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大菱鲆受精卵显微注射技术的建立
崔忠凯1,2, 王 倩1, 刘新富1, 孟 振1, 陈松林1
1.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;2.上海海洋大学水产与生命学院 上海 201306
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。
关键词:  大菱鲆  受精卵  显微注射  GFP
DOI:
分类号:
基金项目:
Establishment of a microinjection technique in zygote of turbot (Scophthalmus maximus).
CUI Zhongkai1,2, WANG Qian1, LIU Xinfu1, MENG Zhen1, CHEN Songlin1
1.Laboratory for Marine Fisheries Science and Food Production Processes, Pilot National Laboratory for Marine Science and Technology (Qingdao), Key Laboratory of Sustainable Development of Marine Fisheries, Ministry of Agriculture and Rural Affairs, Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao 266071;2.College of Fisheries and Life Science, Shanghai Ocean University, Shanghai 201306
Abstract:
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.
Key words:  Scophthalmus maximus  Zygote  Microinjection  GFP