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1 HEREDITAS (Beijing) , 33(5): ISSN 综述 DOI: /SP.J 转基因鱼的研究进展与商业化前景叶星, 田园园, 高风英, 转基因技术为鱼类育种开辟了新的途径目前已培育出转生长激素基因鲤鲑和罗非鱼, 转荧光蛋白基因斑马鱼与唐鱼等可稳定遗传的转基因鱼品系, 其中快长转生长激素基因鱼的获得对于提高水产养殖的产量与养殖效益具有十分重要的意义文章简要综述了转基因鱼应用研究的成就相关技术及生态安全方面的研究进展显微注射仍是目前基因转植的常用方法, 应用转座酶或巨核酸酶介导的转基因新技术可提高基因转植效率与整合率转基因元件的选择应尽量考虑全鱼基因或自源基因, 以减少转基因鱼食用安全方面的顾虑同时也有利于转植基因的表达与生理功效的发挥生态安全是转基因鱼商用化面临的最大问题虽然有研究显示转基因鱼与传统的选育鱼类相比适合度较差, 但由于环境与基因型间的相互作用, 根据实验室获得的转基因鱼对生态影响的结果, 难以预测转基因鱼一旦逃逸会对自然水生态环境产生怎样的影响因此应建立高度自然化的环境以获得可靠的数据客观评价生态风险, 有效的物理拦截不育化处理等生物学控制策略仍是保证转基因鱼安全应用的关键措施转基因鱼 ; 应用研究 ; 生态安全 Progress in transgenic fish techniques and application YE Xing, TIAN Yuan-Yuan, GAO Feng-Ying Pearl River Fishery Research Institute, Chinese academy of fisheries sciences, Guangzhou , China Abstract: Transgenic technique provides a new way for fish breeding. Stable lines of growth hormone gene transfer carps, salmon and tilapia, as well as fluorescence protein gene transfer zebra fish and white cloud mountain minnow have been produced. The fast growth characteristic of GH gene transgenic fish will be of great importance to promote aquaculture production and economic efficiency. This paper summarized the progress in transgenic fish research and ecological assessments. Microinjection is still the most common used method, but often resulted in multi-site and multi-copies integration. Co-injection of transposon or meganuclease will greatly improve the efficiency of gene transfer and integration. All fish gene or auto gene should be considered to produce transgenic fish in order to eliminate misgiving on food safety and to benefit expression of the transferred gene. Environmental risk is the biggest obstacle for transgenic fish to be commercially applied. Data indicates that transgenic fish have inferior fitness compared with the traditional domestic fish. However, because of the genotype-by-environment effects, it is difficult to extrapolate simple phenotypes to the complex ecological interactions that occur in nature based on the ecological consequences of the transgenic fish determined in the laboratory. It is critical to establish highly naturalized environments for acquiring reliable data that can be used to evaluate the environmental risk. Efficacious physical and biological containment strategies remain to be crucial approaches to ensure the safe application of transgenic fish technology. Keywords: transgenic fish; application; environmental risk 收稿日期 : ; 修回日期 : 基金项目 : ( Nycytx-48-4), (863 ), ( 2008A ), ( A200899F01) ( 2009J1-C021; No ) 作者简介 :,,,, gzyexing@163.com

2 5 : [1] 20,,,, ; ; [2], [3, 4] Nature Transgenic fish go large [5], (Food and Drug Administration, FDA) (Salmo salar L.), (Veterinary Medicine Advisory Committee, VMAC) FDA, FDA ( , [6], 鮰 ;,,, 鳉, (GH) GH 5 GH (Cyprinus carpios) [7], GH F ; F 2 1.8~2.5, 10%~13%, [8,9] GH,, 1, [10],, [11~13] 鳚 (Macrozoarces americanus) AFP (Oncorhynchus tshawytscha) GH, 15 GH 1,, FDA, FDA (Oncorhynchus kisutch), (ponmtgh1), (Oncorhynchus nerka) mmt-b GH-I,, 11, 37 [14] GH (CMV-tiGH-SV40) (Oreochromis hornorum) F70, 60%~80%, [15] GH, (Danio rerio) 2004 [4] ;

