食蚊鱼CYP19b基因克隆及环境激素暴露对其表达的影响

赖静萍; 闫月明; 温茹淑; 方展强

doi:10.7541/2016.60

食蚊鱼CYP19b基因克隆及环境激素暴露对其表达的影响

1.
华南师范大学生命科学学院, 广东省高等学校生态与环境科学重点实验室, 广州 510631
2.
嘉应学院生命科学学院, 梅州 514015

基金项目:

广东省科技计划项目(2012B030800006)资助

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出版历程
- 收稿日期: 2015-05-18
- 修回日期: 2015-11-24
- 发布日期: 2016-05-24

MOLECULAR CLONING AND MRNA EXPRESSION OF CYP19B GENE INDUCED BY ENVIRONMENTAL HORMONES IN THE MOSQUITOFISH (GAMBUSIA AFFINIS)

1.
Key Laboratory of Ecology and Environmental Science in Guangdong Higher Education, College of Life Science, South China Normal University, Guangzhou 510631, China;
2.
College of Life Science, Jiaying University, Meizhou 514015, China

Funds:

[Supported by the Science and Technology Project of Guangdong (No. 2012B030800006)

摘要

摘要: 为探讨17-雌二醇(E2)、17-甲基睾酮(MT)和雄烯二酮(AED)暴露对食蚊鱼脑组织CYP19b表达的影响,首次克隆和分析了食蚊鱼(Gambusia affinis) CYP19b cDNA的全系列。采用静水暴露方法, 分别设置2、10、50和100 ng/L E2、MT和AED各4个实验组, 并设置对照组和平行组, 定量测定了1、3、5和7d脑组织中的CYP19b mRNA表达水平的变化。成功克隆了食蚊鱼CYP19b基因全长, 获得基因片段总长1670 bp, ORF(OpenReading Frame)是从52 bp到1554 bp, 共编码501个氨基酸。通过与其他相关鱼类CYP19b基因编码的氨基酸序列相似性作比较, 食蚊鱼CYP19b基因氨基酸序列的同源性很高。结果显示, 短时间的暴露, E2较低浓度组(2和10 ng/L)对食蚊鱼作用不明显, 但E2相对较高浓度组(50和100 ng/L)能显著地上调食蚊鱼CYP19b基因的表达(P0.01)。暴露早期MT对CYP19b表达无明显影响, 随着暴露时间的持续延长, 除了MT较高浓度组(50 ng/L)外, 其他各浓度组MT对CYP19b的表达有显著的抑制作用(P0.01), 并呈一定的剂量相关关系。AED作为雄激素类似物与MT的作用效果相似, 相对较高浓度(50和100 ng/L)的AED抑制CYP19b的表达(P0.01)。研究结果初步表明, 3种典型环境激素对食蚊鱼CYP19b基因表达的影响十分显著, 但不同的暴露浓度及不同的取样时间点可能表现为促进或抑制CYP19b基因的表达。
- 环境激素 /
- CYP19b基因 /
- mRNA表达 /
- 雌二醇 /
- 甲基睾酮 /
- 雄烯二酮 /
- 食蚊鱼
Abstract: The current study investiaged effects of 17-estradiol (E2), 17-methyl testosterone (MT) and androgen analogues androstendione (AED) exposure on brain tissue CYP19b gene expression in Gambusia affinis. The G. affinis CYP19b cDNA of full sequence was cloned and analyzed for the first time. G. affinis were randomly divided into five groups, with one control group and four experimental groups (2, 10, 50 and 100 ng/L E2, MT and AED, respectively). The CYP19b mRNA expression levels in fish brain tissues were determined after 1, 3, 5, and 7d exposure, respectively. The G. affinis CYP19b cDNA of full sequence was cloned, from which 1670 bp nucleotides were obtained, encoding 501 amino acids with an open reading frame (ORF) from 52 bp to 1554 bp. The comparison among the similarity amino acid sequence of CYP19b with other related fishes supports high similarity between species. The results indicated that E2 at high concentrations (50 ng/L and 100 ng/L groups) significantly up-regulated CYP19b gene expression (P0.01) but E2 at low concentration (2 ng/L and 10 ng/L groups) did not. MT had no significant effect on CYP19b expression in early stages, but it significantly inhibited CYP19b expression with prolonged treatment except the relatively high concentrations of MT (50 ng/L group) with the characerisitc of dose correlativity. As androgen analogue, AED was found to have similar effects with MT, and the relatively high concentrations (50 ng/L and 100 ng/L groups) of AED inhibited the CYP19b expression (P0.01). The results indicated the pivotal role of three typical environmental hormones under different concentration and various time points on the expression of CYP19b.
- Environmental hormone /
- CYP19b gene /
- mRNA expression /
- 17-estradiol /
- 17-methyl testosterone /
- Androstendione /
- Gambusia affinis

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参考文献(31)

