金钱鱼卵黄蛋白原基因克隆及EE2诱导卵黄蛋白原合成的研究
发布时间：2018-09-19 19:09
【摘要】：卵黄蛋白原(vitellogenin,Vtg)是卵黄蛋白的前体,通过下丘脑-垂体-性腺(hypothalamic-pituitary-gonadal,HPG)轴激活雌激素受体来合成。卵生脊椎动物的卵可以储蓄大量的卵黄,卵黄的主要成分是被称之为雌性成熟期血清蛋白的卵黄蛋白原。Vtg是合成卵黄过程中的关键性物质,是鱼类生殖过程中重要的生殖蛋白,在卵生动物生殖和发育过程中起重要作用[1]。研究卵黄蛋白原的合成机制不但可以补充完善鱼类生理学、发育生物学等基础研究,同时,掌握卵黄蛋白原体内体外合成方式和机理,可以为鱼类繁殖提供理论依据。为更好地了解金钱鱼(Scatophagus argus)卵黄蛋白原合成机制及能更精确地分析外源性雌激素对鱼类合成卵黄蛋白原的影响,本文以金钱鱼为研究对象,运用基因克隆、荧光定量PCR和H.E.染色等方法,并从泄殖孔腹腔注射EE2(17α-ethynylestradiol)和体外EE2暴露肝原代细胞两个角度对金钱鱼在EE2处理下的合成规律做了进一步的分析。本研究使用RACE克隆得到金钱鱼金钱鱼卵黄蛋白原基因的三种亚型的cDNA序列,金钱鱼vtgAa的GenBank登录号为KY676847,cDNA全长5360 bp,开放阅读框(ORF)为5091 bp,共编码1696个氨基酸。预测金钱鱼VtgAa蛋白分子量为186.07 ku,等电点为9.16,N端的前15个氨基酸为信号肽(图1-3)。金钱鱼vtgAb的GenBank登录号为KY654346,cDNA全长5346 bp,ORF为5100 bp,共编码1699个氨基酸。预测金钱鱼VtgAb蛋白分子量为186.37 ku,等电点为9.24,N端的前15个氨基酸为信号肽(图1-3)。金钱鱼vtgC的GenBank登录号为KY676848,cDNA全长4244 bp,ORF为3828bp,共编码1275个氨基酸。预测蛋白分子量142.87 ku,等电点6.43,信号肽为N端前15个氨基酸(图1-3)。根据H.E.染色的结果显示:本实验所处理的金钱鱼皆处于性腺发育过程中的未成熟期,满足EE2注射实验的前提条件。因此推测注射后合成的卵黄蛋白原主要为注射EE2的诱导结果。本实验使用灌流法从金钱鱼肝脏组织上分离得到金钱鱼肝细胞[2](图3-1),将分离得到的肝细胞进行原代培养至第三代后使用EE2暴露处理。根据RT-qPCR分析结果显示,在不同的采样时间及不同剂量的EE2注射条件下,腹腔注射EE2的肝组织中vtgAb基因表达均高于其他两种亚型,选取vtg Ab作为mRNA表达水平检测的主要基因(图2-5)。根据注射EE2后肝组织中vtgAb的RT-qPCR结果显示,vtgAb在EE2浓度为1.0μg/g和10.0μg/g处理时表达水平较高,而在浓度为0.01μg/g和0.1μg/g时表达水平较低(图2-6)。在表达时间上:24h时vtgAb表达水平较低,48h时表达量逐渐升高并在72h达到最高,在96h时表达量呈下降趋势。金钱鱼肝细胞内vtgAb基因的表达量在EE2暴露浓度为10-8 mol/L、10-7 mol/L、10-6 mol/L、10-5 mol/L时表达量较高,其中10-5 mol/L时的表达量较低于10-6 mol/L时的表达量(图3-2)。而在EE2暴露浓度为10-11 mol/L时,表达量最低。从表达时间上来看:vtg Ab基因从24h开始表达,在48h表达量逐渐升高并在72h时达到最高,在96h时表达量下降至较低于48h表达量,我们推测EE2诱导了肝脏内vtgAb基因的表达。
[Abstract]:Vitellogenin (Vtg) is a precursor of vitellin, synthesized by activating estrogen receptors on the hypothalamic-pituitary-gonadal (HPG) axis. Eggs of oviparous vertebrates can deposit large amounts of yolk, the main component of which is vitellin, known as the serum protein of female maturity. The key substance in the process of yolk synthesis is the important reproductive protein in fish reproduction, which plays an important role in the process of oocyte reproduction and development [1].To study the mechanism of yolk proteogen synthesis can not only complement and perfect the basic research of fish physiology and developmental biology, but also master the synthesis of yolk proteogen in vivo and in vitro. In order to better understand the mechanism of yolk proteogen synthesis in Scatophagus Argus and more accurately analyze the effect of exogenous estrogens on fish yolk proteogen synthesis, we used gene cloning, fluorescence quantitative PCR and H.E. staining to study the effect of