Abstract
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.
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Data Availability Statement
The raw sRNA-seq datasets generated in this study are available from the NCBI SRA database under the accession numbers SRR8389145, SRR8389147, SRR8389148, SRR8389149, SRR8389150, and SRR8389153 and the CNCB GSA database under the accession numbers CRA006740, CRA006741, CRA006742, CRA006743, CRA006744, and CRA006745. The authors declare that all the other data supporting the findings of this study are available within the article and its supplementary files.
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Fan GAO performed the experiments, analyzed the data, and co-wrote the manuscript; Fangru NAN analyzed the data and co-wrote the manuscript; Jia FENG, Junping LÜ, Qi LIU, and Xudong LIU analyzed the data; and Shulian XIE supervised the project and revised the manuscript. All the authors read and approved the final manuscript.
All the samples were obtained from the well-known algae culture and collection, and the sampling procedures in this experiment were performed in accordance with the guidelines for the care and use of laboratory plants of Shanxi University and were approved by the Wildlife Care and Use Committee of Taiyuan Forestry Bureau, Shanxi Province, China.
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Supported by the National Natural Science Foundation of China (No. 32170204), Science and Technology Strategy Research Special Project of Shanxi Province of China (No. 202204031401051), the Basic Research Programs of Shanxi Province of China (No. 202103021224009), the Teaching Reform and Innovation Project of Colleges and Universities in Shanxi of China (No. J20220046), and the Shanxi “1331 Project”. The corresponding author, Shulian XIE, was supported by the National Natural Science Foundation of China and the Shanxi “1331 Project”. The first author, Fan GAO, was supported by the Science and Technology Strategy Research Special Project of Shanxi Province of China, the Teaching Reform and Innovation Project of College and Universities in Shanxi of China, and the Basic Research Programs of Shanxi Province of China
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Gao, F., Nan, F., Feng, J. et al. Identification of novel salt stress-responsive microRNAs through sequencing and bioinformatic analysis in a unique halophilic Dunaliella salina strain. J. Ocean. Limnol. 41, 1558–1574 (2023). https://doi.org/10.1007/s00343-022-2130-1
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DOI: https://doi.org/10.1007/s00343-022-2130-1