金錢魚卵黃蛋白原基因克隆及EE2誘導(dǎo)卵黃蛋白原合成的研究
發(fā)布時(shí)間：2018-09-19 19:09
【摘要】：卵黃蛋白原(vitellogenin,Vtg)是卵黃蛋白的前體,通過下丘腦-垂體-性腺(hypothalamic-pituitary-gonadal,HPG)軸激活雌激素受體來合成。卵生脊椎動(dòng)物的卵可以儲(chǔ)蓄大量的卵黃,卵黃的主要成分是被稱之為雌性成熟期血清蛋白的卵黃蛋白原。Vtg是合成卵黃過程中的關(guān)鍵性物質(zhì),是魚類生殖過程中重要的生殖蛋白,在卵生動(dòng)物生殖和發(fā)育過程中起重要作用[1]。研究卵黃蛋白原的合成機(jī)制不但可以補(bǔ)充完善魚類生理學(xué)、發(fā)育生物學(xué)等基礎(chǔ)研究,同時(shí),掌握卵黃蛋白原體內(nèi)體外合成方式和機(jī)理,可以為魚類繁殖提供理論依據(jù)。為更好地了解金錢魚(Scatophagus argus)卵黃蛋白原合成機(jī)制及能更精確地分析外源性雌激素對(duì)魚類合成卵黃蛋白原的影響,本文以金錢魚為研究對(duì)象,運(yùn)用基因克隆、熒光定量PCR和H.E.染色等方法,并從泄殖孔腹腔注射EE2(17α-ethynylestradiol)和體外EE2暴露肝原代細(xì)胞兩個(gè)角度對(duì)金錢魚在EE2處理下的合成規(guī)律做了進(jìn)一步的分析。本研究使用RACE克隆得到金錢魚金錢魚卵黃蛋白原基因的三種亞型的cDNA序列,金錢魚vtgAa的GenBank登錄號(hào)為KY676847,cDNA全長5360 bp,開放閱讀框(ORF)為5091 bp,共編碼1696個(gè)氨基酸。預(yù)測金錢魚VtgAa蛋白分子量為186.07 ku,等電點(diǎn)為9.16,N端的前15個(gè)氨基酸為信號(hào)肽(圖1-3)。金錢魚vtgAb的GenBank登錄號(hào)為KY654346,cDNA全長5346 bp,ORF為5100 bp,共編碼1699個(gè)氨基酸。預(yù)測金錢魚VtgAb蛋白分子量為186.37 ku,等電點(diǎn)為9.24,N端的前15個(gè)氨基酸為信號(hào)肽(圖1-3)。金錢魚vtgC的GenBank登錄號(hào)為KY676848,cDNA全長4244 bp,ORF為3828bp,共編碼1275個(gè)氨基酸。預(yù)測蛋白分子量142.87 ku,等電點(diǎn)6.43,信號(hào)肽為N端前15個(gè)氨基酸(圖1-3)。根據(jù)H.E.染色的結(jié)果顯示:本實(shí)驗(yàn)所處理的金錢魚皆處于性腺發(fā)育過程中的未成熟期,滿足EE2注射實(shí)驗(yàn)的前提條件。因此推測注射后合成的卵黃蛋白原主要為注射EE2的誘導(dǎo)結(jié)果。本實(shí)驗(yàn)使用灌流法從金錢魚肝臟組織上分離得到金錢魚肝細(xì)胞[2](圖3-1),將分離得到的肝細(xì)胞進(jìn)行原代培養(yǎng)至第三代后使用EE2暴露處理。根據(jù)RT-qPCR分析結(jié)果顯示,在不同的采樣時(shí)間及不同劑量的EE2注射條件下,腹腔注射EE2的肝組織中vtgAb基因表達(dá)均高于其他兩種亞型,選取vtg Ab作為mRNA表達(dá)水平檢測的主要基因(圖2-5)。根據(jù)注射EE2后肝組織中vtgAb的RT-qPCR結(jié)果顯示,vtgAb在EE2濃度為1.0μg/g和10.0μg/g處理時(shí)表達(dá)水平較高,而在濃度為0.01μg/g和0.1μg/g時(shí)表達(dá)水平較低(圖2-6)。在表達(dá)時(shí)間上:24h時(shí)vtgAb表達(dá)水平較低,48h時(shí)表達(dá)量逐漸升高并在72h達(dá)到最高,在96h時(shí)表達(dá)量呈下降趨勢(shì)。金錢魚肝細(xì)胞內(nèi)vtgAb基因的表達(dá)量在EE2暴露濃度為10-8 mol/L、10-7 mol/L、10-6 mol/L、10-5 mol/L時(shí)表達(dá)量較高,其中10-5 mol/L時(shí)的表達(dá)量較低于10-6 mol/L時(shí)的表達(dá)量(圖3-2)。而在EE2暴露濃度為10-11 mol/L時(shí),表達(dá)量最低。從表達(dá)時(shí)間上來看:vtg Ab基因從24h開始表達(dá),在48h表達(dá)量逐漸升高并在72h時(shí)達(dá)到最高,在96h時(shí)表達(dá)量下降至較低于48h表達(dá)量,我們推測EE2誘導(dǎo)了肝臟內(nèi)vtgAb基因的表達(dá)。
[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.
【學(xué)位授予單位】：上海海洋大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：S917.4
本文編號(hào)：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.
【學(xué)位授予單位】：上海海洋大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：S917.4
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