三疣梭子蟹精子塞形成的关键蛋白鉴定及其功能初探

Sperm plugs that male place onto the female genital tract during mating, are a gel-like coagulation. Generally, they are explained as a consequence of male securing paternity and many studies have demonstrated that sperm plugs also have some relationship with species' reproductive success. The swimming crab, Portunus trituberculatus, one of the most valuable marine crustacean species in China, appears sperm plug as well after mating but its precise formation mechanism remains unclear. Combining with the constructed mating male and female individual reproductive system transcriptome libraries, we will first employ Labe-free quantitative proteome sequencing technology in this study to compare and analyze the different proteins presented in the upper and lower parts of sperm plug. Meantime, we continue to identify the possible key proteins that may be involved in sperm plug formation, clone their corresponding entire cDNA sequences and assess the potential formation mechanism through sequence analyses. Then, use real-time quantitative PCR to detect their expression and secretory sites in both male and female reproductive systems; purify these functional proteins and examine the formation mechanism on the method of artificial simulation of sperm plug by mixing one purified protein with the tissue homogenate fluid containing the other functional protein in sequence. If get the artificial sperm plug, we further prepare the antibodies and finally determine them following Western-blot test in order to preliminarily validate the functions of the key proteins in the process of sperm plug formation. Therefore, our study would reveal the significance of sperm plugs in crustacean, deepen the understanding of the process of crab fertilization and provide a sound theorecal base to the subsequent potential work relative to artificial insemination and genetic breeding of P. trituberculatus.

精子塞是动物交配过程中精液在雌性生殖道内生成的一种胶状凝聚物，除了保证父权，大量研究证实精子塞还跟动物的生殖成功相关。三疣梭子蟹（Portunus trituberculatus）是我国重要海产经济蟹类，生殖过程也产生精子塞，但形成机制尚不明确。本项目拟利用Label-free技术，结合已构建的交配个体雌雄生殖系统转录组文库，通过分析精子塞上下部分的蛋白组分，鉴定精子塞形成的关键蛋白；克隆对应基因的cDNA全长，分析潜在机制，并用实时定量PCR考察其在雌雄生殖系统的分泌部位；纯化其中作用蛋白，并与含有底物蛋白的组织匀浆液开展人工模拟精子塞实验；最后，对形成精子塞的组分，制备相应抗体，Western-blot检测，初步验证关键蛋白在精子塞形成中的功能作用。本项目预期成果将有助于揭示甲壳动物精子塞存在的意义，加深对蟹类受精过程的理解，并可为实现三疣梭子蟹的人工授精及遗传育种提供坚实的理论支撑。

精子塞存在于动物多个类群，是动物交配过程中精液在雌性生殖道内生成的一种胶状凝聚物，除了保证父权，大量研究证实精子塞还跟动物的生殖成功相关。三疣梭子蟹（Portunus&nbsp trituberculatus）生殖过程也产生精子塞，但形成机制尚不明确。. 本研究历时3年，主要取得进展、重要结果、关键数据如下：.1）通过室内配对实验，进一步明确了三疣梭子蟹的生殖蜕壳交配规律，可为后续组织样本的获取提供保障，并为开展生殖交配时期的试验奠定基础；.2）利用Illumina HiSeqTM 2500 测序平台，成功构建并公布了三疣梭子蟹交配个体雌雄生殖系统的转录组文库； .3）利用Label free技术，首次对精子塞上下两部分进行了蛋白成分鉴定，结合上面的转录组文库，成功发掘出一批候选功能蛋白基因，可为后续开展类似卵巢发育，精荚裂解及本研究重点关注问题精子塞形成机制奠定重要的分子基础；.4）通过人工模拟精子塞实验及后续的精子塞组化分析，证实了三疣梭子蟹精子塞上下两部分的形成机制不一致，纳精囊分泌物应该参与了精子塞上部分的形成，这可能是与前输精管分泌物共同作用的结果，且相比于上部分，精子塞下部分形成速度相对较慢，推测后者主要由后输精管精液组成；. 三疣梭子蟹是我国重要经济蟹类，对三疣梭子蟹独特的交配习性的研究有助于揭示甲壳动物精子塞存在的意义，进一步加深对蟹类受精过程的理解，并可为资源保护，后续开展的有关三疣梭子蟹人工授精、遗传育种等跟生产密切相关的工作提供必要的理论支撑。