Identification of the RcbZIP gene family in Rubus Chingii and analysis of the regulation of flavonoid synthesis by RcbZIP1 and RcbZIP12

Rubus Chingii is a multi-functional plant that can be used both as medicine and food. The fruit is rich in flavonoids, especially in their unripe fruits, where the content is even higher. This study identified and analyzed the members of the bZIP gene family and explored its regulatory mechanism on flavonoid synthesis. In total, 46 RcbZIP members were identified in this study. They were unevenly distributed across six chromosomes, with the largest number on chromosome 2, reaching 15. Subcellular localization predictions were all located in the nucleus. Based on the evolutionary tree analysis, the 46 members were divided into 13 subfamilies, and the motifs and gene structures of different subfamilies were highly consistent. Analysis of cis -acting elements revealed that their promoters are rich in light-responsive, abscisic acid-responsive, methyl jasmonate-responsive, and other elements. There are eight pairs of collinear genes in the genome, most of which have been purified and selected. WGD and TRD are the main reasons for the expansion of the RcbZIP gene family, and most members exhibit greater collinearity with dicotyledonous plants. Transcriptome analysis revealed that the two RcbZIP s ( RcbZIP1 and RcbZIP12 ), which had high expression levels when the fruit was ripe, but their expression levels decreased when the fruit was ripe. Further analysis revealed that RcbZIP1 and RcbZIP12 were significantly positively correlated with nine flavonol synthesis genes that were differentially expressed in unripe fruits. Notably, the FPKM values of RcbZIP1 and RcbZIP12 were significantly positively correlated with LG05.2134(Rc4CL) , the correlation coefficients of 0.83 for both. RT-qPCR confirmed that RcbZIP1 and RcbZIP12 were highly expressed during the immature stage. Subcellular localization results showed that RcbZIP1 and RcbZIP12 were localized to the nucleus. Yeast monohybrids demonstrated that RcbZIP1 and RcbZIP12 can bind to the LG05.2134(Rc4CL) promoter, and LUC experiments proved that RcbZIP1 and RcbZIP12 can promote the expression of LG05.2134(Rc4CL) . This study lays the foundation for enhancing the medicinal value of Rubus Chingii .