Effects Of SLC8As And ATP2Bs Gene Expression And Polymorphism On Eggshell Quality Of Changshun Blue-Eggshell Chicken

In a variety of epithelial cells,SLC8As and ATP2Bs are located at the apical side of the cell,and serve as highly selective calcium channels to construct the internal circulation network of Ca2+and play an important role in the transcellular transport of calcium ions.So far,there have been few reports on SLC8As and ATP2Bs genes in domestic animals.In order to explore the genetic mechanism of SLC8As and ATP2Bs genes regulating egg shell quality,this experiment took Changshun Blue-eggshell chicken as the research object.The effect mechanism of RT-q PCR,direct DNA sequencing and tissue Ca²⁺concentration determination on eggshell quality of laying hens and the correlation between the expression of SLC8As and ATP2Bs genes and the content of uterine calcium in different stages of laying cycle were used.Through these studies,we aim to reveal the regulatory mechanisms of SLC8As and ATP2Bs genes on the genetic effects of egg shell quality.Through the above study,we hope to clarify the genetic mechanism of SLC8As and ATP2Bs genes regulating egg shell quality,provide reference for the dietary calcium addition level during egg production,and find a new scientific basis for improving egg shell quality.The main results are as follows:1.The results of RT-q PCR showed that SLC8A1 and ATP2B2 mRNA were expressed in different tissues of Changshun Blue-eggshell chicken at 43 weeks of age,and the expression abundance of SLC8A1 gene was in the order of heart>kidney>spleen>uterus>small intestine>pectorals>myogastric>liver>large intestine>adenogastric>duodenum>lung.The expression abundance of ATP2B2 gene was uterus>heart>kidney>small intestine>pectoralis>glandular stomach>spleen>large intestine>lung>muscular stomach>liver>duodenum.2.Three novel SNPS were identified in SLC8A1 gene of Changshun Blue-eggshell chicken by direct PCR sequencing and sequence alignment,all of which were located in the UTR region of exon 11,g.16085681 T>C,g.16085766 C>T and g.16085781 T>C,g.16085681 T>C mutation significantly affected eggshell strength,g.16085766 C>T mutation significantly affected egg shape index.Three SNPs sites produced three haplotypes and six diplotypes,and the combination of three SNPs had significant effects on eggshell strength and eggshell thickness.Three novel SNPs were found in the ATP2B2 gene:the UTR region of Exon 4,the CDS region of exon 15,and the CDS region of exon 25:g.4030584 A>G,g.4144313 C>T and g.4193196 G>A mutation;The effect of g.4030584 A>G and g.4144313 C>T mutation on eggshell strength was significant.There were 6 haplotypes and 10 diplotypes in 3 SNPs sites,and the combination of 3SNPS sites could significantly affect eggshell thickness and eggshell strength.The results revealed that the SNP sites in SLC8A1 and ATP2B2 genes can affect the eggshell strength and egg weight and thickness,which can be used as genetic markers for egg shell quality selection.3.Real-time fluorescence quantitative method was used to detect the relationship between the expression of SLC8A1 and ATP2B2 genes and the content of Ca²⁺in the uterus at different stages of the laying cycle of Changshun Blue-eggshell chicken.The results showed that the maximum value of SLC8A1 and ATP2B2 genes was significantly reached during the eggshell calcification process（P>0.01）,the expression of SLC8A1 gene peaked in the heart,kidney,small intestine and uterus,and was significantly higher than 0～4.5 h（P>0.01）;The expression level of SLC8A1 gene in duodenum at 0 h was significantly higher than that at 2-16 h（P>0.01）;The expression of ATB2B2 gene peaked in the heart,kidney,duodenum and uterus during eggshell calcification,and was significantly higher than that at 0～4.5 h（P>0.01）;The expression level of ATB2B2 gene in small intestine at 0 h was significantly higher than that at 2-16 h（P>0.01）;The Ca²⁺concentration in uterus increased from 0 to 16 h.Correlation results showed that SLC8A1gene expression in kidney at 0 h was negatively correlated with uterine Ca²⁺concentration（P>0.05）;The expression of ATP2B2 gene in duodenum was positively correlated with Ca²⁺concentration in uterus（P>0.05）;The expression level of SLC8A1 gene in small intestine was positively correlated with that in uterus at 2 h（P>0.05）;The expression of SLC8A1 gene in the large intestine at 4.5 h was positively correlated with the concentration of uterine Ca²⁺（P>0.05）;The expression level of SLC8A1 gene in liver,kidney and duodenum at 16 h was positively correlated（P>0.05）.The results showed that SLC8A1 and ATP2B2 genes have certain effects on Ca²⁺transport in the kidney,uterus and intestine during eggshell formation.4.RT-qPCR was used to detect the expression of calcium transport-related genes in tissues at different stages of the laying cycle of Changshun Blue-eggshell chicken.The analysis of gene expression patterns showed that the expression levels of related genes in kidney,large intestine and uterus continued to increase from 0 h to 16 h.In the small intestine and duodenum,the expression of these genes continued to decline.The results showed that all the 10 genes were widely expressed.At 16 h after laying,the expression levels of SLC8A1,SLC8A3,ATP2B1 and ATP2B2 genes in utero also increased significantly and reached the maximum.Ca²⁺transport-related genes increased gradually from 0 h to 16 h in utero.At 0 h after laying,ITPR1 gene was negatively correlated with uterine Ca²⁺concentration（P>0.01）,SLC8A3 gene and ADRA1D gene expression were negatively correlated（P>0.05）;There was a significant negative correlation between the expression of SLC8A1 gene and ITPR2 gene 2 h after laying（P>0.05）,GNA11 was positively correlated with uterine Ca²⁺concentration（P>0.05）;At 4.5 h after laying,ITPR1 gene and ADRA1D gene expression were negatively correlated（P>0.05）,there was a significant negative correlation between ERBB4 and CACNA1E gene expression（P>0.05）;There was a significant negative correlation between ITPR2 and ERBB4 gene expression at 16 h after laying（P>0.05）,SLC8A3 was positively correlated with ATP2B1 gene expression（P>0.05）,there was a significant negative correlation between ATP2B2 and ADRA1D gene expression（P>0.05）,indicating that SLC8As and ATP2Bs genes and ITPR1,ITPR2,CACNA1E,ADRA1D,GNA11,ERBB4 genes synergistically regulate Ca²⁺transport in chicken uterus.In conclusion,SLC8As and ATP2Bs genes may have a synergistic effect in the transport of calcium ions in utero,and there are synergistic and inhibitory effects between related genes on the regulation of calcium ion entry.In the process of eggshell calcification,the expression of SLC8As and ATP2Bs genes in the uterus will have a certain effect on the concentration of uterine Ca²⁺,which will change the expression of intracellular ITPR1,ITPR2,CACNA1E,ADRA1D,GNA11and ERBB4 genes mRNA.