exogenous estrogens on fish yolk proteogen synthesis. In this study, three subtypes of the yolk proteogen gene of goldfish were cloned by RACE and the gene bank of goldfish vtgAa was logged in. It is predicted that the molecular weight of VtgAa protein is 186.07 ku, the isoelectric point is 9.16, and the first 15 amino acids at the N terminal are signal peptides (Fig. 1-3). The GenBank login number of vtgAb is KY654346, the full length of the cDNA is 5346 bp, and the ORF is 5100 bp, which encodes 1699 amino acids. Acid. The predicted molecular weight of VtgAb protein is 186.37 ku, the isoelectric point is 9.24, and the first 15 amino acids at the N-terminal are signal peptides (Fig. 1-3). The GenBank login number of vtgC is KY676848, the full length of the cDNA is 4244 bp, and the ORF is 3828 bp, encoding a total of 1275 amino acids. The predicted molecular weight of VtgAb protein is 142.87 ku, the isoelectric point is 6.43, and the signal peptide is the first 15 amino acids at the N-terminal (Fig. 1-1). 3) According to the results of H.E. staining, the goldfish treated in this experiment were all in the immature stage of gonadal development, which met the precondition of EE2 injection experiment. Therefore, it was speculated that the vitellogenin synthesized after injection was mainly induced by injection of EE2. In this experiment, goldfish was isolated from liver tissue of goldfish by perfusion method. Hepatocytes [2] (Fig. 3-1) were cultured to the third generation and then exposed to EE2. According to the results of RT-q PCR analysis, the expression of vtgAb gene in liver tissue injected with EE2 was higher than that of other two subtypes under different sampling time and different dosage of EE2 injection. VtgAb was selected as mRNA expression. The results of RT-q PCR showed that the expression level of vtgAb in liver tissues was higher at EE2 concentrations of 1.0 and 10.0 ug/g, but lower at concentrations of 0.01 ug/g and 0.1 ug/g (Fig. 2-6). The expression level of vtgAb was lower at 24 h and gradually increased at 48 h. The expression level of vtgAb gene in liver cells was higher at EE2 exposure concentration of 10-8 mol/L, 10-7 mol/L, 10-6 mol/L and 10-5 mol/L, and the expression level at 10-5 mol/L was lower than that at 10-6 mol/L (Fig. 3-2). From the expression time point of view, the expression of VTG Ab gene began at 24 hours, gradually increased at 48 hours and reached the highest at 72 hours, and decreased to less than 48 hours at 96 hours. We speculated that EE2 induced the expression of VTG Ab gene in liver.
