Characterization Of The MiRNA Expressions At Different Embryonic Development Stage In Takifugu Obscurus

MiRNAs, the small non-coding RNAs about 18～22nt in length, can be found in many species, including animals, plants and viruses. They could inhibit the expression of the target genes through combining the 3’UTR of mRNA to regulate the cell behavior. MiRNAs have been shown to play important roles in a wide variety of key biological processes, including cell proliferation, differentiation and apoptosis.In this study, the total RNA from embryos at 32hpf(Fgl) and 50hpf(Fg2) of Takifugu obscurus were extracted and the cDNA library was constructed after reverse transcription. It was sequenced with the HiSeq2500 sequencing platform. The data of sRNA about 18-35nt was filtered and analyzed. The dominant fragment is about 22nt in length, which was proved to be the typical product of Dicer. The sequence data show that 15.03% of the sRNAs is co-expressed both in sample Fgl and Fg2, 41.85% sRNAs specifically expressed in Fgl while 43.2% in Fg2.63.11% sRNAs in Fgl and 47.02% in Fg2 can be positioned in the Takifugu obscurus genome. After blasting with the miRBase Database, there are 95 and 103 known mature miRNAs, and 120 and 127 known miRNA precursors, respectively. There may be 72 novel miRNAs in Fgl and 88 novel miRNAs in Fg2 predicted by software mirdeep2 and miREvo. Through calculating the expression differences of known and novel miRNAs,186 miRNAs were found significantly differentially expressed between two samples, with 99 miRNAs expression up-regulated and 87 miRNAs down-regulated.15 miRNAs with significantly different expression were selected to study the possible regulatory role in the embryonic development, namely miR-7, miR-10d, miR-10c, miR-375, miR-221, miR-222, miR-181b, miR-181amiR-205, miR-200b, miR-429, miR-200a, miR-21, miR-203. The expressions of 15 miRNAs were quantified with β-actin as internal control by qRT-PCR at 4hpf, lOhpf,22hpf,32hpf,50hpf and 148hpf. It was shown that miR-200a and miR-200b reached the maximum expression level at 148hpf, while the others reached the highest level at 50hpf. Besides, miR-200a and miR-200b maintained at a relatively low expression level before 50hpf. These data showed that the miRNAs detected may play important roles in regulation of blastosphere and segmentation period of the embryonic development.Compared to mammals, the studies of fish miRNAs are relatively scare, only limited to some model animals such as zebrafish. Considering the low content of non-coding DNAs in Takifugu genomes, it is necessary to characterize the non-coding sequences like miRNAs to find out their roles in regulating gene expression through epigenetic modification. This study of the miRNAs that differentially expressed at embryonic stages will play fundamental role for further detailed research of its function in regulating embryonic development through targeting genes.