eomesa功能缺失对斑马鱼雌鱼生殖的影响  

The effect of eomesa dysfunction on reproduction in female zebrafish

作　　者：张婷婷[1,2,3,4] 佟广香 孙志鹏[2,3,4] 闫婷 唐国盘[5] 周慧杰 匡友谊 ZHANG Ting-ting;TONG Guang-xiang;SUN Zhi-peng;YAN Ting;TANG Guo-pan;ZHOU Hui-jie;KUANG You-yi(Shanghai Ocean University,Shanghai 201306,China;Heilongjiang River Fisheries Research Institute of Chinese Academy of Fishery Sciences,Harbin 150070,China;Key Laboratory of Freshwater Aquatic Biotechnology and Breeding,Ministry of Agriculture and Rural Affairs,Harbin 150070,China;National and Local Joint Engineering Laboratory for Freshwater Fish Breeding,Harbin 150070,China;Henan University of Animal Husbandry and Economics,Zhengzhou 450046,China;Tianjin Agricultural College,Tianjin 300000,China)

机构地区：[1]上海海洋大学水产与生命学院,上海201306 [2]中国水产科学研究院黑龙江水产研究所,哈尔滨150070 [3]农业农村部淡水水产生物技术与遗传育种重点实验室,哈尔滨150070 [4]淡水鱼类育种国家地方联合工程实验室,哈尔滨150070 [5]河南牧业经济学院,郑州450046 [6]天津农学院,天津300000

出　　处：《淡水渔业》2023年第5期86-96,共11页Freshwater Fisheries

基　　金：国家重点研发计划(2018YFD0900102);中国水产科学研究院黑龙江水产研究所基本科研业务费(HSY202108Q)。

摘　　要：eomesa基因是T-box基因家族重要的转录因子,eomesa突变导致雌性斑马鱼(Danio rerio)生殖障碍,但其机制未知。本研究利用CRISPR/Cas9技术构建了eomesa纯合突变系(eomesa^(-/-)),对其进行了繁殖能力分析、卵巢组织学观察及采用qRT-PCR分析了6个性腺发育相关基因在突变体12 hpf(hours post fertilization)胚胎和45 dpf(days post fertilization)、60 dpf、90 dpf 3个胚后发育阶段中的表达水平变化,以期探讨eomesa影响雌性斑马鱼生殖障碍的机制。研究结果表明,eomesa^(-/-)斑马鱼存在背鳍和臀鳍缺失的表型,雄鱼比例显著增加,可自然繁殖,而雌鱼不能自然排卵,但可通过人工按压腹部排出卵子并以人工授精方式获得正常发育的子代;eomesa^(-/-)斑马鱼卵巢和输卵管形态正常,但卵母细胞发育滞后,胚胎发育观察发现突变体原肠胚外包延迟,且胚胎畸形率显著增加。qRT-PCR结果显示,原始生殖细胞标志基因dnd1和nanos3在胚胎发育体节期(12 hpf)突变体中的表达量显著低于野生型的表达量;dnd1、vasa和nanos3在45 dpf时突变体卵巢组织中的表达量显著高于野生型的表达量;在90 dpf时突变体卵巢组织中cyp17a1和amh基因的表达量显著低于野生型的表达量;而dmrt1基因在突变体中的表达量极显著高于野生型的表达量。推测eomesa基因敲除干扰了生殖细胞的发育和分化,以及排卵机制,并影响了cyp17a1和dmrt1的表达,致使雌雄分化异常。The eomesa is an important transcription factor in the T-box gene family.Previous studies showed that the mutation of eomesa caused reproductive disorders in female Danio rerio,but the mechanism was unknown.The eomesa homozygous mutant line(eomesa^(-/-))was constructed by CRISPR/Cas9 technology and the observation of skeletal phenotype and histological analysis of ovary were carried out to explore the influence of eomsea on female zebrafish′s reproduction.Moreover,the expression of six gonad developmental genes was analyzed using embryos of mutants and wild-type zebrafish at 12 hpf and ovarian tissues at 45,60,and 90 dpf postembryonic developmental stages.The results showed that the knockout of eomesa caused dorsal and anal fins deficiency in D.rerio.The number of male D.rerio in the eomesa^(-/-)homozygous line was significantly increased.The male eomesa^(-/-)could reproduce normally,whereas the female eomesa^(-/-)could not ovulate naturally but could discharge the ovum by pressing the abdomen manually and obtain the typically developed offspring by artificial insemination.The histological analyses showed that the morphologies of the ovary and fallopian tubes of female eomesa^(-/-)were typical.However,the development of oocytes was delayed.The observation of embryo development also showed that the epiboly of the gastrula was postponed.The malformation rate of embryos was significantly increased.The qRT-PCR of 3 marker genes of primordial germ cells showed that the expression levels of dnd1 and nanos3 in eomesa^(-/-)embryos were significantly lower than those in the wild-type embryos at the somite stage(12 hpf).The expression levels of dnd1,vasa,and nanos3 in the ovary of eomesa^(-/-)were significantly higher than those of wild-type D.rerio at 45 dpf.cyp17a1,amh,and dmrt1 were the key genes for developing gonads.The results of qRT-PCR showed that the expression levels of cyp17a1 and amh in the ovary of the mutants were significantly lower than those of the wild-type D.rerio at 90 dpf.In contrast,the expression l

关 键 词：eomesa 斑马鱼(Danio rerio) 卵巢发育 生殖障碍 QRT-PCR 

分 类 号：S965.9[农业科学—水产养殖]