鸡SLIT/ROBO信号通路在前等级卵泡发育中的作用及调节机制

Egg production is the most important economic traits for chicken breeder farms; the production performances are significantly divergent between higher-yield layers and lower-yield layers during their reproductive phases. The remarkable differentiations between the higher and lower layers tightly depend upon the cyclic process of ovarian prehierarchal follicle development, follicle recruitment and maturation. More recent studies have revealed that SLIT/ROBO signaling pathway plays a pivotal role in regulating follicular development. However, the molecular mechanisms of the pathway acting on the cycles remain poorly understood now. Our recent studies and some literatures have indicated that the ligands and their receptors (Slit2、Slit3 and Robo1、Rig-2, et al.) of the Slit/Robo system seem to play an important role in the development of prehierarchal follicles and the recruitment of dominant follicle selection, and the functional genes Slit2、Slit3、 Robo1 and Rig-2are potential candidates associated with laying performance in hen. In this work, following the biological informatics analysis of the candidate genes cloned, the technique of immunohistochemistry is used to localise the ligand Slits and its receptor Robos in ovarian follicles and in situ hybridization is employed to investigate the mRNA expression patterns of the candidate genes in the developing prehierarchal and hierarchal follicles; Northern blotting is utilized to examine the potential alternative splice variants of the Slit or Robo family; method of siRNA interference is designed to prove the functions of the target genes to govern the chicken ovarian follicular development in vitro; and RT-PCR and Real-time quantitative PCR are used to quantify expression levels of the candidate genes involved in the development of the differentiate follicles after and before the siRNA interference. Meanwhile, by imitating the biological effect of SLIT/ROBO signals on chicken prehierarchal follicle development, following TGF-beta and Neureglulin molecules are administrated to incubate the hen granulose cell relative to follicle selection, the differential expressions of the Slits or Robos genes in the cultured ovarian granulose cells are induced, changes of the mRNA expression patterns of the candidate genes detected are mainly to further confirm the different effect of SLIT/ROBO signals on the target follicle development. Taken collectively, the findings of these studies is very helpful in elucidating the role and molecular regulatory mechanism of SLIT/ROBO pathway in prehierarchal follicles development and dominant follicle selection in chicken. This work is expected to provide a novel train of thought and useful data for chicken molecular breeding to improve egg laying performance. It has a very important academic value and economic significance to carry out this study on mechanism of the regulation of follicles development involving of the SLIT/ROBO pathway in this area.

鸡的产蛋性状是一类非常重要的经济性状，在高产和低产鸡之间存在着显著差异，此差异主要决定于鸡卵巢前等级卵泡发育、募集和成熟的循环过程。近年研究表明，SLIT/ROBO信号通路在该过程中起着重要调控作用，然而，其具体作用机制目前尚不清楚。本项目拟对SLIT/ROBO信号通路可能直接参与鸡卵泡前等级化发育调节和优势卵泡选择的重要基因（配体Slit2、Slit3和受体Robo1、Rig-2等）进行克隆及生物信息学分析基础上，利用双重免疫组化、组织原位杂交、siRNA干扰和Real-time qPCR技术等，对配受体互作、前等级卵泡不同发育时段配受体基因时空表达模式和选择剪接体变异等对卵泡发育的作用进行定性和定量分析，通过模拟细胞分化调节因子（如TGF-β）等诱发卵泡颗粒细胞配受体基因生物效应的改变，解析SLIT/ROBO通路调控鸡卵泡发育的作用机制，为提高鸡产蛋性能的分子育种研究提供新思路。

鸡的产蛋性状是一类非常重要的经济性状，在高产和低产鸡之间存在着显著差异，此差异主要决定于鸡卵巢前等级卵泡发育、募集和成熟的循环过程。已有研究表明，SLIT/ROBO信号通路在该过程中起着重要调控作用，然而，其具体作用机制目前尚不清楚。本项目对SLIT/ROBO信号通路可能直接参与调控鸡卵泡前等级化发育和优势卵泡选择的相关基因（配体SLIT2、SLIT3及其受体ROBO1、ROBO2、ROBO3/Rig-1、ROBO4/Magic Robo）全长cDNA序列克隆、结构和功能等生物信息学分析基础上，综合运用免疫组织化学定位、组织原位杂交、Northern杂交、RT-PCR、Real-time qPCR检测和靶向特异性siRNA干扰技术等，重点对不同配体（SLITs）与受体（ROBOs）之间的互作关系、前等级卵泡不同发育时段配体、受体基因的时空表达模式、差异表达动态变化、选择性剪接体变异对卵泡发育的影响进行了研究；并通过模拟多功能性细胞分化调节因子（如TGF-β等）诱发卵泡颗粒细胞中主要配受体基因表达模式的改变，验证其对前等级卵泡组织细胞迁移和细胞分化、发育的神经导向调节所产生的生物学效应，揭示了SLIT/ROBO信号通路在鸡卵泡前等级化发育过程的作用及分子调控机制。此机制的阐明可为深入揭示卵泡发育规律、解析促进卵泡等级发育和优势选择的卵泡募集调控机理提供理论依据；也为发掘鸡的产蛋性状优良基因，开展以提高产蛋性能为目标的育种研究和杂种优势利用提供新的思路，具有十分重要的理论价值和经济意义。