Genome-wide identification and analysis of miR396 family members and their target GRF genes in rubber tree (Hevea brasiliensis)

MicroRNAs (miRNAs) are key regulators of gene expression as they play crucial roles at the post-transcriptional level. In particular, the miR319-GRF module is an important gene regulatory network in plants, extensively involved in processes such as plant growth and development. Although miR396 is one of the most conserved miRNA families, its role in rubber trees remains poorly understood. In this study, bioinformatics analysis, including target prediction, was performed to reveal the evolutionary and expression patterns of the Hbr-miR396 family members. A total of six Hbr-miR396 members were identified, distributed across four chromosomes. Secondary structure analysis revealed that the precursor sequences of the six Hbr-miR396 members could form a typical stem-loop (hairpin) structure. Sequence analysis show that the members of the Hbr-miR396 family form three mature sequences. Furthermore, phylogenetic analysis demonstrated that the Hbr-miR396 family members are closely related to those from cassava. Eight members of the growth regulatory factor (GRF) family were predicted as potential targets of Hbr-miR396. The dual-luciferase assays also confirmed that Hbr-miR396b strongly inhibited the expression of HbrGRF3. Expression analysis of the HbrGRF targets in different tissues revealed that HbrGRFs are mainly expressed in the cambium and flowers. Therefore, Hbr-miR396 may potentially regulate growth and floral organ development in rubber trees by targeting HbrGRFs. The data presented in this study offer valuable insights into the functional and molecular regulatory mechanisms of the miR396-GRFs module in rubber tree growth and development, laying a foundation for further investigation into its biological roles in enhancing both rubber production and timber quality.