Tgf2转座子多克隆位点的克隆与表达

生物技术通报 ›› 2014, Vol. 0 ›› Issue (2): 124-129.

Tgf2转座子多克隆位点的克隆与表达

陶然^1，2, 常玉梅², 梁利群², 唐然², 窦新杰^2，3, 王楠^2，3, 李明云¹

1. 宁波大学海洋学院，宁波 315211；2. 淡水鱼类育种国家地方联合工程实验室农业部淡水水产生物技术与遗传育种重点实验室中国水产科学研究院黑龙江水产研究所，哈尔滨 150070；3. 上海海洋大学水产与生命学院，上海 201306

收稿日期:2013-10-08 出版日期:2014-02-27 发布日期:2014-02-27
作者简介:陶然, 女, 硕士研究生,研究方向: 鱼类基因工程育种; E-mail：taoran0227@163.com
基金资助:
国家重点基础研究发展计划（2010CB126305）, 中央级公益性科研院所基本科研业务费专项资金项目(HSY201205)

Tgf2 Transposon Multiple Cloning Sites Cloning and Expression

Tao Ran^1，2, Chang Yumei², Liang Liqun², Tang Ran², Dou Xinjie^2，3, Wang Nan^2，3, Li Mingyun¹,

（1. College of Ocean，Ningbo University，Ningbo 315211；2.China National & Local United Engineering Laboratory of Freshwater Fish Breeding，Key Laboratory of Freshwater Aquatic Biotechnology and Genetic Breeding，Ministry of Agriculture，Heilongjiang River Fisheries Research Institute，Chinese Academy of Fishery Sciences，Harbin 150070；3. College of Fisheries and Life Science，Shanghai Ocean University，Shanghai 201306）

Received:2013-10-08 Published:2014-02-27 Online:2014-02-27

摘要/Abstract

摘要： 以金鱼pTgf2-EF1α-EGFP转座子为基础，构建含有多克隆位点（Multiple cloning sites，MCS）序列以及外源目的基因肌醇-3-磷酸合成酶（Myo-inositol-3-phosphate synthase，MIPS）的重组表达载体并注射到斑马鱼1-2期受精卵，检测重组表达载体pTgf2-EF1α-MCS-EGFP和pTgf2-EF1α-MCS-MIPS-EGFP在斑马鱼中绿色荧光蛋白基因（EGFP）的表达情况以及外源基因MIPS在斑马鱼体内的整合情况。荧光观察结果显示，两个重组载体均不影响EGFP的表达，只是表达强度存在一定差异，表明对Tgf2转座子的改造是有效的。转基因斑马鱼PCR检测结果显示，靶基因MIPS的编码区能够完整地整合到斑马鱼基因组中，整合效率达31.4%。重组表达载体的成功构建显示金鱼Tgf2转座子可以介导外源基因在斑马鱼中的表达，为以后Tgf2转座子在鱼类基因功能研究方面奠定了基础。

关键词: 金鱼, Tgf2转座子, MCS序列, 转基因斑马鱼

Abstract: The goldfish pTgf2-EF1α-EGFP transposon vector was reconstructed by insertion of artificial synthesized multiple colning sites（MCS）. In order to examine its effectiveness, the recombinant vectors of pTgf2-EF1α-MCS-EGFP and pTgf2-EF1α-MCS-MIPS--EGFP containing the exogenous gene, Myo-inositol-3-phosphate synthase（MIPS）were injected into 1-2 cells of zebrafish embryos. Both fluorescence observation of GFP and PCR detection of exogenous gene sizes verified the effectiveness of the reconstructed vector, in spite of slight differences in fluorescent strength of GFP. Moreover, the reconstructed vector has a higher integration efficiency（31.4%）in the genome of zebrafish based on the results. The successful construction of recombinant expression vector displays goldfish Tgf2 transposon can mediate exogenous gene expression in zebrafish and it would be useful to Tgf2 transposon in gene function research of fish.

陶然, 常玉梅, 梁利群, 唐然, 窦新杰, 王楠, 李明云. Tgf2转座子多克隆位点的克隆与表达[J]. 生物技术通报, 2014, 0(2): 124-129.

Tao Ran, Chang Yumei, Liang Liqun, Tang Ran, Dou Xinjie, Wang Nan, Li Mingyun,. Tgf2 Transposon Multiple Cloning Sites Cloning and Expression[J]. Biotechnology Bulletin, 2014, 0(2): 124-129.

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