IP3Rs通道调控蛋鸭蛋壳品质的效应机制研究

Aims to explain effect mechanism of IP3Rs channel on eggshell quality in poultry, the eggshell traits and calcium content (blood, bone and utrus) of Sansui duck will be measured by energy spectrometer and atomic absorption spectrometry. Histological and histochemical methods, in combination with electron microscopy are used to observe the uterine tissue structure and cell morphology. Furthermore, the haplotypes and expression of the IP3Rs channel genes (IP3R1, IP3R2 and IP3R3), which was measured by the RT-PCR, RT-qPCR and direct sequencing, and its associtation with eggshell traits was studied. Moreover, the pEGFP-N3-IP3Rs recombinant expression vector will be constructed, and its expresion is used to detect by luciferase method after transfecting vector into laying duck uterus cells in vitro. Fluorospectrophotometry was applied to detect Ca2+ concentration loading the Fura-2/AM in tranfected cell, and then analyse association of IP3Rs channel with Ca2+ content. The pcDNA 3.1 expression vector of IP3Rs gene sequence, N-terminal and C-terminal will be constructed, and then be injected into laying duck leg. Flag-Tag kit was used to detect their expression in vivo, in order to explore the relationship between IP3Rs channel gene overexpression and eggshell quality. Our study may provide a new evidence to explain the regulation mechanism of IP3Rs channel on eggshell quality in laying duck.

为了阐释IP3Rs通道调控家禽蛋壳品质的效应机制，项目以三穗鸭为试验素材，采用能谱仪、原子吸收光谱等技术测定蛋壳性状、血浆钙和骨钙及子宫部钙的含量；用组织化学法结合电镜技术等观察子宫部组织结构和细胞形态；用RT-PCR、RT-qPCR、直接测序等技术研究IP3Rs通道基因IP3R1、IP3R2和IP3R3的表达和变异，探讨其与蛋壳品质的关系；构建IP3Rs通道基因全序列、N-端和C-端的pEGFP-N3表达载体，转染子宫上皮细胞，荧光素酶法检测其表达，荧光分光光度法检测负载了Fura-2/AM的细胞内Ca2+浓度，分析IP3Rs通道基因对Ca2+的调控作用；构建IP3Rs通道基因全序列、N-端和C-端的pcDNA3.1表达载体，腿肌注射，Flag-tag试剂盒检测其表达，分析IP3Rs通道基因在蛋鸭体内的过表达与蛋壳品质的关系，探讨并揭示IP3Rs通道对蛋鸭蛋壳品质的效应机制。

现有研究成果表明，三磷酸肌醇受体（IP3Rs）通道是畜禽细胞Ca2+释放的重要通道，并通过调控家禽子宫部Ca2+分泌来影响蛋壳品质，但其效应机制不清。为了阐述IP3Rs通道调控家禽蛋壳品质的效应机制，项目以三穗鸭为试验素材，研究和探讨IP3Rs通道基因遗传变异、体内外表达对蛋壳品质的影响，结果显示，在IP3Rs通道基因及9个阴阳离子转运相关基因中挖掘到65个SNPs位点，其中38个SNPs突变位点对蛋壳品质的影响达到显著水平，各基因中SNPs位点组合产生的双倍型均能显著影响蛋鸭蛋壳品质，可能作为改善蛋鸭蛋壳品质的遗传标记进行利用；IP3Rs通道基因（IP3R1、IP3R2和IP3R3）表达量与蛋壳指标（蛋壳强度、蛋壳厚度、蛋壳重）和血浆钙含量呈正相关；IP3Rs通道基因蛋白功能结构域过表达均能促进蛋鸭子宫上皮细胞中Ca2+向胞外分泌，干扰IP3Rs通道基因表达则导致蛋鸭子宫上皮细胞中钙离子累积，揭示IP3Rs通道基因是参与蛋鸭蛋壳生物矿化的重要基因；产蛋循环中子宫部IP3Rs通道、KCNJ15、TRPV6、SCNN1G、PRKCA、SLC4A9、SCNN1G、PRKCA等基因对Ca2+分泌起重要调节作用。发表SCI收录论文5篇，中文核心期刊11篇，培养硕士研究生6名，申请受理发明专利2件。项目研究结果对深入阐述家禽蛋壳生物矿化机制提供了重要科学资料，对今后解决家禽劣质蛋壳发生问题奠定了基础。