興國紅鯉ERs、AR基因的克隆表達及EE2、MT暴露對幼魚肝中ERs、AR基因表達的影響
發(fā)布時間：2019-06-07 09:24
【摘要】：環(huán)境雌激素/雄激素具有類激素的作用,通過各種途徑進入生物體內能夠模仿機體內源性激素干擾生物包括人類的內分泌活動。乙炔雌二醇(17α-ethynylestradiol,EE2)屬于環(huán)境雌激素,環(huán)境中的甲基睪酮(17α-methyltestosterone,MT)屬于環(huán)境雄激素,這兩種環(huán)境激素進入機體主要是通過雌激素受體(Estrogen Receptors,ERs)和雄激素受體(Androgen Receptor,AR)來干擾機體內正常的分泌等生理活動。本研究克隆了興國紅鯉(Cyprinus carpio var singuonensis)雌激素受體ERs四種亞型ERα、ERβ、ERβ1、ERβ2和雄激素受體AR基因的全長cDNA序列及卵黃蛋白原基因(vitellogenin,vtg)的部分cDNA序列。ERα全長2109 bp,包含189bp的5’非翻譯區(qū)(Untranslated Regions,UTR)和237 bp的3’UTR,開放閱讀框(Opening reading frame,ORF)為1683 bp,編碼560個氨基酸;ERβ全長2147 bp,包含330 bp的5’UTR、編碼559個氨基酸的1680 bp ORF和137 bp的3’UTR;ERβ1全長2300 bp,包含364 bp的5’UTR、編碼585個氨基酸的1758 bp ORF和178 bp的3’UTR;ERβ2全長2007 bp,包含156 bp的5’UTR、編碼544個氨基酸的1635bp ORF和216 bp 3’UTR。AR基因全長3229 bp,包括5’UTR 104 bp,3’UTR 485 bp及2540 bp的ORF。AR編碼區(qū)編碼846個氨基酸。氨基酸序列多重比對分析表明,興國紅鯉ERs和AR基因編碼的氨基酸分別與其它幾種鯉科魚類如鯉(Cyprinus carpio)、彭澤鯽(Carassius auratus var.pengze)、臺灣鏟頜魚(Onychostoma barbatulum)、稀有泩鯽(Gobiocypris rarus)和銀鯽(Carassius auratus gibelio)等的ER和AR氨基酸序列具有較高的相似性。采用N-J法分別對興國紅鯉及其它脊椎動物的ERs和AR進行系統發(fā)生分析。結果表明,在高等脊椎動物和硬骨魚類中ER聚為兩類,魚類及哺乳動物的ERα聚成一類,而ERβ、ERβ1和ERβ2聚為一類;興國紅鯉的四種ER亞型分別與鯉的四種相應亞型的親緣關系最近。AR在分子進化上魚類和高等脊椎動物分別聚為一枝,興國紅鯉的AR與銀鯽、金魚、彭澤鯽聚為一枝,遺傳距離最小,和哺乳動物人、大白鼠在兩個明顯不同的分枝。采用實時熒光定量PCR(Quantitative real-time PCR,qRT-PCR)對各組織中ERs、AR和vtg的表達情況進行檢測,結果表明,四種ERs亞型在雌雄成體組織中呈現差異表達,雌性個體中,肝、卵巢和腸中四種ERs的表達量均較高;雄性個體中,ERα和ERβ主要在肝中表達,ERβ1、ERβ2分別在腸和精巢中的表達量最高。AR在雌性個體的肝、卵巢、腎、肌肉和端腦中有表達,其中肝中的表達量最高;vtg主要在肝、端腦和鰓中表達。將150日齡的興國紅鯉雌性幼魚分別暴露在0.01、0.1和1 nmol/L的17α-乙炔基雌二醇(17α-Ethinylestradiol,EE2)中4周,檢測其肝中四種ERs基因的表達變化。結果表明,在EE2中暴露1~2周后,興國紅鯉雌性幼魚肝中ERα基因的表達水平有極顯著的提升;各濃度EE2能持續(xù)顯著促進其肝中ERβ的表達;在1~2周內各濃度EE2對ERβ1表達量有所抑制;第1周EE2能夠抑制ERβ2基因mRNA的表達,并在0.01 nmol/L時抑制作用達到了顯著的水平。以上結果說明興國紅鯉雌性幼魚受EE2暴露后其肝中不同的雌激素受體亞型表達量的變化不同。且ERα可以作為EE2短期(1~2周)的敏感性生物學標志物。同樣的,將150日齡的興國紅鯉雌性幼魚在50μg/L的甲基睪酮(17α-methyltestosterone,MT)暴露4周后,采用qRT-PCR檢測了肝中AR和vtg的表達情況。在處理第1、2周,AR的表達受到一定的抑制,而在第3、4周其表達量升高,但其表達無顯著性變化;而vtg的表達量在第3、4周均有極顯著的升高。相對于AR基因,vtg可以作為敏感性生物學標志物。本研究首次克隆得到了興國紅鯉雌激素受體ERα、ERβ、ERβ1、ERβ2和雄激素受體AR基因的全長cDNA序列,并通過多重序列比對和系統進化樹分析驗證了我們所得到的序列的正確性。此外,本實驗使用qRT-PCR技術檢測了ERs和AR基因在興國紅鯉各組織中的表達情況,并對興國紅鯉雌性幼魚進行了EE2和MT暴露實驗,檢測了其肝中ERs和AR基因的表達情況。結果表明,EE2暴露對不同亞型的ERs mRNA的表達均有影響,且產生的影響不同,而MT暴露對興國紅鯉幼魚肝AR mRNA的表達量無顯著影響,但對vtg的mRNA表達量有一定的影響。
[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.
【學位授予單位】：上海海洋大學
【學位級別】：碩士
【學位授予年份】：2016
【分類號】：S917.4
本文編號：2494687
[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.
【學位授予單位】：上海海洋大學
【學位級別】：碩士
【學位授予年份】：2016
【分類號】：S917.4
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