rpl15在斑马鱼胚胎中的表达形式探究

Expression of rpl15 in zebrafish embryos

目的探究rpl15基因在野生型斑马鱼胚胎中的表达形式。方法利用NCBI和Ensemble数据库进行rpl15基因的共线性分析和Rpl15蛋白质序列相似性分析,使用MEGA X软件绘制进化树;构建pCS2^(+)-rpl15重组质粒,再利用T3聚合酶制备地高辛标记的rpl15反义RNA探针,用全胚胎原位杂交检测rpl15基因的mRNA水平的表达;通过Western blot检测24、48和72 hpf斑马鱼胚胎中Rpl15蛋白的表达。结果在进化过程中,rpl15基因相对保守。构建了pCS2^(+)-rpl15重组质粒,原位杂交结果显示在24 hpf,rpl15基因在斑马鱼的几乎所有细胞中广泛表达;到48 hpf,rpl15在中枢神经系统的表达最强并且在胰腺的表达也开始显现;72 hpf仍然可见rpl15在后脑中的表达以及胰腺、肝和肠道中的表达。Western blot结果显示24、48和72 hpf斑马鱼胚胎中Rpl15蛋白的相对表达量随时间增加而增多。结论系统观察了斑马鱼发育过程中rpl15的表达部位及表达水平的变化,为斑马鱼rpl15缺失模型的建立提供了实验基础。

Objective To investigate the expression of rpl15 in zebrafish.Methods The NCBI and Ensemble databases were used to analyze the co-collinearity of the rpl15 gene and the sequence similarity of rpl15 protein.MEGA X software was used to align rpl15 in different organizations.The mRNA expression of rpl15 was detected in whole embryos by in situ hybridization with an rpl15 antisense RNA probe,and the expression of rpl15 protein in 24,48,and 72 hpf zebrafish embryos was detected by western blotting.Results The rpl15 gene is relatively conserved during evolution.A pCS2^(+)-rpl15 recombinant plasmid was constructed,and in situ hybridization result showed that at 24 hpf,rpl15 was widely expressed in most cells of zebrafish.At 48 hpf,rpl15 expression was highest in the central nervous system and it was also present in the pancreas.At 72 hpf,rpl15 expression was still present in the posterior brain,as well as the pancreas,liver,and intestine.The relative protein expressions of Rpl15 in 24,48,and 72 hpf zebrafish embryos increased with increasing time.Conclusions Changes in the expression sites and expression levels of rpl15 in zebrafish during development were systematically observed,which provide the experimental basis for establishing a model of rpl15 deletion in zebrafish.