piwi调控青鳉生殖细胞和眼睛发育的机理研究

RNA binding protein Piwi is essential for stem cell maintenance, primordial germ cell (PGC) specification, germ stem cell division and differentiation in diverse organisms. Our previous results showed that medaka piwi RNA was present throughout cleavages stages and widely distributed to every cell until the blastula stage, and concentrated in PGCs, brain and eye during somitogenesis stage; piwi knock down by morpholino (MO) reduced the PGC number and affected PGC migration. This project will continue to study the expression pattern of piwi RNA and protein by fluorescent in situ hybridization and immunohistochemistry. We will further investigate the role of piwi in embryonic PGC and eye development by MO knock down and RNA overexpression. In addition, we will establish piwi null mutant by making use of activator-like effector nucleases (TALENs) mediated genome editing to study the function of piwi in adult germ cells and eye development. Furthermore, we will isolate piwi interacting RNAs (piRNA). Moreover，pull down and immuno-precipitation will be used to screen and identify genes that specifically interact with piwi and the mechanism involved. This study will enhance the understanding of the function and mechanism of piwi in regulating germ cells and eye development, and the establishment of TALENs technique with genome editing will open a new avenue for fish gene function study and the production of food fish with new traits such as virus resistance.

Piwi是RNA结合蛋白，在动物的干细胞特性维持、原始生殖细胞（PGC)形成、生殖细胞增殖和分化等方面起重要作用。前期研究表明，青鳉piwi RNA 分布于囊胚期前的每个细胞中，体节期后则定位于PGC、眼和脑；敲降piwi基因影响PGC的增殖和迁移。本研究拟通过RNA荧光原位杂交和免疫组化研究piwi在胚胎和成体的表达模式；通过RNA过表达和敲降研究piwi在胚胎PGC和眼睛发育中的功能；通过 TALENs技术获得piwi敲除的突变体，研究 piwi在成体生殖细胞和眼睛发育中的功能。分析与piwi结合的小RNA（piRNA）；还将通过pull down和免疫共沉淀等技术手段筛选与Piwi蛋白相互作用的基因，揭示Piwi与基因互作的机理。本项目将揭示piwi在生殖细胞和眼睛发育中的功能和作用机理；同时，TALENs技术的建立将为鱼类基因功能研究、遗传育种如抗病品种的培育开拓新的途径。

Piwi是RNA 结合蛋白，在动物的干细胞特性维持、原始生殖细胞（PGC)形成、生殖细胞增殖和分化等方面起重要作用。本项目在青鳉中研究了piwi和其它生殖发育相关基因的表达模式和功能：分析了Piwi蛋白的在胚胎发育、成体组织的表达模式；证实了piwi 3’UTR对PGC能有效标记；验证了过表达全长或缺失功能区的正负显性RNA对PGC发育的影响；构建了高效的CRISP/Cas9大片段基因敲除技术平台，在F0突变体中PGC数目大大减少，甚至完全缺失，在F0就能获得2个位点敲除的纯合体，其突变纯合体性腺大大萎缩、退化；目前Piwi调控生殖发育的分子机制还在进行中。我们还发现母源性dazl基因影响PGC的形成；而dnd基因除了影响PGC形成外，过表达dnd则导致PGC数目大大增加；以敲降dnd缺失PGC而不育的囊胚胚胎为受体，以过表达dnd的囊胚期细胞为供体，实现了囊胚细胞移植的高效的借腹怀胎技术。此外我们对性腺发育相关的miRNA和igf1进行了鉴定和表达验证。这些研究将为经济鱼类的相关研究奠定基础。