外源性红色荧光蛋白基因(RFP)在转基因唐鱼中的整合分析  

Integration Analysis of Red Fluorescent Protein Gene(RFP) in Transgenic Tanichthys albonubes

作　　者：姜鹏[1,2] 陈敏[1,2] 白俊杰[1] 李胜杰[1] 叶星[1] 简清[1] 

机构地区：[1]中国水产科学研究院珠江水产研究所,中国水产科学研究院热带亚热带鱼类选育与养殖重点开放实验室,广州510380 [2]大连海洋大学生命科学与技术学院,大连116023

出　　处：《农业生物技术学报》2010年第5期968-974,共7页Journal of Agricultural Biotechnology

基　　金：国家高技术研究发展计划(863)(No.2009AA10Z105);广东省海洋渔业科技推广专项项目(No.A200899F01);广东省科技计划项目(No.2005B20301018)共同资助

摘　　要：转红色荧光蛋白(red fluorescent protein,RFP)基因唐鱼(Tanichthys albonubes)的遗传符合孟德尔分离定律,为进一步了解RFP基因在转基因唐鱼基因组上的整合特点,采用PCR和Southern印迹检测转植基因的拷贝数和连接方式。结果表明,转植基因是以单位点、多拷贝(2～3个拷贝)、头尾相接(head-to-tail)的串联体形式整合。应用基因组步移技术分别克隆出1.0和1.2kb的转植基因整合位点5'和3'侧翼区序列。序列分析表明,5'侧翼区为宿主唐鱼基因组序列,在GenBank数据库中经BLAST分析后未发现有同源序列;3'侧翼序列与P1噬菌体部分基因组序列同源性达到99%,非转基因唐鱼基因组中未检测到该侧翼序列。研究结果为转红色荧光蛋白基因唐鱼的遗传稳定提供了评价依据。A stable line of transgenic Tanichthys albonubes containing red fluorescent protein（RFP） gene was produced previously and RFP gene is proved transmitting in a Mendelian fashion.For further reveals the integration characteristics of RFP gene in transgenic fish,the number of construct copies,the arrangement of constructs and the nearby sequence of foreign gene inserted were detected using PCR,Southern blotting and genomic walking.The results showed that the integration pattern of exogenous gene was single locus,multi-copy（2 or 3） and head-to-tail multimers.Both fragments of 1.0 and 1.2 kb were amplified by genomic walking from 5＇-and 3＇-flanking sequence,respectively.Sequence analysis showed that 5＇-flanking sequence existed in wild Tanichthys albonubes genome sequence,and there was no homologous sequence in the GenBank database,the 3＇-flanking sequence was 99%identical to that of phage P1 partial genome,but this sequence could not be detected in wild Tanichthys albonubes genome.These result provide a evaluation basis for the genetic stability of RFP transgenic T.albonubes.

关 键 词：转基因鱼 唐鱼 红色荧光蛋白 整合 侧翼序列 

分 类 号：S513[农业科学—作物学]