Please wait a minute...
浙江大学学报（医学版）
J Zhejiang Univ (Med Sci)  2018, Vol. 47 Issue (1): 57-63    DOI: 10.3785/j.issn.1008-9292.2018.02.08
    
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
Download: HTML( 8 )   PDF(1153KB)
Export: BibTeX | EndNote (RIS)      

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     
Received: 20 October 2017      Published: 12 June 2018
CLC:  R394  
Corresponding Authors: HE Zhixu,SHU Liping     E-mail: 912500243@qq.com;hzx@gmc.edu.cn;gyslp456@gmc.edu.cn
Service
E-mail this article
Add to my bookshelf
Add to citation manager
E-mail Alert
RSS
Articles by authors
XIA Haixiong
LI Li
ZHOU Yanhua
REN Pingping
HE Zhixu
SHU Liping
Cite this article:

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.

URL:

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


g6pd基因在野生型斑马鱼胚胎发育过程中的表达

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 
Fig 1 The synteny analysis of the g6pd gene among homo sapiens and mus musculus
Fig 2 Results of in situ hybridization of g6pd gene in wild-type zebrafish
Fig 3 Expression of green fluorescent protein in wild-type zebrafish embryos after microinjection of g6pd-EGFP-pCS2+ plasmid
Fig 4 G6pd protein expression in zebrafish embryos at different time points
[1]   TZOUNAKAS V L , KRIEBARDIS A G , GEORGATZAKOU H T et al. Glucose 6-phosphate dehydrogenase deficient subjects may be better "storers" than donors of red bloodcells[J]. Free Radic Biol Med, 2016, 96 152- 165
doi: 10.1016/j.freeradbiomed.2016.04.005
[2]   NGUETSE C N , MEYER C G , ADEGNIKA A A et al. Glucose-6-phosphate dehydrogenase deficiency and reduced haemoglobin levels in African children with severe malaria[J]. Malar J, 2016, 15 (1): 346
doi: 10.1186/s12936-016-1396-1
[3]   KOTAKA M , GOVER S , VANDEPUTTE-RUTTEN L et al. Structural studies of glucose-6-phosphate and NADP+ binding to human glucose-6-phosphate dehydrogenase[J]. Acta Crystallogr D Biol Crystallogr, 2005, 61 (Pt 5): 495- 504
[4]   TANG J , JIANG C , XIAO X et al. Changes in red blood cell membrane structure in G6PD deficiency:an atomic force microscopy study[J]. Clin Chim Acta, 2015, 444 264- 270
doi: 10.1016/j.cca.2015.02.042
[5]   YANG H C , CHEN T L , WU Y H et al. Glucose 6-phosphate dehydrogenase deficiency enhances germ cell apoptosis and causes defective embryogenesis in Caenorhabditis elegans[J]. Cell Death Dis, 2013, 4 e616
doi: 10.1038/cddis.2013.132
[6]   ROCHFORD R , OHRT C , BARESEL P C et al. Humanized mouse model of glucose 6-phosphate dehydrogenase deficiency for in vivo assessment of hemolytic toxicity[J]. Proc Natl Acad Sci U S A, 2013, 110 (43): 17486- 17491
doi: 10.1073/pnas.1310402110
[7]   CVEJIC A , SERBANOVIC-CANIC J , STEMPLE D L et al. The role of meis1 in primitive and definitive hematopoiesis during zebrafish development[J]. Haematologica, 2011, 96 (2): 190- 198
doi: 10.3324/haematol.2010.027698
[8]   HARRISON N R , LAROCHE F J , GUTIERREZ A et al. Zebrafish models of human leukemia:technological advances and mechanistic insights[J]. Adv Exp Med Biol, 2016, 916 335- 369
doi: 10.1007/978-3-319-30654-4
[9]   SHU L P , ZHOU Z W , ZHOU T et al. Ectopic expression of Hoxb4a in hemangioblasts promotes hematopoietic development in early embryogenesis of zebrafish[J]. Clin Exp Pharmacol Physiol, 2015, 42 (12): 1275- 1286
doi: 10.1111/1440-1681.12483
[10]   候仕芳, 王志华, 王珺 et al. 下调lmna基因对斑马鱼胚胎髓系和红系造血干细胞发育的影响[J]. 浙江大学学报(医学版), 2016, 45 (6): 620- 625
HOU Shifang , WANG Zhihua , WANG Jun et al. Myeloid and erythroid hematopoietic transcription factor expression decline after knockdown of lmna genes in zebrafish embryos[J]. Journal of Zhejiang University(Medical Science), 2016, 45 (6): 620- 625
[11]   LU X , HUA L , ZHANG T et al. A reverse dot blot assay for the expanded screening of eleven Chinese G6PD mutations[J]. Clin Chim Acta, 2013, 418 45- 49
