文章摘要
崔忠凯,王 倩,刘新富,孟 振,陈松林.大菱鲆受精卵显微注射技术的建立.渔业科学进展,2018,39(4):30-36
大菱鲆受精卵显微注射技术的建立
Establishment of a microinjection technique in zygote of turbot (Scophthalmus maximus).
投稿时间:2017-06-01  修订日期:2017-07-10
DOI:
中文关键词: 大菱鲆  受精卵  显微注射  GFP
英文关键词: Scophthalmus maximus  Zygote  Microinjection  GFP
基金项目:
作者单位
崔忠凯 青岛海洋科学与技术试点国家实验室海洋渔业科学与食物产出过程功能实验室 农业农村部海洋渔业可持续发展重点实验室 中国水产科学研究院黄海水产研究所 青岛 266071 上海海洋大学水产与生命学院 上海 201306 
王 倩 青岛海洋科学与技术试点国家实验室海洋渔业科学与食物产出过程功能实验室 农业农村部海洋渔业可持续发展重点实验室 中国水产科学研究院黄海水产研究所 青岛 266071 
刘新富 青岛海洋科学与技术试点国家实验室海洋渔业科学与食物产出过程功能实验室 农业农村部海洋渔业可持续发展重点实验室 中国水产科学研究院黄海水产研究所 青岛 266071 
孟 振 青岛海洋科学与技术试点国家实验室海洋渔业科学与食物产出过程功能实验室 农业农村部海洋渔业可持续发展重点实验室 中国水产科学研究院黄海水产研究所 青岛 266071 
陈松林 青岛海洋科学与技术试点国家实验室海洋渔业科学与食物产出过程功能实验室 农业农村部海洋渔业可持续发展重点实验室 中国水产科学研究院黄海水产研究所 青岛 266071 
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中文摘要:
      本研究将100~300 pg含有肌肉特异表达启动子和绿色荧光蛋白(Green fluorescent protein, GFP)基因的重组质粒(smyd1 : gfp)显微注射到大菱鲆(Scophthalmus maximus)受精卵动物极细胞中,通过细心培育,成功孵化出鱼苗约120尾。统计分析显示,显微注射后,大菱鲆胚胎存活率为4.8%。利用荧光显微镜观察大菱鲆胚胎及仔鱼,只在注射smyd1 : gfp质粒的胚胎及仔鱼的肌肉中发现有绿色荧光。通过进一步PCR扩增检测,在注射的大菱鲆胚胎及仔鱼DNA中扩增出了GFP特异片段,大小约为340 bp。研究表明,本研究成功建立了大菱鲆显微注射技术,可为大菱鲆基因功能研究和遗传育种奠定基础。
英文摘要:
      Genome editing in fish has many potential applications in aquaculture and is important for gene functional analyses and genetic engineering breeding. Furthermore, genome editing in aquaculture animals is useful for cultivating new varieties and researching disease resistance and aquaculture growth. However, the difficulty of microinjection and cultivation in mariculture fish embryos seriously hampers the application of genome editing technology. Many methods are used to introduce DNA, RNA, and protein to fish genomes, such as pulsed electric, retroviral transfection, and laser-mediated technologies. Microinjection is the most widely used method. Turbot (Scophthalmus maximus) is one of the most important mariculture fish in Chinese aquaculture. To our knowledge, there have been no reports on the success of the microinjection technique in turbots. In this study, the smyd1 : gfp plasmid (50 ng/µl) with green fluorescent protein (GFP) expression by a 5.3 kb muscle-specific smyd1 promoter fragment was successfully microinjected and expressed in the cytoplasm of newly zygote of turbot at the 1~4 cell stage. The effects of microinjection on the survival rate of turbot embryos were studied. The survival rate of microinjected embryos was lower than that of uninjected embryos. Overall, 120 microinjected fishes (4.8%) survived and 190 (19.0%) uninjected survived. The GFP expression in the embryos and larvae was observed using a fluorescence microscope (Nikon Eclipse 80i). In total, 56% (28/50) of microinjected fishes expressed GFP. A specific GFP-encoding segment (346 bp) was amplified from the DNA of microinjected embryos and larvae by polymerase chain reaction, showing that the GFP gene had been introduced into the turbot embryos and confirming successful microinjection technique of turbot.
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