天然三倍体鲫鱼萍乡红鲫精卵识别相关因子SP56-like与ZP3功能互作研究

A form reproduction in which the egg can inhibit heterologous sperm from transforming into male pronucleus so that ontogenesis is completely controlled by the female nucleus is thought as gynogenesis. A red-transparent population, distributed in Pingxiang of Jiangxi Province(termed as Pingxiang Red crucian carp), was identified to be natural triploid Carassius auratus by our group.The species exists a bisexual population, female outnumbering male by about six to one in their natural habits,and was also demonstrated that it has two different reproductive development modes including bisexual reproduction involving the fusion of male and female gametes and gynogenesis reproduction by means of artificial propagation experiments in our previous study. In comparision with naturally gynogenetic silver crucian carp, no evidences have not yet for the present been found the obvious allogynogenetic biological effects in growth, rate of fertilization and other aspects exist in Pingxiang Red crucian carp, which suggested that this red-transparent triploid Carassius auratus possesses more complicated molecular mechanism and regulative pathway in the reproduction and development..To better understanding the special genetic mechanism in Pingxiang Red crucian carp, in this study, the homologous genes to sperm protein 56(SP56-like), known as the receptor of zona pellucida 3(ZP3) involving in the recognition, combination and penetration of sperms and oocytes in mammal would be cloned from Pingxiang Red crucian carp, and their spatio-temporal expression would be also analyzed.In addition, using Co-Immunoprecipitation and competitive receptors experiment, we would investigate from in vivo and in vitro studies that whether SP56-like interacts with ZP3, if ture, then whether the interaction affects the recognition of sperms and oocytes and gynogenesis on the foundation of our previous work that the ZP3 gene has already been cloned from Pingxiang Red crucian carp and its polyclonal antibody to ZP3 has also been prepared. The study would contribute to exploring and revealing the molecular mechanism of gynogenesis in naturally gynogenetic red-transparent triploid Carassius auratus var. Pingxiangnensis.

雌核发育是卵子在精子的刺激下，依靠自身的细胞核发育成个体的一种有性生殖方式，精子仅起到刺激卵核发育并不参与卵核融合。萍乡红鲫是江西萍乡地区发现的一种天然的野生型三倍体鲫鱼突变体，是行雌核发育和两性生殖的两性型种群，且种群内暂未发现异精效应，与银鲫不尽相同，暗示着该鲫鱼突变体具有较复杂的生殖调控机制。为进一步了解其生殖遗传机制，本研究结合文献报道在哺乳动物中SP56作为卵透明带蛋白ZP3的受体参与精卵识别的基础上，将从萍乡红鲫中克隆其ZP3的受体SP56-like基因，分析其时空表达特征；并结合已有的工作基础（已克隆到ZP3基因并获得其抗体），采用免疫共沉淀技术和竞争受体试验从体外和体内两方面研究萍乡红鲫SP56-like是否与ZP3相互作用，以及它们与受精识别和雌核发育的关系。其研究对于探索和揭示萍乡红鲫受精生殖分子调控机制具有重要意义。

萍乡红鲫是行雌核发育和两性生殖的两性型种群，为进一步了解其生殖遗传机制，本研究克隆了萍乡红鲫ZP3(JN669426)和ZP2（JX984601）基因的cDNA全长。ZP3和ZP2基因在性腺组织中的表达，显著高于其他组织。ZP2和ZP3基因具有相同的表达模式，在4-8月龄的卵巢中表达量显著增加，在9-12月龄的卵巢中表达量显著下降。以ZP2基因RNA序列设计杂交探针，强的杂交信号在早期发育的卵母细胞中被检测到，很弱的杂交信号在晚期发育的卵母细胞中被检测到。原核表达并纯化ZP3蛋白，将得到的ZP3蛋白免疫家兔并得到多克隆抗体。利用免疫荧光技术检测，在I和II时相卵母细胞中，没有明显的荧光信号被检测到。荧光信号在III时相的卵母细胞中被检测到，且随着卵母细胞发育进入IV时相，荧光信号逐渐加强。步移获得三个ZP基因DNA序列。三个ZP基因外显子和内含子的基因结构是完全相同的，都包含8个外显子。剥取卵母细胞的卵壳，提取卵壳蛋白。SDS-PAGE胶检测显示，10条特异性的蛋白条带在35-170KD之间。根据分子量的大小推测，在40-70KD之间的4条蛋白可能是ZP蛋白。获得一段新奇的启动子序列，该序列利用顺反方向上不同的调控元件，启动了CaP_ZP2和CaP_ZP3.2基因的表达。对卵母细胞内，可能与ZP蛋白有相互作用的Hira蛋白进行研究。克隆得到Hira(HMO67834)基因cDNA序列全长。Hira基因在卵巢中大量表达,在其余组织中存在少量表达或是不表达。Hira基因在卵巢组织中表达的周年内总体表现为上升趋势,出苗后1～5月龄表达量较小,在第6月龄突然增加,之后急剧减少。第7月龄～第10月龄,Hira基因的表达量相对第6月龄时表现为急剧减少趋势。第11月龄时又突然增加到全年中表达量最大值,之后在第12月龄时又有减少情况。原核表达并纯化Hira蛋白，将获得的Hira蛋白作为抗原，获得兔抗抗体。Elisa结果显示获得了较高效价的兔抗体。该抗体可用于后续的蛋白互作实验。最后，比对常见物种的SP56基因序列，设计简并引物，对萍乡红鲫的SP56基因进行筛选和克隆，目前为止获得部分SP56基因序列，以上的研究为进一步研究萍乡红鲫生殖遗传机制奠定基础。