Objective  To predict, mine and analyze the miRNAs involved in low temperature stress response of Eucalyptus camaldulensis, and lay a foundation for further study of the molecular network of regulating low temperature stress response.

Method  Small RNAs were sequenced by high-throughput sequencing using the shoot tips of the tissue cultured seedlings of E. camaldulensis from the low temperature treatment group and the control group (CK). The miRBase21.0, Rfam14.1 and E. grandis genome were taken as reference databases. Bowtie, miREAP as well as miRDeep2 software were used for miRNA prediction. RNAfold was used to fold the secondary structure of the predicted miRNA precursors. psRNATarget was used to predict target genes. The miRNAs with differential expression were analyzed through DEGSeq package, and GO annotation and KEGG enrichment analysis were further performed.

Result  A total of 392 known miRNAs and 97 novel miRNAs belonging to 54 families were predicted in E. camaldulensis. The 282 known miRNAs and 65 novel miRNAs were predicted in CK, while 329 known miRNAs and 51 novel miRNAs were predicted in the low temperature treatment group. At the same time, 80 significantly differentially expressed miRNAs in low temperature treatment group were mined, including 55 up-regulated and 25 down-regulated. The results of GO annotation and KEGG enrichment analysis indicated that these differentially expressed miRNAs might respond to low temperature stress by participating in metabolic pathways, biosynthesis of secondary metabolites, cell membrane changes, signal transduction, and biological regulation. In addition, we found 25 miRNAs that might be associated with the ICE1-CBFs-COR pathway.

Conclusion  The differentially expressed miRNAs are initially obtained by high-throughput sequencing and bioinformatics software under low temperature stress, which can provide some references for further analysis of the molecular functions of these miRNAs in E. camaldulensis under low temperature stress.