Effect Mechanism Of IP3R2 Protein Domain On Duck Eggshell Quality

Eggshell damages lead to economic losses in the egg production industry.Mineralization of the avian eggshell is a complex process involving the precipitation of calcium carbonate that results from interaction between this mineral and the organic matrix during the nucleation and growth phases.The inositol 1,4,5-trisphosphate（IP3）receptor（IP3R）type 2（IP3R2）is an intracellular Ca²⁺-release channel located on the endoplasmic reticulum（ER）,and allowing the release of Ca²⁺from this organelle.But little is known about the function of IP3R2 or the regulatory effect of eggshell quality of the IP3R2 gene in avian species.Therefore,in this study,RT-qPCR,PCR direct sequencing,gene cloning,ELISA and Fluo-3/AM calcium fluorescence labeling were used to investigate:The tissue-specific expression of IP3R2 gene in various organs of the peak laying period ducks,identify polymorphisms in IP3R2 and to study their association with duck eggshell quality traits;The regulatory effect of IP3R2 gene protein domain on Ca²⁺in duck uterine epithelial cells;Effect of overexpression of protein domain of IP3R2 gene on the expression of 20 anion and cation transport related genes m RNA in uterine epithelial cells.The main results are as follows:1.RT-qPCR was applied to investigate the distribution of IP3R2 m RNA expression in 11tissues and the results demonstrated that IP3R2 m RNA was widely expressed in the 11 examined tissues from the 6 female ducks aged 45 weeks,with expression being the highest in pancreas and the lowest in the gizzard.The IP3R2 m RNA level in different tissues was as follows:pancreas>uterus>glandular stomach>kidney>chest muscle>spleen>small intestine>lung>liver>heart>gizzard.Three novel SNPs,the synonymous mutation of g.128662092 C>T in exon 16,g.9583222 C>T in intron 14 and g.128767757 A>G in in intron 37,were found in the IP3R2 gene,giving 4 haplotypes and 10 diplotypes.The g.128662092 C>T mutation were significantly associated with eggshell strength,TT genotype is favorable for increasing eggshell strength.The combination of the three SNP loci was significantly associated with egg shape index,shell thickness and strength（P<0.05）,the egg shape index of diplotypes H3H3,H4H4 and H1H2 being significantly higher than diplotypes H2H2（P<0.05）,the eggshell thickness of diplotypes H1H1 and H1H2 was significantly higher than diplotypes H2H2,and the eggshell strength of diplotypes H1H1,H1H2and H1H3 was significantly higher than diplotypes H3H4 and H4H4.Thus,the three SNPs of IP3R2gene may be used as markers in selection for improved eggshell quality in layer ducks.2.The pcDNA3.1+Flag expression vectors of protein domains MIR,RYDR＿ITPR（1）,RYDR＿ITPR（2）,RIH＿assoc and Ion＿trans of IP3R2 gene were successfully constructed and transfected into primary duck uterine epithelial cells.The overexpression of five domains of IP3R2gene and intracellular Ca²⁺concentration was detected by ELISA and Fura-3/AM,respectively.Experiments revealed that the overexpression of MIR,RYDR＿ITPR（1）,RYDR＿ITPR（2）,RIH＿assoc and Ion＿trans domains decreased the concentration of Ca²⁺in uterine epithelial cells.The expression of RYDR＿ITPR（1）,RYDR＿ITPR（2）,RIH＿assoc and Ion＿trans domains was negatively correlated with the concentration of intracellular Ca²⁺.The RYDR＿ITPR（2）domain had the strongest regulatory effect on Ca²⁺in uterine epithelial cells.The results showed that the overexpression of IP3R2 gene five protein domains would lead to the release of Ca²⁺from duck uterine epithelial cells.3.RT-qPCR was performed to detect the effect of IP3R2 protein domain overexpression on the m RNA expression levels of 20 ion transporter genes in epithelial cells,and the results revealed that the MIR protein domain overexpression was positively related to IP3R1,CALB1 and CA2 m RNA expression（P<0.05）,and the overexpression of two RYDR＿ITPR domains was positively correlated with the KCNJ15,RYR2 and CA2 expression（P<0.05）,the RIH＿assoc domain overexpression was positively correlated with the expression of IP3R2 and ORAI1 genes（P<0.05）,and the overexpression of Ion＿trans domain was positively correlated with the expression of CALB1,SLC8A1 and CA2 genes（P<0.05）.The expression of five domains overexpression was positively correlated with the expression of CFTR and negatively correlated with TRPV6 gene（P<0.05）,demonstrating that the overexpression of the five structural domains of the IP3R2 gene differentially regulated the m RNA expression levels of 20 ion transport genes in uterine epithelial cells.4.The correlation between the m RNA expression levels of 20 intracellular ion transport genes after overexpression of the five protein domains of the IP3R2 gene in uterine epithelial cells was analyzed,and the results showed that there was a significant positive correlation between IP3R2gene expression and IP3R3,RYR2,SLC4A4,SLC4A9 and ATP2B1 gene expression in un-transfected group;There was a significant negative correlation between IP3R2 gene expression and TRPV6expression after overexpression of RYDR＿ITPR（1）domain,a significant positive correlation between IP3R2 and CALB1 and RYR2 expression after overexpression of RYDR＿ITPR（2）domain,a significant positive correlation between IP3R2 gene expression and ORAI1 gene expression after overexpression of RIH＿assoc domain,and a positive correlation between IP3R2 and KCNJ15 gene expression after overexpression of Ion＿trans domain.After overexpression of five domains,the expression of IP3R2 was positively correlated with CALB1,SLC8A1,CA2 and SLC4A9 genes,and negatively correlated with RYR2 gene expression.It was demonstrated that overexpression of the IP3R2 gene 5 protein domains in duck epithelial cells altered the correlation coefficients of the m RNA expression levels of the 20 ion transport genes detected intracellularly,further revealing that overexpression of the IP3R2 gene 5 protein structural domain regulates the mechanism of Ca²⁺release to the extracellular in duck epithelial cells differently.