摘要
为探究中华鳖(Pelodiscus sinensis)卵黄蛋白原Vtg基因的生物学信息与表达及其作为生理与毒理研究标志物的可行性,本实验克隆获得VtgAB1、VtgAB2、VtgAB3(GenBank登录号OK287927、OK287929、OK287928)基因序列,其cDNA长分别为5055、5537、5306 bp,分别编码了1684、1760、1725个氨基酸.三种亚型Vtg蛋白均由脂蛋白N-末端(Vitellogenin_N)、1943DUF、1944DUF、PV、D型血管血友病因子(VWD)5个保守结构域构成,为完整型Vtg.同源性分析发现皆与龟鳖目具有更近的亲缘关系,同时在3种亚型中VtgAB2与VtgAB3更相似.荧光定量PCR显示,3种亚型Vtg只在雌性肝脏中强烈表达.在60日龄、2龄、4龄雌性个体肝脏中,Vtg表达量随年龄呈上升趋势,VtgAB1、VtgAB2为主要表达亚型.原位杂交结果显示,3种亚型Vtg在卵原细胞内未发现杂交信号;在生长期卵母细胞胞质和滤泡细胞中发现杂交信号;成熟期卵母细胞胞质中未发现杂交信号,在滤泡细胞和周围的网状组织中发现杂交信号.信号在初级卵母细胞的细胞质中分布较为均匀且表达最强,随着卵母细胞的发育信号聚集在核周区且信号逐渐减弱.以雄性中华鳖为对象,注射雌激素E2,3种亚型Vtg表达量表现出明显的时间和剂量相关性.研究结果表明,在中华鳖卵黄积累过程中Vtg发挥重要作用,首次证实其Vtg存在内源性合成,并发现其可以作为雌激素暴露的生物标志物.
Abstract
Vitellogenin (Vtg) is the precursor of vitellin (Vn) in oviparous animals,a large lipoprotein contai-ning sugar and phosphorus.In oviparous vertebrates, Vtg is synthesized in the liver by regulating various hor-mones and released into the bloodstream.It is transported to the ovary through blood circulation, endocytosed by specific receptors on the surface of oocytes (VtgR),and then cleaved into vitellin lipoprotein (Lv) and vitel-lin high-phosphorus protein (Pv) by cathepsin D to provide nutrients for embryonic development.So far, cD-NAs encoding the Vtg gene have been cloned and identified from nematodes, crustaceans, fish, amphibians, reptiles and birds.However, the sequence and expression pattern of the Vtg gene in Chinese Soft-shell Turtle (Pelodiscus sinensis) have not been reported.In vertebrates, the liver has long been considered the site of Vtg synthesis.However, more and more studies have shown that tissues outside the liver also participate in Vtg synthesis.Whether endogenous synthesis exists in the testudo formes and its precise location in ovarian tissues remains to be studied further.Environmental estrogens, as endocrine disruptors, have received widespread at-tention.The concentration of estrogens in the natural environment is low, and it is difficult to detect through routine testing.Therefore, exploring the effects of environmental estrogens can only be revealed through a se-ries of sensitive in vitro and in vivo ecological toxicological tests.Vtg is considered to be a sensitive estrogen bio-marker.This experiment studies the cloning and expression of the P.sinensis Vtg gene and the effects of exoge-nous E2, to provide basic information for the biological laws of vertebrate egg development, deepen the un-derstanding of Vtg production in the testudoformes, and exploring its feasibility as an essential biomarker for reproductive physiology and toxicology in P.sinensis.The gene sequences of VtgAB1, VtgAB2, and VtgAB3 (GenBank accession numbers OK287927, OK287929, and OK287928) were cloned in this experiment.The cDNA lengths were 5055, 5537, and 5306 bp, respectively, encoding 1684, 1760, and 1725 amino acids.The three subtypes of Vtg proteins are all composed of five conserved domains of lipoprotein N-terminal (Vitellogenin_N),1943DUF, 1944DUF, PV, and von Willebrand factor D (VWD),which are the com-plete Vtg.Homology analysis showed that it had a closer genetic relationship with Trionycis, and VtgAB2 was more similar to VtgAB3 in the three subtypes.Fluorescence quantitative PCR showed that three subtypes of Vtg were strongly expressed only in the female liver.In the liver of 60-day-old, 2-year-old and 4-year-old fe-male individuals, the expression of Vtg increased with age, and VtgAB1 and VtgAB2 were the main expression subtypes.The in situ hybridization results showed that no hybridization signal was found in the oogonia of the three subtypes of Vtg.Hybridization signals were found in the cytoplasm and follicular cells of growing oo-cytes.No hybridization signal was found in the cytoplasm of mature oocytes but in follicular cells and surround-ing reticular tissues.The signal was evenly distributed in the cytoplasm of primary oocytes and the expression was the strongest.With the development of oocytes, the signal gathered in the perinuclear region gradually weakened.Male P.sinensis injected with estrogen E2, the expression of three subtypes of Vtg showed a signifi-cant time and dose correlation.The results showed that Vtg played an essential role in the process of yolk accu-mulation in P.sinensis, and it was confirmed for the first time that Vtg had endogenous synthesis and found that it can be used as a biomarker of estrogen exposure.
基金项目
重庆市农业委员会"生态渔产业技术体系"(2021-4322000121)
维普
万方数据