Liu J, Sun Y H, Wang Y W, Wang N, Pei D S, Wang Y P, Hu W, Zhu Z Y
State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Luojiashan, Wuhan 430072, China.
Theriogenology. 2008 Dec;70(9):1525-35. doi: 10.1016/j.theriogenology.2008.07.001. Epub 2008 Aug 9.
The crosstalk between naive nucleus and maternal factors deposited in egg cytoplasm before zygotic genome activation is crucial for early development. In this study, we utilized two laboratory fishes, zebrafish (Danio rerio) and Chinese rare minnow (Gobiocypris rarus), to obtain mutual crossbred embryos and examine the interaction between nucleus and egg cytoplasm from different species. Although these two types of crossbred embryos originated from common nuclei, various developmental capacities were gained due to different origins of the egg cytoplasm. Using cDNA amplified fragment length polymorphism (cDNA-AFLP), we compared transcript profiles between the mutual crossbred embryos at two developmental stages (50%- and 90%-epiboly). Three thousand cDNA fragments were generated in four cDNA pools with 64 primer combinations. All differentially displayed transcript-derived fragments (TDFs) were screened by dot blot hybridization, and the selected sequences were further analyzed by semi-quantitative RT-PCR and quantitative real-time RT-PCR. Compared with ZR embryos, 12 genes were up-regulated and 12 were down-regulated in RZ embryos. The gene fragments were sequenced and subjected to BLASTN analysis. The sequences encoded various proteins which functioned at various levels of proliferation, growth, and development. One gene (ZR6), dramatically down-regulated in RZ embryos, was chosen for loss-of-function study; the knockdown of ZR6 gave rise to the phenotype resembling that of RZ embryos.
在合子基因组激活之前，幼稚细胞核与卵细胞质中储存的母体因子之间的相互作用对于早期发育至关重要。在本研究中，我们利用两种实验鱼类，斑马鱼（Danio rerio）和稀有鮈鲫（Gobiocypris rarus），获得相互杂交的胚胎，并研究来自不同物种的细胞核与卵细胞质之间的相互作用。尽管这两种类型的杂交胚胎起源于共同的细胞核，但由于卵细胞质的不同来源，它们获得了不同的发育能力。利用cDNA扩增片段长度多态性（cDNA-AFLP）技术，我们比较了两个发育阶段（50%-和90%-外包期）相互杂交胚胎之间的转录谱。使用64种引物组合在四个cDNA池中产生了3000个cDNA片段。通过斑点杂交筛选所有差异显示的转录本衍生片段（TDF），并通过半定量RT-PCR和定量实时RT-PCR对所选序列进行进一步分析。与ZR胚胎相比，RZ胚胎中有12个基因上调，12个基因下调。对这些基因片段进行测序并进行BLASTN分析。这些序列编码了在增殖、生长和发育的各个水平上发挥作用的各种蛋白质。选择一个在RZ胚胎中显著下调的基因（ZR6）进行功能丧失研究；敲低ZR6会产生类似于RZ胚胎的表型。
