Functional Characterization Of An R2R3-MYB Transcription Factor AbMYB1 In Regulating The Anthocyanin Biosynthesis In Atropa Belladonna

Many studies have shown that anthocyanins has anti-cancer,anti-inflammatory and other functions,widely used in aspects of food,medicine and so on.At the same time,anthocyanin plays an important role in plant coloration,so the research on the regulation of anthocyanin biosynthesis has become a hot spot in recent years.Anthocyanin biosynthesis is influenced not only by the key enzyme genes in its pathway,but also by the transcription factors that regulate the anthocyanin biosynthesis.The MYB class is the largest family of transcription factors in plants.Previous studies have indicated that MYB is involved in the regulation of primary metabolism and the regulation of secondary metabolism.Among them,R2R3-MYB class is an important transcription factor regulating anthocyanin biosynthesis in plants.There are many economically valuable crops in Solanaceous plants.For example,potatoes,tomatoes,belladonna and so on.Atropa belladonna belongs to the Solanaceae family of plants.Its fruit appears violet black after maturing.The content of anthocyanins in fruit is highest after ripening.The peel and flesh of ripe fruit are violet black owing to high content of anthocyanin.The flesh of other Solanaceae plants contain a little or none anthocyanin,such as tomatoes,peppers and eggplants.It is suggested that specific R2R3-MYB transcription factors may be involved in the regulation of anthocyanin biosynthesis in the medicinal plant Atropa belladonna.In order to explore the MYB transcription factor that can be used in the biosynthetic engineering of anthocyanin from Solanaceae plants,this study identified an R2R3-MYB transcription factor from Atropa belladonna,named AbMYB1.The function of AbMYB1 in regulating anthocyanin was confirmed by gene expression analysis and transgenic plants.The results of this study are mainly as follows:1.The full-length c DNA of AbMYB1 was cloned by PCR technology,which was 1035 bp in total,containing a 762 bp opening reading frame(ORF)encoding 254 amino acids.Phylogenetic tree analysis showed that AbMYB1 was more closely related to MYB transcription factors Sn MYB,Ph DPL and Ph PHZ that involved in regulating anthocyanin biosynthesis.Multiple alignment analysis of the amino acid sequence indicated that AbMYB1 had R2 and R3 conserved domains and that belongs to the R2R3-MYB type transcription factor.2.The expression levels of AbMYB1 in roots,stems,leaves,flowers,green fruits and mature fruits of wild-type Atropa belladonna were analyzed by fluorescence quantitative PCR.The results revealed that AbMYB1 was highly expressed in flowers,green fruits and mature fruits,and the expression was highest in mature fruits,but hardly in roots,stems and leaves.3.In order to analyze the expression changes of related genes in anthocyanin biosynthesis during the development process of Atropa belladonna fruit,this study selected five fruits of different stages including young fruit,green fruit,broken fruit,purple fruit and mature fruit used for fluorescence quantitative PCR.The results showed that the expression levels of AbMYB1,Ab CHS,Ab CHI,Ab F3 H,Ab F3'H,Ab F3'5'H,Ab DFR and Ab ANS were the highest when the fruit is coloring,and the expression levels were still high in the mature stage.The expression level of Ab3 GT was also high at the fruit in Veraison,but reached the maximum at the stage of fruit maturity.These results indicated that the expression change of AbMYB1 in Atropa belladonna fruits at different development stages were positively correlated with the accumulation of anthocyanin,and the expression pattern was similar to that of anthocyanin biosynthesis pathway genes.4.In order to further explore the regulatory function of AbMYB1 in anthocyanin biosynthesis,the AbMYB1 overexpression/interference vector were constructed in this study.Through agrobacterium-mediated transformation of the explant of belladonna,the regenerated seedlings were obtained.Through genomic PCR and fluorescence quantitative PCR,relevant transgenic materials that overexpressing/interfering with AbMYB1 were detected.Anthocyanin was extracted from leaves,flowers and mature fruits of the wild type and transgenic lines,and the content of anthocyanin was determined by uv spectrophotometer.At the molecular level,fluorescence quantitative PCR was used to identify the changes of the gene expression related to the biosynthesis pathway of anthocyanin in transgenic Atropa belladonna plants.The results are summed up as follows:1)Compared with wild-type belladonna lines,the anthocyanin content of overexpressed AbMYB1 lines was significantly increased in flowers and mature fruits by 3-5 times and 1-2 times,respectively.And anthocyanin was detected in the leaves.Compared with the wild-type lines,the expression levels of anthocyanin biosynthesis related genes AbMYB1,Ab F3 H,Ab F3'5'H,Ab DFR,Ab ANS and Ab3 GT were significantly increased in the leaves of overexpressing AbMYB1 lines.The expression levels of Ab CHS,Ab CHI,Ab F3 H,Ab F3'5'H,Ab DFR,Ab ANS and Ab3 GT were significantly increased in the flowers of overexpressing AbMYB1 lines.The expression levels of Ab CHS,Ab CHI,Ab F3 H,Ab F3'5'H,Ab DFR,Ab ANS and Ab3 GT were all up-regulated in the fruit growing for 13 days of the AbMYB1 overexpressing lines.However,in mature fruits,only AbMYB1 and Ab3 GT were significantly increased in overexpressing AbMYB1 lines.2)The flowers and ripe fruits of the AbMYB1 interfering Atropa belladonna presented light yellow color,and the anthocyanin content was very low,which was lower than that of wild type Atropa belladonna.The expression levels of AbMYB1,Ab F3'5'H,Ab DFR and Ab3 GT were decreased in the flowers interfering AbMYB1 lines.The expression levels of AbMYB1,Ab DFR,Ab F3 H,Ab F3'5'H and Ab ANS were significantly reduced in the fruit growing for 13 days of the interfering AbMYB1 lines.The expression levels of AbMYB1,Ab CHS,Ab CHI,Ab F3 H,Ab F3'5'H,Ab DFR,Ab ANS and Ab3 GT were significantly decreased in mature fruits of the interfering AbMYB1 lines.In brief,AbMYB1 is an R2R3-MYB transcription factor.It mainly regulates the expression of anthocyanin biosynthesis pathway genes in the flowers and fruits of Atropa belladonna,thereby affecting the biosynthesis of anthocyanin.This study provides a theoretical basis for further exploring the biosynthetic pathway of anthocyanin in Solanaceae plants.And it also provides a candidate gene for anthocyanin metabolism.