Pax5基因在野生型斑马鱼全胚胎发育早期的表达

Expression of pax5 gene in wildtype zebrafish embryos

目的为了深入了解在胚胎发育过程中,尤其是在早期B淋巴细胞的生成和中脑的发育过程中pax5基因可能的作用及其机制,选择斑马鱼作为实验动物,构建野生型斑马鱼pCS2+-pax5重组质粒,并观察pax5基因在野生型斑马鱼胚胎中的表达。方法提取斑马鱼胚胎总RNA后经RT-PCR克隆斑马鱼pax5基因片段,将得到的pax5基因片段和pCS2+质粒经EcoRⅠ、XbaⅠ双酶切后,在T4DNA连接酶的作用下插入pCS2+质粒中,通过对重组质粒进行双酶切、菌落PCR以及插入片段序列测定鉴定后,经T3RNA体外转录体系合成地高辛标记的pax5基因的反义mRNA探针。将得到的探针用全胚胎原位杂交技术检测pax5基因在斑马鱼早期发育过程中的表达。结果成功构建pCS2+-pax5重组质粒并制备了pax5基因的反义mRNA探针,通过斑马鱼原位杂交技术,发现在斑马鱼受精后18～72小时(18～72hpf)胚胎头部的小脑、中脑后脑交界处均可观察到pax5基因阳性杂交信号;在24～72hpf胚胎的耳蜗均可观察到pax5基因的阳性杂交信号,且随着时相的增长其表达逐渐增多;在18～48hpf胚胎的脊索部位可见pax5基因的阳性杂交信号。结论明确了pax5基因在斑马鱼胚胎的脑部、脊索神经系统高表达,并首次证实其在耳蜗听神经的早期发育过程中有表达,可能对斑马鱼早期胚胎神经系统的发育起着某些至关重要的作用。

Objective To study the role and related mechanisms of pax5 gene during the development of the embryos, especially in the early development of B cell and midbrain, and to observe the expression of pax5 gene in wildtype zebrafish embryos. Methods Total RNA of Tuebingen wildtype zebrafish embryos was extracted to obtain cDNA of pax5 gene by RT- PCR with specific primers. The cDNA of pax5 gene and pCS^z＋ vector were double digested with EcoR Ⅰ and XbaⅠ , and then ligated by T4 DNA ligase. The recombinant vector was verified by double digestion, colony PCR screening and sequencing. The verified recombinant vector was then used to synthesize digoxin labeled anti-sense mRNA probe of pax5 gene using T3 RNA polymerase in vitro transcription system. The generated probes were used to detect pax5 gene expression in zebrafish embryos by whole-mount in situ hybridization. Results The pCS^z＋-pax5 recombinant plasmid and the probe of digoxin-labeled anti-sense mRNA of pax5 gene was successfully constructed. Whole-mount in situ hybridization showed that pax5 gene was expressed in the cerebellum and midbrain hindbrain boundary from 18-72 h post-fertilization（hpf）. In the cochlea pax5 gene was expressed from 24-72 hpf and the expression was increased as time went by. From 18-48 hpf pax5 gene is found in the notoehord. Conclusion Pax5 gene is highly expressed in the brain and notochord of zebrafish embryos. Pax5 gene expression is found in the cochlea of the Tuebingen wildtype zebrafish＇s embryos for the first time. It is suggested that pax5 may play an important role inthe development of the nervous system in the early zebrafish embryos.