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

黄伟; 周莉; 刘静霞; 李志; 桂建芳

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

黄伟^1,2,
周莉¹,
刘静霞¹,
李志¹,
桂建芳^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

基金项目:

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

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出版历程
- 收稿日期: 2009-05-13
- 修回日期: 2009-10-09
- 发布日期: 2010-05-24

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

State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072;
2. Graduate University of the Chinese Academy of Sciences, Beijing 100049

摘要

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。
- 赤点石斑鱼 /
- 卵型芳香化酶 /
- 启动子 /
- 绿色荧光蛋白 /
- 斑马鱼
Abstract: 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 EaCyp19a1a promoter in ovary specific expression of EaCyp19a1a. 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 zebrafish and the embryos were observed termly with fluorescent microscopy for the green fluorescent protein (GFP) expression. 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 development 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.
- Red-spotted grouper /
- Ovarian aromatase /
- Promoter /
- Green fluorescent protein /
- Zebrafish

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参考文献(28)

[1]	[3] Xia W, Zhou L, Gui J F. The linkage structure and phylogeny of DMRT gene family in the teleost fish [J]. Acta Hydrobiologica Sinica, 2009, 33(2): 254-263 [夏伟, 周莉, 桂建芳. 真骨鱼类DMRT 基因家族的连锁结构及其系统发生. 水生生物学报, 2009, 33(2): 254-263]
[1]	Li G L, Liu X C, Lin H R. Effects of 17α-methyltestosterone on sex reversal in red-spotted grouper, Epinephelus akaara [J]. Journal of Fisheries of China, 2006, 30(2): 145-150 [李广丽, 刘晓春, 林浩然. 17α-甲基睾酮对赤点石斑鱼性逆转的影响. 水产学报, 2006, 30(2): 145-150]
[2]	Hong W S, Zhang Q Y, Gong M J, et al. Masculinization of red grouper (Epinephelus akaara) induced by exogenous hormone [J] Journal of Oceanography in Taiwan Strait. 1994, 13(4): 375-381 [洪万树, 张其永, 公茂军, 等. 外源激素诱导赤点石斑鱼雄性化. 台湾海峡, 1994, 13(4): 375-381]
[2]	Xing C G, Jin S, Yuan S P. Sex reversal of Grouper (Epinephelus sp.) [J]. Fisheries Science, 2006, 25(4): 214- 216 [刑晨光, 金珊, 袁思平. 石斑鱼性逆转研究现状. 水产科学, 2006, 25(4): 214-216]
[3]	[5] Li G L, Liu X C, Lin H R. Aromatase inhibitor letrozole induces sex inversion in the protogynous red spotted grouper (Epinephelus akaara) [J]. Acta Physiological Sinica, 2005 57(4): 473-479 [李广丽, 刘晓春, 林浩然. 芳香化酶抑制剂letrozole 对赤点石斑鱼(Epinephelus akaara)性逆转的作用. 生理学报, 2005, 57(4): 473-479]
[4]	[6] Guiguen Y, Fostier A, Piferrer F, et al. Ovarian aromatase and estrogens: A pivotal role for gonadal sex differentiation and sex change in fish [J]. Gen. Comp. Endocrinol, 2009, doi: 10.1016/j.ygcen.2009.03.002
[5]	[7] Piferrer F, Blazquez M. Aromatase distribution and regulation in fish [J]. Fish Physiol. Biochem., 2005, 31: 215-226
[6]	[8] Tchoudakova A, Kishida M, Wood E, et al. Promoter characteristics of two cyp19 genes differentially expressed in the brain and ovary of teleost fish [J]. J. Steroid. Biochem. Mol. Biol., 2001, 78: 427-439
[7]	[9] Tong S K, Mouriec K, Kuo M W, et al. A Cyp19a1b-GFP (Aromatase B) transgenic zebrafish line that expresses GFP in radial glial cells [J]. Genesis. 2009, 47(2): 67-73
[8]	[10] Tanaka M, Fukada S, Matsuyama M, et al. Structure and promoter analysis of the cytochrome P450 aromatase gene of the teleost fish, Medaka, Oriyzias latipes [J]. J. Biochem., 1995, 117: 719-725
[9]	[11] Kazeto Y, Ijiri S, Place A R, et al. The 5′-flanking regions of CYP19A1A1 and CYP19A1A2 in zebrafish [J]. Biochem. Biophys. Res. Commun., 2001, 288: 503-508
[10]	[12] Yoshiura Y, Senthilkumaran B, Watanabe M, et al. Synergistic expression of Ad4BP/SF-1 and cytochrome P-450 aromatase (ovarian type) in the ovary of Nile tilapia, Oreochromis niloticus, during vitellogenesis suggests transcriptional interaction [J]. Biol. Reprod., 2003, 68: 1545-1553
