斑马鱼小头震颤突变体mise的鉴定与基因定位

Identification and gene localization of a microcephaly and microcephaly and suspected epilepsy mutant mise in zebrafish

目的以自发突变导致的小头、震颤(microcephaly and seizure,mise)斑马鱼为模型,研究其遗传方式和突变基因定位。方法通过比较正常和突变体斑马鱼的头宽和眼球面积,鉴定突变体的小头小眼性状;通过构建家系确定突变遗传方式。对极端表型群体进行基因组重测序,随后利用分离群体分组分析(bulked segregation analysis,BSA)和竞争性等位基因特异性PCR(kompetitive allele specific PCR,KASP),一代测序等方法获得突变基因定位。结果与野生型相比,突变体mise受精后3 d(3 days post fertilization,3 dpf)表现为小头小眼震颤表型。经基因定位及验证明确与表型相关的突变基因为terfa。结论本研究通过全基因组重测序结合连锁分析的方法快速定位突变基因,可为斑马鱼突变体的基因定位提供参考,为神经系统发育异常的相关基因功能分析奠定基础。

Objective Taking the congenital microcephaly and suspected epilepsy(mise)zebrafish mutant caused by spontaneous as a model,to study its inheritance pattern and phenotype-related mutation gene location.Methods By comparing the head width and eye area of normal and mutant zebrafish,the microcephaly and ommatidia traits of mutants were identified;the inheritance pattern of the mutation was determined by constructing a family.Genome resequencing combined with bulked segregation analysis(BSA)and kompetitive allele specific PCR(kompetitive allele specific PCR,KASP)and Sanger sequencing were used to obtain the location of mutant genes.Results Compared with the wild type,the head and eye were significantly reduced at 3 dpf,accompanied by convulsions and tremors.The mutant gene associated with phenotype was identified as terfa by gene mapping and verification.Conclusions This study uses whole-genome resequencing combined with linkage analysis to quickly locate mutant genes.It laid the foundation for the locational cloning of zebrafish mutants and the functional analysis of genes related to abnormal development of the nervous system.