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。
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 80 i). 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.
引文
Ansai S,Kinoshita M.Targeted mutagenesis using CRISPR/Cas system in medaka.Biology Open,2014,3(5):362-371
Chen SL,Zhang G,Shao CW,et al.Whole-genome sequence of a flatfish provides insights into ZW sex chromosome evolution and adaptation to a benthic lifestyle.Nature Genetics,2014,46(3):253-260
Cui ZK,Liu Y,Wang WW,et al.Genome editing reveals dmrt1as an essential male sex-determining gene in Chinese tongue sole(Cynoglossus semilaevis).Scientific Reports,2017,7:42213
Du SJ,Rotllant J,Tan XG.Muscle-specific expression of the smyd1 gene is controlled by its 5.3-kb promoter and 5'-flanking sequence in zebrafish embryos.Developmental Dynamics,2006,235(12):3306-3315
Dunham RA.Transgenic fish resistant to infectious diseases,their risk and prevention of escape into the environment and future candidate genes for disease transgene manipulation.Comparative Immunology Microbiology and Infectious Disease,2009,32(2):139-161
Grabher C,Wittbrodt J.Recent advances in meganuclease-and transposon-mediated transgenesis of medaka and zebrafish.Methods Molecular Biology,2008,461:521-539
Heather AH,Stephanie LP,William MM.High efficiency production of germ-line transgenic Japanese medaka(Oryzias latipes)by electroporation with direct currentshifted radio frequency pulses.Transgenic Research,2003,12:413-424
Hwang WY,Fu Y,Reyon D,et al.Efficient genome editing in zebrafish using a CRISPR-Cas system.Nature Biotechnology,2013,31(3):227-229
Kato K,Takagi M,Tamaru Y,et al.Construction of an expression vector containing aβ-actin promoter region for gene transfer by microinjection in red sea bream Pagrus major.Fisherues Science,2007,73(2):440-445
Lei JL,Ma AJ,Liu XF,et al.Study on the development of embryo,larval and juvenile of turbot Scophthalmus maximus L.Oceanologia et Limnologia Sinica,2003,34(1):9-18[雷霁霖,马爱军,刘新富,等.大菱鲆胚胎及仔稚幼鱼发育研究.海洋与湖沼,2003,34(1):9-18]
Li MH,Yang HH,Li MR,et al.Antagonistic roles of Dmrt1 and Foxl2 in sex differentiation via estrogen production in tilapia as demonstrated by TALENs.Endocrinology,2013,154(12):4814-4825
Liu Y,Sha ZX,Chen SL,et al.Expression of green fluorescent protein gene transferred into newly fertilized sea perch(Lateolabrax japonicus)embryos by microinjection.Journal of Fishery Sciences of China,2004,11(5):385-390[刘洋,沙珍霞,陈松林,等.绿色荧光蛋白基因向花鲈胚胎的转移及其表达.中国水产科学,2004,11(5):385-390]
Li Z,Yu S,Du TG,et al.Effect analysis of transgenic microinjection dose.Chinese Journal of Laboratory Animal Science,1998,8(2):65-69[李智,俞生,都同功,等.转基因显微注射的剂量控制与效果分析.中国实验动物学杂志,1998,8(2):65-69]
Otani S,Ohara M,Miyashita S,et al.A method for the microinjection into naturally spawned eggs of marine fish,especially cultured Pacific bluefin tuna Thunnus orientalis.Fisheries Science,2008,74(1):208-210
Qin ZK,Li Y,Su BF,et al.Editing of the luteinizing hormone gene to sterilize channel catfish,Ictalurus punctatus,using a modified zinc finger nuclease technology with electroporation.Marine Biotechnology(NY),2016,18(2):255-263
Rembold M,Lahiri K,Foulkes NS,et al.Transgenesis in fish:Efficient selection of transgenic fish by co-injection with a fluorescent reporter construct.Nature Protocols,2006,1(3):1133-1139
Sha ZX,Chen SL,Liu Y,et al.Progress on the application of microinjection technique in fish cell transplantation and in transgenic marine fish.Marine Fisheries Research,2005,26(3):86-90[沙珍霞,陈松林,刘洋,等.显微注射技术在制备鱼类嵌合体和转基因海水鱼上的应用.海洋水产研究,2005,26(3):86-90]
Shao CW,Bao BL,Xie ZY,et al.The genome and transcriptome of Japanese flounder provide insights into flatfish asymmetry.Nature Genetics,2016,49(1):119-124
Shen Y,Huang P,Zhang B.A protocol for TALEN contruction and gene targeting in zebrafish.Hereditas(Beijing),2013,35(4):533-544[沈延,黄鹏,张博.TALEN构建与斑马鱼基因组定点突变的实验方法与流程.遗传(北京),2013,35(4):533-544]
Tong DZ,Wu SQ,Ye YF,et al.Migration of fish nuclei.Chinese Science Bulletin,1963,13(7):60-61[童第周,吴尚懃,叶毓芬,等.鱼类细胞核的移植.科学通报,1963,13(7):60-61]
Wang YL,Wang SS,Zhang L,et al.Effect of microinjection dose of transgene on transgenic efficiency and embryo development of chicken.China Poultry,2017,39(4):4-7[王亚丽,王赛赛,张丽,等.转基因显微注射剂量对鸡胚转基因效率和胚胎发育的影响.中国家禽,2017,39(4):4-7]
Yoji Y,Naoki K,Yutaka T,et al.Establishment of a stable transgenic strain in a pelagic egg spawning marine teleost,Nibe croaker Nibea mitsukurii.Aquaculture,2011,313(1-4):42-49
Zhu Z,Li G,He L,et al.Novel gene transfer into the fertilized eggs of goldfish(Carassius auratus L.1758).Journal of Applied Ichthyology,1985,1(1):31-34
Zhu ZY,Wang YP.Transgenic fish.Bulletin of Biology,1999,34(5):1-3[朱作言,汪亚平.转基因鱼.生物学通报,1999,34(5):1-3]
Zhu ZY,Xu KS,Xie YF,et al.A model of transgnic fish.Scientia Sinica(Ser B).1989,19(2):147-155[朱作言,许克圣,谢岳峰,等.转基因模型鱼的建立.中国科学B辑,1989,19(2):147-155]