Prediction Of MiRNA Precursors And Analysis Of Target Genes In Cynoglossus Semilaevis

Cynoglossus semilaevis is an important aquaculture fish in China,and the scale of farming has become larger and larger.In order to increase the yield,in addition to improving cultivation techniques,it is necessary to carry out varieties improvement and disease prevention.Cynoglossus semilaevis often cause various diseases,especially viral diseases.Although some drugs can treat diseases,the long-term use of drugs in large areas will worsen the deterioration of water bodies.Therefore,cultivating new varieties and increasing the fish's own resistance against pathogenic microorganisms is the fundamental solution to increase the production of Cynoglossus semilaevis.The miRNAs were first discovered in nematodes.The 22-nucleotide,single-stranded,endogenous,non-coding,small RNA molecules have regulatory functions.Many genes regulated by miRNAs play an important role in immunity.There are many genes involved in the development and morphogenesis of organisms.miRBase has not miRNAs of Cynoglossus semilaevis,and there are few studies on miRNAs in Cynoglossus semilaevis.In this study,bioinformatics methods were used.We downloaded the miRNA sequences of all species from the miRBase database.Perl scripts were written to extract the 2938 mature miRNA sequences of humans and zebrafish.After elimination of redundancy,we used the local Blastn to compare them to Cynoglossus semilaevis genome.437 sequences were screened out.We extended the matching site at both ends by 75 bp?65 bases before the matching site and 10bases after the matching site,10 bases before the matching site and 65 bases after the matching site?,and used the prepared Perl script to extract the sequence as the candidate sequence.A total of 874 miRNA precursors were obtained.RNAfold was used to fold the precursor candidate sequence and predict its minimum free energy?MFE?.Then we used Triple-SVM-classifier and LibSVM machine learning methods to process 874 candidate miRNA precursors secondary structure files generated by RNAfold,and 291 miRNA precursors were predicted.The phylogenetic analysis of mir-20 and mir-133 families showing that the mir-20 may be highly conserved in fish,and the mir-133a of Cynoglossus semilaevis is closely related to the Gallus gallus and Cyprinus carpio.The miRNA of interest was used to perform target gene prediction and functional annotation.miRanda was used for target gene prediction and a total of 155 target genes were obtained.Among them,28 target gene transcripts did not match the NR library,and 127 target gene transcripts were found homologous proteins.55 out of 127 target genes were annotated successfully.We used WEGO software to perform GO function categorization statistics and visual display on a macro scale to understand the target gene function distribution.mi RNA target gene annotated results:59 genes were annotated into 6 subclasses of molecular function,and the function is mainly related to molecular binding?23?,catalytic activity?12?and signal transduction activity?10?;232 genes were annotated into 11 subclasses of cell components,and the function is mainly related to cells?44?,cell parts?42?and membrane?31?;272 genes were annotated into 22 subclasses of biological processes,and the functions are mainly about single biological processes?32?and cellular processes?31?.A series of bioinformatics software were used to perform physicochemical properties analysis and high-level structural prediction of the expression product of the target gene WSB1.The WSB1 transcript protein has a molecular formula of C₂₁₁₇H₃₂₉₁N₅₈₉O₆₁₀S₂₀,a molecular weight of 47.4 kDa,and an isoelectric point of 7.56.The number of negatively charged residue?Asp+GLU?is 45,and the number of positively charged residues?Arg+Lys?is 46.Its instability coefficient is 39.50.It is determined as a stable protein.Its average hydrophilicity coefficient,GRAVY,is-0.144 and it is judged as a water-soluble protein.The protein has no signal peptide,indicating that the protein is a cytoplasmic protein,and is not a secreted protein,which is consistent with the functional properties of WSB1 protein family.Through subcellular localization,the protein is localized in the cytoplasm and the nucleus.The domain analysis using CDD?Cnserved Domain Database?demonstrated that the protein has two characteristics:first,it has a wide range of WD40 repeat domain;second,and it has a SOCS box at the C-terminus.The tertiary structure prediction showed that WSB1 contains a ring structure.In this study,miRNA precursors were predicted by using bioinformatics methods,and target genes were predicted and annotated.Target gene WSB1 analysis were performed.This study provides references and theoretical basis for the study of the specific mechanism of miRNAs,disease prevention,and variety improvement of Cynoglossus semilaevis.The results of the calculation and analysis need to be further verified by molecular biology experiments.