兴国红鲤ERs、AR基因的克隆表达及EE2、MT暴露对幼鱼肝中ERs、AR基因表达的影响

发布时间：2019-06-07 09:24

【摘要】：环境雌激素/雄激素具有类激素的作用,通过各种途径进入生物体内能够模仿机体内源性激素干扰生物包括人类的内分泌活动。乙炔雌二醇(17α-ethynylestradiol,EE2)属于环境雌激素,环境中的甲基睾酮(17α-methyltestosterone,MT)属于环境雄激素,这两种环境激素进入机体主要是通过雌激素受体(Estrogen Receptors,ERs)和雄激素受体(Androgen Receptor,AR)来干扰机体内正常的分泌等生理活动。本研究克隆了兴国红鲤(Cyprinus carpio var singuonensis)雌激素受体ERs四种亚型ERα、ERβ、ERβ1、ERβ2和雄激素受体AR基因的全长cDNA序列及卵黄蛋白原基因(vitellogenin,vtg)的部分cDNA序列。ERα全长2109 bp,包含189bp的5’非翻译区(Untranslated Regions,UTR)和237 bp的3’UTR,开放阅读框(Opening reading frame,ORF)为1683 bp,编码560个氨基酸;ERβ全长2147 bp,包含330 bp的5’UTR、编码559个氨基酸的1680 bp ORF和137 bp的3’UTR;ERβ1全长2300 bp,包含364 bp的5’UTR、编码585个氨基酸的1758 bp ORF和178 bp的3’UTR;ERβ2全长2007 bp,包含156 bp的5’UTR、编码544个氨基酸的1635bp ORF和216 bp 3’UTR。AR基因全长3229 bp,包括5’UTR 104 bp,3’UTR 485 bp及2540 bp的ORF。AR编码区编码846个氨基酸。氨基酸序列多重比对分析表明,兴国红鲤ERs和AR基因编码的氨基酸分别与其它几种鲤科鱼类如鲤(Cyprinus carpio)、彭泽鲫(Carassius auratus var.pengze)、台湾铲颌鱼(Onychostoma barbatulum)、稀有泩鲫(Gobiocypris rarus)和银鲫(Carassius auratus gibelio)等的ER和AR氨基酸序列具有较高的相似性。采用N-J法分别对兴国红鲤及其它脊椎动物的ERs和AR进行系统发生分析。结果表明,在高等脊椎动物和硬骨鱼类中ER聚为两类,鱼类及哺乳动物的ERα聚成一类,而ERβ、ERβ1和ERβ2聚为一类;兴国红鲤的四种ER亚型分别与鲤的四种相应亚型的亲缘关系最近。AR在分子进化上鱼类和高等脊椎动物分别聚为一枝,兴国红鲤的AR与银鲫、金鱼、彭泽鲫聚为一枝,遗传距离最小,和哺乳动物人、大白鼠在两个明显不同的分枝。采用实时荧光定量PCR(Quantitative real-time PCR,qRT-PCR)对各组织中ERs、AR和vtg的表达情况进行检测,结果表明,四种ERs亚型在雌雄成体组织中呈现差异表达,雌性个体中,肝、卵巢和肠中四种ERs的表达量均较高;雄性个体中,ERα和ERβ主要在肝中表达,ERβ1、ERβ2分别在肠和精巢中的表达量最高。AR在雌性个体的肝、卵巢、肾、肌肉和端脑中有表达,其中肝中的表达量最高;vtg主要在肝、端脑和鳃中表达。将150日龄的兴国红鲤雌性幼鱼分别暴露在0.01、0.1和1 nmol/L的17α-乙炔基雌二醇(17α-Ethinylestradiol,EE2)中4周,检测其肝中四种ERs基因的表达变化。结果表明,在EE2中暴露1~2周后,兴国红鲤雌性幼鱼肝中ERα基因的表达水平有极显著的提升;各浓度EE2能持续显著促进其肝中ERβ的表达;在1~2周内各浓度EE2对ERβ1表达量有所抑制;第1周EE2能够抑制ERβ2基因mRNA的表达,并在0.01 nmol/L时抑制作用达到了显著的水平。以上结果说明兴国红鲤雌性幼鱼受EE2暴露后其肝中不同的雌激素受体亚型表达量的变化不同。且ERα可以作为EE2短期(1~2周)的敏感性生物学标志物。同样的,将150日龄的兴国红鲤雌性幼鱼在50μg/L的甲基睾酮(17α-methyltestosterone,MT)暴露4周后,采用qRT-PCR检测了肝中AR和vtg的表达情况。在处理第1、2周,AR的表达受到一定的抑制,而在第3、4周其表达量升高,但其表达无显著性变化;而vtg的表达量在第3、4周均有极显著的升高。相对于AR基因,vtg可以作为敏感性生物学标志物。本研究首次克隆得到了兴国红鲤雌激素受体ERα、ERβ、ERβ1、ERβ2和雄激素受体AR基因的全长cDNA序列,并通过多重序列比对和系统进化树分析验证了我们所得到的序列的正确性。此外,本实验使用qRT-PCR技术检测了ERs和AR基因在兴国红鲤各组织中的表达情况,并对兴国红鲤雌性幼鱼进行了EE2和MT暴露实验,检测了其肝中ERs和AR基因的表达情况。结果表明,EE2暴露对不同亚型的ERs mRNA的表达均有影响,且产生的影响不同,而MT暴露对兴国红鲤幼鱼肝AR mRNA的表达量无显著影响,但对vtg的mRNA表达量有一定的影响。
[Abstract]:The environmental estrogen/ androgen has the role of a hormone, and can imitate the endocrine activity of the organism, including human, through various routes into the organism. ethinyl estradiol (EE2) is an environmental estrogen, and methyltestosterone (MT) in the environment belongs to the environment, and the two environmental hormones enter the body mainly through the estrogen receptors (ERs) and the androgen receptor (Androgen Receptor, AR) to interfere with physiological activities such as normal secretion in the body of the machine. The full-length cDNA sequence of ER, ER, ER-1, ER-1, ER-2 and androgen receptor AR gene, and the partial cDNA sequence of vitellogen, vtg, were cloned. The total length of ER was 2109 bp, which contained 188bp of 5 'non-translated region (UTR) and 237 bp of 3' UTR. The open reading frame (ORF) was 1683 bp, encoding 560 amino acids. The total length of ER was 2147 bp, containing 330 bp of 5 'UTR, encoding the 1680 bp ORF of 559 amino acids and 3' UTR of 137 bp; The total length of ER-1 is 2300 bp, which contains 364 bp of 5 'UTR, a 1758bp ORF that encodes 585 amino acids, and a 3' UTR of 178 bp; the total length of ER-2 is 2007 bp, comprising a 156bp 5 'UTR, a 1635 bp ORF encoding 544 amino acids, and a 216 bp 3' UTR. The full length of the AR gene is 3229 bp, including 5 'UTR 104 bp, The 3 'UTR 485 bp and 2540 bp ORF. The AR coding region encodes 846 amino acids. The analysis of the multiple ratios of the amino acid sequence showed that the amino acids encoded by the ERs and AR genes of the