[1]	Seralin G E, Moslemi S. Aromatase inhibitors: past, present and future [J]. Molecular and Cellular Endocrinology, 2001, 178(12): 117131
[2]	John M T, Sonja G, Jared A, et al. Developmental expression of cytochrome P450 aromatase genes (CYP19a and CYP19b) in zebrafish fry (Danio rerio) [J]. Journal of Experimental Zoology, 2001, 290(5): 475483
[3]	Yu J H, Tang Y K, Li J L. Cloning, structure and expression pattern of the P450 aromatase genes in rice field eel (Monopterus albus) [J]. Biochemical Genetics, 2008, 46(5): 267280
[4]	Yang Y P. Studies on reproduction physiological function of the P450aron in rockfish (Sebactes schlegeli) [D]. Thesis for Master of Science. Ocean University of China, Qingdao. 2010 [杨艳平. P450芳香化酶在许氏平鲥繁殖生理中的作用研究. 硕士学位论文. 中国海洋大学, 青岛. 2010]
[5]	Halm S, Pounds N, Maddix S. Exposure to exogenous 17 beta-oestradiol disrupts P450aromB mRNA expression in the brain and gonad of adult fathead minnows (Pimephales promelas) [J]. Aquatic Toxicology, 2002, 60(34): 285299
[6]	Contractor R G, Foran C M, Li S F, et al. Evidence of gender- and tissue-specific promoter methylation and the potential for ethinylestradiol-induced changes in japanese medaka (Oryzias latipes) estrogen receptor and aromatase genes [J]. Journal of Toxicology and Environmental Health Part A, 2004, 67(1): 122
[7]	Kishida M, McLellan M, Miranda J A, et al. Estrogen and xenoestrogens upregulate the brain aromatase isoform (P450aromB) and perturb markers of early development in zebrafish (Danio rerio) [J]. Comparative Biochemistry and Physiology B-Biochemistry Molecular Biology, 2000, 129(23): 261268
[8]	Xie Y P, Fang Z Q. Evaluation of estrogen/androgen pollution by using the mosquitofish target gene expression in Hanxi River, Dongguan City [J]. Acta Hydrobiologica Sinica, 2013, 37(4): 691697 [谢勇平, 方展强. 利用食蚊鱼目标基因转录水平评价东莞寒溪河雌/雄激素物质污染现状. 水生生物学报, 2013, 37(4): 691697]
[9]	Xie Y P, Fang Z Q. Urban sewage induces morphological feminization/masculinization in mosquitofish (Gambusia affinis) [J]. Biological Disaster Science, 2015, 38(2): 106113 [谢勇平, 方展强. 城市废水暴露致食蚊鱼形态雌/雄性化的生物学效应. 生物灾害科学, 2015, 38(2): 106113]
[10]	Yan Y M. Effects of PCBs (Aroclor 1248) exposure on the expression of gonadal cytochmme P450 aromatase (CYP19a) gene and the toxic effect in mosquitofish (Gambusia affinis) [D]. Thesis for Master of Science. South China Normal University, Guangzhou. 2013 [闫月明. 多氯联苯(PCB 1248)暴露对食蚊鱼CYP19a mRNA表达的影响及毒性效应研究.硕士学位论文, 华南师范大学, 广州. 2013]
[11]	Leusch F D L, Vanden Heuvel M R, Laurie A D, et al. Quantification of vitellogenin mRNA induction in mosquitofish (Gambusia affinis) by reverse transcription real-time polymerase chain reaction (RT-PCR) [J]. Biomarkers, 2005, 10(6): 429438
[12]	Gan W. Molecular cloning and mRNA expression of CYP19a gene induced by tributyltin in the mosquitofish (Gambusia affinis) [D]. Thesis for Master of Science. South China Normal University, Guangzhou. 2015 [甘为. 食蚊鱼CYP19a基因的克隆及三丁基锡暴露对其表达的影响. 硕士学位论文. 华南师范大学, 广州. 2015]
[13]	Zhang Y, Zhang L H, Zhang W M. CYP19 genes in Teleosts: origin, evolution, and expression regulation. Chinese Scientific Papers Online, fist time published: 201012-29 [张扬, 张利红, 张为民. 硬骨鱼类芳香化酶基因起源、进化和表达调控. 中国科技论文在线, 首发时间：201012-29]
[14]	Cao M X, Yang Y H, Jiang W B, et al. Cloning and expressional analysis of Cyp19b gene fragment in Gobiocypris rarus [J]. Acta Hydrobiologica Sinica, 2009, 33(4): 749755 [曹梦西, 杨玉慧, 江文波, 等. 稀有逗鲫脑芳香化酶 cDNA片段的克隆与表达分析. 水生生物学报, 2009, 33(4): 749755]
[15]	Xu P, Yu J H, Tang Y K, et al. Molecular cloning of ovarian P450arom gene and its expression in male and female [J]. Journal of Fishery Sciences of China, 2005, 12(5): 541548 [徐跑, 俞菊华, 唐永凯, 等. 黄颡鱼卵巢 P-450arom 基因的克隆及组织表达.中国水产科学, 2005, 12(5): 541548]
[16]	Graham-Lorence S, Amameh B, White R E. A three-dimentional model of aromatase cytochome P450 [J]. Protein Science, 1995, 4(6): 10651080
