斑马鱼mcm3合子基因敲除实验模型的构建与鉴定  

Construction and Identification of Zebrafish mcm3 Zygote Gene Knockout Experimental Model

作　　者：夏佳敏 张可 陈炳宇 黄四洲[2,3] 刘敏 Xia Jiamin;Zhang Ke;Chen Bingyu;Huang Sizhou;Liu Min(School of Basic Medicine, Chengdu Medical College, Chengdu 610500, China;Department of Human Anatomy, Histology and Embryology, Chengdu Medical College, Chengdu 610500, China;Key Laboratory of Development and Regeneration of Sichuan Province, Chengdu Medical College, Chengdu 610500, China;The First Affiliated Hospital of Chengdu Medical College, Chengdu 610500,China)

机构地区：[1]成都医学院基础医学院,成都610500 [2]成都医学院人体解剖与组织胚胎学教研室,成都610500 [3]成都医学院发育与再生四川省重点实验室,成都610500 [4]成都医学院第一附属医院,成都610500

出　　处：《成都医学院学报》2021年第4期409-414,共6页Journal of Chengdu Medical College

基　　金：国家自然科学基金(No:32070805);四川省科技厅基础研究项目(No:20YYJC3369);发育与再生四川省重点实验室项目(No:20180498);成都医学院创新团队项目(No:CYTD19-01);成都医学院研究生科研创新基金(No:YCX2020-06)。

摘　　要：目的构建斑马鱼mcm3合子基因突变体,研究mcm3合子基因在斑马鱼胚胎早期发育中的作用。方法利用系统发育树分析不同物种间mcm3基因的保守性;利用国家生物技术信息中心(NCBI)网站及Jalview软件序列比对,分析斑马鱼MCM3蛋白与人类MCM3蛋白之间的氨基酸同源性;用CRISPR/Cas9技术构建可稳定遗传的斑马鱼mcm3合子基因缺失突变体;提取胚胎的基因组DNA后进行基因测序,检测斑马鱼mcm3合子基因突变类型;利用原位杂交检测mcm3突变体中mcm3 mRNA的表达水平。结果斑马鱼合子基因mcm3与人类mcm3基因同源,且高度保守;可稳定遗传的mcm3合子基因突变体成功构建;在mcm3突变体斑马鱼中,胚胎从第3天开始出现头和眼睛,其外部表型较野生型斑马鱼小,且mcm3纯合子胚胎在第8天后相继死亡;运用整胚原位杂交发现,mcm3在野生型斑马鱼的头部、眼睛、中后脑边缘、鳃、胸腺及内胚层组织中高表达,但在mcm3突变体斑马鱼中,mcm3 mRNA的表达水平明显降低。结论斑马鱼mcm3基因缺失突变体成功构建,并且mcm3基因在斑马鱼的早期胚胎发育中起重要作用。Objective To construct zebrafish mcm3 zygotic gene mutant,and study the role of mcm3 zygotic gene in early embryonic development of zebrafish.Methods Phylogenetic tree was used to analyze the conservation of mcm3 zygotes among different species.The website of National Center of Biotechnology Information(NCBI)and Jalview software sequence alignment were used to analyze the amino acid homology between zebrafish MCM3 protein and human MCM3 protein.And CRISPR/Cas9 technique was used to construct stable genetic mcm3 zygotes gene deletion mutants.After the extraction of embryonic genomic DNA,gene sequencing was performed to detect the type of zebrafish mcm3 zygotic gene mutation.The expression level of mcm3 mRNA in mcm3 mutants was detected by in situ hybridization.Results Zebrafish zygotic gene mcm3 is homologous to human mcm3 gene and is highly conserved.Stable genetic zebrafish mcm3 zygotic gene mutant was successfully constructed.In mcm3 mutants,embryos developed an external phenotype with a relatively smaller head and eyes from 3 day post-fertilization(dpf)compared with wild-type embryos,and the mcm3 homozygous embryos died after 8 dpf.In situ hybridization of whole embryo showed that mcm3 was highly expressed in head,eyes,middle and posterior brain margin,gill,thymus and endoderm tissues of wild-type zebrafish,but the expression level of mcm3 mRNA was significantly decreased in mcm3 mutants.Conclusion mcm3 gene deletion mutant was successfully constructed in zebrafish.And mcm3 gene plays an important role in the early embryonic development of zebrafish.

关 键 词：斑马鱼 mcm3 突变体 CRISPR/Cas9 

分 类 号：Q95-33[生物学—动物学]