Analysis Of Mirnas And Verification Of Target Genes In Goats During The Follicular And Luterous Phases Of Hypothalamic-pituitary-ovarian

The difference of miRNA expression changes in the follicular phase and luteal phase may help us find these miRNA that regulate goat estrous cycle.In order to find the key pathway that regulate goat estrous cycle,GO and KEGG enrichment analysis would be used to analysis the target gene of differnened expression miRNA between the follicular phase and luteal phase.This study will expoud the molecular mechanism of goat estrous cyclicity from perspective of miRNA and benefit to select the population with high fecundity and molecular breeding.(1)Hypothalamus,pituitary and ovarian tissues were collected from Anhuai goat and immedieatly sequenced by high throughput way.We had got 12125000,11696869,11319069,11432846,11727490,12209928 original sequcences from FH、LH、FP、LP、FO and LO six libraries.Then filtered out low quality sequence,11788536,11344763,11162888,11016438,11546193,12013471 pure sequences were got from six libraries.(2)In order to obtain the miRNA expression profiles of six libraries and their specific information in the goat genome,we will perform predictions of target genes for miRNAs in six libraries and perform related bioinformatics analysis.390,384,368,371,342,and 352 known miRNAs and 159,201,75,125,70,94 new miRNAs were identified in the FH,LH,FP,LP,FO,and LO,of which 302 known miRNA and 20 new miRNA were co-expressed in these 6 libraries compared with goat genome in NCBI.(3)The cell composition,molecular function,and biological processes involved in the differentially expressed miRNA target genes in the 6 library were analyzed by GO and KEGG enrichment.(4)Several miRNAs were randomly selected from 6 libraries and U6 was used as the reference gene to verify the sequencing results by qPCR.The results showed that the results of qPCR results were basically consistent with the results of miRNA sequencing.(5)Chi-miR-144-3p was selected from differentially expressed miRNAs in FO and LO libraries,and its target gene was Follistain predicted by bioinformatics analysis.The relationship between miR-144-3p and Follistain was verified by dual luciferase reporter assay.The viral vectors of over-expression and interference Follistain expression were successfully constructed for next step research.