心血管发育相关基因mpx在斑马鱼早期胚胎发育中的表达

Expression of Cardiovascular Development-Related Gene mpx in Early Embryo Development of Zebrafish

目的:探讨斑马鱼体内髓样特异性过氧化物酶(mpx)在胚胎发育过程中的表达模式。方法:按Trizol法提取斑马鱼总RNA后逆转录成cDNA,再以cDNA为模板RT-PCR扩增mpx基因片段;用基因重组技术构建pCS2^+-mpx重组质粒后将质粒酶切线性化,T3聚合酶制备mpx反义RNA探针,用全胚胎原位杂交检测mpx基因的表达。结果:成功构建了pCS2^+-mpx重组质粒,原位杂交结果显示在24 hpf,mpx基因在斑马鱼胚胎中间细胞团表达;从24 hpf开始,mpx基因在斑马鱼胚胎卵黄囊表面、心脏和轴向脉管系统中高表达,一直持续到72 hpf;尤其是在72 hpf,mpx基因在整个胚胎头部、卵黄囊表面、尾部中弥散表达。结论:mpx基因在斑马鱼心血管系统发育的时间和部位中都有表达,mpx基因可能参与调控斑马鱼心血管系统发育。

Objective:To investigate the expression pattern of myeloid-specific peroxidase(mpx)in embryonic development in zebrafish.Methods:The total RNA of zebrafish was extracted by Trizol method and then reversely transcribed into cDNA,which served as a template to amplify mpx gene fragment by RT-PCR.mpx gene fragment was ligated into pCS2 to obtain the recombinant plasmid pCS2+-mpx,which was further linearized.Digoxigenin-labeled mpx antisense RNA probe was synthesized in vitro by T3 RNA polymerase.After the embryos of different phases were fixed,the expression of mpx gene in zebrafish embryos was detected by whole embryo in situ hybridization.Results:The pCS2+-mpx recombinant plasmid was successfully constructed.In situ hybridization showed that the mpx gene was expressed in intermediate cell mass at 24 hpf and highly expressed in the zebrafish embryonic yolk sac surface,heart and axial vasculature from 24 to 72 hpf.Especially,mpx gene is expressed throughout the embryonic head,the surface of the yolk sac,and the tail at 72 hpf.Conclusion:mpx gene expresses at the time and location of zebrafish cardiovascular system development,suggesting that mpx gene may be involved in the regulation of zebrafish cardiovascular system development.