3 496 HEREDITAS (Beijing) (Tanichthys atbonubes), Mylz2, [16], (Pterophyllums carale), 1.2, ( ) ( DNA ) DEAE- [17],,, 5%,,,, [18, 19],, 11, 37 [14],, (Border elements),, 1, -, [1, 20, 21], GH 2 35 bp [22],, ( - ) [23], [21,24], [7] (Labeo rohita) [25], (hot-spots) I-SceⅠ, I-Sc Ⅰ,, I-SceⅠ, [26] 鳉 (Oryzias latipes)tol2, hat [27, 28], Tol2 DNA, Tol2,, 鳉 Tol2,,, 鳉 Tol2 10 [28] (Carassius auratus) Tgf2,, Tgf2, 鳉 Tol2 [29] I-Sce I, (Saccharomyces

4 5 : 497 cerevisiae) (, homing endonuclease), 18 bp, I-SceⅠ,,,, [30] (GH)- Ⅰ (GH-IGF-I axis) GH, [31, 32], GH GH, GH, GH [21, 33] GH GH GH,, GH 1, [34], GH,, [14, 31] 鳚 AFP GH,, 15 10, 30 ; (Oncorhynchus mykiss) (Oncorhynchus clarki) [35] (Orechromis niloticus),, GH 7, 2.5 [36] ; CMV GH cdna [37] ; MT GH, 2,, [10], GH, GH, GH 84%~93%, 73%, 66%, 43.4%~82.5% [32] GH,,, (mmt) (SV40) (RSV),, β (β-actin) (AFP) (mmt) [38] β-actin,,, β-actin [39], 100 bp CAAT TATA, β-actin,, β-actin 1.5 kb β-actin (cβap) 4.7 kb β-actin (cβap) [40,41], 3.0 kb β-actin 1.7 kb

5 498 HEREDITAS (Beijing) [42] 鳚 op5a GH bp bp GH, 266 bp,, bp 70%, 10%, 5 5 [43], [44, 45], mmt-b GH-I, [11] ; β-actin GH, F 1 [46] (Autotransgenic) (Allotransgeneic) [6], (Misgurnus mizolepis) β-actin GH,, 30 [47] histone 3 GH, [25] β-actin GH GH , 3.0 [48] SV40, pdsred2-1 pegfp β-actin GH, GH, GH β-actin [49],,,, GH IGF-1 IGF-1 IGF-1 mrna, mrna 2 ; IGF-1, IGF-1 IGF-1, IGF-1 [50], GH IGF-1 [32] ; IGF-1 mrna, IGF-1 [51], GH /, GH GH, GH, GH- GH, IGF GH IGF GH GH, GH [51], GH 2 GH,,,,,,

6 5 : 499,, ;,, (Fitness), [52] GH 鳉鳉 [53] ; GH,,, [54] ; GH,, [55],,, [56], ;,, GH, [52] ; F 3 GH 22% 24% [57] ; ; [58] ; 鮰 [56, 59] GH,, GH,, ;, [60] ; GH,,,, 20%, [61] GH, GH GH (ADS), [62] ; GH ( 12 ),,, GH [63],,,

7 500 HEREDITAS (Beijing) [56] (Genotype-byenvironment effects, GXE),,,, [61, 64],, 863 [65] ; 100%,,, [66], ( RNA ) (GnRHs) RNA (RNA interference, RNAi) [67, 68] ,, 10% [69], 2010, ,,,,,,,,,, 参考文献 (References): [1] Text Zhu ZY, Li G, He L, Chen S. Novel gene transfer into the fertilized eggs of the goldfish (Carassius acuratus L. 1758). J Appl Ichthyol, 1985, 1(1): [2] Zbikowska HM. Fish can be first-advances in fish transgenesis for commercial application. Transgenic Res, 2003, 12(4): [3],.., 2000, (1): 1 6. [4] Beardmore JA, Porter JS. Genetically modified organisms and aquaculture. FAO Fish Circ, 2003, 989: [5] Marris E. Transgenic fish go large. Nature, 2010, 467(7313): [6] Beardmore JA. Transgenics: autotransgenics and allotransgenics. Transgenic Res, 1997, 6(1): [7] Hu W, Zhu ZY. Integration mechanisms of transgenes and population fitness of GH transgenic fish. Sci China Lift Sci, 2010, 53(4): [8],,,,,,,,. F1

