Identification And Analysis Of The Sexual Differentially Expressed MiRNA In Chicken Embryos Before And After Sexual Differentiation

Chicken is known as an important agricultural animal and model animal, and its sex is an important economic trait and growth trait. The sex-determining mechanism in chicken is still poorly understood. Recently, the CASI(cell autonomous sex identity) hypothesis of chicken found that sex has been determined before gonadal differentiation, and the sex determinant may locate on sex chromosome and regulate in the upstream of testis determining factor DMRT1. Based on this point of view and the important function of mi RNA in development of animals, the mi RNAs expressed in chicken embryos(E2.5 and E4.5) were identified by high throughput sequencing technology, and their expression patterns and the function of target genes were analyzed, then the expression levels of partial differentially expressed mi RNAs were validated by real time quantitative RT-PCR. This study aims to search the sex determining factor which functioned at early stage from mi RNA level, and provide basic data to the furhter study for important candidate mi RNA.. The main results are indicated as follows:(1) The mi RDeep* software was used to make the alignment of mi RNAs with chicken genome sequence, the results indicated that the distribution of mi RNAs in each chromosome in different samples were shown in a similar manner, but obviously there are more Z-chromosome mi RNAs in male chicken embryos at E2.5. Using the multiple sequence alignment method, 1687, 1791, 1288 and 1355 mi RNAs were identified in male chicken embryos(E2.5), female chicken embryos(E2.5), male chicken embryos(E4.5), female chicken embryos(E4.5), respectively, among them 1687, 1791, 1288 and 1355 mi RNAs are given the informations on mi RBase in chicken.(2) The DESeq software was used to identify the different expression pattern of known mi RNAs between male and female chicken embryos at E2.5 and E4.5. As the result, 42 differentially expressed mi RNAs were identified, among which 3 mi RNAs were differentially expressed both at E2.5 and E4.5, gga-mi R-2954 was expressed higher in male chicken embryos at both stages, gga-mi R-2954 was expressed higher in female chicken embryos than male(E2.5 and E4.5), while gga-mi R-6552-3p was expressed higher in male chicken embryos at E2.5 and lower in male at E4.5.(3) 42 differentially expressed mi RNAs were analysed by Target gene screening, GO gene function enrichment analysis and KEGG signaling pathway, we found that wnt signaling pathway, Ubiquitin mediated proteolysis, TGF-beta signaling pathway, Focal adhesion, Vascular smooth muscle contraction and MAPK signaling pathway are Possible regulation of sex differentiation. There were 20 GO classifications with significant enrichment analysis in two stages, including phosphorus metabolism, gene regulation, biosynthesis regulation, development, protein/enzyme activity.(4) Compared with sequencing results, the expressions of 16 mi RNAs confirmed by q PCR showed 13 and 10 of them are the same tendency at E2.5 and E4.5.