Molecular Mechanism Of ChMYB1 Regulating Cerasus Humilis Anthocyanin Synthesis

Cerasus humilis is a unique economic tree in China,belonging to Cherry of Rosceae family in plant taxonomy.Its fruit has a special flavor and rich nutrition.And it is also called calcium fruit due to its high calcium content.As one of the crucial characteristics of fruit,fruit color has long been favored by breeders.Bright colors can not only be used as an indicator of fruit maturity but also increase the commerciality of fruit.Many studies have shown that anthocyanins are closely related to the formation of fruit color.Therefore,it is significant to study the molecular mechanisms related to anthocyanin biosynthesis in order to improve the fruit color and economic value of C.humilis.In this study,we studied the biosynthesis and regulation mechanism of anthocyanins in C.humilis fruit using transcriptome,metabolomics,bioinformatics,and molecular biology,using the "Zhangwu" provenance C.humilis,which is unique in the northeast of China,as the research material.The main results are as follows:1.Qualitative and quantitative analysis of anthocyanins in four different development stages of C.humilis fruit were carried out by UPLC-MS/MS technology.A total of 16 anthocyanin metabolites were detected in four development stages,and 11 anthocyanin metabolites showed significant changes at different development stages.Among them,the content changes of cyanidin O-syringic acid and pelargonidin 3-O-beta-D-glucoside were most active with fruit development.Transcriptome data showed that most anthocyanin biosynthesis genes were significantly up-regulated,and the results of q RT-PCR were basically consistent with those of transcriptome sequencing.Co-expression analysis showed that ANS and UFGT may play a crucial role in anthocyanin accumulation.2.128 R2R3-MYB transcription factors were screened from the genome and transcriptome database of C.humilis.The primary characteristics of the R2R3-MYB transcription factor family were identified through phylogenetic relationship,conservative motif,promoter analysis,and collinearity analysis.QRT-PCR was used to study the expression patterns of the R2R3-MYB transcription factor in C.humilis at different developmental stages.Among them,the expression of the ChMYB1(ChMYB90)gene located in the S6 subgroup increased significantly with fruit development,especially in accordance with the trend of anthocyanin accumulation in fruit,suggesting that the gene plays a vital role in anthocyanin synthesis of C.humilis fruit.3.Through phylogenetic analysis with other species,the selected R2R3-MYB transcription factor was officially named ChMYB1.The ChMYB1 gene of C.humilis was cloned.Its CDS region was 732 bp,encoding 243 amino acids.ChMYB1 protein has two conservative MYB domains and one b HLH binding site.The subcellular localization and transcriptional activation experiments found that ChMYB1 is a typical transcription factor with a nuclear-localized transcriptional activation region located at the C-terminal.Moreover,pro ChMYB1 drives the specific expression of the GUS gene in Arabidopsis.4.Construction of 35S::ChMYB1 overexpression vector and its heterologous transformation into apple calli and Arabidopsis by agrobacterium-mediated technology.It was found that overexpression of ChMYB1 can promote the accumulation of anthocyanins in apple calli and Arabidopsis,while the up-regulation of anthocyanin-related structural gene expression.Overexpression of ChMYB1 in C.humilis fruit promoted the accumulation of anthocyanin.TRV2-ChMYB1 was transiently expressed in C.humilis fruit,which inhibited the accumulation of anthocyanins.The Yeast one-hybrid,double luciferase reporter assay,and GUS staining experiments proved that ChMYB1 could promote the expression of Ch CHS and Ch UFGT by combining with MBS cis-acting elements.5.Chb HLH42 and Ch TTG1 were screened from the genome database of C.humilis by phylogenetic relationship.The interaction of ChMYB1 with Chb HLH42 and Ch TTG1 was proved by Yeast two-hybrid,Bi FC,LCI,and Pull-down experiments to form MBW complex,and the expression of Ch UFGT was further promoted by ChMYB1-Chb HLH42-Ch TTG1 complex through GUS staining.The overexpression vectors of Chb HLH42 and Ch TTG1 were heterologously transformed into Arabidopsis by agrobacterium-mediated technology.The results showed that both of them could promote the accumulation of anthocyanins in Arabidopsis.6.Through the analysis of the pro ChMYB1 sequence,it was found that pro ChMYB1 contains abscisic acid response elements.External application of ABA promoted the expression of ChMYB1 and enhance the activity of pro ChMYB1.ABA treatment promoted the anthocyanin accumulation of C.humilis fruit.However,ABA treatment inhibited the expression of Ch ABI5 and the activity of pro Ch ABI5.The interaction between ChMYB1 and Ch ABI5 was demonstrated through Yeast two-hybrid,Bi FC,LCI,and Pull-down experiments.Through the LCI experiment,it was found that ABA treatment enhanced the interaction between ChMYB1 and Chb HLH42 while adding Ch ABI5 inhibited the interaction between ChMYB1 and Chb HLH42;In addition,the inhibition of Ch ABI5 was relieved after ABA application.The above results showed that ChMYB1 promoted the biosynthesis of cryogenic by promoting the expression of Ch CHS directly.It also promoted the expression of Ch UFGT through the formation of the ChMYB1-Chb HLH42-Ch TTG1 complex,thus promoting the biosynthesis of C.humilis.ABA treatment promoted the accumulation of anthocyanins and the expression of ChMYB1 in C.humilis fruit,while Ch ABI5 inhibits the interaction between ChMYB1-Chb HLH42.Our data clarified the primary molecular mechanism of anthocyanin biosynthesis,deepened the understanding of the regulatory network of anthocyanin metabolism in C.humilis,and laid a theoretical foundation for further exploring how hormones and environmental factors regulate anthocyanin biosynthesis.