8 5 : 501., 2001, 46(3): [9],,,,,.., 2007, 52(8): [10],,,.., 2002, 26(5): [11],,,.., 2005, 13(4): [12],,,,,.., 2007, 27(3): [13],,,,,,.., 2010, 31(5): [14] Devlin RH, Yesakl TY, Blagl CA, Donaldson EM, Swanson P, Chan WK. Extraordinary salmon growth. Nature, 1994, 371(6494): [15] Guillén II, Berlanga J, Valenzuela CM, Morales A, Toledo J, Estrada MP, Puentes P, Hayes O, de la Fuente J. Safety evaluation of transgenic tilapia with accelerated growth. Mar Biotechnol (NY), 1999, 1(1): [16],,,,,.., 2004, 9(1): [17].., 1997, 6(1): [18] Gross ML, Schneider JF, Moav N, Moav B, Alvarez C, Myster SH, Liu ZJ, Hallerman EM, Hackett PB, Guise KS, Faras AJ, Kapuscinski AR. Molecular analysis and growth evaluation of northern pike (Esox lucius) microinjected with growth hormone genes. Aquaculture, 1992, 103(3 4): [19] Hackett PB. The molecular biology of transgenic fish. In: Hochachka PW, Mommsen TP, eds. Biochemistry and Molecular Biology of Fishes, Molecular Biology Frontiers. Amsterdam: Elsevier,, 1993, 2: [20] Tewari R, Michard-Vanhée C, Perrot E, Chourrout D, Mendelian transmission, structure and expression of transgenes following their injection into the cytoplasm of trout eggs. Transgenic Res, 1992, 1(6): [21] Devlin RH, Sakhrani D, Tymchuk WE, Rise ML, Goh B. Domestication and growth hormone transgenesis cause similar changes in gene expression in coho salmon (Oncorhynchus kisutch), Proc Natl Acad Sci USA, 2009, 106(9): [22] Yaskowiak ES, Shears MA, Agarwal-Mawal A, Fletcher GL. Characterization and multi-generational stability of the growth hormone transgene (EO-1α) responsible for enhanced growth rates in Atlantic salmon. Transgenic Res, 2006, 15(4): Erratum in: Transgenic Res, 2007, 16(2): [23] Uh M, Khattra J, Devlin RH. Transgene constructs in coho salmon (Oncorhynchus kisutch) are repeated in a head-to-tail fashion and can be integrated adjacent to horizontally-ransmitted parasite DNA. Transgenic Res, 2006, 15(6): [24] Garrick D, Fiering S, Martin DIK, Whitelaw E. Repeat-induced gene silencing in mammals. Nat Genet, 1998, 18(1): [25] Rajesh R, Majumdar KC. Transgene integration - an analysis in autotransgenic Labeo rohita Hamilton (Pisces: Cyprinidae). Fish Physiol Biochem, 2005, 31(2 3): [26] Soroldoni D, Hogan BM, Oates AC. Simple and efficient transgenesis with meganuclease constructs in zebrafish. Methods Mol Biol, 2009, 546: [27] Koga A, Suzuki M, Inagaki H, Bessho Y, Hori H. Transposable element in fish. Nature, 1996, 383(6595): 30. [28] Kawakami K, Shima A. Identification of the Tol2 transposase of the medaka fish Oryzias latipes that catalyzes excision of a nonautonomous Tol2 element in zebrafish Danio rerio. Gene, 1999, 240(1): [29],,,. hat Tgl2., 2010, 32(12): [30] Babaryka A, Kühn E, Köster RW. In vivo synthesis of meganuclease for generating transgenic zebrafish Danio rerio. J Fish Biol, 2009, 74(2): [31],,,,.., 1989, 19(2): [32]. ( )., [33] Guyomard R, Chourrout D, Leroux C, Houdebine LM, Pourrain F. Integration and germ line transmission of foreign genes microinjected into fertilized trout eggs. Biochimie, 1989, 71(7): [34] Devlin RH, Yesaki TY, Donaldson EM, Du SJ, Hew CL. Production of germline transgenic Pacific salmonids with dramatically increased growth performance. Can J Fish Aquat Sci, 1995, 52(7): [35] Rahman MA, Mak R, Ayad H, Smith A, Maclean N. Expression of a novel piscine growth hormone gene results in growth enhancement in transgenic tilapia (Orechromis niloticus). Transgenic Res, 1998, 7(5):

