Identification Of MicroRNAs In The Toxigenic Dinoflagellate Alexandrium Catenella By High-throughput Illumina Sequencing And Bioinformatic Analysis

Micro ribonucleic acids (miRNAs) are a large group of endogenous, tiny, non-coding RNAs consisting of 19-25 nucleotides. that regulate gene expression at either the transcriptional or post-transcriptional level in eukaryotes. As a class of relatively conservative regulator, accumulated reports demonstrated that miRNAs play an important role in a large variety of biological and metabolic processes, including cell proliferation, organ maturation, signal transduction and responseto stress,’red tides’ or harmful algal blooms (HABs) is an abnormal ecological phenomenon and natural disaster, which had serious impact on the human productive activity, human life and ecological environment globally. The occurrence,distribution and damage of HABs has dramatically increased since the reform and opening-up policy in China. Researchers had made much efforts to investigate the occurrence mechanism and prevention of HABs. Dinoflagellates is a kind of important organisms that can cause HABs, much researches have been conducted on dinoflagellates by physiology and ecology. However, the research in the field of molecular mechanism is limited. Alexandrium catenella is a representative of dinoflagellates that is widely spread all over the world and has the ability of producing PSTs. In this paper, high-throughput sequencing was performed on A.catenella to identify and quantitatively profile the repertoire of small RNAs from two different growth phases. A total of 38,092,056 and 32,969,156 raw reads were obtained from two small RNA libraries, respectively.88 mature miRNAs belonging to 32 miRNA families were identified. A total of 15 potentially novel miRNAs were identified.83 miRNA precursors were also detected. Comparative profiling showed that 12 known miRNAs exhibited differential expression between the lag phase and the logarithmic phase. Target predictions of the 12 differentially expressed miRNAs resulted in 1813 target genes. GO and KEGG annotations revealed that some microRNAs (such as osa-miR2876, rgl-miR5139, aca-miR-3p-456915 and ae-miR159a) were associated with DNA duplication and the speedy growth of the algae. The cell cycle, the metabolism process of purine and pyrimidine, mismatch repair are included. Moreover, rgl-miR5139 is related to the phagocytosis of dinoflagellates, which provided evidence for the amphitophy.qPCR was performed to confirm the expression of the miRNAs identified using the high-throughput sequence approach, including three up-regulated osa-miR2876, rgl-miR5139 and aca-miR-3p-456915, one down-regulated conserved tae-miR159a. qPCR assay was performed by chosen the lag phase,the logarithmic phase, and the induced high N,P, Mn logarithmic phase. The reference gene was 5.8sRNA and the fluorochrome was SYBR Green I. The results that the expression of osa-miR2876、 rgl-miR5139、aca-miR456915-3p in the lag phase was extremely higher than that in the both f/2 logarithmic phase and the induced logarithmic phase, the expression of tae-miR159a in the both f/2 logarithmic phase and the induced logarithmic phase was extremely higher than that in the lag phase were generally consistent with the deep sequencing result.In this study, we combined the the high-throughput sequence approach and the biological experiment which further confirmed the existence of microRNA in dinoflagellates and the roles that miRNAs play in the growth of A. catenella. It also lay the foundation for the research of blooming mechanism in organism causing HABs and the prevention of HABs.