【作者】 谢松；

【导师】 宋大祥；

【作者基本信息】 河北大学 ， 动物学， 2006， 博士

【摘要】 卵黄蛋白(Vitellin, Vn)是卵黄的主要成分,在雌性对虾性腺成熟过程中合成,为胚胎和幼体早期发育提供营养。本文对中国明对虾卵黄蛋白及其前体卵黄蛋白原(Vitellogenin, Vg)的生化特征进行了研究,并克隆了Vg的全长cDNA序列。利用凝胶过滤(SephadexG-150)、离子交换(DEAE-Cellulose-32)以及电洗脱等方法纯化了中国明对虾Vn和Vg。Vn是一种糖脂磷类胡萝卜素蛋白,总分子量409 ku,等电点5.4,含糖量8.7%,具有5个亚基(202 ku、171 ku、105ku、80 ku和73 ku),亚基之间有1个二硫键。Vg也是一种糖脂磷类胡萝卜素蛋白,总分子量476 ku,等电点5.2,含糖量11.6%,亚基之间未发现二硫键。利用纯化的Vn免疫家兔制备抗血清。此抗血清能与对虾属内不同个体Vn或Vg发生免疫反应,但与属外个体间无此特性。免疫组织学观察证明对虾外源性Vg产生于肝胰腺,经血淋巴、滤泡细胞的转运,最终被卵母细胞吸收,加工成Vn。采用ELISA方法检测了对虾卵及无节幼体对Vn的利用过程。卵子排出后卵及幼体体内卵黄蛋白含量不断减少,N3-4至N5-6期和N5-6至Z1期两个时期Vn的消耗量最大,分别为1322 ng／mL和1213 ng／mL,占卵中卵黄蛋白总量的38.9%和35.7%,Z1期以后体内基本不含卵黄蛋白。通过同源克隆和RACE等方法从成熟中国明对虾卵巢中克隆到了一条VgcDNA序列。全长7956 bp,含有一个7761 bp的开放阅读框(ORF),编码2587个氨基酸残基。推导的氨基酸序列与其他已知甲壳动物Vg具有80-95%的同一性(identity)。其中,中国明对虾和墨吉明对虾Vg分子的亲缘关系最近。SingalP程序预测Vg前体N末端含有18个氨基酸残基的信号肽。其余2569个氨基酸中含有一个RXRR分裂位点,此位点能被枯草杆菌样蛋白内切酶(subtilisin-like endoproteases)所识别并裂解。推导的Vg的理论分子量是282 ku,理论等电点为6.13。氨基酸含量分析显示,Vg蛋白含有较多的Ala(11.56%)、Glu(8.99%)、Val(8.52%)和Ile(7.12%)。RT-PCR方法检测到Vg基因同时存在于雌性和雄性对虾基因组中,但其转录只发生在卵黄形成期的雌性对虾的卵巢和肝胰腺中。更多还原

【Abstract】 Vitellin (Vn), the major egg yolk protein synthesized in female shrimp during gonad maturation, provides nourishing substance to the growing and developing of embryo and larva. Biochemical character of Vn and its precursor vitellogenin (Vg) from Chinese shrimp were studied and a full-length cDNA of Vg was cloned.Vn and Vg were isolatied from female shrimp with gel filtration chromatography (SephadexG-150), ion exchange chromatography (DEAE-Cellulose-32) and electroelution methods jointly. Vn is a lipo-glyco-phospho-caroprotein with an apparent molecular weight of 409 ku, whose pⅠis 5.4. The molecule of Vn contains 8.7% oligosaccharide. Five subunits(202 ku,171 ku,105 ku,80 ku and 73 ku) and one disulfide bond were detected. Vg is a lipo-glyco-phospho-caroprotein too. Its molecular weight is 476 ku, pⅠis 5.2. About 11.6% oligosaccharide was detected in the whole Vg molecule. Nondisulfide bond was found in the binding of polypeptide subunits.Antiserum was gained from a rabbit with purified Vn as antigen, which can be used to detect Vn or Vg of shrimp from the identical genus but not from other genus of crustacean. It was proved with immunohistochemical methods that extraovarian original Vg is synthesized in hepatopancreas and secreted into hemolymph. Vg is absorbed by follicular cells from hemolymph and then transferred into oocytes where Vg is made into Vn. ELISA was introduced to detect the change of Vn content in the eggs and larva of different stage. The content of Vn decrease along with the development of larval prawn, Vn is digested very quickly from N3-4 to N5-6 and from N5-6 to Z1, the decrease of Vn are 1322 ng/mL and 1213 ng/mL, it occupy gross of Vn content of eggs 38.9% and 35.7%, respectively. After the stage of Z1, the content of Vn was not detected.In the present study, a full-length cDNA sequence encoding Vg was cloned by homologous clone and RACE method from mature ovary of Chinese shrimp Fenneropenaeus chinensis, an important commercial species in aquaculture. This cDNA sequence is 7956 bp in length, containing a 7761 bp open reading frame, which encodes 2587 amino acid residues. The deduced amino acid sequence showed 80-95% identity with other known crustacean Vgs. Multiple alignment of amino acid sequences of Vgs from different crustaceans shows that Vg of F. chinensis possesses the highest identity with that of F. merguiensis. Deduced Vg contains a putative N-terminal signal sequence (1-18) followed by a mature peptide including 2569 amino acid residues. A potential cleavage site (RXRR) that is recognized by subtilisin-like endoproteases was identified in the deduced Vg precursor. The deduced peptide, with a predicted molecular weight of 282 ku, and a pⅠof 6.13, has more negatively charged amino acids. Vg contains more alanine, glutamic acid, valine and isoleucine (Ala 11.56%, Glu 8.99%, Val 8.52% and Ile 7.12%) by amino acids composition analysis. Vg gene was detected both in female and male shrimp by RT-PCR method. But transcriptions happen exceptionally in ovary and hepatopancreas of vitellogensis females.更多还原