9 502 HEREDITAS (Beijing) [36] Martínez R, Estrada MP, Berlanga J, Guillén I, Hernández O, Cabrera E, Pimentel R, Morales R, Herrera F, Morales A, Piña JC, Abad Z, Sánchez V, Melamed P, Lleonart R, de la Fuente J. Growth enhancement in transgenic tilapia by ectopic expression of tilapia growth hormone. Mol Mar Biol Biotechnol, 1996, 5(1): [37],,,,,,. β-., 2008, 15(1): [38] Liu Z, Zhu Z, Roberg K, Faras AJ, Guise KS, Kapuscinski AR, Hackett PB. The β-actin gene of carp (Ctenopharyngodon idella). Nucleic Acids Res, 1989, 17(14): [39] Moav B, Hinits Y, Groll Y, Rothbard S. Inheritance of recombinant carp β-actin/gh cdna gene intransgenic carp. Aquaculture, 1995, 137(1 4): [40] Liu ZJ, Moav B, Faras AJ, Guise KS, Kapuscinski AR, Hackett PB. Functional analysis of elements affecting expression of the β-actin gene of carp. Mol Cell Biol, 1990, 10(7): [41] Gyu-Lin H, Azizur RM, Shaherudin AR, Frédéric S, Hamid F, Alan S, Carly B, Norman M. Isolation and characterization of tilapia β-actin promoter and comparison of its activity with carp beta-actin promoter. Biochim Biophys Acta, 2003, 1625 (1): [42],,,,,. β-actin., 2008, 24(10): [43] Butler TM, Fletcher GL. Promoter analysis of a growth hormone transgene in Atlantic salmon. Theriogenology, 2009, 72(1): [44] Higashijima S, Okamato H, Ueno N, Hotta Y, Eguchi G. High-frequency generation of transgenic zebrafish which reliably expression GFP in whole muscles or the whole body by using promoters of zebrafish origin. Dev Biol, 1997, 192(2): [45] Kinoshita M, Yamauchi M, Sasanuma M, Ishikawa Y, Osada T, Inoue K, Wakamatsu Y, Ozato K. A transgene and its expression profile are stably transmitted to offspring in transgenic medaka generated by the particle gun method. Zoolog Sci, 2003, 20(7): [46] Morales R, Herrera MT, Arenal A, Cruz A, Hernández O, Pimentel R, Guillén I, Martínez R, Estrada MP. Tilapia chromosomal growth hormone gene expression accelerates growth in transgenic zebrafish (Danio rerio). Electronic J of Biotechnol, 2001, 4(2): [47] Nam YK, Noh JK, Cho YS, Cho HJ, Cho KN, Kim CG, Kim DS. Dramatically accelerated growth and extraordinary gigantism of transgenic mud loach Misgurnus mizolepis. Transgenic Res, 2001, 10(4): [48] Yu EM, Ye X, Wang HY, Bai JJ, Xia SL, Lao HH, Jian Q. Isolation of Tanichthys albonubes β-actin gene and production of transgenic Tanichthys albonubes. Fish Physiol Biochem, 2010, 36(2): [49],,,,,.., 2009, 31(8): [50] Eppler E, Berishvili G, Mazel P, Caelers A, Hwang G, Maclean N, Reinecke M. Distinct organ-specific up- and down-regulation of IGF-I and IGF-II mrna in various organs of a GH-overexpressing transgenic Nile tilapia. Transgenic Res, 2010, 19(2): [51] Raven PA, Uh M, Sakhrani D, Beckman BR, Cooper K, Pinter J, Leder EH, Silverstein J, Devlin RH. Endocrine effects of growth hormone overexpression in transgenic coho salmon. Gen Comp Endocrinol, 2008, 159(1): [52] Sundström LF, Lohmus M, Johnsson JI, Devlin RH. Growth hormone transgenic salmon pay for growth potential with increased predation mortality. Proc Biol Sci, 2004, 271(Suppl.): S350 S352. [53] Howard RD, DeWoody JA, Muir WM. Transgenic male mating advantage provides opportunity for Trojan gene effect in a fish. Proc Natl Acad Sci USA, 2004, 101(9): [54] Bessey C, Devlin RH, Liley NR, Biagi CA. Reproductive performance of growth-enhanced transgenic coho salmon (Oncorhynchus kisutch). Trans Am Fish Soc, 2004, 133(5): [55] Tymchuk WEV, Abrahams MV, Devlin RH. Competitive ability and mortality of growth-enhanced transgenic coho salmon fry and parr when foraging for food. Trans Am Fish Soc, 2005, 134(2): [56] 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. Comp Immunol Microbiol Infect Dis, 2009, 32(2): [57],,,,,.., 2007, 52(8): [58] Lee CG, Devlin RH, Farrell AP. Swimming performance, oxygen consumption and excess post-exercise oxygen consumption in adult transgenic and ocean-ranched coho salmon. J Fish Biol, 2003, 62(4): [59] Dunham RA, Chen TT, Powers DA, Nichols A, Argue B, Chitmanat C. Predator avoidance, spawning and foraging ability of transgenic catfish. In: Levin M, Grim C, Angle JS, ed. Proceedings Biotechnology Risk Assessment Symposium, 1994, USDA, EPA, College Park, MD, [60] Sundström LF Lõhmus M, Devlin RH. Migration and growth potential of coho salmon smolts: implications for ecological impacts from growth-enhanced fish. Ecol Appl, 2010, 20(5):

