【作者】 许燕；

【导师】 邓凤姣； 程汉华；

【作者基本信息】 武汉大学 ， 细胞生物学， 2010， 博士

【摘要】 原始生殖细胞（Primordial germ cells, PGCs）是多细胞生物体中生殖细胞的祖先,同时也是研究细胞迁移的一个重要的模型。在果蝇、线虫和小鼠等物种中,nanos在PGCs迁移过程中发挥作用,干涉该基因后,PGCs的迁移不正常。尽管Nanos蛋白在脊椎动物和无脊椎动物的PGCs的迁移中起着举足轻重的作用,然而在斑马鱼中,Nanos如何调节PCGs的迁移的分子机制目前还不清楚。为了探讨这一问题,我们以全长的Nanos为诱饵蛋白,通过酵母双杂交系统筛选了一个新的与Nanos相互作用的蛋白,即肌球蛋白轻链2（Myosin light chain II, Mylz2）; Nanos的锌指结构域和C末端的14个氨基酸残基是Nanos-Mylz2相互作用所必需,缺少这两个部分中的任何一个,Nanos都不能与Mylz2相互作用。采用免疫共沉淀和GST pull-down方法,在体内、外证明了Nanos蛋白与Mylz2蛋白能形成一个稳定的复合体。用Mylz2磷酸化的抗体检测,进一步证明了Nanos在人的293T细胞中下调Mylz2蛋白的磷酸化。结果分析Nanos可能通过调控Mylz2的磷酸化来调节斑马鱼PGCs的发育和迁移。Pumilio和Nanos相互作用最早在果蝇中发现,其功能是调控胚胎的背、腹轴形成。随后研究同样发现,人的Pumilio2与Nanos的同源蛋白Nanos 1相互作用,在生殖细胞的发育和维持中发挥作用。在斑马鱼中,pumilio2的研究未见报道。本文通过序列比对,在斑马鱼EST数据库中,发现了一个与pumilio2同源的EST序列。采用3’和5’末端快速扩增cDNA （RACE）方法,从斑马鱼卵巢中获得了该基因的cDNA;通过测序和分析,该基因与pumilio2同源,因此命名为pum-like。斑马鱼pum-like的cDNA含有3861个碱基,编码1106个氨基酸,通过与斑马鱼基因组数据库比对,发现其定位于第25号染色体上,含有21个外显子和20个内含子。同时将斑马鱼pum-like与人、果蝇等物种pumilio基因进行比对构建进化树,结果表明,pum-like与其他物种pumilio2的同源性高于pumiliol。Pum-like的C端含有pumilio基因家族的5个PUF （Pumilio-family RNA binding repeat）结构域。免疫组织化学显示,Pum-like在卵母细胞中进行表达。整胚原位杂交结果证明,pum-like mRNA在2细胞和4细胞的分裂沟中表达,随着胚胎发育,其mRNA主要集中在脑和神经管中。因此,我们推测pum-like可能参与早期胚胎的细胞分裂,并与神经系统的发育和形成有关。卵黄膜是位于卵母细胞外的一层膜结构,在动物的受精和胚胎发育过程中起着十分重要的作用。通过NCBI的数据库和电子差减杂交的方法,在GeneBank斑马鱼卵巢的cDNA文库中获得一条EST序列（XM₀01340234.1）；利用其EST序列,进行3’、5’RACE,获得该基因的cDNA全长；通过PCR和测序验证该基因的cDNA全长为2720bp,它编码761个氨基酸。通过蛋白比对,发现该蛋白与已知的卵黄膜蛋白没有同源性,因此我们认为它是一个新的卵黄膜蛋白,命名为斑马鱼卵黄膜蛋白zvep （zebrafish vitelline envelope protein）。通过RT-PCR和western blot检测,zvep在卵巢和脑中表达,在其他组织中不表达。采用组织原位杂交和免疫组织化学证明zvep mRNA在卵母细胞的Ⅰ期,Ⅱ期,Ⅲ期转录,蛋白在Ⅲ期卵母细胞的卵黄膜上特异性的表达（阳性信号为2条细线）。以上结果表明,ZVEP在早期卵母细胞中合成,是卵黄膜中的一个新成员,在卵黄膜形成过程中发挥了重要作用。更多还原

【Abstract】 Primordial germ cells （PGCs） are the progenitors of reproductive cells in metazoans and are an important model for the study of cell migration in vivo. Previous reports have suggested that the protein Nanos plays critical roles in the migration and survival of PGCs in vertebrates and invertebrates. The molecular mechanisms by which Nanos governs PGCs migration are largely unknown. In this study, we report a novel interaction between zebrafish Nanos and myosin light chainⅡ（Mylz2）. This stable complex, which is formed through the RNA-binding zinc finger domain and fourteen amino acids at the C-terminus of Nanos, was confirmed both in vitro and in vivo. Both of these two parts are required for the interaction with Mylz2, but nity-one amio acids at N-terminus of Nanos are not required. Using a phospho-myosin light chain 2 （Serl9） antibody, we demonstrate that Nanos regulate the phosphorylation of the Mylz2. We discuss the biological roles of this Nanos-regulated phosphorylation of the Mylz2 in PGCs and soma. Based on these data, we suggest that the interaction between Nanos and Mylz2 and the Nanos regulated phosphorylation of the Mylz2 may play an essential role in germ cell development and PGC migration in zebrafish.NOS1 interacting with Pumilio2 has been found in drosophila and human. Using the zebrafish EST database from NCBI, we found an EST which has more homology to pumilio2. The full-length cDNA of this transcript was obtained by 3’and 5’ RACE and further confirmed by PCR and sequencing, and it was named pum-like. The full-length cDNA of this gene is 3861bp and encodes a protein of 1106 amino acids, which locating at the twenty-fifth chromosome and containing twenty-one extrons and twenty introns. It shows much more homology to pumilio2 than pumiliol by phylogenetic construction, and contains five PUF repeats at the C-terminal. RT-PCR and western blot analysis showed that pum-like’s mRNA transcribed at the early stage of oocyte, and its protein located in the cytoplasm of oocyte which may be a component of the yolk. Whole-mount in situ hybridization at the 2-cell and 4-cell stage of embryos revealed the remarkable signals in the cleavage furrows which formed distinct dots. It suggested that the gene regulated the cell divisions at the early stage of embryo. Along with the development of the embryo, its expression was found mainly in caudal spinal chord and brain. So we guessed it had some functions in the nervous system.Egg envelope is a specialized extracellular matrix that surrounds and protects the oocyte and plays significant roles in animal reproductive and developmental processes. Using the NCBI digital differential display program we identified an EST sequence （XM₀01340234.1） acquired from zebrafish ovary cDNA libraries in GeneBank. The full-length cDNA of this transcript was obtained by 3’and 5’ RACE and further confirmed by PCR and sequencing. The full-length cDNA of the novel gene is 2720bp and encodes a protein of 761 amino acids. RT-PCR and western blot analysis showed its expression in ovary and brain but not in other tissues. In situ hybridization demonstrates that the mRNA is transcribed in ooplasm of stage I, II and III oocytes. Interestingly, immunohistochemistry on zebrafish ovarian sections showed that protein expression in the vitelline envelope was located to two thin positive lines in the stage III oocytes. These ovarian expression patterns show that this is a new component of the vitelline envelope that is synthesized during early developing oocytes. This protein was named ZVEP （zebrafish vitelline envelope protein） and it did not have any homology with other known vitelline envelope genes. Thus, we found that zvep is a novel gene related to the vitelline envelope in zebrafish.更多还原