Irx5a在野生型斑马鱼早期发育中的表达

Expression of Irx5a Gene in Early Development of Wide Type Zebrafish

目的:观测Irx5a基因在Tuebingen野生型斑马鱼胚胎发育过程中的表达。方法:利用Trizol法提取斑马鱼胚胎的总RNA,应用RT-PCR方法扩增Irx5a基因,将其克隆入pCS^(2+)质粒中,经菌落PCR、双酶切法及DNA测序鉴定正确后,利用体外转录体系制备Irx5a的反义mRNA探针;用该反义mRNA探针进行斑马鱼全胚胎原位杂交。结果:构建和鉴定了Irx5a-pCS^(2+)重组质粒,经斑马鱼全胚胎原位杂交检测Irx5a基因的表达,发现从3.7 hpf开始有表达,并且在胚胎神经系统和造血系统都有明显表达。结论:Irx5a基因可能参与了Tuebingen野生型斑马鱼胚胎发育早期的神经系统和造血系统的发育。

Objective：To observe the expression of Irx5a gene in early development of the wild type zebrafish embryo of with whole mount in situ hybridization（ WISH）. Methods：Total RNA of zebrafish embryos in different development stage was extracted by Trizol. The cDNA of Irx5a gene was amplified by RT-PCR,and then cloned into pCS^2＋ vector by BamH I and EcoR I restrictive enzyme sites. The recombinant gene was identified and confirmed by bacterial colony PCR,double enzyme digestion and DNA sequencing. Antisense mRNA probes of Irx5a gene was prepared by in vitro transcription system and underwent whole mount in situ hybridization with zebrafish embryos. Results：Irx5a-pCS^2＋ recom-binant plasmid was constructed and identified. After whole mount in situ hybridization,it was observed that Irx5 a mRNA was expressed from 3.7 hpf and highly expressed in embryonic nervous system and hematopoietic system. Conclusion：Irx5a gene may be involved in the development of nervous system and hematopoietic system in the early stage of Tuebingen wild type zebrafish embryo development.