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脊尾白虾翻译控制肿瘤蛋白基因TCTP克隆及自噬调控相关基因在卵巢发育期的表达
马骊1,2, 葛倩倩2, 许杨1,2, 彭素晓1,2, 李健2,3
1.上海海洋大学水产与生命学院 上海 201306;2.农业农村部海洋渔业可持续发展重点实验室 中国水产科学研究院黄海水产研究所 青岛 266071;3.青岛海洋科学与技术试点国家实验室海洋 渔业科学与食物产出过程功能实验室 青岛 266071
摘要:
为深入了解脊尾白虾(Exopalaemon carinicauda)卵巢发育和卵黄蛋白原发生的分子机制,本研究克隆得到脊尾白虾翻译控制肿瘤蛋白基因TCTP,并结合自噬调控基因TCTP、Hif-1α、Beclin1和Bcl-2在卵巢发育期的表达水平,阐释脊尾白虾卵黄蛋白原合成过程中的分子调控特征。研究显示,脊尾白虾TCTP基因cDNA全长为732 bp,编码168个氨基酸,具有典型的TCTP1和TCTP2功能域以及PKC和TKⅡ等mTOR信号通路相关的磷酸化位点。同时发现,甲壳动物TCTP普遍缺乏在其他动植物中高度保守的C末端的cys残基。进化分析显示,脊尾白虾TCTP与中华绒螯蟹(Eriocheir sinensis)亲缘关系最近。4种自噬调控基因在脊尾白虾卵巢发育期的表达结果显示,肝胰腺TCTP基因从增殖期到产后恢复期呈递减趋势;肝胰腺Hif-1α、Beclin1和Bcl-2基因表达趋势相似,即从增殖期到小生长期高表达,大生长期极显著下降,表达量最低(P<0.01),这与外源性卵黄蛋白原的合成趋势大致相反。这些自噬调控基因可能通过自噬作用共同调节外源性卵黄蛋白原的合成。卵巢TCTP基因在小生长期表达量最高;卵巢Hif-1α基因从增殖期到产后恢复期持续升高,这与内源性卵黄蛋白原表达趋势相似;卵巢Beclin1基因在大生长期表达量最高,与脊尾白虾卵巢EcR表达趋势相似,与卵巢Bcl-2基因表达趋势相反,这些自噬调控基因可能通过自噬作用共同促进内源性卵黄蛋白的合成。本研究表明,自噬调控基因TCTP、Hif-1α、Beclin1和Bcl-2在脊尾白虾卵巢发育时期相互协调共同作用,可能通过自噬作用调节脊尾白虾卵黄蛋白原的合成和卵巢发育。
关键词:  脊尾白虾  TCTP  细胞自噬  卵黄蛋白  表达分析
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Cloning of the translationally controlled tumor protein gene (TCTP) and expression analysis of autophagy regulatory related genes during the development of ovary in Exopalaemon carinicauda
MA Li1,2, GE Qianqian2, XU Yang1,2, PENG Suxiao1,2, LI Jian2,3
1.College of Fisheries and Life Science, Shanghai Ocean University, Shanghai 201306;2.Key Laboratory of Sustainable Development of Marine Fisheries, Ministry of Agriculture and Rural Affairs, Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao 266071;3.Laboratory for Marine Fisheries Science and Food Production Processes, Pilot National Laboratory for Marine Science and Technology (Qingdao), Qingdao 266071
Abstract:
In order to understand the regulatory mechanism of ovarian development in Exopalaemon carinicauda, we used RACE (Rapid amplification of cDNA ends) technology to clone the translationally controlled tumor protein (TCTP) gene. The full-length cDNA of TCTP gene in E. carinicauda (EcTCTP) is 732 bp long with an intact open reading frame of 507 bp encoding a polypeptide of 168 amino acids. Six kinds of functional sites were identified in EcTCTP, including one N-glycosylation site, three protein kinase C phosphorylation sites, six casein kinase Ⅱ phosphorylation sites, three N-myristoylation sites, TCTP domain signature 1, and TCTP domain signature 2. Multiple alignment analysis and phylogenetic tree revealed that EcTCTP has the highest similarity to TCTP from Eriocheir sinensis. The results demonstrated that the cysteine (Cys) residue at the C terminal is highly conserved in all the animals, except the crustaceans. The lack of Cys residue might affect the oxidation resistance and the formation of TCTP dimers in crustaceans. The expression profile of 4 kinds of autophagy-related genes during the ovarian development period showed that: (1) EcTCTP gene in the hepatopancreas from proliferative stage to postpartum recovery period decreased. (2) Expression of EcTCTP gene in the ovary was the highest during the minor growth phase. Furthermore, the expression profile of Hif-1α, Beclin1, and Bcl-2 in hepatopancreas showed a similar trend, that is, high expression from proliferative period to minor growth phase, the lowest expression during the major growth phase with an increase during the mature period. The expression level of the gene Hif-1 in the ovary was similar to that of the gene vitellogenin in the ovary, which increased from the proliferative phase to the postpartum recovery period. The expression level of the gene Beclin1 in the ovarian cells increased from the proliferative stage to the major growth phase, and reached the highest level during the major growth phase, which was consistent with the synthesis of endogenous vitellogenin during the late development stage in prawn. The expression of the gene Bcl-2 in the ovary was the lowest during the major growth phase. The results illustrated that the autophagy-related genes might function together in the ovarian development of E. carinicauda by regulating autophagy during the synthesis of vitellogenin.
Key words:  Exopalaemon carinicauda  TCTP  Autophagy  Vitellogenin  Expression analysis