Background Blueberries are rich in anthocyanins, which have antioxidant properties. Anthocyanin content is an important indicator for evaluating blueberry quality. The mechanism of anthocyanin synthesis requires further clarification for blueberry breeding and cultivation regulation. Results This study mined the blueberry ERF gene family based on single-molecule real-time (SMRT) and Illumina transcriptome sequencing results, and further explored the gene function of VcERF061. In this study, a transient injection experiment was conducted on blueberry leaves and fruits. Compared to the control, the injection sites of leaves and fruits overexpressing VcERF061 showed significant anthocyanin accumulation. Similarly, in the blueberry transgenic adventitious shoot experiment, the anthocyanin content in the blueberry shoots and lateral buds of adventitious buds overexpressing the VcERF061 gene was significantly higher than that of transgenic adventitious shoots transformed with the empty vector, and a significant increase in the expression of VcMYB1, VcF3'5'H, VcDFR, VcANS, and VcUFGT. We then speculated whether VcERF061 interacts with structural genes to activate the expression of the structural gene VcANS promoter and promote anthocyanin synthesis. Unfortunately, after conducting a Y1H assay, we found that VcERF061 did not bind to the VcANS promoter, and there was no interaction between the two. Exogenous abscisic acid (ABA) and ethephon (ETH) treatment of blueberry fruit upregulated VcERF061 expression. Our previous study verified that VcMYB1 promotes the accumulation of blueberry anthocyanins, therefore, this study also analyzed VcMYB1 expression in ABA- and ETH-treated blueberry fruit, and found that the expression of VcMYB1 was also up-regulated in ABA- and ETH-treated blueberry fruits. The expression of VcMYB1 and VcERF061 were both up-regulated in ABA- and ETH-treated blueberry fruits, and both reached the maximum proportion after 12 h of treatment. The yeast two-hybrid (Y2H) experiment showed that VcMYB1 and VcERF061 did not interact with each other at the protein–protein level, but yeast one-hybrid (Y1H) experiment and tobacco leaf transient injection experiment demonstrated that VcMYB1 transcription factor can bind to the promoter sequence of VcERF061 and promote the expression of the VcERF061 gene. Conclusion In this study, we found that VcERF061 promoted blueberry anthocyanin accumulation, but did not directly bind to the promoters of structural genes such as VcANS. Both VcMYB1 and VcERF061 genes responded to exogenous ABA and ETH signals and exhibited similar expression patterns. There was an interaction between VcMYB1 and VcERF061. The findings of this study enrich the regulatory network of anthocyanin synthesis in plants and provide theoretical support for blueberry quality improvement.

Molecular mechanism of ethylene signaling response gene VcERF061 to promote anthocyanin synthesis in blueberry (Vaccinium corymbosum)

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2025年12月2日

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Shurui Ma,
Qi Tang,
Xuan Wang,
Fumei Chi,
Yang Song

摘要

Background

Blueberries are rich in anthocyanins, which have antioxidant properties. Anthocyanin content is an important indicator for evaluating blueberry quality. The mechanism of anthocyanin synthesis requires further clarification for blueberry breeding and cultivation regulation.

Results

This study mined the blueberry ERF gene family based on single-molecule real-time (SMRT) and Illumina transcriptome sequencing results, and further explored the gene function of VcERF061. In this study, a transient injection experiment was conducted on blueberry leaves and fruits. Compared to the control, the injection sites of leaves and fruits overexpressing VcERF061 showed significant anthocyanin accumulation. Similarly, in the blueberry transgenic adventitious shoot experiment, the anthocyanin content in the blueberry shoots and lateral buds of adventitious buds overexpressing the VcERF061 gene was significantly higher than that of transgenic adventitious shoots transformed with the empty vector, and a significant increase in the expression of VcMYB1, VcF3'5'H, VcDFR, VcANS, and VcUFGT. We then speculated whether VcERF061 interacts with structural genes to activate the expression of the structural gene VcANS promoter and promote anthocyanin synthesis. Unfortunately, after conducting a Y1H assay, we found that VcERF061 did not bind to the VcANS promoter, and there was no interaction between the two. Exogenous abscisic acid (ABA) and ethephon (ETH) treatment of blueberry fruit upregulated VcERF061 expression. Our previous study verified that VcMYB1 promotes the accumulation of blueberry anthocyanins, therefore, this study also analyzed VcMYB1 expression in ABA- and ETH-treated blueberry fruit, and found that the expression of VcMYB1 was also up-regulated in ABA- and ETH-treated blueberry fruits. The expression of VcMYB1 and VcERF061 were both up-regulated in ABA- and ETH-treated blueberry fruits, and both reached the maximum proportion after 12 h of treatment. The yeast two-hybrid (Y2H) experiment showed that VcMYB1 and VcERF061 did not interact with each other at the protein–protein level, but yeast one-hybrid (Y1H) experiment and tobacco leaf transient injection experiment demonstrated that VcMYB1 transcription factor can bind to the promoter sequence of VcERF061 and promote the expression of the VcERF061 gene.

Conclusion

In this study, we found that VcERF061 promoted blueberry anthocyanin accumulation, but did not directly bind to the promoters of structural genes such as VcANS. Both VcMYB1 and VcERF061 genes responded to exogenous ABA and ETH signals and exhibited similar expression patterns. There was an interaction between VcMYB1 and VcERF061. The findings of this study enrich the regulatory network of anthocyanin synthesis in plants and provide theoretical support for blueberry quality improvement.