Effect Of Methylation Of ZP2 Gene Promoter Regions On Gene Expression And Number Of Laying Eggs In Jinghai Yellow Chicken

Small,high quality,early maturing and strong resistance are four traits of Jinghai Yellow Chicken.In the production records,we found there were special individuals with high and low egg production in 300-day-old.Interestingly,number of eggs produced are quantitative traits,which are not only susceptible to the effects of the environment,but also controlled by micro multi-genes.In this paper,four genes(ZP2 gene,Stat5b gene,AMH gene,MDH1 gene),which could influence number of eggs produced of Jinghai Yellow Chicken,were selected.This study detected mRNA expression of these four genes in 300-day-old Jinghai Yellow Chicken by qRT-PCR,then detected the core promoter region of ZP2 gene by double luciferase gene reporting system.In order to discuss the regulatory mechanism of the ZP2 gene,in this paper,combined with the prediction of the potential transcription factor binding site in the promoter region,we detected the methylation levels of the ZP2 gene's promoter CpG island in ovary by BSP(Bisulfite Sequencing PCR),and analyzed the correlation between methylation level and mRNA expression.The main results are:1.qRT-PCR was used to detect the 4 genes mRNA expression in 9 tissues,we found that the expression of ZP2 gene in ovary was the highest,followed by kidney;the expression of Stat5b gene in leg muscle was the highest,and that was low in ovary;the expression of AMH gene was highest in ovary than the other tissues;the expression of MDH1 gene was highest in heart,followed by leg muscle,and that was low in ovary.Further analysis of expression in high-yield group and low-yield group showed that the expression of ZP2 gene in high-yield group was very significantly higher than that in low-yield group(P<0.01),and the expression of Stat5b gene and AMH gene in high-yield group was lower than that in low-yield group with no significant difference,while that was almost the same for MDHlgene in two groups.The results showed that the up-regulation of ZP2 gene may affect the number of eggs produced of Jinghai Yellow Chicken.Stat5b gene and MDH1 gene may had no direct effect on the number of eggs produced of Jinghai Yellow Chicken.AMH gene had a high expression level in ovary,but the expression of AMH gene in two groups had no significant difference,and its role in ovary of Jinghai yellow chicken needs further study.2.The results of double luciferase gene reporting system showed that the promoter fragment of ZP2 gene had different activity in DF-1 cells,and the activity of recombinant plasmid pGL3-P6 was the highest,while the activity of pGL3-P5 was significantly decreased,indicating that the core region of ZP2 gene promoter was between-1552 and-1348,whicih was consistent with the results predicted by the Neural Network Promoter Predietion online site.3.Four CpG islands were found in ZP2 gene' promoter regions by methprimer.The methylation status of CpG island was detected by BSP.The results showed the overall methylation of ZP2-1 amplified fragment was negatively correlated with mRNA expression(R =-0.197,P = 0.672),but there was no significant correlation between methylation degree and mRNA expression in all single loci.The overall methylation of ZP2-2 amplified fragment was also negatively correlated with mRNA expression(R =-0.264,P = 0.567),and the methylation degree of mC-9 was very significantly negatively correlated mRNA expression(P<0.01),but it wasn't located at the binding site of transcription factor.There was a significantly negative correlation between the mC-20 and mRNA expression as well as the mC-21(P<0.05),which were located at the binding site of Sp1 transcription factor,indicating that the above-mentioned sites may be the main site of transcriptional regulation and may inhibit the transcription of the ZP2 gene by inhibiting the binding of Spl.