光裸星虫Hsp90基因的全长克隆及其在全组织和卵母细胞中的表达分析

Molecular Cloning and Expression Analysis of HSP90 of Peanut Worm Sipunculus nudus

中文关键词: 光裸星虫  热休克蛋白Hsp90  基因克隆  表达分析  卵母细胞

英文关键词: Sipunculus nudus  Oocyte  HSP90  Gene cloning  Expression analysis

中文摘要:

      热休克蛋白Hsp90在蛋白质正确折叠、胞内物质运输、细胞增殖调控、配子发生等过程中发挥重要的作用。本研究利用RACE技术克隆获得了光裸星虫热休克蛋白Hsp90 (SnHsp90) cDNA全长序列。SnHsp90全长3110 bp，其中，3´UTR为582 bp，5´UTR为72 bp，开放阅读框为2456 bp，编码818个氨基酸。序列分析结果显示，SnHsp90蛋白具有1个信号肽，存在B型Hsp90的3个标签序列FLREL、IGQFGVGFYS、LPLNVSRE以及保守模块GxxGxG，表明SnHsp90属于Hsp90B亚族。与其他物种的Hsp90氨基酸序列比对发现，SnHsp90与鸭嘴海豆芽(Lingula anatine) Hsp90相似度最高，为77.72%。三维结构建模发现，Hsp90在不同物种之间的空间构象具有较高保守性。系统进化树分析显示，SnHsp90先与小头虫(Capitella teleta)、水蛭(Helobdella robusta)等环节动物的Hsp90聚为一支，再与鸭嘴海豆芽等其他无脊椎动物聚为一大支，而脊椎动物聚为另一大支，表明SnHsp90与小头虫Hsp90亲缘关系最近。RT-PCR结果显示，SnHsp90在光裸星虫的体壁、收吻肌、肾管等组织中均有表达。SnHsp90也可在卵母细胞不同发育时期表达，其中，在卵黄旺盛合成前期(Egg2)和外排卵母细胞期(Egg6)显著高表达(P<0.05)，暗示，SnHsp90可能参与到光裸星虫卵母细胞的蛋白质正确折叠、卵黄合成与环境抗逆等过程。研究结果为进一步探究光裸星虫卵母细胞发育的分子机制积累了基础资料。

英文摘要:

      Heat shock protein 90 (Hsp90) plays important roles in correct protein folding, intracellular material transport, cell proliferation regulation, and gametogenesis. In this study, the full-length cDNA of Hsp90 (SnHsp90) of Sipunculus nudus was cloned using RACE technology. The results showed that SnHsp90 is 3110 bp in length, including a 3´ UTR of 582 bp and a 5´ UTR of 72 bp. The open reading frame is 2456 bp in length and it encodes 818 amino acids. Sequence analysis showed that SnHsp90 has a signal peptide, with three tag sequences FLREL, IGQFGVGFYS, and LPLNVSRE and a conserved module, GxGxG, of type B Hsp90, thereby indicating that SnHsp90 belongs to the Hsp90B subgroup. Compared with other species, SnHsp90 showed the highest similarity (77.72%) with the Hsp90 of duck-billed bean sprouts. Three-dimensional structural modeling showed that the spatial conformation of Hsp90 among different species is highly conserved. Phylogenetic tree analysis showed that SnHsp90 was clustered into one branch of Hsp90 of annelids such as Capitella teleta and Helobdella robusta. The Hsp90 of all invertebrates was clustered into one branch, whereas that of the vertebrates was clustered into another branch, thus suggesting that SnHsp90 is closely related to the Hsp90 of Capitella teleta. RT-PCR results showed that SnHsp90 was expressed in different tissues of S. nudus, such as those of the body wall, muscles, and kidney tubes. SnHsp90 can also be expressed at different developmental stages of oocytes, and it is significantly expressed in Egg2 (oocyte in early stage of vigorous yolk synthesis) and Egg6 (excretive oocyte) (P<0.05). This result suggests the involvement of SnHsp90 in correct protein folding, yolk synthesis, and environmental stress resistance of S. nudus. The above results provide a basis for further exploration of the developmental mechanism of Sipunculus nudus oocytes.