Identification Of Egg-laying Related Genes In Hypothalamus Of Local Chickens And The Effect Of CNR1 Expression On GnRH Synthesis

Local chicken is favored by the majority of consumers because of its excellent meat and egg quality,but the poor laying performance of most local chicken breeds affects the economic benefits of local chicken breeding to a great extent.Therefore,it is an urgent need for local chicken breeding to mine the key genes and their molecular markers related to egg production performance,and then improve egg production performance through genetic selection.In this study,differentially expressed genes were identified through transcriptome sequencing analysis of the hypothalamus of Lushi chickens in the prelaying period（15 weeks）and peak laying period（30 weeks）.Then,Gene Ontology enrichment analysis was used to screen for differentially expressed genes.Genes involved in the biological process of reproductive regulation;then,through protein interaction network analysis,potential core candidate genes that regulate the reproductive process are screened.On this basis,qRT-PCR was used to analyze the change trend of the expression levels of potential core candidate genes in the hypothalamus of the two local chicken breeds during the pre-laying period（15w）and the peak laying period（30w）,and then the gene expression level was analyzed.Correlation analysis with 30-week-old egg production and blood reproductive hormone levels to screen key genes affecting local chicken egg production performance;screen single nucleotide polymorphism loci from these key genes and correlate with egg production in different periods.Association analysis was performed to further identify potential SNP loci in these key genes that affect egg production.Finally,gain-of-function and loss-of-function assays were used to analyze the effect of the key gene CNR1 on GnRH expression and GnRH hormone levels in hypothalamic neurons.The result is as follows:1.By comparing the ovarian morphology and reproductive hormone levels of Lu’s chickens in the early laying period and the peak laying period,it was found that the serum follicle-stimulating hormone（FSH）,luteinizing hormone（LH）and estrogen（E₂）at the peak laying period and progesterone（PROG）levels were significantly higher than pre-laying,while prolactin（PRL）levels were significantly lower than prelaying.The hypothalamus tissue of 3 individuals of Lushi chickens in the pre-laying period（15 weeks）and the egg-laying peak period（30 weeks）were collected for transcriptome sequencing,and a total of 518 differentially expressed genes were identified;GO enrichment analysis of differentially expressed genes The results showed that these 518 genes were significantly enriched in 340 GO Terms,including247 biological processes,64 cellular components and 29 molecular functions.Among them,10 GO Terms are enriched in neurotransmitter level regulation,neurotransmitter transport,neurotransmitter transport regulation,hormone transport,folliclestimulating hormone secretion,hormone level regulation,synaptic vesicle cycle,and vesicle-mediated synapse Transport,transsynaptic signaling,and establishment of predicted vesicle localization are involved in biological processes related to the regulation of reproduction in the chicken hypothalamus.A protein interaction network was constructed for the 64 differentially expressed genes in the enriched 10 GO Terms,and a total of 16 genes with high degree of connectivity（Degree≥12）were screened（APP,SNAP25,SYT1,POMC,TRH,ANXA5,SYNJ1,PENK,NRXN1,TTR,SLC1A2,DNM1,AP2M1,HIP1 R,GABRA1,CNR1）,which were considered as potential candidate genes involved in reproductive regulation.2.The expression patterns of the 16 potential candidate genes identified above in the hypothalamus at the early laying（15w）and peak laying periods（30w）of Lushi chickens and Gushi chickens were analyzed.There was a significant difference in the hypothalamus between the early laying period and the peak laying period（P<0.05）,and HIP1 R was only significantly different in the two periods of Lushi chicken;The expression in the hypothalamus at peak was not different,and the remaining 11 genes（SNAP25,POMC,TRH,PENK,NRXN1,TTR,SLC1A2,AP2M1,GABRA1,ANXA5 and CNR1）had the same expression trend in the two local chicken breeds.There were significant differences between 15 w and 30 w hypothalamus（P<0.05）.The expression trend of the above 11 genes in the hypothalamus of high-yield and low-yield Gushi chickens at 30 weeks was analyzed,and 8 genes（ANXA5,NRXN1,PENK,SLC1A2,SNAP25,TRH,AP2M1,CNR1）in high-yield and low-yield Gushi Differential expression in chicken hypothalamus（P<0.05）;further analysis of egg production and reproductive hormone levels of 30 w high-yield and low-yield Gushi chickens showed that the serum levels of FSH,LH,E₂ and PROG in high-yield Gushi chickens were significantly higher than those of low-yield Gushi chickens,while the level of PRL was significantly lower than that of low-yield Gushi chickens;then the correlation analysis of the expression of these 8 genes with the egg production and reproductive hormone levels of 30 w Gushi chickens showed that,The expression levels of CNR1 and AP2M1 genes were negatively correlated with egg production,FSH,LH,E₂,and PROG,but positively correlated with PRL.LH,E₂,PROG were positively correlated to varying degrees,and negatively correlated with PRL.The SNP loci of these 8 genes were scanned in the Gushi chicken group,and a total of 11 SNP loci were screened,of which 8 SNP loci can be used for high-throughput genotyping using KASP technology.Analysis: The association analysis between the different genotypes of these 8 loci and egg-laying traits showed that the different genotypes of these 8 SNP loci were significantly correlated with the number of eggs laid at different stages（P<0.05）.3.The expression of CNR1 gene in Gushi chickens was detected by q PCR.The results showed that the expression of CNR1 gene was high in pituitary and hypothalamus,but low in pectoral muscle.The overexpression vector of CNR1 gene was constructed and the corresponding interference fragment was synthesized.and analyzed the expression and hormone levels of GnRH in hypothalamic neuron cells by CNR1 gene.The results showed that CNR1 gene inhibited GnRH expression and GnRH synthesis in hypothalamic neuron cells.In summary,this study identified a total of 518 differentially expressed genes in the hypothalamus transcriptome during the pre-and peak egg-laying periods,of which64 genes were significantly enriched in the biological processes involved in the regulation of reproduction in the hypothalamus;The PPI interaction network was constructed,and 16 genes involved in reproductive regulation were further screened.By analyzing the expression of the above 16 genes in the hypothalamus of 15 w and30w Lushi chickens and Gushi chickens,it was found that 11 genes were in the two breeds.The expression trends were the same between the two groups,and they were differentially expressed in the early and peak periods of laying eggs;the expression levels of these 11 genes in the hypothalamus of 30 w high-yielding and low-yielding Gushi chickens were further analyzed,and 8 genes were identified in the highyielding and low-yielding Gushi chickens.Key genes significantly expressed in chicken hypothalamus;8 SNP loci were successfully identified from these 8 genes,and association analysis showed that different genotypes of SNP loci of these 8 key genes were significantly associated with egg production at different stages related.Overexpression and interference of CNR1 gene in hypothalamic neurons showed that CNR1 gene inhibited the expression of GnRH gene and the synthesis of GnRH hormone in the hypothalamus.These findings provide new insights into the further understanding of the regulation mechanism of egg laying performance in Lushi chickens,and provide possible molecular markers for breeding performance of local chickens.