Tgf2转座子介导鲤Fst1基因元件在鲤中的转基因效率研究
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中国水产科学研究院黑龙江水产研究所 淡水鱼类育种国家地方联合工程实验室 淡水水产生物技术与遗传育种重点实验室 黑龙江 哈尔滨,上海海洋大学农业部淡水水产种质资源重点实验室,中国水产科学研究院,上海海洋大学农业部淡水水产种质资源重点实验室,中国水产科学研究院黑龙江水产研究所 淡水鱼类育种国家地方联合工程实验室 淡水水产生物技术与遗传育种重点实验室 黑龙江 哈尔滨

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国家高技术研究发展计划项目(2011AA100404);国家高技术研究发展计划项目(2009AA10Z105)


Study on transgenic efficiency of Fst1 gene element mediated by Tgf2 transposon in common carp
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Heilongjiang River Fisheries Research Institute,Chinese Academy of Fishery Sciences,National Local Joint Engineering Laboratory of Freshwater Fish Breeding,Key Laboratory of Freshwater Aquatic Biotechnology and Genetic Breeding,Key Laboratory of Freshwater Aquatic Genetic Resources,Shanghai Ocean University,Ministry of Agriculture,Shanghai,The Centre of Applied Aquatic Genomics,Chinese Academy of Fishery Sciences,Beijing,Key Laboratory of Freshwater Aquatic Genetic Resources,Shanghai Ocean University,Ministry of Agriculture,Shanghai,Heilongjiang River Fisheries Research Institute,Chinese Academy of Fishery Sciences,National Local Joint Engineering Laboratory of Freshwater Fish Breeding,Key Laboratory of Freshwater Aquatic Biotechnology and Genetic Breeding

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    摘要:

    卵泡抑素(follistatin,Fst)蛋白具有拮抗TGF-β超家族许多成员的功能,通过直接的蛋白结合可抑制肌肉抑制素(myostatin)的活性,从而恢复肌肉的生长,表现出增肌效应。参照鲤高通量转录组测序数据的拼接结果,筛选出鲤卵泡抑素Fst1基因的编码框(ORF)序列(全长1 260 bp,编码320个氨基酸),构建了包含金鱼Tgf2转座子左臂(220 bp)、右臂(185 bp)、斑马鱼Mylz2启动子和鲤Fst1基因ORF的供体质粒pTgf2-Mylz2-ccfst1。通过显微注射方法,并在体外合成的Tgf2转座酶5’加帽mRNA的介导下,获得了一批转鲤Fst1基因的转基因鲤。经PCR检测,转基因鲤Fst1外源基因的整合率平均为44.7%,对其中4尾转基因阳性鲤的扩增产物进行回收、克隆和测序验证,结果表明阳性鲤中均含有转基因目的片段。说明Tgf2转座子转基因系统可在鲤中实现较高的转座效率,为进一步研究卵泡抑素在鲤肌肉发育中的功能奠定了基础。

    Abstract:

    Follistatin (FST) could antagonize the TGF-β superfamily members and inhibit the activity of endo-myostatin via direct protein-protein interaction, thereby restoring muscle growth. Here, according to the splicing results of the high-throughput transcriptome sequencing data for Cyprinus carpio, we cloned the open reading frame (ORF) sequence of the Fst1 gene, which was 1 260 bp in length and encodes a protein of 320 amino acids. We then constructed a pTgf2-Mylz2-ccfst1 donor plasmid carrying both the left (220 bp) and right (185 bp) arms of goldfish Tgf2 transposon, the promoter of zebrafish Mylz2 and the ORF of carp Fst1. Transgenic carps were obtained via co-microinjection of the donor and the capped mRNA of the Tgf2 transposase synthesized in vitro. The average integration rate of the exogenous gene in transgenic carp was 44.7% as measured by PCR. The amplified products of four positive transgenic carp were recovered, cloned and sequenced to confirm that all of them contained the target fragment. Our results demonstrated the high transgenic efficiency of the Tgf2 transposon in C. carpio, which laid the foundation for further studies on the function of FST during carp muscle development.

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闫学春,钟莎莎,徐鹏,邹曙明,孙效文.Tgf2转座子介导鲤Fst1基因元件在鲤中的转基因效率研究[J].上海海洋大学学报,2014,23(1):1-7.
YAN Xue-chun, ZHONG Sha-sha, XU Peng, ZOU Shu-ming, SUN Xiao-wen. Study on transgenic efficiency of Fst1 gene element mediated by Tgf2 transposon in common carp[J]. Journal of Shanghai Ocean University,2014,23(1):1-7.

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  • 收稿日期:2013-09-03
  • 最后修改日期:2013-10-25
  • 录用日期:2013-11-11
  • 在线发布日期: 2014-01-16
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