光裸星虫SERCA基因克隆及在卵母细胞中的表达分析

Cloning and expression analysis of SERCA gene of Sipunculus nudus in oocytes

细胞内钙离子(Ca^(2+))浓度的变化是介导卵母细胞成熟的重要因素。肌质网/内质网Ca^(2+)-ATP酶(Sarco/endoplasmic reticulum calcium adenosine triphosphatase,SERCA)属P型ATP酶家族成员,是细胞内Ca^(2+)转运的重要调控蛋白。前期转录组分析显示,光裸星虫(Sipunculus nudus)SERCA(Sn-SERCA)的表达量在卵母细胞发育过程中差异显著,为进一步研究SERCA在卵母细胞不同发育阶段中的作用,利用RACE技术得到Sn-SERCA cDNA全长,采用实时荧光定量PCR检测Sn-SERCA在各卵母细胞发育时期的相对表达量。结果表明,Sn-SERCA全长为3840 bp,5'非编码区(UTR)为196 bp,3'UTR为581 bp,开放阅读框3060 bp,编码1020个氨基酸,Sn-SERCA具有P型ATP酶家族进行催化反应所需的两个保守基序“TGES”和“DKTGT”。多序列比对、Motif分析及三级结构预测结果显示SERCA同源蛋白具有较高保守性。系统进化树分析表明Sn-SERCA与粉正蚓(Lumbricus rubellus)、鸭嘴海豆芽(Lingula anatine)等无脊椎动物同源蛋白序列聚为一支。荧光定量结果显示:卵母细胞在体腔液中发育时,Sn-SERCA在卵黄旺盛合成后期高表达;当卵母细胞进入肾管后,Sn-SERCA的表达量大幅上升,显著高于其他时期(P<0.05)。结合前期卵母细胞发育的超微结构观察,研究结果表明Sn-SERCA在光裸星虫卵母细胞的卵黄积累和生发泡破裂发生过程中起重要作用。

The change of intracellular calcium(Ca^(2+))concentration is an important factor in oocyte maturation.As a member of the P-type ATPase family and a crucial regulator of intracellular calcium transport,sarco/endoplasmic reticulum calcium adenosine triphosphatase(SERCA)is a key enzyme.To investigate the function of SERCA in the development of the oocytes of Sipunculus nudus,we obtained the full length of S.nudus SERCA(Sn-SERCA)cDNA by using RACE technique,and determined the Sn-SERCA relative expression level in different oocyte developmental periods by using real-time fluorescence quantitative PCR(qRT-PCR).The results indicate that the overall length of the Sn-SERCA was 3840 bp,the 5'UTR was 196 bp,the 3'UTR was 581 bp,and the open reading frame was 3060 bp,encoding 1020 amino acids.Sn-SERCA had TGES and DKTGT which were the P-type ATPase family's two conserved motifs and were necessary for catalytic reactions.According to multiple sequence alignment,motif analysis,and predictions of tertiary structures,the SERCA homologous proteins exhibited great conservatism.The phylogenetic tree analysis shows that the Sn-SERCA formed a broad branch with homologous protein sequences from invertebrates such as Lumbricus rubellus and Lingula anatine.The result of qRT-PCR demonstrates that the Sn-SERCA was significantly expressed in the late yolk vigorous synthesis period and had the maximum value during coelomic fluid development.When the oocytes moved to the nephridioduct,the Sn-SERCA expression was considerably higher than that at other time(P<0.05).The variable expression of Sn-SERCA in different periods of oocyte development suggests that Sn-SERCA is crucial in the development and germinal vesicle breakdown in S.nudus oocytes.