【学位授予单位】：上海海洋大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S917.4
本文编号：2251057
[Abstract]:Vitellogenin (Vtg) is a precursor of vitellin, synthesized by activating estrogen receptors on the hypothalamic-pituitary-gonadal (HPG) axis. Eggs of oviparous vertebrates can deposit large amounts of yolk, the main component of which is vitellin, known as the serum protein of female maturity. The key substance in the process of yolk synthesis is the important reproductive protein in fish reproduction, which plays an important role in the process of oocyte reproduction and development [1].To study the mechanism of yolk proteogen synthesis can not only complement and perfect the basic research of fish physiology and developmental biology, but also master the synthesis of yolk proteogen in vivo and in vitro. In order to better understand the mechanism of yolk proteogen synthesis in Scatophagus Argus and more accurately analyze the effect of exogenous estrogens on fish yolk proteogen synthesis, we used gene cloning, fluorescence quantitative PCR and H.E. staining to study the effect of exogenous estrogens on fish yolk proteogen synthesis. In this study, three subtypes of the yolk proteogen gene of goldfish were cloned by RACE and the gene bank of goldfish vtgAa was logged in. It is predicted that the molecular weight of VtgAa protein is 186.07 ku, the isoelectric point is 9.16, and the first 15 amino acids at the N terminal are signal peptides (Fig. 1-3). The GenBank login number of vtgAb is KY654346, the full length of the cDNA is 5346 bp, and the ORF is 5100 bp, which encodes 1699 amino acids. Acid. The predicted molecular weight of VtgAb protein is 186.37 ku, the isoelectric point is 9.24, and the first 15 amino acids at the N-terminal are signal peptides (Fig. 1-3). The GenBank login number of vtgC is KY676848, the full length of the cDNA is 4244 bp, and the ORF is 3828 bp, encoding a total of 1275 amino acids. The predicted molecular weight of VtgAb protein is 142.87 ku, the isoelectric point is 6.43, and the signal peptide is the first 15 amino acids at the N-terminal (Fig. 1-1). 3) According to the results of H.E. staining, the goldfish treated in this experiment were all in the immature stage of gonadal development, which met the precondition of EE2 injection experiment. Therefore, it was speculated that the vitellogenin synthesized after injection was mainly induced by injection of EE2. In this experiment, goldfish was isolated from liver tissue of goldfish by perfusion method. Hepatocytes [2] (Fig. 3-1) were cultured to the third generation and then exposed to EE2. According to the results of RT-q PCR analysis, the expression of vtgAb gene in liver tissue injected with EE2 was higher than that of other two subtypes under different sampling time and different dosage of EE2 injection. VtgAb was selected as mRNA expression. The results of RT-q PCR showed that the expression level of vtgAb in liver tissues was higher at EE2 concentrations of 1.0 and 10.0 ug/g, but lower at concentrations of 0.01 ug/g and 0.1 ug/g (Fig. 2-6). The expression level of vtgAb was lower at 24 h and gradually increased at 48 h. The expression level of vtgAb gene in liver cells was higher at EE2 exposure concentration of 10-8 mol/L, 10-7 mol/L, 10-6 mol/L and 10-5 mol/L, and the expression level at 10-5 mol/L was lower than that at 10-6 mol/L (Fig. 3-2). From the expression time point of view, the expression of VTG Ab gene began at 24 hours, gradually increased at 48 hours and reached the highest at 72 hours, and decreased to less than 48 hours at 96 hours. We speculated that EE2 induced the expression of VTG Ab gene in liver.
【学位授予单位】：上海海洋大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S917.4
【参考文献】
相关期刊论文 前9条
1 张莹莹;梁雪梅;曾文刚;张俊彬;;金钱鱼肾细胞系的建立及生长特性研究[J];海洋与湖沼;2014年03期
2 崔丹;刘志伟;刘南希;张莹莹;张俊彬;;金钱鱼性腺发育及其组织结构观察[J];水产学报;2013年05期
3 宋双双;安立会;郑丙辉;赵艳民;李子成;陈浩;赵兴茹;刘代成;;浑河流域野生鲫鱼卵黄蛋白原基因表达[J];生态毒理学报;2013年01期
4 刘春;李凯彬;王芳;王庆;聂湘平;王英英;吴淑勤;;剑尾鱼卵黄蛋白原C(Vg C)全长cDNA的克隆及表达[J];水产学报;2011年10期
5 陈琳琳;张高生;陈静;任宗明;;汞、硒暴露对紫贻贝(Mytilus edulis)抗氧化酶系统的影响[J];生态毒理学报;2011年04期
6 刘春;李凯彬;耿冬雨;聂湘平;王芳;吴淑勤;;剑尾鱼2种卵黄蛋白原全长cDNA的克隆及序列分析[J];中国水产科学;2010年01期
7 洪万树;吴秋艳;张其永;;中华乌塘鳢血清性类固醇激素含量与性腺发育的关系及其季节变化[J];厦门大学学报(自然科学版);2009年02期
8 沈卓坤;郑剑辉;陈怀定;赵会宏;;双棘黄姑鱼血清性类固醇激素的周年变化[J];广东海洋大学学报;2007年03期
9 马陶武,王子健;环境内分泌干扰物筛选和测试研究中的鱼类实验动物[J];环境科学学报;2005年02期
，本文编号：2251057
本文链接：https://www.wllwen.com/shoufeilunwen/zaizhiyanjiusheng/2251057.html