[11]	[13] Chang X, Kobayashi T, Senthilkumaran B, et al. Two types of aromatase with different encoding genes, tissue distribution and developmental expression in Nile tilapia (Oreochromis niloticus) [J]. Gen. Comp. Endocrinol., 2005, 141: 101-115
[12]	[14] Galay-Burgos M, Gealy C, Navarro-Martín L, et al. Cloning of the promoter from the gonadal aromatase gene of the European sea bass and identification of single nucleotide polymorphisms [J]. Comp. Biochem. Physiol. A. Mol. Integr. Physiol., 2006, 145: 47-53 [15] Nocillado J N, Elizur A, Avitan A, et al. Cytochrome P450 aromatase in grey mullet: cDNA and promoter isolation; brain, pituitary and ovarian expression during puberty [J]. Mol. Cell. Endocrinol., 2006, 263: 65-78
[13]	[16] Tzchori I, Degani G, Hurvitz A, et al. Cloning and developmental expression of the cytochrome P450 aromatase gene (CYP19) in the European eel (Anguilla anguilla) [J]. Gen. Comp. Endocrinol., 2004, 138: 271-280
[14]	[17] Zhang Y, Zhang W, Zhang L, et al. Two distinct cytochrome P450 aromatases in the orange-spotted grouper (Epinephelus coioides): cDNA cloning and differential mRNA expression [J]. J. Steroid. Biochem. Mol. Biol., 2004, 92: 39-50
[15]	[18] Wang Y, Zhou L, Yao B, et al. Differential expression of thyroid-stimulating hormone beta subunit in gonads during sex reversal of orange-spotted and red-spotted grouper [J]. Mol. Cell. Endocrinol., 2004, 220: 77-88
[16]	[19] Li C J, Zhou L, Wang Y, et al. Molecular and expression characterization of three gonadotropin subunits common α, FSHβ and LHβ in groupers [J]. Mol. Cell. Endocrinol., 2005, 233: 33-46
[17]	[20] Yao B, Zhou L, Wang Y, et al. Differential expression and dynamic changes of SOX3 during gametogenesis and sex reversal in protogynous hermaphroditic fish [J]. J. Exp. Zool. Part. A. Ecol. Genet. Physiol., 2007, 307: 207-219
[18]	[21] Xia W, Zhou L, Yao B, et al. Differential and spermatogenic cell-specific expression of DMRT1 during sex reversal in protogynous hermaphroditic groupers [J]. Mol. Cell. Endocrinol., 2007, 263: 156-172
[19]	[22] Zhou L, Li C J, Wang Y, et al. Identification and characterization of a MBP isoform specific to hypothalamus in orange- spotted grouper (Epinephelus coioides) [J]. J. Chem. Neuroanat., 2007, 34: 47-59
[20]	[23] Wang Y, Zhou L, Li Z, et al. Molecular cloning and expression characterization of ApoC-I in the orange-spotted grouper [J]. Fish Physiol. Biochem., 2008, 34: 339-348
[21]	[24] Huang W, Zhou L, Li Z, et al. Expression pattern, cellular localization and promoter activity analysis of ovarian aromatase (Cyp19a1a) in protogynous hermaphrodite red-spotted grouper [J]. Mol. Cell. Endocrinol., 2009, 307: 224-236
[22]	[25] Bulun S E, Sebastian S, Takayama K, et al. The human Cyp19 (aromatase P450) gene: update on physiologic roles and genomic organization of promoters [J]. J. Steroid. Biochem. Mol. Biol., 2003, 86: 219-224
[23]	[26] Hinshelwood M M, Smith M E, Murry B A, et al. A 278 bp region just upstream of the human CYP19 (aromatase) gene mediates ovary-specific expression in transgenic mice [J]. Endocrinology, 2000, 141: 2050-2053
[24]	[27] Stocco C. Aromatase expression in the ovary: Hormonal and molecular regulation [J]. Steroids, 2008, 73: 473-487
[25]	[28] Meng A, Tang H, Ong B A, et al. Promoter analysis in living zebrafish embryos identifies a cis-acting motif required for neuronal expression of GATA-2 [J]. Proc. Natl. Acad. Sci. U. S. A., 1997, 94: 6267-6272
[26]	[29] Long Q, Meng A, Wang H, et al. GATA-1 expression pattern can be recapitulated in living transgenic zebrafish using GFP reporter gene [J]. Development, 1997, 124: 4105-4111

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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

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

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