转红色荧光蛋白基因唐鱼肌肉中抗生素标记基因(NPTⅡ)检测和表达量分析  

Detection and Expression Analysis of Neomycin Phosphotransferase ⅡGene (NPTⅡ) in Muscle of Transgenic Tanichthys albonubes Expressing the Red Fluorescent Protein

作　　者：樊佳佳[1] 白俊杰[1] 简清[1] 于凌云[1] 

机构地区：[1]中国水产科学研究院珠江水产研究所/农业部热带亚热带水产资源利用与养殖重点实验室,广州510380

出　　处：《农业生物技术学报》2012年第4期420-425,共6页Journal of Agricultural Biotechnology

基　　金：国家高技术研究发展计划(863)项目(No.2009AA10Z105);广东省科技计划项目(No.2010B020308002);广东省海洋渔业科技推广专项子项目(No.A200899F01)

摘　　要：为了获得有特色的观赏鱼,本实验室在2003年将含有海葵红色荧光蛋白基因的表达载体（同时含有标记基因新霉素磷酸转移酶基因（NPTⅡ））转入唐鱼（Tanichthys albonubes）的受精卵,获得转红色荧光蛋白基因唐鱼,对其外源标记基因NPTⅡ的表达产物和存在时间进行分析,为评价转基因鱼的生物安全提供依据。本实验对转基因唐鱼和非转基因唐鱼肌肉中抗生素标记基因（NPTⅡ）进行PCR检测和NPTⅡ表达产物的免疫检测试剂条检测,结果显示,转基因唐鱼检测到NPTⅡ基因和表达产物,而非转基因唐鱼中均没检测到该基因和表达产物存在。同时在唐鱼死亡后（水温20~25℃）,应用酶联免疫吸附测定法（ELISA）对死亡0（鲜活肌肉）、24、48、72、96、120和144h组的转基因唐鱼肌肉中NPTⅡ蛋白的含量进行定量检测,结果表明,转基因唐鱼鲜活肌肉中NPTⅡ蛋白在肌肉中的含量为9.12ng/g,随着转基因唐鱼死亡时间增加肌肉中NPTⅡ蛋白含量逐渐减少,死亡96h组转基因唐鱼肌肉中NPTⅡ蛋白已经接近为0。结果表明,转基因唐鱼肌肉中NPTⅡ蛋白在自然环境中容易降解,推测其对环境安全隐患相对较低。For omamental purpose, our laboratory chose fertilized egg of Tanichthys albonubes used as a receptor to transfer red fluorescent protein vector of sea anemone in 2003. Transgenic T. albonubes expressing the red fluorescent protein was obtained. This paper aimed at evaluating its biological safety by analyzing expression and existence time of neomycin phosphotransferase Ⅱ gene（NPT Ⅱ ）. PCR amplification and rapid diagnosis strip were applied on selectable marker gene detection and NPT Ⅱ express detection in transgenic T. albonubes and non-transgenic T. albonubes. The results showed that NPT Ⅱ gene and protein product were detected in transgenic T. albonubes, but that were not detected in non-transgenic T. albonubes. To quantify the expression of NPT Ⅱ protein in muscle of transgenic T. albonubes, enzyme linked immunosorbent assay （ELISA） was performed at different death time in natural water （water temperature at 20-25 ~C）. The death times are 0（fresh muscle）, 24, 48, 72, 96, 120 and 144 h group, respectively. The result showed that the NPTll protein content in the fresh muscle oftransgenic T. albonubes was 9.12 ng/g, and the amount ofNPT Ⅱ protein in the muscle of transgenic T. albonubes was decreased with death time prolonging. At the NPT Ⅱ protein content of the muscle was close to zero at 96 h after death. The results indicate that the NPT Ⅱ protein in transgenic T. albonubes muscle is so easy to be degraded under nature conditions and we speculate that it has low security risk to environmen.

关 键 词：转基因唐鱼 红色荧光蛋白 新霉素磷酸转移酶 酶联免疫吸附测定 

分 类 号：S562[农业科学—作物学]