synthesis in other species,three candidate MYBs were finally selected,and named GbMYB4?GbMYB6 and GbMYB11 respectively.(2)Through gene cloning analysis,it was found that the alignment rate of three MYB genes sequence to the genome was 100%,and the structure of genes was approximately the same,containing three exons and two introns.We further found that the amino sequence of three MYBs contained typical R2R3 repeats,and the predicted tertiary structure of the MYB proteins contained helix-turn-helix structure.In addition,the subcellular localization experiment of tobacco showed that GbMYB4?GbMYB6 and GbMYB11 were all localized on the nucleus.(3)The analysis of cis-elements of promoter showed that there were low temperature,drought,jasmonate acid and gibberellin response elements in the promoter region of GbMYB4;Cis-elements mainly related to defense and stress were found in the GbMYB6 promoter region;Response elements such as low temperature,abscisic acid,jasmonate acid and auxin were found in the promoter region of GbMYB11.In addition,some light-responsive elements were found in the promoter region of three MYB genes.These results suggestted that candidate MYBs may be regulated by hormones and stress-related factors.(4)The overexpression vector of MYB genes was constructed and transferred into the callus of G.biloba.It was found that the total flavonoids contents in OE-GbMYB4 callus significantly decreased,while increased in OE-GbMYB6 and OE-GbMYB11 callus.Through RNA-Seq,We found that the expression of PAL,C4H,4CL and lignin synthesis gene CAD were significantly up-regulated in the OE-GbMYB4 and OE-GbMYB11 callus.The expression of FLS involved in the flavonol synthesis pathway and the HID involved in the isflavone synthesis was highly expressed in the transgenic callus,while the expression of DFR,ANS,LAR and ANR involved in the anthocyanin and proanthocyanin pathways were significantly decreased.These results indicated that flavonoid synthesis pathway could be activated by overexpressing GbMYB11 in the callus of G.biloba,and promoting the accumulation of flavonoids.Overexpressing GbMYB4 may reduce the content of total flavonoids by inhibiting the the synthesis pathway of anthocyanin.(5)In ABA signal transduction pathway,most of PYL genes and SnRK2 genes were up-regulated in the OE-GbMYB4 and OE-GbMYB11 callus.The expression of genes LOX,4CL8,ACX,MFP2 and PKT involved in the JA synthesis pathway were significantly increased,and the genes encoding JAZ and MYC,which are involved in the JA signal transduction pathway,were also up-regulated in transgenic callus.In addition,the expression of genes involved in the ethylene and salicylic acid signal transduction pathway exhibited an increasing trend compared with the control group,and the related pathway was activated.These results showed that overexpressing GbMYB4 and GbMYB1 could induce the hormone signaling transduction pathways in the callus of G.biloba,which may affect the synthesis of flavonoids by regulating hormones.(6)In the present study,we found that AP2,WRKY,bHLH,WD40,NAC and bZIP transcription factors were differentially expressed in transgenic callus.Most of the WRKY,NAC and bZIP genes were up-regualted,while the expression of some members of the bHLH and WD40 genes were promoted or inhibited in transgenic callus.It showed that these TFs may participate in the regulation of flavonoids together with MYBs.(7)Transforming the constructed vector 35S::GbMYB4,35S::GbMYB6 and 35S::GbMYB11 into A.thaliana plants.The overexpression plasmids were transferred into A.thaliana through agrobacterium GV3101 by using Floral-dip methods,and the T2 generation transgenic plants were obtained through the resistance screening of T1 generation on the medium.The positive plants were identified by extracting DNA from each lines and PCR amplification.At present,12 positive T2 generation plants were identified and used for measuring the content of total flavonoids.Comparing with the control,it was found that the total flavonoids content in OE-GbMYB6 and OE-GbMYB11 transgenic A.thaliana was increased,while the total flavonoids content in OE-GbMYB4 transgenic A.thaliana was significantly reduced.It showed that GbMYB6 and GbMYB11 positively regulated the synthesis of flavonoids,while GbMYB4 inhibited the accumulation of flavonoids.