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浙江大学学报（医学版）
浙江大学学报（医学版）  2018, Vol. 47 Issue (1): 57-63    DOI: 10.3785/j.issn.1008-9292.2018.02.08
原著     
g6pd基因在野生型斑马鱼胚胎发育过程中的表达
夏海雄1,2(),李莉1,周艳华1,任平平1,3,何志旭1,3,*(),舒莉萍1,2,4,*()
1. 贵州医科大学组织工程与干细胞实验中心, 贵州 贵阳 550004
2. 贵州医科大学基础医学院免疫学教研室, 贵州 贵阳 550004
3. 贵州医科大学儿科学教研室, 贵州 贵阳 550004
4. 贵州医科大学实验动物中心, 贵州 贵阳 550004
Expression of g6pd gene in wild type zebrafish embryos of early development
XIA Haixiong1,2(),LI Li1,ZHOU Yanhua1,REN Pingping1,3,HE Zhixu1,3,*(),SHU Liping1,2,4,*()
1. Tissue Engineering and Stem Cell Research Center, Guizhou Medical University, Guiyang 550004, China
2. Department of Immunology, Basic Medical College, Guizhou Medical University, Guiyang 550004, China
3. Department of Pediatrics, Guizhou Medical University, Guiyang 550004, China
4. Laboratory Animal Center, Guizhou Medical University, Guiyang 550004, China
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摘要：

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-pCS2+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-pCS2+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。
关键词： 葡糖磷酸脱氢酶缺乏;  葡糖磷酸脱氢酶;  原位杂交;  DNA, 重组;  质粒;  基因表达;  斑马鱼;  胚胎发育    
Abstract:

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). Conclusion: 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.
Key words: Glucosephosphate dehydrogenase deficiency    Glucosephosphate dehydrogenase    In situ hybridization    DNA, recombinant    Plasmids    Gene expression    Zebrafish    Embryonic development
收稿日期: 2017-10-20 出版日期: 2018-06-12
CLC:  R394  
基金资助: 国家自然科学基金(31360285)
通讯作者: 何志旭,舒莉萍     E-mail: 912500243@qq.com;hzx@gmc.edu.cn;gyslp456@gmc.edu.cn
作者简介: 夏海雄(1990-), 男, 硕士研究生, 主要从事医学免疫学研究; E-mail:912500243@qq.com; https://orcid.org/0000-0002-6776-9725
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引用本文:

夏海雄,李莉,周艳华,任平平,何志旭,舒莉萍. g6pd基因在野生型斑马鱼胚胎发育过程中的表达[J]. 浙江大学学报（医学版）, 2018, 47(1): 57-63.

XIA Haixiong,LI Li,ZHOU Yanhua,REN Pingping,HE Zhixu,SHU Liping. Expression of g6pd gene in wild type zebrafish embryos of early development. J Zhejiang Univ (Med Sci), 2018, 47(1): 57-63.

链接本文:

http://www.zjujournals.com/med/CN/10.3785/j.issn.1008-9292.2018.02.08    或    http://www.zjujournals.com/med/CN/Y2018/V47/I1/57

图 1  g6pd基因在斑马鱼、人类和小鼠中的共线性分析结果
图 2  原位杂交技术检测g6pd基因在野生型斑马鱼胚胎发育过程中的表达情况
图 3  显微注射g6pd-EGFP-pCS2+质粒后斑马鱼胚胎发育不同时期绿色荧光蛋白的表达
图 4  不同时相斑马鱼胚胎中G6pd蛋白电泳图
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