Tgf2转座子在团头鲂基因组中的插入效率

doi:10.3724/SP.J.1005.2013.00999

遗传 ›› 2013, Vol. 35 ›› Issue (8): 999-1006.doi: 10.3724/SP.J.1005.2013.00999

Tgf2转座子在团头鲂基因组中的插入效率

郭秀明, 黄创新, 沈睿杰, 蒋霞云, 陈杰, 邹曙明

上海海洋大学, 农业部淡水水产种质资源重点实验室, 上海201306

收稿日期:2013-01-21 修回日期:2013-02-07 出版日期:2013-08-20 发布日期:2013-08-25
通讯作者: 邹曙明 E-mail:smzou@shou.edu.cn
基金资助:
“十二五”国家支撑计划(编号：2012BAD26B02)、国家自然科学基金项目(编号：31272633, 31201760)和上海高校知识服务平台(ZF1206)资助

Insertion efficiency of Tgf2 transposon in the genome of Megalo-brama amblycephala

GUO Xiu-Ming, HUANG Chuang-Xin, SHEN Rui-Jie, JIANG Xia-Yun, CHEN Jie, ZOU Shu-Ming

Key Laboratory of Freshwater Aquatic Genetic Resources, Ministry of Agriculture, Shanghai Ocean University, Shanghai 201306, China

Received:2013-01-21 Revised:2013-02-07 Online:2013-08-20 Published:2013-08-25

摘要/Abstract

摘要：

文章通过构建带金鱼Tgf2转座子左右臂、斑马鱼肌球蛋白轻链2(Mlyz2)启动子和红色荧光蛋白(RFP)的供体质粒Tgf2-Mlyz2-RFP, 与Tgf2转座酶mRNA共同显微注射入团头鲂1~2细胞期受精卵, 检测金鱼Tgf2转座子在团头鲂基因组中的整合效率。在团头鲂出膜仔鱼、30 d和180 d幼鱼阶段, 可在鱼体背部和侧面肌肉观察到荧光, 红色荧光蛋白的表达率为48.1%, PCR检测结果显示, 金鱼Tgf2转座系统在团头鲂成鱼基因组中的整合效率为31.5%; 对5尾阳性团头鲂进行了RT-PCR检测, 3尾团头鲂在12个组织均能检测到较高的RFP基因的表达, 2尾团头鲂仅在肌肉、皮和肾脏中存在较高的RFP基因的表达, 显示RFP基因在不同转基因团头鲂个体中的组织表达存在一定差异; 通过检测Tgf2转座子在团头鲂基因组插入位置5′端的侧翼序列, 检测出金鱼Tgf2转座系统在转基因团头鲂中的拷贝数至少为2个, 每尾鱼的平均拷贝数大约为5个, 50%以上插入位点的侧翼序列可找出其它脊椎动物的相关同源性序列。研究结果显示金鱼Tgf2转座子可高效介导基因在团头鲂基因组中插入, 为开展团头鲂转基因和基因捕获研究奠定了一定的基础。

关键词: 插入效率, Tgf2转座子, 团头鲂, 基因组

Abstract:

To study insertion efficiency of goldfish Tgf2 transposon in the genome of Megalobrama amblycephala, we built Tgf2-Mlyz2-RFP donor plasmid with goldfish Tgf2 transposon left and right arms, and then co-injected with goldfish Tgf2 transposase mRNA into the 1–2 cell stage fertilized eggs of M. amblycephala. In 30 d- and 180 d-stage larval, RFP fluorescence can be observed in back and side muscle of the fish. The rate of RFP fluorescence expression was 48.1%. In adult fish, PCR results demonstrated that integration efficiency of goldfish Tgf2 transposition system was 31.5% in M. am-blycephala genome. RT-PCR analysis showed that RFP RNAs were highly transcribed among all the 12 tissues in three transgenic fishes, while it could be highly detected only in muscle, skin, and kidney in another two individuals. Our results suggested that RFP expression in tissues vaied among different M. amblycephala. By means of the inverse PCR, the copy numbers of Tgf2 transposon were at least 2 in transgenic M. amblycephala. The average copy number of each fish was about 5. Over 50% of flanking sequences at the insertion site have homologous sequence in other vertebrate species. Our data suggest that goldfish Tgf2 transposon can efficiently mediate gene insertion in M. amblycephala, which could been used in transgene and gene trap in M. amblycephala.

