Identification of novel salt stress-responsive microRNAs through sequencing and bioinformatic analysis in a unique halophilic Dunaliella salina strain

Dunaliella salina is a classic halophilic alga. However, its molecular mechanisms in response to high salinity at the post transcriptional level remain unknown. A unique halophilic alga strain, DS-CN1, was screened from four D. salina strains via cell biological, physiological, and biochemical methods. High-throughput sequencing of small RNAs (sRNAs) of DS-CN1 in culture medium containing 3.42-mol/L NaCl (SS group) or 0.05-mol/L NaCl (CO group) was performed on the BGISEQ-500 platform. The annotation and sequences of D. salina sRNAs were profiled. Altogether, 44 novel salt stress-responsive microRNAs (miRNAs) with a relatively high C content, with the majority of them being 24 nt in length, were identified and characterized in DS-CN1. Twenty-one differentially expressed miRNAs (DEMs) in SS and CO were screened via bioinformatic analysis. A total of 319 putative salt stress-related genes targeted (104 overlapping genes) by novel miRNAs in this alga were screened based on our previous transcriptome sequencing research. Furthermore, these target genes were classified and enriched by GO and KEGG pathway analysis. Moreover, 5 novel DEMs (dsa-mir3, dsa-mir16, dsa-mir17, and dsa-mir26 were significantly upregulated, and dsa-mir40 was significantly downregulated) and their corresponding 10 target genes involved in the 6 significantly enriched metabolic pathways were verified by quantitative real-time PCR. Next, their regulatory relationships were comprehensively analyzed. Lastly, a unique salt stress response metabolic network was constructed based on the novel DEM-target gene pairs. Taken together, our results suggest that 44 novel salt stress-responsive microRNAs were identified, and 4 of them might play important roles in D. salina upon salinity stress and contribute to clarify its distinctive halophilic feature. Our study will shed light on the regulatory mechanisms of salt stress responses.