Cloning and Functional Analysis of the VfRR17 Gene from tung tree (Vernicia fordii)

Tung tree (Vernicia fordii) is one of the four major woody oilseed species in China. However, its fruit yield is severely hampered by the low number of female flowers and the imbalanced male-to-female flower ratio, which is a problem that restricts the development of the oilseed industry. Previous research has demonstrated that the exogenous application of cytokinins can significantly augment the number of female flowers, although the underlying regulatory mechanism remains elusive. To elucidate the involvement of VfRR17, a member of the A-type ARRs family, in the exogenous cytokinin regulation of flower sexual differentiation in tung tree, this study conducted a comprehensive bioinformatic analysis of the physicochemical properties, structural characteristics, and evolutionary relationships of the protein encoded by VfRR17. Additionally, gene function analysis was performed using subcellular localization, qRT-PCR, and genetic transformation techniques. The findings revealed that the VfRR17 gene’s coding region spanned 471 bp, encoding an unstable protein of 156 amino acids with a relative molecular mass of 17.4 kDa. Phylogenetic analysis demonstrated a higher similarity between VfRR17 of the tung tree and the RR17 gene of Jatropha curcas, Ricinus communis, Hevea brasiliensis, and other species within the Euphorbiaceae family compared to other species, with the greatest similarity of 86% observed with the RR17 gene of Jatropha curcas. The qRT-PCR analysis indicated that VfRR17 exhibited high expression levels during the early stage of tung tree inflorescence buds following 6-BA treatment, peaking at 24 h and displaying a 3.47-fold increase compared to that at 0 h. In female and male flowers of the tung tree, the expression in female flowers during the 1 DBF period was significantly higher than in male flowers, exhibiting a difference of approximately 47.91-fold. Furthermore, notable differential expression was observed in the root, leaf, and petiole segments of the tung tree under low-temperature stress at the 12-h time point. In transgenic Arabidopsis, the VfRR17 lines and wild-type lines exhibited significantly different flowering times under an exogenous 6-BA treatment at a concentration of 2 mg/L, with the VfRR17 lines experiencing an 11-day delay compared to the wild-type lines. Additionally, the number of fruit pods in VfRR17 transgenic Arabidopsis lines was significantly reduced by 28 compared to the wild-type lines at a 6-BA concentration of 3 mg/L. These findings suggest that VfRR17 likely plays a critical role in regulating flower development in response to exogenous 6-BA, providing valuable insights into the mechanisms underlying exogenous 6-BA-mediated regulation of female flower development in the tung tree.