Key words: Tgf2 transposon, Megalobrama amblycephala, insertion efficiency, genome

郭秀明黄创新沈睿杰蒋霞云陈杰邹曙明. Tgf2转座子在团头鲂基因组中的插入效率[J]. 遗传, 2013, 35(8): 999-1006.

GUO Xiu-Ming, HUANG Chuang-Xin, SHEN Rui-Jie, JIANG Xia-Yun, CHEN Jie, ZOU Shu-Ming. Insertion efficiency of Tgf2 transposon in the genome of Megalo-brama amblycephala[J]. HEREDITAS, 2013, 35(8): 999-1006.

参考文献

[1] Csaba M, Zsuzsanna I, Ronald HP, Zoltan I. The Frog Prince: a reconstructed transposon from Rana pipiens with high transpositional activity in vertebrate cells. Nucleic Acids Res, 2003, 31(23): 6873-6881.

[2] Kawakami K. Tol2: a versatile gene transfer vector in vertebrates. Genome Biol, 2007, 8(Suppl. 1): S7.

[3] 邹曙明, 杜雪地, 蒋霞云. 鱼类活性DNA转座子的发掘与应用概况. 上海海洋大学学报, 2012, 21(5): 656-661.

[4] Standford WL, Cohn JB, Cordes SP. Gene-trap mutagenesis: past, present and beyond. Nat Rev Genet, 2001, 2(10): 756-768.

[5] 朱作言, 许克圣, 谢岳峰, 李国华, 何玲. 转基因鱼模型的建立. 中国科学(B), 1989, (2): 147-155.

[6] Urasaki A, Mito T, Noji S, Ueda R, Kawakami K. Trans-position of the vertebrate Tol2 transposable element in Drosophila melanogaster. Gene, 2008, 425(1-2): 64-68.

[7] Kawakami K, Shima A. Identification of the Tol2 transpo-sase of the medaka fish Oryzias latipes that catalyzes excision of a nonautonomous Tol2 element in zebrafish Danio rerio. Gene, 1999, 240(1): 239-244.

[8] Kawakami K, Koga A, Hori H, Shima A. Excision of the Tol2 transposable element of the medaka fish, Oryzias latipes, in zebrafish, Danio rerio. Gene, 1998, 225(1-2): 17-22.

[9] Kawakami K, Shima A, Kawakami N. Identification of a functional transposase of the Tol2 element, an Ac-like element from the Japanese medaka fish, and its transposition in the zebrafish germ lineage. Proc Natl Acad Sci USA, 2000, 97(21): 11403-11408.

[10] Kawakami K, Imanaka K, Itoh M, Taira M. Excision of the Tol2 transposable element of the medaka fish Oryzias latipes in Xenopus laevis and Xenopus tropicalis. Gene, 2004, 338(1): 93-98.

[11] Sato Y, Kasai T, Nakagawa S, Tanabe K, Watanabe T, Kawakami K, Takahashi Y. Stable integration and conditional expression of electroporated transgenes in chicken embryos. Dev Biol, 2007, 305(2): 616-624.

[12] Kawakami K, Noda T. Transposition of the Tol2 element, an Ac-like element from the Japanese medaka fish Oryzias latipes, in mouse embryonic stem cells. Genetics, 2004, 166(2): 895-899.

[13] Balciunas D, Wangensteen KJ, Wilber A, Bell J, Geurts A, Sivasubbu S, Wang X, Hackett PB, Largaespada DA, McIvor RS, Ekker SC. Harnessing a high cargo-capacity transposon for genetic applications in vertebrates. PLoS Genet, 2006, 2(11): e169.

[14] 邹曙明, 杜雪地, 袁剑, 蒋霞云. 金鱼 hAT 家族转座子Tgf2 的克隆及其结构. 遗传, 2010, 32(12): 1263-1268.

