斑马鱼和稀有鲫的SCAR标记建立及其异种克隆胚的检测

Identification and application of SCAR markers in detecting cross-species cloned embryos between zebrafish and rare minnow

以斑马鱼和稀有（鱼句）鲫的基因组DNA进行RAPD分析,获得特异的RAPD标记,经克隆、测序,合成特异性引物并优化SCAR-PCR反应条件,将四个特异的RAPD标记转化成稳定的SCAR标记.SCAR1、SCAR3和SCAR2、SCAR4分别在斑马鱼与稀有（鱼句）鲫基因组DNA扩增中产生单一的条带,可作为区分斑马鱼和稀有（鱼句）鲫的分子标记.此外,SCAR3和SCAR4还可以分别将斑马鱼和稀有（鱼句）鲫从鲫鱼、白鲢、鲤鱼、鳊鱼等其它鱼类中鉴别出来,从而成为良好的种质鉴定分子标记.在此基础上,选用SCAR标记进行了斑马鱼与稀有（鱼句）鲫配合的鱼类异种克隆胚胎鉴定研究,结果发现,克隆胚由供体核支持发育而来.SCAR标记的获得,为鱼类异种间克隆胚胎和克隆鱼的检验,以及细胞核再程序化机制等重大理论问题的探索提供了重要的技术支持.

A randomly amplified polymorphic DNA （RAPD） analysis was used to identify the zebmfish （Danio rerio ） and rare minnow （ Gobiocypris rarus ）. Four specific RAPD markers were identified, cloned and sequenced. According to the nucleotide sequence, four pairs of primers in sequence characterized amplified regions （SCAR） were designed and synthesized to distinguish the specific fragments from either zebrafish or rare minnow. Then, RAPD markers were successfully converted into SCAR markers. Additionally, there were two SCAR markers, which could distinguish either zebrafish or rare minnow from Carassius auratus, Hypophthalmichthys molitrix, Cyprinus carpio, and Megalobrama amblycephala. Subsequently, crosss-species cloned embryos derived from zebrafish embryonic nuclei and rare minnow enucleated eggs or from rare minnow embryonic nuclei and zebrafish enucleated eggs were screened with such SCAR markers. The results showed that the SCAR patterns of cloned embryos were consistent with those of the nuclear donor species. The SCAR marker offers a powerful approach to distinguish the cloned embryos and cloned fish from different species. Furthermore, it will be helpful to study of the nuclear- reprogramming mechanisms in fish.