斑马鱼eomesb基因突变体的构建及其功能分析  

Mutant Construction and Functional Analysis of eomesb Gene in Zebrafish

作　　者：胡金萌 杜博博 宋诗颖 张庆华[1,2,3] 任建峰 Hu Jinmeng;Du Bobo;Song Shiying;Zhang Qinghua;Ren Jianfeng(International Research Center of Ministry of Science and Technology for Marine Biosciences,Shanghai Ocean University,Shanghai,201306;Key Lab of Ministry of Education for Exploration and Utilization of Aquatic Genetic Resources,Shanghai Ocean University,Shanghai,201306;National Demonstration Center for Experimental Fisheries Science Education,Shanghai Ocean University,Shanghai,201306)

机构地区：[1]上海海洋大学,科技部海洋生物科学国际联合研究中心,上海201306 [2]上海海洋大学,水产种质资源发掘与利用教育部重点实验室,上海201306 [3]上海海洋大学,水产科学国家级实验教学示范中心,上海201306

出　　处：《基因组学与应用生物学》2021年第3期1047-1054,共8页Genomics and Applied Biology

基　　金：上海海洋大学一流学科和上海市科委国际科技合作项目(15410723300)共同资助

摘　　要：Eomesodermin(Eomes)是T-box转录因子基因家族的成员,在脊椎动物胚胎发育、模式形成和形态发生过程中发挥重要作用。斑马鱼(Danio rerio)eomesa基因的一个点突变导致其母源合子突变体胚胎发育延迟而大量死亡,而eomesa合子突变体可正常发育至性成熟,但缺失背鳍。本研究利用CRISPR/Cas9技术构建了斑马鱼eomesa旁系同源基因eomesb的突变体,eomesb纯合突变体不仅可正常发育至性成熟,而且鳍的发育也完全正常。q-PCR和整体原位杂交实验结果显示,eomesa和eomesb在脑部组织具有重叠的表达域,eomesb在中脑的表达略高于eomesa,故推测两基因在脑部可能行使相似的功能,存在一定的功能冗余。对2,4,6 dpf(Days post fertilization)和6 dpf的WT和突变体q-PCR基因表达分析显示,eomesb突变对eomesa的表达没有明显影响。eomesa^(-/-);eomesb^(-/-)双突变体未出现背鳍缺失以外的附加表型,推测斑马鱼eomesa和eomesb在背鳍发育过程中可能不存在功能冗余,eomesb不参与背鳍的发育过程。此外,整体原位杂交实验结果显示,eomesb突变也不影响其同源基因tbr1a和tbr1b的早期表达。eomesb纯合突变体未观察到明显表型,可能与其影响的功能不引起明显表型有关,也可能与其它基因的遗传补偿效应有关。本研究结果为深入研究斑马鱼eomesb基因的功能提供了实验材料,同时对研究eomesa与eomesb基因功能分化具有参考价值。Eomesodermin(Eomes)is a member of the T-box transcription factor gene family and plays an important role in vertebrate embryonic development,pattern formation and morphogenesis.In zebrafish(Danio rerio),a point mutation in the eomesa gene caused delayed and fatal embryonic development of the maternal zygotic mutant,while the eomesa zygotic mutant could normally develop to sexual maturity with lack of the dorsal fin.In this study,we constructed a mutant of eomesb gene,the paralogous gene of eomesa in zebrafish by CRISPR/Cas9 technology.The eomesb homozygous mutants could not only develop normally to sexual maturity,but also develop completely normally in fin.The results of q-PCR and overall in situ hybridization showed that eomesa and eomesb had overlapping expression domains in brain tissues,and the expression of eomesb in the midbrain was slightly higher than that of eomesa.Therefore,it is speculated that the two genes might perform similar functions in the brain and have certain functional redundancy.Gene expression analysis of WT and mutants by q-PCR at 2,4,6 dpf(Days post fertilization)showed that eomesb mutation had no significant effect on eomesa expression.The eomesa^(-/-);eomesb^(-/-)double mutant did not show any additional phenotypes,thus,we speculated that there was no functional redundancy between eomesa and eomesb during the dorsal fin developmental process and eomesb may not be involved in dorsal fin development.In addition,the result from the whole embryo in situ hybridization experiments showed that the eomesb mutation did not affect the early expression of its homologous genes tbr1a and tbr1b.The reason why the homozygous mutant of eomesb did not present any phenotype may be because either the function affected by the mutation did not result in obvious phenotype at all or other genes had the effect of genetic compensation response to the mutation.The results in this study provided not only the materials for further study on the function of eomesb gene in zebrafish,but also a good reference for th

关 键 词：斑马鱼 CRISPR/Cas9 Eomesa Eomesb Q-PCR 

分 类 号：Q953[生物学—动物学]