一种新的斑马鱼cloche突变体亚型的基因鉴定

Positional cloning of a novel allele of zebrafish cloche mutant

目的斑马鱼cloche172突变体的基因鉴定。方法采用化学诱变剂N-乙基-N-亚硝基脲(N-ethyl-N-nitrosourea,ENU)诱变雄性野生型AB斑马鱼,实施大规模正向遗传学方法筛选髓系细胞标记物lysozymeC(lyC)表达缺失突变体,并对其中一个名为cloche172突变体进行大体形态学观察,基因定位克隆和遗传学互补实验。结果大规模正向遗传学筛选出4个lyc表达缺失突变体,其中一个cloche172突变体受精后3d(day post fertilization,dpf)时期大体形态改变与已知但基因不明的cloche突变体相似。遗传学互补实验观察到有1/4胚胎出现类似cloche突变体的表型。同时,cloche172突变体基因定位在13号染色体近端粒的区域,与cloche突变体基因定位区域一致。而o-dianisdine实验证实cloche172突变体有较多红细胞聚集,cloche突变体仅有少量在尾部存在。结论cloche172基因与cloche基因定位一致,cloche172突变体是一个新的点突变位点的cloche突变体。

Objective To perform the genetic idencification of cloche172 mutant zebrafish. Methods The chemical mutagen N-ethyl-N-nitrosourea （ENU） was used to treat the AB stain male fish. Large-scale forward genetic screening was carried out to search for lyC-deficient zebrafish mutant by WISH. The morphology changes of the embryos at 3 days postfertilization （3dpf） stage were observed and the cloche172 gene was identified by mapping and complementation test. Results We selected 4 lyC-deficient zebrafish by WISH. cloche172 mutant showed morphological changes similar to cloche mutant in 3dpf stage. One fourth of the embryos showed cloche phenotype as found in complementation test, and the cloche172 gene was mapped on the telomere of zebrafish 13 chromosome where cloche gene was located. Numerous red blood cells were observed in the cloche172 mutant, while only a few cells were found in the cloche mutant in the tail region by o-dianisdine staining. Conclusion cloche172 gene which is responsible for the phenotype of cloche mutant may be a novel point mutation allele of the cloche mutant.