赤点石斑鱼卵型芳香化酶基因启动子调控绿色荧光蛋白在斑马鱼早期生殖腺中的表达  

THE EACYP19A1A PROMOTER OF RED-SPOTTED GROUPER DIRECTS THE EXPRESSION OF GFP IN EARLY PRIMARY GONAD CELLS OF ZEBRAFISH

作　　者：黄伟[1,2] 周莉[1] 刘静霞[1] 李志[1] 桂建芳[1] 

机构地区：[1]中国科学院水生生物研究所,淡水生态与生物技术国家重点实验室,武汉430072 [2]中国科学院研究生院,北京100049

出　　处：《水生生物学报》2010年第3期502-508,共7页Acta Hydrobiologica Sinica

基　　金：国家973计划(2004CB117401);中国科学院知识创新工程重要方向项目(KSCX2-YW-N-020)资助

摘　　要：P450芳香化酶(P450arom)是生物体内合成雌激素的关键酶。为研究赤点石斑鱼(Epinephelus akaara)卵型芳香化酶基因(EaCyp19a1a)启动子的调控作用,构建了EaCyp19a1a启动子9个不同的5′端缺失片段与绿色荧光蛋白(Green fluorescent protein,GFP)组成的表达载体,分别命名为pEaCyp19a1a-EGFP1-9。在COS-7细胞中的瞬时转染实验证明载体pEaCyp19a1a-EGFP7具有很强的转录活性。通过显微注射,将线性化的pEaCyp19a1a-EGFP7载体注入斑马鱼单细胞期受精卵中,定期在荧光显微镜下观察GFP的表达。结果发现GFP在胚胎期(受精后约48h)开始表达,主要发生在斑马鱼早期生殖腺细胞中,具有明显的性腺特异性。这说明EaCyp19a1a的启动子序列中含有调控其性腺特异表达的顺势作用原件。研究为进一步探明芳香化酶在鱼类性别分化和性别转变过程中的作用奠定了基础。Cytochrome P450 aromatase （P450arom） is the key enzyme responsible for estrogen biosynthesis in many species, and is believed to participate in the regulation of sexual differentiation of teleost fishes. However, the regulation mechanisms of aromatase genes remain unknown. In this study, we examined the role of EaCyp 19a1a promoter in ovary specific expression of EaCypl9ala. Firstly, nine 5＇-deletion promoter region were obtained by PCR amplification using primers designed according to the accurate localizations of the special positive regulatory elements in the isolated 5＇-flanking sequence, such as SOX5, CREB, SF-1, and so on. Then, these nine deletion mutants covering EaCyp19a1a promoter were constructed into the recombinant pEGFP-N1 （4.1 kb） vector, and examined their transcription activities. The construct of pEaCyp19a1a-EGFP7 vector was proved to possess the highest activity by transient transfection in COS-7 cells. Subsequently, the linearized pEaCyp19a1a-EGFP7 DNA was microinjected into fertilization eggs of ze-brafish and the embryos were observed termly with fluorescent microscopy for the green fluorescent protein （GFP） ex-pression. Specific green fluorescence was found to be restricted in the early primary gonad cells as early as zebrafish embryos at the hatching stage （about 48 hpf） and extend gradually along with the direction of germinal ridge develop-ment in larvae. These results indicated that some cis-acting elements were binding to EaCyp19a1a promoter and had the ability to regulate EaCyp19a1a gene expression specificly in the gonad. This research laid a foundation for further study on the role of aromatase in sex differentiation and sex inversion of fish.

关 键 词：赤点石斑鱼 卵型芳香化酶 启动子 绿色荧光蛋白 斑马鱼 

分 类 号：Q786[生物学—分子生物学]