The Screening And Identification Of MiRNA In Ovarian And Testis Of Adult Alpine Fine Wool Sheep

MiRNAs is a class of short endogenous, single stranded, non-coding functionalRNAmolecules, about19-25nucleotides（nt） in length, and widely exist in eukaryoteand prokaryote, which regulate the transcription levels expression of target genes byspecific binding to the3’UTR region of target gene mRNA. MiRNAs have animportant role in regulating on cell division, differentiation and apoptosis, formationand development of tissues and organs, metabolism, disease occurrence anddevelopment. Up to now, there are more than28,645miRNAs which have beendiscovered in over130species of animals, plants by using various experimentalapproaches and computational predictions （http://www.mirbase.org/index.shtml;7.1.2014）.But the research of Ovis aries miRNAis relatively lagging behind,especially in functional studies.According to the latest miRNARegistry Database（http://www.mirbase.org/index.shtml, June2014）, only153digits miRNAs of Ovisaries have been reported, far lower than other species. In this study, we predictedcandidate miRNAs using high-throughput sequencing and bioinformatics on adultAlpine fine wool sheep ovarian and testis, and predicted target genes throughbiological software and online software, also did functional analysis. Meanwhile, wedetected the expression levels of miRNAon ovary and testis by real-time PCR andNorthern blot.The experimental results were as follows:（1）Successful built two miRNAcDNAlibrarys of Ovis aries ovaries and testis inadultAlpine fine wool sheep by High-throughput sequencing technology, andrespectively obtained9,321,775and9,511,538clean reads （18-30nt）.（2）Atotal of486candidate miRNAs （267and218respective in ovary and testis）for Ovis aries have been indentified. The length of majority miRNAwere distributedwithin20-24nt, and22nt proportion was highest （respectively56.06%and61.09%inovary and testis）.All new candidate miRNAprecursors have a typical collarsecondary structure and minimum free energy.（3）A total of42candidate miRNAs of Ovis aries have been predicted withbioinformatics, and the length was both in18-26nt, and the proportion of20nt wasmaximum （52.4%）, followed by21nt （21.47%）,22nt （14.3%）,19nt （4.8%）.Evolutionary analysis indicated that candidate miRNAs of Ovis aries were in thesame evolutionary branch with people, pigs, dogs, cows, mice, etc. This proved thatthey were homologous sequences. （4）The expression levels of five known high-frequency and30new candidatemiRNAs （reproduction-related） have been detecte in the ovary and testis of Ovis ariesby Real time PCR. The results showed that31genes were detected,4were notdetected. And this found the different expression patterns of the same gene on ovaryand testis.（5）The experiment detected five miRNAs by northern blot, but only4werefound including a known miRNA（oar_miR₃958₃p）,1ovarian candidate miRNA（Ovis_aries_ovary-m0033₃p）,2testis candidate miRNAs（Ovis_aries_testis-m0052₅p, Ovis_aries_testis-m0165₅p）. Oar_miR₃958₃p wasthe highest expression abundance.（6）We found that three new miRNAs respectively belong to miR-214family,miR-34family, miR-145family by bioinformatics analysis, and named them asoar-miR-214₃p, oar-miR-34-5p and oar-miR-145-5p combined with the namingrules. The three miRNAs were the first reported on the Ovis aries.（7）The experiments predicted1,821,503of total mRNAtarget genes for486new candidate miRNAs by biological software.GO annotation and KEGG pathwayanalysis showed that the new candidate miRNAs may be related to ovary and testisdevelopment and function, cell differentiation and apoptosis, the occurrence anddevelopment of disease and so on.The research is the first reports on studying miRNAs and its target genes of theadultAlpine fine wool sheep ovaries and testes, and exploring the miRNAsexpression levels. Especially, the three new miRNAs were the first reported on theOvis aries. The research will play an important role on the regulation mechanism ofmiRNAs to Ovis aries reproductive organs and reproductive activity.