Objective: : To observe the expression of g6pd gene in the early development stage of wild zebrafish embryos.
Methods: : The collinearity of g6pd gene and the sequence similarity of G6pd protein were analyzed with gene database and BLAST software, respectively. Expression of g6pd gene in different development stages of zebrafish embryos was detected by in situ hybridization. The g6pd-EGFP-pCS2+ recombinant plasmids were microinjected into zebrafish embryos, and fluorescence was observed under a fluorescence microscope. The expression of G6pd protein at 24, 48 and 72 hour post fertilization (hpf) zebrafish embryos was detected by Western blotting; the enzyme activity of G6pd at 24, 48 and 72 hpf zebrafish embryos was detected by modified G6pd quantitative ratio method.
Results: : The G6pd protein similarity of zebrafish and human was 88%, and that of zebrafish and mouse was 87%. The results of in situ hybridization showed that the g6pd gene was mainly expressed in the hematopoietic tissues of zebrafish; the results observed after microinjection of g6pd-EGFP-pCS2+ recombinant plasmid were consistent with the results of in situ hybridization. At 24, 48 and 72 hpf, the relative expression levels of G6pd protein in zebrafish embryos were 1.44±0.03, 1.47±0.05, and 1.54±0.02, respectively(P>0.05); the G6pd enzyme activity levels were 1.74±0.17, 1.75±0.12, 1.71±0.22, respectively (P>0.05).
Conclusions: : The study has observed the expression of g6pd gene and G6pd protein, and G6pd enzyme activity in zebrafish embryos at different development phases, which provides a reference for the establishment of a zebrafish G6PD deficiency model.
目的: 观察 g6pd基因在野生型斑马鱼胚胎早期发育过程中的表达情况。
方法: 利用基因数据库和BLAST软件分别进行 g6pd基因的共线性分析和G6pd蛋白质序列相似性分析。原位杂交技术观察不同时相斑马鱼胚胎中 g6pd基因的表达；构建 g6pd-EGFP-pCS 2+重组质粒，并将其显微注射入斑马鱼胚胎内，荧光显微镜下观察绿色荧光蛋白表达；蛋白质印迹法检测24、48、72 hpf斑马鱼胚胎中G6pd蛋白的表达量；改良G6pd定量比值法检测24、48、72 hpf斑马鱼胚胎中G6pd酶活性。
结果: g6pd基因在进化过程中相对保守，斑马鱼与人类的G6pd蛋白质相似度为88%，斑马鱼与小鼠的G6pd蛋白质相似度为87%。原位杂交试验结果显示， g6pd基因主要表达于斑马鱼的造血组织，显微注射 g6pd-EGFP-pCS 2+重组质粒后观察的结果与原位杂交试验结果一致。24、48、72 hpf斑马鱼胚胎中G6pd蛋白相对表达量分别为1.44±0.03、1.47±0.05、1.54±0.02，差异无统计学意义（ P > 0.05）；G6pd酶活性分别为1.74±0.17、1.75±0.12、1.71±0.22，差异无统计学意义（ P > 0.05）。
结论: 成功观察到斑马鱼体内 g6pd基因表达部位及其变化规律，并测定了不同发育时相斑马鱼胚胎中G6pd蛋白表达和酶活性，为建立斑马鱼G6PD缺乏症模型奠定了基础。