贵州医科大学学报

2016, v.41;No.190(07) 755-759

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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

舒莉萍;何志旭;许威;丁可军;
SHU Liping;HE Zhixu;XU Wei;DING Kejun;Tissue Engineering and Stem Cell Research Center,Guizhou Medical University;Experimental Animal Center,Guizhou Medical University;Department of Immunology,Guizhou Medical University;Department of Pediatrics,Guizhou Medical University;

摘要(Abstract):

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 Irx5 a 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 c DNA of Irx5 a gene was amplified by RT-PCR,and then cloned into pCS~(2+)vector by Bam H 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 Irx5 a gene was prepared by in vitro transcription system and underwent whole mount in situ hybridization with zebrafish embryos. Results: Irx5a-pCS~(2+) +recombinant plasmid was constructed and identified. After whole mount in situ hybridization,it was observed that Irx5 a mRNA was expressed from 3. 7hpf and highly expressed in embryonic nervous system and hematopoietic system. Conclusion: Irx5 a gene may be involved in the development of nervous system and hematopoietic system in the early stage of Tuebingen wild type zebrafish embryo development.

关键词(KeyWords): Irx5a;pCS2+质粒;DNA,重组;RNA探针;全胚胎原位杂交技术
Irx5a;pCS2+plasmid;DNA,recombination;RNA probe;whole mount in situ hybridization

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基金项目(Foundation): 国家自然科学基金(31260284);; 贵州省科技基础条件平台项目[(2009)4005];; 贵州省优秀科技省长专项基金(S2008-4)

作者(Author): 舒莉萍;何志旭;许威;丁可军;
SHU Liping;HE Zhixu;XU Wei;DING Kejun;Tissue Engineering and Stem Cell Research Center,Guizhou Medical University;Experimental Animal Center,Guizhou Medical University;Department of Immunology,Guizhou Medical University;Department of Pediatrics,Guizhou Medical University;

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DOI: 10.19367/j.cnki.1000-2707.2016.07.003

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