[17]	Cui J L. Molecular cloning of CYP19 gene and their expression of adult and embryo in two apecies of Loaches [D]. Thesis for Master of Science. Henan Normal University, Xinxiang. 2013 [崔俊莉. 两种泥鳅中CYP19基因的克隆及表达分析. 硕士学位论文. 河南师范大学, 新乡. 2011]
[18]	Chesheko K, Brion F, Le Page, et al. Expression of zebra fish aromatase CYP19a and CYP19b genes in response to the ligands of estrogen receptor and arylhydrocarbon receptor [J]. Toxicological Sciences, 2007, 96(2): 255267
[19]	Menuet A, Pellegrini E, Brion F, et al. Expression and estrogen-dependent regulation of the zebrafish brain aromatase gene [J]. Journal of Comparative Neurology, 2006, 485(4): 304320
[20]	Hinfray N, Palluel O, Turies C, et al. Brain and gonadal aromatase as potential targets of endocrine disrupting chemicals in a model species, the zebrafish (Danio rerio) [J]. Environmental Toxicology, 2006, 21(4): 332337
[21]	Lyssimachou A, Jenssen B M, Arukwe A. Brain cytochrome P450 aromatase gene isoforms and activity levels in Atlantic salmon after waterborne exposure to nominal environmental concentrations of the pharmaceutical enhinylestradiol and antifoulant tributyltin [J]. Toxicological Sciences, 2006, 91(1): 8292
[22]	Wu P. Effects and mechanisms of tributyltin on sexual chatacteristics and reproduction of guppy (Poecilia reticulate) [D]. Thesis for Master of Science. Ocean University of China, Qingdao. 2013 [吴鹏. 三丁基锡对孔雀鱼(Poecilia reticulata)性征和生殖的影响. 硕士学位论文. 中国海洋大学, 青岛. 2013]
[23]	Xiong T T, Fang Z Q. Target gene expression in Gambusia affinis exposed to 17-estradiol, Bisphenol A and Benzo [a] Pyrene [J]. Journal of Biology, 2015, 32(5): 1924 [熊甜甜, 方展强. 雌二醇、双酚A和苯并[a]芘混合物对食蚊鱼目标基因表达的影响. 生物学杂志, 2015, 32(5): 1924]
[24]	Kazeto Y, Trant J M. Molecular biology of channel catfish brain cytochrome P450 aromatase (CYP19A2): cloning, preovulatory induction of gene expression, hormonal gene regulation and analysis of promoter region [J]. Journal of Molecular Endocrinology, 2005, 35(3): 571583
[25]	Gloria V Callard, Anna V Tchoudakova, Mitsuyo Kishida, et al. Differential tissue distribution, developmental programming, estrogen regulation and promoter characteristics of CYP19 genes in teleost fish [J]. Journal of Steroid Biochemistry Molecular Biology, 2001, 79(15): 305314
[26]	Fan J J, Xu S Q, Fang Z Q. Target gene expression and morphological masculinization in mosquitofish (Gambusia affinis) exposed to 17-methyl testosterone [J]. Journal of Fisheries of China, 2013, 37(1): 915 [范俊杰, 徐少群, 方展强, 等. 17-甲基睾酮对食蚊鱼形态雄性化及目标基因表达的影响. 水产学报, 2013, 37(1): 915]
[27]	Martina Fenske, Helmut Segner. Aromatase modulation alters gonadal differentiation in developing zebrafish (Danio rerio) [J]. Aquatic Toxicology, 2004, 67(2): 105126
[28]	Zhang Y. Cytochrome P450aromatases in the orange-spotted grouper (Epinephelus coioides): cDNA cloning and regulation of expression [D]. Thesis for Doctor of Philosophy. Sun Yat-sen University, Guangzhou. 2011 [张勇. 斜带石斑鱼P450芳香化酶基因克隆及其表达调控的研究. 博士学位论文. 中山大学, 广州.2004]
[29]	Denton E W, Howell W M. Allison J J, et al. Masculinization of female mosquitofish by exposure to plant sterols and Mycobacterium smegmatis [J]. Bulletin of Environmental Contamination and Toxicology, 1985, 35(4): 627632
[30]	Stanko J P, Angus R A. In vivo assessment of the capacity of androstenedione to masculinize female mosquitofish (Gambusia affinis) exposed through dietary and static renewal methods [J]. Environmental Toxicology and Chemistry, 2007, 26(5): 920926.
[31]	Fan J J. Vitellogenin gene expression and morphological masculinization in female western mosquitofish (Gambusia affinis) exposed to androgens and progesterone [D]. Thesis for Master of Science. South China Normal University, Guangzhou. 2012 [范俊杰. 雄激素和孕激素暴露对雌性食蚊鱼形态雄性化和生殖基因的影响. 硕士学位论文, 华南师范大学, 广州. 2012]