red carp of the Xingguo were the same as the other species of Cyprinus carpio, Carassius auratus var.pengze and Onychostoma barbatum, respectively. The ER and AR amino acid sequences of the rare Carassius auratus and the Carassius auratus gibelio have high similarity. The ERs and AR of Xingguo red carp and other vertebrates were analyzed by N-J method. The results showed that ER, ER, ER and ER-1 and ER-1, ER-1, ER-2 and ER-1, ER-1, ER-1, ER-2, and ER-1, ER-1, ER-2, and ER-1, and ER-1, ER-1 and ER-2 in high-and hard-bone fish were one class, and the relationship between the four ER subtypes of Xingguo red carp and the four corresponding subtypes of Cyprinus carpio was the most recent. AR is the one of the fish and the higher vertebrates in the molecular evolution, and the AR of the Xingguo red carp is the same as that of the crucian carp, the goldfish and the penser, the genetic distance is the smallest, and the mammal and the rat are branched in two distinct branches. The expression of ERs, AR and vtg in tissues was detected by real-time fluorescence quantitative PCR (qRT-PCR). The results showed that the expression of four types of ERs in male and female adult tissues was higher, and in the female, the expression of the four ERs in the liver, the ovary and the intestine was higher; in the male, ER-1 and ER-2 were mainly expressed in the liver, and the expression of ER-1 and ER-2 in the intestine and testis was the highest. AR is expressed in the liver, the ovary, the kidney, the muscle and the head of the female, with the highest expression in the liver; vtg is mainly expressed in the liver, the brain and the brain. The expression of four ERs genes in the liver was detected by exposure to 17-ethinyl estradiol (EE2) of 0.01, 0.1 and 1 nmol/ L, respectively, at the age of 150 days. The results showed that the expression level of ER-1 gene in the female juvenile of Xingguo red carp was significantly improved after 1-2 weeks of exposure in EE2. In the first week, EE2 could inhibit the expression of ER-2 gene mRNA and inhibit the expression of ER-2 gene at 0.01 nmol/ L. The above results show that the changes of the expression of different estrogen receptor subtypes in the liver of the female juvenile of Xingguo red carp after exposure to EE2 are different. And the ER antigen can be used as the sensitive biological marker of the EE2 short-term (1-2 weeks). Similarly, the expression of AR and vtg in the liver was detected by qRT-PCR after 4 weeks of exposure to 50. m u.g/ L of methyltestosterone (MT). In the first and second week, the expression of AR was inhibited, while in the third and fourth weeks, the expression of AR was increased, but the expression of vtg was significantly increased in the 3rd and 4th week. Vtg can be used as a sensitive biological marker with respect to the ar gene. The full-length cDNA sequence of ER-, ER-1, ER-1, ER-2 and androgen receptor AR gene was obtained for the first time in this study, and the correctness of the sequence obtained was verified by multi-sequence comparison and phylogenetic tree analysis. In addition, the expression of ERs and AR gene in the tissues of Xingguo red carp was detected by using qRT-PCR technique, and the expression of ERs and AR genes in the liver of the red carp of Xingguo was detected by EE2 and MT exposure experiments. The results showed that EE2 exposure had an effect on the expression of ERs mRNA in different subtypes, and the effect of MT exposure on the expression of the liver AR mRNA in the young Chinese carp was not significantly affected, but the expression of the mRNA of vtg had a certain effect.
【学位授予单位】：上海海洋大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S917.4

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