异源和同源dmrt1基因过表达载体在青鳉体内的整合和表达  

Integration and expression comparative analysis of heterologous and homologous dmrt1 expression vector in Medaka, Oryzias latipe

作　　者：李亚亚[1,2] 陈松林[1] 林帆[1] 王娜[1] 刘洋 黄进强[1] 

机构地区：[1]中国水产科学研究院黄海水产研究所农业部海洋渔业可持续发展重点实验室,山东青岛266071 [2]上海海洋大学水产与生命学院,上海201306 [3]大连海洋大学水产与生命学院,辽宁大连116023

出　　处：《中国水产科学》2014年第3期425-431,共7页Journal of Fishery Sciences of China

基　　金：国家自然科学基金项目(31130057;31072202);国家公益性行业(农业)科研专项(200903046);山东省泰山学者工程专项

摘　　要：利用半滑舌鳎(Cynoglossus semilaevis)dmrt1基因和青鳉(Oryzias latipe)dmrt1基因构建过表达载体,采用显微注射技术将两种载体分别导入青鳉受精卵中,比较异源和同源dmrt1基因对青鳉胚胎孵化率的影响以及两种dmrt1基因在青鳉体内的基因整合率、基因表达率及对芳香化酶基因(cyp19a)表达的影响。结果表明,注射48 h后,两种载体均在胚胎内大量表达,注射青鳉同源性dmrt1载体的受精卵孵化率(69.5%)要显著高于注射异源性dmrt1载体的受精卵(36.5%)。30 dph(days post hatching)两组被注射稚鱼均能检测到基因整合,注射同源dmrt1的整合率(30%)要高于异源dmrt1(10%)。30 dph稚鱼体内检测不到异源性dmrt1载体的表达,但能检测到同源dmrt1载体的mRNA表达,同时芳香化酶基因的表达受到了抑制。本研究为研究舌鳎性别分化相关基因功能提供了基础,并为在模式鱼类中研究海水鱼相关基因功能累积了必要的资料。Fish are an ideal model for research on functional genes. Transgenic fish have many important applications in aquaculture. Recently, a number of researchers have evaluated the use of transgenic fish to improve the commercial value of a species by manipulating traits such as growth, sex, and disease resistance. In particular, the function of sex-determining genes has received significant attention. The current studies of gene function, which are related to sex differentiation, are largely confined to a few fresh water fish species. In contrast, little effort has been made to study gene function in marine fish because of logistical difficulties, such as the presence of a hard fertilized egg membrane, low survival rate, and/or long period of a generation. To address this gap, we studied the function of a homologous gene in a model marine fish species. A number of methods have been used to modify the fish genome, among which microinjection is the most commonly used approach. We constructed expression vectors by subcloning Cynoglossus semilaevis and Medaka dmrtl into a plRES- hrGFP-la vector. In vivo over-expression of dmrtl in Medaka was induced by injecting these GFP constructs into the cytoplasm of fertilized Medaka embryos at the one cell stage. We then measured GFP expression and hatching rate. Additionally, we quantified the level of gene integration, expression, and the effect on cyp19a expression. GFP was expressed at high levels 48 h after injection and the number of embryos expressing GFP decreased with the development of embryos. The hatching rate and integration efficiency of embryos injected with the homologous dmrtl expression vector was significantly higher than those injected with the heterologous dmrtl expression vector. We observed the RNA expression of Medaka dmrtl but not Cynoglossus semilaevis dmrtl. Furthermore, in fishes with dmrtl expression, the expression of cyp19a was down-regulated. Our research provides a foundation for the study of Cynoglossus semilaevis sex differentiation and contribute

关 键 词：半滑舌鳎 青鳉 DMRT1 转基因鱼 显微注射 

分 类 号：S917[农业科学—水产科学]