doi: 10.1016/j.cca.2012.12.023
[12]   PINNA A , CONTINI E L , CARRU C et al. Glucose-6-phosphate dehydrogenase deficiency and diabetes mellitus with severe retinal complications in a Sardinian population, Italy[J]. Int J Med Sci, 2013, 10 (13): 1907- 1913
doi: 10.7150/ijms.6776
[13]   BALDERIA P G , WONGRAKPANICH S , PATEL M et al. Healing the orphaned heart:heart failure in a patient with glucose-6-phosphate dehydrogenase deficiency[J]. BMJ Case Rep, 2015, 2015 pii2015209365
doi: 10.1136/bcr-2015-209365
[1] PAN Zongfu, FANG Qilu, ZHANG Yiwen, LI Li, HUANG Ping. Identification of key pathways and drug repurposing for anaplastic thyroid carcinoma by integrated bioinformatics analysis[J]. J Zhejiang Univ (Med Sci), 2018, 47(2): 187-193.
[2] CHEN Dahua, LI Youming. Construction of all-in-one CRISPR/Cas9 vector system targeting miR-101a gene in mouse hepatic cell line AML12[J]. J Zhejiang Univ (Med Sci), 2017, 46(4): 427-432.
[3] REN Xiaomei, XIN Bao, QIAN Wenwen, ZHANG Rongqiang. Effects of low salt diet on gene expression in dog's heart[J]. J Zhejiang Univ (Med Sci), 2017, 46(4): 433-438.
[4] SUN Yixi, LUO Yuqin, QIAN Yeqing, DONG Minyue, JIN Fan. Single nucleotide polymorphism-array in genetic analysis of chorionic villi from early spontaneous miscarriages[J]. J Zhejiang Univ (Med Sci), 2017, 46(3): 262-267.
[5] LI Enshu, YE Xiaoqun, FANG Li, YE Yinghui. Effect of oxygen concentration on outcome of in-vitro fertilization-embryo transfer[J]. J Zhejiang Univ (Med Sci), 2017, 46(3): 290-294.
[6] WANG Liya, QIAN Yeqing, JIN Fan. Research progress on the safety of offsprings conceived by assisted reproductive technology[J]. J Zhejiang Univ (Med Sci), 2017, 46(3): 279-284.
[7] HOU Shifang, WANG Zhihua, WANG Jun, HE Zhixu, SHU Liping. Myeloid and erythroid hematopoietic transcription factor expression decline after knockdown of lmna genes in zebrafish embryos[J]. J Zhejiang Univ (Med Sci), 2016, 45(6): 620-625.
[8] WU Zhihua, JING Min, LIANG Hanying, YANG Rong, HUANG Yaping, CHEN Xiaoming, HU Jianhua, FAN Jun. T cell receptor β-chain CDR3 spectratyping and cytomegalovirus activation in allogeneic hematopoietic stem cell transplant recipients[J]. J Zhejiang Univ (Med Sci), 2016, 45(5): 515-521.
[9] CHEN Xiaojing, XU Junfen, YE Jing, CHENG Xiaodong, XIE Xing, LYU Weiguo. Expression of miR-let-7e-3p in cervical intraepithelial neoplasm and cervix carcinoma and its clinical significance[J]. J Zhejiang Univ (Med Sci), 2016, 45(4): 342-348.
[10] WANG Cheng, WANG Wenjun, YANG Wei, YU Xiaohua, YAN Yiguo, ZHANG Jian, JIANG Zhisheng. MicroRNAs: a type of novel regulative factor for intervertebral disc degeneration[J]. J Zhejiang Univ (Med Sci), 2016, 45(2): 170-178.
[11] WANG Jing, HU Yan, TAN Bi-qin, WANG Jia-jia, ZHAO Meng-ting, WENG Qin-jie, ZHU Di-feng, WANG Hui-ying. Construction of expression vector pLCK-CD69-IRES-EGFP and generation of CD69 transgenic mice[J]. J Zhejiang Univ (Med Sci), 2015, 44(5): 511-516.
[12] YANG Yan, LI Yu-mei, ZHANG Na, LI Wan-yun, OU Yu-rong, WANG Rui, ZHAO Fu-you, WU Qiong. Expression of gap junction protein connexin 26 in human hepatocellular carcinoma and its significance[J]. J Zhejiang Univ (Med Sci), 2015, 44(5): 517-524.
[13] LIU Yan-ru, ZHANG Rui-feng, YING Ke-jing. Roles of long noncoding RNA in lung cancer[J]. J Zhejiang Univ (Med Sci), 2014, 43(5): 607-.
[14] OU Yu-rong，et al. Expression of secreted frizzled related protein 1，β-catenin and E-cadherin in colorectal carcinoma and its clinicopathological significances [J]. J Zhejiang Univ (Med Sci), 2014, 43(4): 397-405.
[15] JIN Shu-qing，et al. Correlation of PBX2/ELF2 expression with prognosis of non-small cell lung cancer[J]. J Zhejiang Univ (Med Sci), 2014, 43(4): 413-419.