[15] Jiang XY, Du XD, Tian YM, Shen RJ, Sun CF, Zou SM. Gold?sh transposase Tgf2 presumably from recent horizontal transfer is active. FASEB J, 2012, 26(7): 2743-2752.

[16] Ministry of Agriculture of the People’s Republic of China. Chinese Fisheries Yearbook. Beijing: Chinese Agricultural Press, 2012: 30-31.

[17] Shen RJ, Jiang XY, Pu JW, Zou SM. HIF-1α and -2α genes in a hypoxia-sensitive teleost species Megalobrama am-blycephala: cDNA cloning, expression and different responses to hypoxia. Comp Biochem Physiol B Biochem Mol Biol, 2010, 157(3): 273-280.

[18] 孙效文, 徐鹏. 水产基因组技术与研究进展. 北京: 海洋出版社, 2011: 188-202.

[19] 简清, 白俊杰, 叶星, 夏仕玲, 梁旭方, 罗建仁. 斑马鱼Mylz2启动子的克隆与转绿色荧光蛋白基因鱼的构建. 中国水产科学, 2004, 11(5): 391-395.

[20] Ju BS, Chong SW, He JY, Wang XK, Xu YF, Wan HY, Tong Y, Yan T, Korzh V, Gong ZY. Recapitulation of fast skeletal muscle development in zebrafish by transgenic expression of GFP under the Mylz2 promoter. Dev Dyn, 2003, 227(1): 14-26.

[21] Sambrook J, Fritsch EF, Maniatis T. Molecular cloning: A laboratory manual. 2nd ed. New York: Cold Spring Harbor Laboratory Press, 1989.

[22] Chun KT, Edenberg HJ, Kelley MR, Goebl MG. Rapid amplification of uncharacterized transposon-tagged DNA sequences from genomic DNA. Yeast, 1997, 13(3): 233-240.

[23] Uren AG, Mikkers H, Kool J, van der Weyden L, Lund AH, Wilson CH, Rance R, Jonkers J, van Lohuizen M, Berns A, Adams DJ. A high-throughput splinkerette-PCR method for the isolation and sequencing of retroviral insertion sites. Nat Protoc, 2009, 4(5): 789-798.

[24] Koga A, Suzuki M, Inagaki H, Bessho Y, Hori H. Trans-posable element in fish. Nature, 1996, 383(6595): 30.

[25] Li YY, Zhang JP. Gene trapping techniques and current progress. Acta Genet Sin, 2006, 33(3): 189-198.

[26] Fujimura K, Kocher TD. Tol2-mediated transgenesis in ti-lapia (Oreochromis niloticus). Aquaculture, 2011, 319(3-4): 342-346.

[27] Golling G, Amsterdam A, Sun Z, Antonelli M, Maldonado E, Chen W, Burgess S, Haldi M, Artzt K, Farrington S, Lin SY, Nissen RM, Hopkins N. Insertional mutagenesis in zebrafish rapidly identifies genes essential for early vertebrate development. Nat Genet, 2002, 31(2):135-140.

[28] 龙华, 尾里建二郎, 若松佑子, 松岛良次. 红色荧光蛋白(RFP)基因在转基因青鳉中的表达.中国水产科学, 2002, 9(2): 97-99.

[29] Moutou KA, Canario AV, Mamuris Z, Power DM. Molecular cloning and sequence of Sparus aurata skeletal myosin light chains expressed in white muscle: developmental expression and thyroid regulation. J Exp Biol, 2001, 204(17): 3009-3018.

[30] Gothilf Y, Toyama R, Coon SL, Du SJ, Dawid IB, Klein DC. Pineal-specific expression of green fluorescent pro-tein under the control of the serotonin-N-acetyltransferase gene regulatory regions in transgenic zebrafish. Dev Dyn, 2002, 225(3): 241-249.

[31] 陈敏, 白俊杰, 姜鹏, 叶星. 红色荧光蛋白基因在转基因唐鱼中的表达.大连水产学院学报, 2009, 24(增刊): 59-63.

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Tgf2转座子在团头鲂基因组中的插入效率

Insertion efficiency of Tgf2 transposon in the genome of Megalo-brama amblycephala

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