miR396 family identification and ethylene responses in Hevea brasiliensis

【Objective】 This study aimed to identify miR396 family members in Hevea brasiliensis and analyze their ethylene response patterns， thereby providing theoretical basis for elucidating the molecular mechanisms of ethylene in latex production and flow in Hevea brasiliensis laticifer cells and regulatory role of miRNAs related to ethylene response in latex metabolism of Hevea brasiliensis. 【Method】 The key response factor miR396 was identified based on transcriptomic sequencing data， and the miR396 family members were identified in Hevea brasiliensis genome using their mature sequences. Bioinformatics methods were employed to predict and analyze the secondary structure， phylogenetic relationships， and cis-acting elements in promoter regions of these family members， and real-time fluorescence quantitative PCR was used to detect expression levels of miR396 at different time under ethrel treatment. 【Result】 A total of 14 miR396 family members were identified in Hevea brasiliensis genome through genome-wide screening， whose mature forms were named hbr-miR396a to hbr-miR396l. The hbr-miR396b contained two mature forms hbr-miR396b-5p and hbr-miR396b-3p， while hbr-miR396k contained two mature forms hbr-miR396k-5p and hbr-miR396k-3p. All these precursors of miR396 family members could form typical stem-loop structures and unevenly distributed across nine chromosomes. Phylogenetic analysis showed that the precursor sequences of this family exhibited genetic diversity， whilst the mature sequences within the same species showed a certain degree of conservation during evolution. The promoter regions of 14 miR396 family members contained numerous hormone response elements， defence and stress response elements， and light response elements， suggesting their involvement in hormone response， stress response， and developmental regulation. Among the miR396 family members in Hevea brasiliensis， only hbr-miR396b-5p， hbr-miR396e， and hbr-miR396k-5p were highly expressed in latex. The expression of hbr-miR396e and hbr-miR396k-5p continued to increase with prolonged ethephon treatment， while the expression of hbr-miR396b-5p was down-regulated after 6 h post-treatment and up-regulated again. Target gene prediction results indicated that the expression of most target genes was down-regulated during ethephon treatment， and this was contrary to the expression trends of the highly abundant expression of Hevea brasiliensis miR396 family members， which was consistent with the miRNA silencing mechanism.GO functional annotation and KEGG signaling pathway enrichment analysis results confirmed that these target genes played their roles in brassinosterorid biosynthesis， leaf development， as well as fructose and mannose metabolism， and so on. 【Conclusion】 After ethephon treatment of tapping panel， Hevea brasiliensis miR396 family members regulate key biological processes such as stress response， latex synthesis， and hormone response through a complex multi-level regulatory network， exerting a great influence on latex metabolism. Among these， hbr-miR396b-5p， hbr-miR396e， hbr-miR396k-5p may participate in ethylene response pathway in Hevea brasiliensis laticifers via differentiated regulatory mechanisms.