Genetic Effects Of TRPV6 Channels In The Regulation Of Duck Egg Shell Quality

The TRPV6 channel is highly selective for Ca²⁺transport from extracellular to intracellular,thus maintaining calcium stability in the body.However,calcium is the most important mineral element in the biological mineralization of poultry eggshell,which can directly affect the quality of eggshell.However,there are few reports on the TRPV6 pathway in poultry,and only a few studies on chicken.Therefore,in this study,the tissue expression profile of TRPV6 gene and the effect of SNPs on eggshell quality were studied by means of RT-qPCR,direct sequencing after PCR amplification,and Ca²⁺concentration determination of uterine tissue.In addition,the tissue expression level of TRPV6 gene and its effect on uterine Ca²⁺concentration of ducks during laying cycle were studied,and the regulation of TRPV6 gene expression on calcium transport-related gene expression level was also studied.The main results are as follows:1.The results of RT-qPCR showed that TRPV6 and SLC4A4 genes were expressed in different tissues of 43 week-old Sansui ducks.The expression abundance of TRPV6 gene was as follows:lung>uterus>large intestine>heart>pancreas>liver>pectoral muscle>adenogastric>kidney>duodenum>small intestine>myogastric>spleen.The expression abundance of SLC4A4 gene was as follows:lung>pancreas>uterus>large intestine>heart>kidney>liver>spleen>duodenum>glandular stomach>small intestine>myogastric.2.The second intron of TRPV6 gene was detected by PCR amplification and direct sequencing.The g.81465153 C>G and g.81465176 A>G mutations had significant effects on eggshell weight and egg weight（P<0.05）,and the synonymous mutation g.81465450 T>C was found in exon 3.The combination of three SNPS resulted in 5 haplotypes and 7 diplotypes.The combination of SNPs had significant effects on eggshell weight（P<0.05）,and had extremely significant effects on egg weight（P<0.01）.A synonymous mutation g.15125097 C>T was found in exon 24 of the SLC4A4 gene,the g.15132523 G>A mutation was found to have a highly significant effect on egg shape index in intron25（P<0.01）and the g.15135079 G>A mutation in intron 26.The combination of 3 SNP sites produced 4 haplotypes and 7 diplotypes,and the combination of sites had a significant effect on eggshell weight（P<0.01）.After the 2 genes were polymerized,6 SNPs combined to produce 8haplotypes and 7 diplotypes.The combination of SNPs had a significant effect on eggshell weight and egg weight（P<0.05）;It was showed that the combination of three newly identified SNP loci in the TRPV6 and SLC4A4 genes and the aggregation of six SNP loci in both genes had significant effects on egg and shell weights,which can be used as a genetic marker for duck egg shell quality selection.3.The expression level of TRPV6 gene in tissues of ducks at different stages of laying cycle was detected by RT-qPCR,and the Ca²⁺concentration of uterine tissues at different stages was determined,and the correlation analysis was conducted.The results showed that the expression of the TRPV6 gene in the large intestine and uterus reached a highly significant maximum（P<0.01）during eggshell calcification（16 h）,the expression of the TRPV6 gene in the small intestine and duodenum was significantly higher at 0 h than at 2 h to 16 h（P<0.01）,expression of TRPV6 gene in kidney was significantly higher at 0 h and 5 h than at 2 h and 16 h（P<0.05）;In contrast,Ca²⁺concentration in the uterus tended to rise from 0 h to 16 h,reaching a highly significant maximum（P<0.01）during eggshell calcification（16 h）;correlation results showed that 2 h uterine TRPV6gene expression was significantly negatively correlated with uterine Ca²⁺concentration（P<0.05）,5h TRPV6 gene expression in the heart and lungs was significantly and positively correlated with uterine Ca²⁺concentration（P<0.05）,16 h uterine Ca²⁺concentration was significantly negatively correlated with TRPV6 gene expression in the heart,kidney and small intestine（P<0.05）,the TRPV6gene has been shown to play a role in calcium transport in the uterus,kidney and intestine during duck egg shell formation,which provided a reference and theoretical basis for further studies on the regulatory mechanism of the TRPV6 gene affecting calcium ion transport during eggshell formation.4.RT-qPCR results showed that calcium transport-related genes were expressed in tissues of ducks at different stages of laying cycle.The expression of related genes increased from 0 h to 16 h in kidney,large intestine and uterus,and decreased from 0 h to 16 h in the small intestine and duodenum.The correlation results showed that there was a significant negative correlation between Ca²⁺concentration and the expression levels of IP3R2 and KCNJ15 genes at 0 h（P>0.05）,IP3R2was significantly positively correlated with KCNJ15 gene expression（P<0.05）.TRPV6 was negatively correlated with KCNJ15 gene expression and uterine Ca²⁺concentration at 2 h（P<0.05）,IP3R3 was negatively correlated with the expression of SCNN1G gene（P<0.05）.The expression level of KCNJ15 and SCNN1G gene was positively correlated at 5 h（P<0.01）;There was a significant negative correlation between Ca²⁺concentration and SCNN1G gene expression at 16 h（P<0.05）,Ca²⁺concentration was significantly positively correlated with SLC4A9 gene expression（P<0.05）,the expression levels of IP3R1 and IP3R3 genes were significantly negatively correlated（P<0.05）,the expression levels of PRKCA and SCNN1G genes were significantly positively correlated（P<0.05）,there was a significant negative correlation between PRKCA and SLC4A9 gene expression（P<0.05）,the expression levels of SCNN1G and SLC4A9 genes were significantly negatively correlated（P<0.05）.The results showed that the expression of TRPV6 gene in duck uterus affected the concentration of Ca²⁺,and then changed the m RNA expressions of IP3R1,IP3R2,IP3R3,KCNJ15,SCNN1G,SLC4A9 and PRKCA genes.It further revealed that TRPV6 gene expression regulated the mechanism of Ca²⁺release in ducks.