Research On Regulation Of IP3R3 On Duck Eggshell Quality And Ca(2+) Release From The Epithelial Cells Of The Eggshell Gland

IP3R3,a member of the IP3Rs family,plays an important role in regulating Ca²⁺transport in the endoplasmic reticulum of cells.Ca is a restrictive mineral element that determines the quality of poultry eggshells.Eggshell glands are the parts where eggshells are formed.IP3R3 may affect eggshell’s quality by regulating the secretion of Ca²⁺of poultry eggshell glands.In order to explore the genetic effects of IP3R3 on poultry eggshell quality and Ca²⁺release from eggshell glandular epithelial cells,Sansui duck was used as the experimental material to study the effects of genetic variation of IP3R3 gene on duck eggshell quality and the regulatory effects of overexpression of different functional domains of IP3R3 gene on Ca²⁺secretion in duck shell gland epithelial cells.The findings are as follows:1.The mRNA tissue expression profile and SNPs of Sansui duck IP3R3 gene were examined by direct sequencing methods of RT-qPCR and PCR’s product.The results show that in exon 8 of the gene IP3R3 g.16067 C>T synonymous mutations,exon 49g.35195 T>C,and g.35207 G>A synonymous mutations as well as exon 50 of the g.35425 C>A synonymous mutations,producing a total of 5 haplotypes and 15 diplotypes,and the effect of g.16067 C>T site on eggshell’s thickness is significant（P<0.05）;double-type H1H1 individuals had the highest eggshell thickness,while double-type H5H5 had the lowest;the m RNA expression of IP3R3 gene is on different levels in 11tissues of 45-week-old Sansui duck,and the expression abundance is in the order of eggshell gland>heart>spleen>glandular stomach>pancreas>kidney>liver>small intestine>lung>pectoral muscle>myogastric.2.The IP3R3 gene MIR,RYDR-ITPR-1,RYDR-ITPR-2,RIH-assoc,and Ion-trans functional structural domain expression vectors,pcDNA3.1（+）-Flag-structural domain and p EGFP-N3-structural domain were constructed and transfected with EK-293T cells and duck eggshell gland epithelial cells,and immunofluorescence The results showed that the vectors were successfully transfected and all five functional domains were localized in the nucleus and cytoplasm of EK-293T cells and duck eggshell gland epithelial cells.3.The pcDNA3.1（+）-Flag-domain expression vector was used to transfect duck eggshell gland epithelial cells.CCK8 kit was used to detect the proliferation of the transfected cells.The results show that overexpression of all 5 functional structural domains of the IP3R3 gene inhibited the proliferation of duck eggshell gland epithelial cells,with the strongest inhibitory effect of the RIH-assoc protein domain.4.Duck egg shell gland epithelial cells were transfected using pcDNA3.1（+）-Flag-domain expression vector.Ca²⁺was loaded with Fluo-3/AM fluorescence labeling.The results show that overexpression of 5 functional protein domains of IP3R3gene caused significantly lower Ca²⁺content in duck eggshell gland epithelial cells than the blank control group（P<0.05）,revealing that overexpression of all five functional domains could promote Ca²⁺release in duck eggshell gland epithelial cells.Overexpression of the RIH-assoc functional protein domain had the strongest effect on Ca²⁺release in duck eggshell gland epithelial cells.5.By studying the effects of overexpression of 5 functional domains of IP3R3gene in duck eggshell gland epithelial cells on the expression of 9 Ca²⁺transport-related genes CALB1,ITPR1,ITPR2,KCNJ15,PRKCA,SCNN1G,SLC4A4,SLC4A9,TRPV6 m RNA,the results showed:That the overexpression of MIR protein domain was negatively correlated with the expression of CALB1,ITPR1,PRKCA and SLC4A9and positively correlated with the expression of other genes;RYDR-ITPR-1 protein domain overexpression was negatively correlated with the expression of CALB1,SCNN1G,SLC4A4 and SLC4A9 and positively correlated with the expression of other genes;RYDR-ITPR-2 protein domain overexpression was negatively correlated with ITPR2,KCNJ15 and SCNN1G expression,significantly positively correlated with PRKCA expression（P<0.05）,and significantly negatively correlated with SLC4A4expression（P<0.05）;RIH-assoc protein domain overexpression was negatively correlated with KCNJ15,SLC4A4,SLC4A9 and TRPV6 expression The expression of IP3R3 gene was negatively correlated with the expression of CALB1,PRKCA,SLC4A4 and TRPV6 in the blank group,revealing that overexpression of five functional protein domains of IP3R3 gene regulated the extracellular transport of Ca²⁺in duck egg shell gland epithelial cells.The mechanism of Ca²⁺transport to the extracellular gland of duck egg shell gland is differentially regulated by the overexpression of 5 functional protein domains of IP3R3 gene.