Cloning Of Epigenetic D Gene SmMYB1 Controlling Peel Color And Its Function In Anthocyanin Synthesis In Eggplant

Anthocyanins are a kind of water-soluble natural pigments widely found in the petals,fruits,stems,and leaves of plants in nature.Anthocyanins have antioxidant effects,play a beneficial role in plant growth and development,and are beneficial to human health.Eggplant occupies an important position in the production of solanaceous vegetables.China is the country with the largest planting area of eggplant in the world.Eating purple eggplant rich in anthocyanins is beneficial to human health and makes purple eggplant popular.Vegetable Research Institute,Guangdong Academy of Agricultural Sciences found two eggplant fruit color inheritance phenomena controlled by epistatic effect genes in eggplant breeding practice.Using SLAF-Seq and BSA association analysis technology,the epistatic gene D that controls the fruit color of eggplant was located on chromosome 10,and gene function annotation was performed in the closely related region.A candidate gene for MYB transcription factor D was found.To further study the effect of SmMYB1 gene on anthocyanin synthesis in eggplant peel.The SmMYB1 gene was overexpressed in male parent of D gene mutant with white peel color and silenced 3316F₁ with purple red peel color by VIGS technology.The main findings are as follows:1.SmMYB1 gene promoter,full-length DNA sequence and m RNA sequence were obtained through gene cloning.The results showed that the length of the paternal promoter sequence was 1601 bp,the length of the maternal promoter sequence was 1600 bp,and there was an extra‘G’base insertion at-1153 site in promoter of paternal SmMYB1.The promoter sequence of maternal SmMYB1 was the same as that of the reference genome（KT727965）.The maternal SmMYB1 gene had a total length of 1230 bp and consists of three exons with lengths of 136bp,130bp,and 505bp,respectively,and two introns with lengths of 88 bp and 371bp,respectively.The paternal SmMYB1 gene DNA was 1,224 bp in length,consisting of three exons,130 bp,130 bp,and 505 bp in length,and two introns,88bp and 371 bp in length.Compared with female parent,the length of SmMYB1 c DNA of male parent was 765 bp,with a missing of 6 bases（GCTAGA）in the 1st exon,coding the43th and 44th amino acids,and the two missing amino acids were located in the highly conserved R2 DNA binding domain of MYB protein,which affected the transcriptional activation function of MYB protein,eventually anthocyanins couldn’t be synthesized normally.2.Using real-time quantitative PCR technology to analyze the expression of the candidate gene SmMYB1,it was found that the expression patterns of SmMYB1 gene were different in different tissues and different developmental stages.In the peel,the expression of SmMYB1 gene in female parent was significantly higher than that in male parent,the expression level in male parent was very low（or not expressed）,and the expression level of F₁tends to be medium,which indicates that the deletion mutation of the male parent SmMYB1 causes the gene expression in the peel to be down-regulated.The expression of SmMYB1 gene is higher in maternal commercial flesh and flowers,and the expression level in male stems is higher.The expression of F₁ SmMYB1 gene is higher in stems and flowers.The expression level of SmMYB1 was positively correlated with the accumulation of anthocyanins in eggplant.3.The VIGS verification of the candidate gene SmMYB1 showed that two different vectors,p TRV2-SmMYB1-1 and p TRV2-SmMYB1-2,could effectively inhibit the synthesis of anthocyanins,resulting in bleaching of skin color.Real-time quantitative PCR expression analysis was performed on different vector-treated materials.The results showed that the relative expression of SmMYB1 in VIGS-treated materials was significantly lower than that of the negative control.4.The p BWA（V）HS-SmMYB1 eggplant overexpression vector was constructed and the explants were infected by Agrobacterium-mediated method.The purple-red tissues were observed on the transformed callus of paternal mutants,which indicated overexpression of the SmMYB1 gene could restore and improve anthocyanin synthesis in paternal mutants with white peel color.Thus,it was proved that SmMYB1 was a candidate gene for the epistatic D gene locus controlling eggplant skin color.