Study On The Regulatory Mechanism Of BMP15 Gene On Follicle Selection At Late Stage Of Laying In Chickens

The laying traits of poultry are an important economic trait.Maintaining the laying rate in the late laying period is the key to improving the utilization rate of hens and prolonging the laying cycle.In addition to external environmental factors,the egg-laying traits of poultry are more importantly the continuous ovulation of the poultry,and the selection of follicles is an important part of determining the continuous ovulation of the poultry.Bone morphogenetic protein-15(BMP15)is an important member of the TGF-β family and plays an important role in the development and selection of poultry follicles.However,domestic and foreign studies have not yet clarified the mechanism of BMP 15 gene regulation of follicle selection.In this study,the small yolk follicles of 43-week-old native chickens were used as the research material,and the differential genes were screened through transcriptome sequencing technology.Use RACE technology to clone the full length of BMP 15 gene and analyze the genetic structure;carry out BMP 15 conditional culture on granulosa cells cultured in vitro,explore the mechanism of BMP15 gene to explore the mechanism of follicle selection;use dual luciferase reporter technology to identify the transcriptional active region of BMP15,based on the analysis of mutation information,screen the impact of laying hens effective molecular markers in the promoter region of the BMP15 gene at a high rate,expounding the transcriptional regulation mechanism of the differential expression of the BMP15 gene,in order to deeply analyze the internal relationship between BMP 15 and follicle selection,reveal the molecular mechanism of BMP15 gene regulating chicken follicle selection.The main research work and results carried out are as follows:1.Transcriptome analysis of small yolk follicles of native chickens in late laying periodTranscriptome sequencing of small yellow follicles from 43-week-old local chickens screened out 687 differentially expressed genes,167 up-regulated genes,and 520 down-regulated genes.Differential genes were enriched in Wnt signaling pathway,MAPK signaling pathway,and PPAR signaling.Pathway,Foxo signaling pathway,steroid hormone synthesis signaling pathway,among which the BMP15 gene was significantly down-regulated in the small yolk follicles of high-producing chickens.2.Analysis of genetic characteristics of BMP 15 geneTwo transcripts of BMP 15 gene were obtained using RACE technology.The lengths of the two transcripts were 1865 and 1707 bp,respectively.Transcript 1 included 5’UTR 217bp,3’UTR 595bp,CDS 1050bp,and transcript 2 included 5’UTR 62bp,3’UTR 595bp,CDS 1050bp,can encode 350 amino acids.The phylogenetic tree shows that the chicken BMP15 gene is closely related to turkey and is highly conserved in mammals.The results of tissue expression showed that BMP15 was specifically and highly expressed in chicken ovary,and the expression level in ovarian granulosa cells was much higher than that in membrane cells.3.The mechanism of BMP15 gene regulating follicle selection in chickenThe granulosa cells cultured in vitro were cultured under BMP15 condition.The results showed that BMP15 accelerated granulosa cell aggregation,up-regulated the expression of BMPR1B,FSHR and intracellular cAMP levels,and activated the Smadl signaling pathway,indicating that BMP15 promoted follicular selection;co-processing BMP15 with FSH,FSHR The expression of genes related to steroid hormone synthesis was up-regulated,and the level of intracellular cAMP and progesterone synthesis in the supernatant of granulosa cells increased,indicating that FSH promotes follicular selection on the basis of BMP15.4.Cloning and activity analysis of BMP 15 gene promoterDifferent truncations of the promoter region of the BMP 15 gene were successfully constructed.The dual luciferase reporter technology was used to identify that the core promoter region of the chicken is located at-1bp～-153bp,and there are trans-acting factors in the region of-153bp～-691bp,-691bp～-1153bp region has cis-regulatory elements.SNP scanning was performed on the functional promoter region,and 9 SNP sites were identified,including Spl,ER,GCN4 and other transcription factor binding sites;3 SNP sites in the core promoter region were compared with Taihang chicken population for laying eggs.The traits were subjected to association analysis,and none of the three loci were found to be significantly related to egg-laying traits.In summary,in this study,687 differentially expressed genes were screened through transcriptome sequencing of small yolk follicle tissues in late laying stage of local chickens.Among them,BMP15 was significantly down-regulated in small yolk follicles of high-laying hens,and this gene was specific in chicken ovarian tissues.The expression level in granulosa cells is much higher than that in membrane cells;BMP15 gene promotes follicle selection,and there is a complex interaction between BMP15 and FSH in the follicle selection process;the core promoter region of BMP 15 gene is located at-1bp～-153bp,of which 3 SNP sites in the core promoter region were not found to be significantly related to egg production traits.This experiment studied the molecular mechanism of BMP 15 gene regulating follicle selection in the late laying period of chickens,which can provide an important theoretical basis and breeding basis for improving the laying rate of poultry in the late laying period.