Function And Mechanism Of The MrMYB1/2 Gene Cluster Members And MrGST1 In Anthocyanin Accumulation In Morella Rubra Fruit

Chinese bayberry(Morella rubra)is a fruit tree with a remarkable variation in fruit color,rich in nutrition and has important economic and medicinal value.Color is one of the key traits for evaluating fruit quality,and anthocyanin content is the dominant factor for determining the fruit color of Chinese bayberry.In past decades,researches involved regulation of anthocyanin in Chinese bayberry fruit mainly focused on the identification of anthocyanin biosynthesis genes and transcription factors,but the internal molecular mechanism of fruit color variation among different cultivars has not been thoroughly studied.In this study,several Chinese bayberry cultivars with different fruit colors were used as research materials to explore the structure characteristics,expression patterns,functional differenciations and regulatory mechanisms of MrMYB1/2 gene cluster members,as well as the function and mechanism of MrGST1in anthocyanin transport of Chinese bayberry.The main results were as follows:1.A MrMYB1/2 gene cluster associated with anthocyanin accumulation was identified,including four members:MrMYB1.1,MrMYB1.2,MrMYB1.3 and MrMYB2,with two or three alleles for each member.The genotypes of MrMYB1.1 in different cultivars were closely related to fruit color,and there were two alleles:MrMYB1.1-1(MrMYB1.1~n)and MrMYB1.1-2(MrMYB1.1~d).The former was a complete R2R3-MYB gene and homozygous in the purple-black fruit cultivar’Biqi’.The latter was a truncated MYB gene with only the R3 domain remains due to the deletion of one base and homozygous in the white fruit cultivar’Shuijing’.MrMYB1.1~n and MrMYB1.1~d were heterozygous in the red fruit cultivar’Dongkui’and the pink fruit cultivar’Fenhong’.CAPS markers developed based on MrMYB1.1~n/MrMYB1.1~d genotypes can accurately and rapidly identify Chinese bayberry cultivars with different fruit colors.There was no correlation between allele genotypes of MrMYB1.2,MrMYB1.3 and MrMYB2 with fruit color.The expression levels of MrMYB1.1,MrMYB1.2 and MrMYB2 were similar and increased gradually during fruit ripening of four cultivars.MrMYB1.3 was only expressed in the purple black fruit cultivar’Biqi’,and the expression level was correlated with the content of anthocyanins during fruit ripening.Collinear analysis indicated that anthocyanin-related MYB tandem gene cluster was highly conserved in eudicot genomes and may play unique evolutionary importance.2.Transient ectopic expression analysis of tobacco leaves showed that MrMYB1.1~nand MrMYB1.3-1 could induce anthocyanin accumulation,while MrMYB1.1~d,MrMYB1.2-1 and MrMYB2-1 could not.Stable transformation of MrMYB1.3-1 in tobacco significantly increased the anthocyanin content in flower and pericarp.Dual-luciferase,yeast one-hybrid and electrophoretic mobility shift assays showed that MrMYB1.1~n and MrMYB1.3-1 could directly bind and activate the promoters of anthocyanin biosynthesis genes,while MrMYB1.1~d,MrMYB1.2-1,and MrMYB2-1could not.Firefly luciferase complementation imaging and yeast two-hybrid assays showed that MrMYB1.1~n,MrMYB1.3-1 and MrMYB2-1 could interact with MrbHLH1,while MrMYB1.1~d,and MrMYB1.2-1 could not.3.Genome-wide analysis of MrGST gene family was performed,and a candidate MrGST1 involved in anthocyanin transport was identified.The expression level of MrGST1 was positively correlated with anthocyanin content in different tissues and different fruit developmental stages of‘Biqi’cultivar as well as fruits of 12 Chinese bayberry cultivars.Functional complementation on Arabidopsis tt19 mutants of MrGST1 showed that MrGST1 was responsible for anthocyanin transport,but not for proanthocyanidin transport.Dual-luciferase and yeast one-hybrid assays showed that MrMYB1.1~n and MrMYB1.3-1 could directly bind and activate the transcription of MrGST1.Cis-element analysis showed that the MrGST1 promoter had eight MYB binding sites.Dual-luciferase and yeast one-hybrid assays showed that MrMYB1.1~nactivated the MrGST1 promoter by identifying the fourth MYB binding site before the ATG start codon.In summary,this study systematically explored the differences in gene structure,expression patterns,functional and regulatory mechanisms of MrMYB1/2 gene cluster members,and identified the activators MrMYB1.1~n and MrMYB1.3 that regulate anthocyanin accumulation in Chinese bayberry fruit.It was proved that the variation of fruit color among different cultivars was due to the different MrMYB1.1~n/MrMYB1.1~dgenotypes and the exclusive expression of MrMYB1.3 in specific cultivar.At the same time,MrGST1 involved in anthocyanin transport of Chinese bayberry was identified.Results provided new understanding for the regulatory mechanisms of anthocyanin biosynthesis and transport,and provided theoretical basis for the improvement of fruit color quality.