鹅就巢期卵巢机能退化相关miRNA鉴定及其作用机理解析

The industrial production of geese has been considerably constrained by their relatively poor egg-laying performance. Broodiness, a unique feature of birds including geese, has a major impact on their egg-laying performance. Until now, the molecular mechanism underlying the selection of ovarian follicles is not known in mammals or birds. However, involvement of miRNAs in the regulation of ovarian function has recently been indicated in some species (cattle, swine, mice and chicken). The broodiness in geese biologically features follicle atrophy. Therefore, we propose the use of deep-sequencing by Solexa to systematically investigate the miRNA profiles in ovarian cortex and follicles at the different times of geese broodiness. The differential spatial and (or) temporal expression patterns of miRNAs will be revealed by data mining. Cell lines (theca of follicles and granular cells) will be established and in vivo analysis of prospective miRNAs in those cell lines will be performed using hit miRNA mimics and antagomirs technologies. The following analyses based on quantitative PCR and immunoblotting tests will be done in order to identify target mRNA(s) in the miRNA-mediated regulatory pathway, and therefore the regulatory factor(s) involved in the follicle atrophy during the broodiness. Finally, the function of identified target mRNA(s) will be further validated in individual level. The purpose of this project is to explore the mechanisms whereby the programmed follicle atrophy occurs based on the analyses from the miRNA mediated post-transcriptional levels. This will finally improve our understanding of regulatory network related to broodiness and the development of ovarian follicles and lay a scientific basis for the improvement of egg production performance.

鹅产蛋性能较低，成为制约产业发展的关键。就巢性是禽类特有的习性，是影响产蛋性能的主要原因之一。但迄今为止，国内外对禽类和哺乳动物卵泡选择的分子调控机制尚不清楚。本项目利用鹅就巢期卵泡萎缩的生物学特征，通过Solexa深度测序，研究鹅就巢行为的不同时期与卵泡萎缩直接相关的卵巢皮质和卵泡的miRNAs表达谱，进行差异表达分析；建立卵泡膜细胞和颗粒细胞培养体系，转入hit miRNA mimics和antagomirs，利用qPCR和免疫印迹技术，在细胞水平对hit mRNA功能及其信号通路进行分析，获得就巢期与鹅卵泡萎缩密切相关的调控因子，并在鹅活体水平验证其功能。研究目标是从miRNA介导的基因转录后调控水平探讨鹅就巢期卵泡程序性萎缩的调控机理，为最终阐明就巢性和卵泡发育的调控机理，以及通过调控手段或分子育种途径改良鹅的产蛋性能提供研究基础。

鹅产蛋性能较低，成为制约产业发展的关键。就巢性是禽类特有的习性，是影响产蛋性能的主要原因之一。但目前国内外对禽类和哺乳动物卵泡选择的分子调控机制尚不清楚。本项目利用鹅就巢期卵泡萎缩的生物学特征，研究鹅就巢行为产生过程中卵泡萎缩不同时期差异表达miRNAs及其靶基因，并进行表达水平的关联分析，同时在细胞水平进行差异表达基因的功能验证，解析与卵泡萎缩密切相关的调控因子和信号通路。项目执行期主要开展了以下三方面研究。（1）就巢鹅的判定标准制定：采集及鉴定产蛋期和就巢期的卵泡样品，根据血清雌二醇、孕酮和催乳素建立了激素判定鹅就巢行为标准。（2）自噬对卵泡发育和闭锁的调控作用：通过电镜发现就巢期鹅卵泡内自噬体增加；QPCR和Western blot分析自噬标记蛋白表明，就巢与自噬直接存在密切的联系；发现鹅卵泡自噬主要发生在大白卵泡和小黄卵泡阶段，推测自噬的过量发生影响了卵泡的发育，从而导致就巢鹅不能产蛋或少产蛋；并证实鹅卵泡颗粒细胞自噬主要是通过mTOR通路调节。（3）不同阶段等级前卵泡转录组和miRNA测序分析：根据标准采集不同时期的卵泡样品，针对小白卵泡、大白卵泡和小黄卵泡进行了miRNA和转录组测序，分析两个转录组解析鹅就巢的调控网络体系。联合分析发现23个miRNAs在就巢期鹅卵泡上调，21个miRNAs下调，GO和KEGG分析揭示这些miRNA可能与细胞骨架、细胞连接以及与癌症通路相关。而转录组差异mRNA的GO注释和KEGG分析表明，差异基因主要与激素、细胞联系、细胞骨架、癌症通路以及吞噬小泡的形成密切相关，也进一步证明自噬的发生与抱窝密切关联。根据上述生理生化和分子生物学的分析结果，初步推测鹅的抱窝主要与卵泡的自噬和凋亡相关，在自噬与凋亡相关蛋白的一系列作用下，miRNAs和基因相互调控，最终影响到鹅卵泡的发育和命运，从而改变鹅的生理状态，是鹅抱窝和产蛋的重要调节因素。以上研究结果对最终阐明鹅就巢性和卵泡发育的调控机理，以及通过调控手段或分子育种途径改良鹅的产蛋性能提供了研究基础。