| Chicken and quail are poultry production’s common species, chicken and quail are different genera, hybridization between chicken and quail are more typical distant hybridization, good resources of studies genes function and comparative genomics; while chicken-quail hybrids are deaths large number in the incubation process, studies have shown that incomplete development of tissues, organs and sexual differentiation are lead that hybrid embryonic death, so many genes and signaling pathways involved in the process of embryonic development and gender differentiation processsexual differentiation. In this study, use the female,male chicken and hybrid DGE were screened embryonic development, sex differentiation related genes; Construction of gene overexpression and RNA interference vector, as chick embryo cells are experimental material, regulation of target gene expression, Using real-time q RT-PCR techniques to detect changes in gene expression, related to explore the abnormal expression of target genes in embryonic development, and lay the foundation for further study of female hybrids reason early embryonic death.1. Through bioinformatic analysis for male,female chicken and hybrids DGE libraries screening of embryo development and sexual differentiation relate significantly enriched in signaling pathways such as MPKA, Hedgehog, Wnt, Notch, etc; significantly differentially expressed related genes FGF8, DACH, LFNG, SOX1, SOX2, SHH, FOXL2, GPR149, CCND1.2. Construction of differences in gene FGF8, SHH, FOXL2 overexpression and interference vectors, reference Gallus on the NCBI published FGF8, SHH gene m RNA sequences in the coding region using the gene primers were designed to amplify the full-length, PEGFP-C2 as a carrier to build chicken FGF8 and SHH gene overexpression vector; by bacteria PCR, restriction enzyme digestion and sequencing, overexpression vector was successfully constructed. According to the NCBI Gallus published FGF8, SHH and Foxl2 gene m RNA sequence were based on the characteristics of the coding region of the gene designed sh RNA interference of primers to pmi RZip as the carrier, to build targeted FGF8, SHH, Foxl2 gene carrier interference; verified by sequencing interference vector was successfully constructed.Build successful FGF8, SHH overexpression vector and FGF8, SHH, Foxl2 interference vector transfected chicken embryo cells, expression vectors and verification carrier interference effects.3. Analysis FGF8, SHH, Foxl2 gene influence on embryonic development, respectively FGF8, SHH, FOXL2 gene overexpression and RNAi vector transfected chicken embryo cells to detect the expression of related genes, the experimental results obtained: When FGF8 gene expression changes, in the MAPK signaling pathway, reducing the protein Grb2 expression level, thereby affecting the activation and the subsequent series of signal transduction pathways, so that the embryo can not be normal growth and development. In the Hedgehog signaling pathway, when SHH expression of the interference, the Hedgehog signaling pathway can’t be activated while in the inhibition, so that early embryos can not develop normally; when SHH overexpression, activation of the Hedgehog signaling pathway can be successful, but it’s overd, that also have a negative impact on the embryo development, such as polydactyly phenomenon. It interfere Foxl2 gene expression was reduced, resulting in DMRT1,SOX9, CYP19 genes of early embryonic development involved in sex differentiation gene expression disorder, which led to the development of abnormal sexual differentiation process, so that the embryo can not be normal growth and development... |
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