10 5 : [61] Sundström LF, Lõhmus M, Tymchuk WE, Devlin RH. Gene-environment interactions influence ecological consequences of transgenic animals. Proc Natl Acad Sci USA, 2007, 104(10): [62] da Rosa CE, Figueiredo MA, Lanes CF, Almeida DV, Marins LF. Genotype-dependent gene expression profile of the antioxidant defense system (ADS) in the liver of a GH-transgenic zebrafish model. Transgenic Res, (1): [63] Devlin RH, Sakhrani D, Tymchuk WE, Rise ML, Goh B. Domestication and growth hormone transgenesis cause similar changes in gene expression in coho salmon (Oncorhynchus kisutch). Proc Natl Acad Sci USA, 2009, 106(9): [64] Devlin RH, Sundström LF, Muir WM. Interface of biotechnology and ecology for environmental risk assessments of transgenic fish. Trends Biotechnol, 2006, 24(2): [65].., 2001, 18(1): [66],,.. C :, 2007, 37(4): [67] Uzbekova S, Chyb J, Ferrière F, Bailhache T, Prunet P, Alestrom P, Breton B. Transgenic rainbow trout expressed sgnrh-antisense RNA under the control of sgnrh promoter of Atlantic salmon. J Mol Endocrnol, 2000, 25(3): [68] Huang WT, Hsieh JC, Chiou M J, Chen JY, Wu JL, Kuo CM. Application of RNAi technology to the inhibition of zebrafish GtHα, FSHβ, and LHβ expression and to functional analyses. Zoolog Sci, 2008, 25(6): [69],,.., 2009, (7): ˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇ 科学新闻从基因组计划到个体化医疗 [ ] , Life Technologies Life Technologies Siddhartha Kadia 40,, 1100, 2500, 650, ,, , 160,,,,,,,,,,,, 1, 200,,, 1000,, 3-5, 1000 Kadia, Life Technologies 2,,,, ,,,,

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