VcNF-YC9 Interacts With VcMYB21/5/3 Transcription Factors And Regulates Anthocyanin Biosynthesis During Fruit Development In Blueberry

Due to the rich content of anthocyanins in fruit,blueberry is benefit to our health and do well to the economy development.Transcription factor regulation of target genes is an important way for plants to regulate growth and physiological metabolism.In recent years,many MYB family genes in plants have been shown to be associated with anthocyanin biosynthesis.Some NF-Y family members have also been cloned and identified and have been involved in the process of plant growth and stress response.However,whether NF-YC is involved in plant anthocyanin biosynthesis and its specific regulatory mechanism is not clear.In this study,one NF-YC gene and three MYB genes were identified from blueberries,named VcNF-YC9 and VcMYB3,VcMYB5 and VcMYB21,respectively.The relationship between VIGS gene silencing and transient overexpression was performed in the blueberry fruit after the interaction relationships between the corresponding proteins was established by yeast two-hybrid experiment.Results are as follows:1?the relationship between VcNF-YC9,VcMYB3,VcMYB5 and VcMYB214 proteins was detected by yeast two-hybrid assay.The results showed that VcMYB5 protein had strong self-activating activity,VcMYB21 protein could form its own homodimer,and VcNF-YC9 interacted with VcMYB3,VcMYB5 and VcMYB21 proteins respectively.2?The expression of VcMYB3.VcMYB5,VcMYB21 and VcNF-YC9 genes in blueberry tissues and corresponding UV stress were analyzed by fluorescence quantitative PCR(RT-qPCR).The results showed that VcMYB3,VcMYBS,VcMYB21 and VcNF-YC9 genes' expression level was the highest in blueberry peel and was significantly higher than that in other tissues.The expression of VcMYB21 gene in blueberry leaves was lower than that in blueberry peel,and was significantly higher than that in other tissues except peel.When the blueberry leaves were treated with UV-B,the expression of VcMYB21 gene increased firstly and then decreased,and the accumulation of anthocyanins was rapid with the decrease of gene expression.Suggesting that VcMYB3,VcMYB5,VcMYB21 and VcNF-YC9 genes may play a regulatory role in anthocyanin biosynthesis,in which the VcMYB21 gene may negatively regulate anthocyanin biosynthesis.3?The expression changes of VcMYB3,VcMYB5.VcMYB21 and VcNF-YC9 genes were observed in different fruit development stages,including pro-green fruit,green fruit,white fruit,semi-red fruit,full red fruit and purple fruit.The results showed that the expression of VcMYB5 gene was not significantly different at different stages of fruit development.The expression level of VcMYB3 gene was the highest in pro-green fruit and reached the lowest at white fruit period,but then the expression level of VcMYB3 gene hardly rose to the level of pro-green fruit at full red fruit period.VcMYB21 and VcNF-YC9 genes expression was the highest in the snall green fruit period,and significantly higher than the other period,and the expression slightly increased in the semi-red fruit and full red fruit period,but the rise was not significant.4?VcMYB3,VcMYB5,VcMYB21 and VcNF-YC9 genes were used to perform VIGS silencing experiments every sigle or every double interacted genes respectively.The results showed that the blueberry fruit was mature in advance and the blueberry flesh was turned from green to green in the blueberry peel and pulp after gene silencing,especially in the flesh of fruit that the corresponding gene expression was significantly lower than the normal level.The expression of related genes of the anthocyanin biosynthesis pathway also showed a significant increase in blueberry pulp,but there were differences after VIGS.Anthocyanin content,especially in the blueberry pulp,significantly higher than the control group.5?VcMYB3,VcMYB5,VcMYB21 and VcNF-YC9 genes were used to perform genes overexpression experiments every sigle or every double interacted genes respectively.The results showed that the blueberry fruit was prolonged and the blueberry flesh was changed from blue to white when the gene was over-expression.And the expression of the corresponding genes in the fruit,especially the flesh,were significantly higher than the normal level.The expression of related genes of the anthocyanin biosynthesis pathway also showed a significant decrease in blueberry pulp,but there were differences after OE.Anthocyanin content,especially in the blueberry pulp,was significantly lower than the control group.It was proved that VcMYB3,VcMYB5,VcMYB21 and VcNF-YC9 genes play an important negative regulatory role in blueberries anthocyanin biosynthesis.6?The results of other physiological indexes showed that the content of polyphenol and flavonoids in blueberry fruit after VIGS gene silencing or overexpression was not significantly different from that in normal blueberry.The results of soluble sugar content showed that VIGS gene silencing or overexpression the content of sucrose in the blueberry fruit was lower than that in the control blueberry fruit,but the monosaccharide content increased slightly,but the rising trend was not significant.7?The results showed that the content of hormone in the blueberry fruit was not significantly different from that of the control group,and there was no regularity in the treatment of the blueberry fruit treated with abscisic acid,salicylic acid,brassinolide,jasmonic acid and auxin.