BMP15基因调控鸡产蛋后期卵泡选择的分子机制研究

High level of laying rate at late stage could improve the utilization rate of laying hens and prolong the laying period. Follicle selection is the pivotal process for persistent ovulation in poultry. Published data surveyed that BMP15 gene was significantly associated with egg production, which also functioned in the follicle selection process. However, the regulatory mechanism of BMP15 gene on follicle selection hasn’t been elucidated yet. Our previous results demonstrated that the expression level of BMP15 presented significant difference between high and low egg production group. And a positive correlation was observed between the expression level of BMP15 and egg production. The aim of this study is going to further investigate the mechanism of avian follicle selection mediated by BMP15: (1) Identifying the upstream regulatory element and genetic markers which have effects on the transcriptional activity of BMP15 gene, accompanying with the expression pattern of BMP15 gene, further to elucidate the regulatory mechanism of BMP15 transcription in F6 follicle. (2) Depending on two conditional culture systems mediated by BMP15, validating the dynamic changes of marker phenotypes and downstream signaling pathways during follicle selection, finally to investigate the regulatory mechanism of BMP15 gene on follicle selection during the late laying stage. Above results will provide important evidence and breeding basis for improving laying rate at late stage of laying hens.

维持产蛋后期产蛋率是提高母鸡利用率和延长产蛋周期的关键，而卵泡选择是决定家禽持续排卵的重要环节。多项研究发现BMP15基因与产蛋量显著相关且参与卵泡选择过程，但BMP15基因调控卵泡选择的分子机制尚未阐明。申请人前期研究发现产蛋后期高低产蛋鸡组BMP15基因表达存在显著差异，且与产蛋量呈正相关。本项目拟在前期基础上进一步开展由BMP15基因介导的卵泡选择的研究：(1)鉴定影响BMP15基因转录活性的上游调控元件及效应遗传标记，协同BMP15基因的表达模式，研究产蛋后期F6级卵泡中BMP15基因的转录调控机制；(2)依赖两种BMP15介导的条件培养系统，分别验证卵泡选择的“标志”表型及下游信号通路的变化模式，探究BMP15基因在产蛋后期调控卵泡选择的分子机制。上述研究结果可为提高家禽产蛋后期的产蛋率提供重要的理论依据和育种基础。

家禽的产蛋性状是一类重要的经济性状，维持产蛋后期的产蛋率是提高母鸡利用率和延长产蛋周期的关键。家禽的产蛋性能除了受外界环境的因素影响，更重要的是家禽的持续排卵，而卵泡选择是决定家禽持续排卵的重要环节。骨形态发生蛋白15(bone morphogenetic protein-15, BMP15) 是TGF-β家族的重要成员，对家禽卵泡发育和选择起着重要作用。然而，目前国内外研究尚未阐明鸡BMP15基因调控卵泡选择的机制。本研究以43周龄地方鸡产蛋后期小黄卵泡为研究材料，通过转录组测序技术，筛选出差异基因。利用RACE技术克隆鸡BMP15基因全长，进行遗传结构解析；对体外培养的等级前卵泡颗粒细胞进行BMP15条件培养，探究BMP15基因调控鸡卵泡选择的作用机制；利用双荧光素酶报告技术鉴定BMP15转录活性区域，基于对变异信息的分析，筛选影响蛋鸡产蛋率的BMP15基因启动子区的效应分子标记，阐述鸡BMP15基因差异表达的转录调控机制。从而深入解析BMP15与卵泡选择的内在联系，揭示鸡BMP15基因调控鸡卵泡选择的分子机理。.(1)高低产地方鸡F6级卵泡转录组测序共筛选出687个差异表达基因，上调基因167个，下调基因520个，差异基因富集在Wnt信号通路、MAPK 信号通路、类固醇激素合成信号通路，其中BMP15基因在高产鸡的小黄卵泡中显著下调。.(2)利用 RACE 技术获得了鸡BMP15基因的全长转录本，编码350个氨基酸。基于全长转录本物理位置，分析并验证了BMP15基因核心启动子区位于-1~-153bp。组织表达结果表明和BMP15基因在卵巢组织中特异性高表达，卵巢颗粒细胞表达显著高于膜细胞。.(3) 对体外培养的颗粒细胞进行BMP15条件培养，结果显示，BMP15加速颗粒细胞聚集，上调BMPR1B、FSHR表达和胞内cAMP水平，激活Smad1信号通路，表明BMP15促进卵泡选择；将BMP15与FSH共处理，FSHR和类固醇激素合成相关基因表达上调，胞内cAMP与颗粒细胞上清中孕酮合成水平上升，说明FSH在BMP15基础上促进卵泡选择。.本试验研究了BMP15基因在鸡产蛋后期调控卵泡选择的分子机制，可为提高家禽产蛋后期的产蛋率提供重要的理论依据和育种基础。