宋锦;姬牧远;吴西军;周艳华;舒莉萍;何志旭;
SONG Jin;JI Muyuan;WU Xijun;ZHOU Yanhua;SHU Liping;HE Zhixu;Department of Immunology,Guizhou Medical University;The Center of Stem Cell and Tissue Engineering Research ,Guizhou Medical University;Department of Paediatrics,Affiliated Hospital of Guizhou Medical University;Laboratory Animal Center,Guizhou Medical University;

• 1:贵州医科大学免疫学教研室
• 2:贵州医科大学干细胞与组织工程实验中心
• 3:贵州医科大学
• 4:贵州医科大学附院儿科学教研室

摘要(Abstract)：

目的:探究p21基因在斑马鱼早期发育过程中的表达。方法:Trizol法提取野生型斑马鱼胚胎总RNA,RT-PCR法扩增p21基因片段,与p~(CS2+)载体重组构建p21-p~(CS2+)重组质粒,以T3 RNA聚合酶制备地高辛标记的反义mRNA探针,通过斑马鱼全胚胎原位杂交检测p21基因在野生型斑马鱼胚胎早期发育过程中的表达。结果:成功构建了p21-p~(CS2+)重组质粒,并制备反义mRNA探针;原位杂交结果显示野生型斑马鱼0.5~18 hpf的胚胎均未发现p21基因阳性杂交信号,24~36 hpf胚胎可在脊索部位观察到阳性信号,48~72 hpf胚胎可在头部胸腺附近观察到阳性信号,表达量均较低。结论:成功制备了p21-p~(CS2+)重组质粒及斑马鱼p21基因反义mRNA探针,p21基因在野生型斑马鱼胚胎不同发育时相中表达水平不同。
Objective: To explore the expression of p21 in zebrafish. Methods: Extract 0. 75 hpf ~120 hpf wild-type Zebrafish embryos in multiple phase and total RNA by Trizol,amplificating p21 gene by RT-PCR,and constructing p21-pCS~(2+)plasmid. Preparing antisense mRNA probe with T3 RNA polymerase,using zebrafish whole-mount in situ hybridization to examine the p21 expression pattern in early embryo development. Results: Successfully constructed p21-pCS~(2+)plasmid,preparing antisense mRNA probe; within situ hybridization,there is no expression of p21 in wild-type zebrafish at 0. 5 hpf~ 18 hpf; positive signal in notochord was found in 24 hpf ~ 36 hpf of the wild-type Zebrafish embryos,and positive signal could be found near head and thymus,but indicating low expression. Conclusion: In this research,it has been successfully constructed p21-pCS~(2+)plasmid,antisense mRNA probe preparation,and have a preliminary observation in the expression of p21 gene in Zebrafish embryos in different periods.

关键词(KeyWords)： p21;斑马鱼;胚胎;发育;探针;质粒
p21;zebrafish;embryo;development;probe;plasmid

Abstract:

Keywords:

基金项目(Foundation): 国家自然科学基金项目(30960412);; 贵州省科技基础条件平台项目[(2009)4005];; 贵州省优秀科技教育人才省长专项资金(S2008-4)

作者(Author): 宋锦;姬牧远;吴西军;周艳华;舒莉萍;何志旭;
SONG Jin;JI Muyuan;WU Xijun;ZHOU Yanhua;SHU Liping;HE Zhixu;Department of Immunology,Guizhou Medical University;The Center of Stem Cell and Tissue Engineering Research ,Guizhou Medical University;Department of Paediatrics,Affiliated Hospital of Guizhou Medical University;Laboratory Animal Center,Guizhou Medical University;

Email:

DOI: 10.19367/j.cnki.1000-2707.2016.07.005

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