短蛸原生殖细胞的迁移图式及分子基础研究

Ontogenesis of germ cells is an important issue in developmental biology. The leaing studies in model animals are revealing the molecular mechanisms of germ cell origin and development, some functionally conserved genes through evolution have been discovered. These genes might be useful as markers for an extensive investigation on germ cells development of non-model animals. The most extensively studied of these functionally conserved genes is vasa, which is a broadly conserved DEAD-box RNA helicase associated with germ line development and is expressed in multipotent cells in many animals. Octopus ocellatus is a new important economic species in north part of China, however, report on the development of germ line cells and the occurrence of gonad on O. ocellatus ever cephalopod is seldom..In this project, cDNAs of reproduction-related gene of O. ocellatus vasa-like gene (Oovlg) of DEAD-box family will be isolated, using a homologous cloning strategy and applying rapid-amplification of cDNA ends (RACE) technology, followed by analysis of characterization and spatio-temporal expression. Whole-mount in situ RNA hybridization and immunohistochemistry will be used in which Oovlg mRNA and Anti-Oovlg antibodies as markers is to establish the early developmental pattern of O. ocellatus primordial germ cells (PGCs) including the model and timing of PGCs origin and the migration in early embryo development. Localized Oovlg protein expression within the embryo at some point during development is a common feature in all animals studied and it appears that animals accomplish this by utilizing several independent regulatory mechanisms, such as, post-transcriptional. Selective Vasa proteolysis may contribute to the formation of PGCs, in this progress the ubiquitin modi?cation is an important factor. In this project microinjection and RNA interruption technologies will be applied to analysis how the ubiquitin gustavus gene interact with Oovlg gene. Finally, formation of gonad anlage during embryonic development and PGCs migration pattern as well as gonad formation during larva to mature process will be studied. The results of this project will provide important theoretical foundations on O. ocellatus, including PGCs original pattern, differentiation of germ line cells, occurrence of gonad, sexual determination and reproductive regulation etc.

模式动物生殖细胞特异性功能保守基因，诸如vasa基因的发现为大量研究非模式动物配子发生和原生殖细胞(PGCs)起源、迁移、分化提供了条件。短蛸是我国北部沿海蛸类中新兴的重要的经济种之一。然而，在国内外迄今尚未见对短蛸甚至是对头足类生殖系起源和性腺发生的研究报道。本研究采用同源克隆策略和cDNA末端快速扩增(RACE)技术，克隆获得短蛸vasa-like基因(Oovlg)的cDNA，对其进行时空表达分析；并以Oovlg mRNA和抗体蛋白为标记，通过整体原位杂交技术研究PGCs起源的方式和时间；以及通过显微注射和RNA干扰技术研究泛素蛋白gustavus基因对Oovlg基因的调控机制；探索短蛸胚胎发育过程中性腺原基的发育过程和幼体生长过程中PGCs迁移路线、性腺形成的机制。研究结果将为短蛸PGCs的起源模式、生殖细胞分化、生殖腺的发生、性别决定以及生殖调控等提供重要的理论。

（1）本项目通过构建了短蛸cDNA文库，利用分子生物学和生物信息学手段进行了EST序列测定和序列分析，获得了包括DDX、PL10和VASA-like在内的生殖相关基因20余条；利用实时定量PCR技术检测了DDX、PL10和VASA-like基因在健康短蛸各个组织中的表达，以及其在短蛸胚胎发育各个阶段的表达。研究发现：①DDX和VASA-like基因分别在雄性和雌性性腺中表达量最高，而PL10在鳃心中表达量最高；②DDX基因在受精卵发育的前15天表达量较高，第19-29天表达量低，第33天升高至较高水平；③PL10基因在前15天表达量低，第19天开始升高，一直到出膜前都保持高水平；④VASA-like基因在胚胎发育的前9天表达较低，至15天时表达量稍有增加，其后除在第25和33天降低外，一直保持较高的表达量。（2）本项目采用生物统计学的方法研究了短蛸发育的生物学零度、有效积温、胚胎发育的温度系数和胚胎发育时期与生长积温的相关性，受精卵发育的生物学零度为5.15℃，受精卵发育到初孵幼体的有效积温为589.45℃•d，胚胎发育最适温度范围为19.46-23.06 ℃，胚胎发育阶段所处时期和发育生长积温呈线性函数关系：K = 26.72Ts + 55.34 (R2 = 0.996)。（3）分析了短蛸体重、胴长、性腺指数和生殖系统参数的周年变化。利用组织学方法研究了短蛸性腺发育过程，确定了短蛸卵巢和精巢形成过程及其发育分期，总结出短蛸性腺发育的周年变化和发育阶段包含的细胞类型；将短蛸的性腺发育过程分成5个阶段，并阐述了每个发育分期的特征。研究结果为短蛸生殖腺的发生、性别决定以及生殖调控等提供了重要的理论依据。