IP3R2结构域对三穗鸭子宫内膜上皮细胞Ca<sup>2+</sup>分泌的影响

doi:10.3969/j.issn.1674-7968.2022.06.011

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摘要肌醇1, 4, 5-三磷酸受体2型(inositol 1,4,5-triphosphate receptor type 2, IP3R2)是位于内质网的细胞内Ca²⁺释放通道,通过调控Ca²⁺释放而影响蛋壳品质。为阐明IP3R2蛋白结构域对鸭(Anas platyrhyncha domestica)子宫内膜上皮细胞Ca²⁺分泌的调控效应及其对钙转运相关基因的影响,本研究以蛋鸭品种三穗鸭为研究对象,构建IP3R2蛋白结构域的表达载体pCDNA3.1-IP3R2,转染至鸭子宫内膜上皮细胞,以Flag标签ELISA检测试剂盒确定IP3R2蛋白结构域的表达量;以Fluo-3/AM钙离子荧光标记检测胞内Ca²⁺含量;以qPCR检测不同结构域过表达量及离子转运相关基因的mRNA水平。结果显示,MIR、RYDR_ITPR(1)、RYDR_ITPR(2)、RIH_assoc和Ion_trans结构域过表达均引起鸭子宫内膜上皮细胞内Ca²⁺浓度下降;RYDR_ITPR(1)和IP3R2-Ion_trans的表达量与胞内Ca²⁺浓度显著负相关(P<0.05);RYDR_ITPR(2)、RIH_assoc和5个结构域共转染的表达量与胞内Ca²⁺浓度极显著负相关(P<0.01);RYDR_ITPR(2)结构域表达量与RYR2、KCNJ15和CA2基因表达量呈显著正相关(P<0.05);MIR结构域过表达量与IP3R1、CALB1和CA2的基因表达量呈现显著正相关(P<0.05),2个RYDR_ITPR结构域过表达量均与KCNJ15和RYR2基因表达量呈显著正相关(P<0.05);RIH_assoc结构域过表达量与IP3R2和ORAI1基因表达量呈显著正相关(P<0.05);Ion_trans结构域过表达量与CALB1、SLC8A1 和CA2基因表达量呈显著正相关(P<0.05);5个结构域共转染后的表达量与CFTR基因表达量呈显著正相关、与TRPV6呈显著负相关(P<0.05)。上述结果提示,5个IP3R2蛋白结构域的过表达可能引起鸭子宫内膜上皮细胞内Ca²⁺向胞外释放,对细胞中20个离子转运基因表达水平的调控效应存在差异。本研究可为分子遗传标记选择和鸭蛋壳品质提高提供参考依据。

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	杨酸
	谭光辉
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	游敏芳
	廖朝美
	谭元成
	王盈童

关键词 ：三穗鸭, 肌醇1,4,5-三磷酸受体2型(IP3R2)蛋白结构域, 子宫内膜上皮细胞, Ca²⁺分泌, 蛋壳品质

Abstract：The inositol 1,4,5-trisphosphate receptor type 2 (IP3R2) is an intracellular Ca²⁺-release channel located on the endoplasmic reticulum (ER), which can regulate Ca²⁺ release and affect eggshell quality. To elucidate the regulatory effect of IP3R2 protein domain on Ca²⁺ secretion of duck (Anas platyrhyncha domestica) endometrial epithelial cells and its effect on calcium transport-related genes, in this study, the IP3R2 protein domain expression vector PCDNA3.1-IP3R2 was constructed in laying duck (Sansui duck) and transfected into duck endometrial epithelial cells. The expression of IP3R2 protein domain was detected by Flag label detection kit (ELISA); Fluo-3/AM calcium fluorescence label was used to detect intracellular Ca²⁺ content; qPCR was used to detect the overexpression levels of different domains and mRNA levels of ion transport-related genes. The results showed that overexpression of MIR, RYDR_ITPR (1), RYDR_ITPR(2), RIH_assoc and Ion_trans domains all caused the decrease of Ca²⁺ concentration in duck endometrial epithelial cells; The expression levels of RYDR_ITPR(1) and IP3R2-Ion_trans were significantly negatively correlated with intracellular Ca²⁺ concentration (P<0.05); The expression levels of RYDR_ITPR(2), RIH_assoc and 5 domains were significantly negatively correlated with intracellular Ca²⁺ concentration (P<0.01); The expression of RYDR_ITPR(2) domain was significantly positively correlated with the expression of RYR2, KCNJ15 and CA2 genes (P<0.05); The overexpression levels of MIR-domain were significantly positively correlated with the gene expression levels of IP3R1, CALB1 and CA2 (P<0.05), and the overexpression levels of the 2 RYDR_ITPR domains were significantly positively correlated with the gene expression levels of KCNJ15 and RYR2 (P<0.05); The overexpression of RIH_assoc domain was significantly positively correlated with the expression of IP3R2 and ORAI1 genes (P<0.05); and the overexpression of Ion_trans domain was significantly positively correlated with the expression of CALB1, SLC8A1 and CA2 genes (P<0.05). The co-transfection level of the five domains were significantly positively correlated with CFTR gene expression, and negatively correlated with TRPV6 gene expression (P<0.05). These results suggest that overexpression of 5 IP3R2 protein domains can cause extracellular Ca²⁺ release in duck endometrial epithelial cells, and have different regulatory effects on the expression levels of 20 ion transport genes. This study provides a reference for improving eggshell quality of ducks in terms of molecular genetic marker selection.

Key words： Sansui duck Inositol 1,4,5-triphosphate receptor type 2 (IP3R2) protein domain Endometrial epithelial cell Ca²⁺ secretion Eggshell quality

收稿日期: 2021-08-23

ZTFLH:

S834

基金资助:国家自然科学基金(31760663); 贵州省科技人才补助项目(黔科合平台人才[2017])(5788); 贵州省千层次创新人才项目(贵大千层次[2015]01)

通讯作者: * zyy8yyc@163.com

引用本文:

杨酸, 谭光辉, 张依裕, 游敏芳, 廖朝美, 谭元成, 王盈童. IP3R2结构域对三穗鸭子宫内膜上皮细胞Ca²⁺分泌的影响[J]. 农业生物技术学报, 2022, 30(6): 1153-1165.
YANG Suan, TAN Guang-Hui, ZHANG Yi-Yu, YOU Min-Fang, LIAO Chao-Mei, TAN Yuan-Cheng, WANG Ying-Tong. Effect of IP3R2 Domains on Ca²⁺ Secretion of Sansui Duck (Anas platyrhyncha domestica) Endometrial Epithelial Cells. 农业生物技术学报, 2022, 30(6): 1153-1165.

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http://journal05.magtech.org.cn/Jwk_ny/CN/10.3969/j.issn.1674-7968.2022.06.011 或 http://journal05.magtech.org.cn/Jwk_ny/CN/Y2022/V30/I6/1153

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