文章摘要
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
投稿时间:2019-04-16  修订日期:2019-06-04
DOI:
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, HSP90, Gene cloning, Expression analysis,Oocyte
基金项目:广东省大学生创新创业训练计划(201810566049)和广东省科技计划(2016A020209010,2017A030303075)
作者单位E-mail
周丹 广东海洋大学水产学院 1334285134@qq.com 
苏泳霖 广东海洋大学水产学院  
钟如卓 广东海洋大学水产学院  
郭志诚 广东海洋大学水产学院  
邬婧 广东海洋大学水产学院  
郑哲 广东海洋大学水产学院 haidazhengzhe@163.com 
王庆恒 广东海洋大学水产学院  
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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、配子发生等过程中起重要的作用。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 582 bp,5’UTR 72 bp,开放阅读框为2456 bp,编码818个氨基酸?序列分析结果表明,SnHsp90具有一个信号肽,同时存在B型Hsp90的三个标签序列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Hsp90相似度最高(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。系统进化树分析表明, SnHsp90先与小头虫,水蛭等环节动物的Hsp90聚为一支,再与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Egg2和Egg6期显著高表达(P<0.05),该结果暗示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) play important roles in the process of correct protein folding, intracellular material transport, cell proliferation regulation and gametogenesis. In this study, the full-length cDNA of Hsp90 (SnHsp90) was cloned from S. nudus using RACE technology. The results showed that SnHsp90 is 3110 bp in length, including 3' UTR 580 bp and 5' UTR 74 bp. The Open reading frame (ORF) is 2456 bp and encodes 818 amino acids. Sequence analysis showed that SnHsp90 had a signal peptide, and ther e were three tag sequences FLREL, IGQFGVGFYS, LPLNVSRE and conservative module GxGxG of type B Hsp90, which indicated SnHsp90 belonged to Hsp90B subgroup. Compared with other species, SnHsp90 had the highest similarity with duck-billed bean sprouts Hsp90 (77.72%). Three-dimensional structural modeling shows that the spatial conformation of Hsp90 among different species is highly conservative. Phylogenetic tree analysis showed that SnHsp90 was clustered into one branch of Hsp90 from annelids such as cephalococcus and leeches, all invertebrates Hsp90 was clustered into one branch, while vertebrates Hsp90 were clustered into another branch, which suggesting that SnHsp90 was closely related to cephalococcus Hsp90. RT-PCR results showed that SnHsp90 was expressed in differently tissues, such as the body wall, muscles, kidney tubes of S. nudus. SnHsp90 can also be expressed in different developmental stages of oocytes, and it is Significantly expressed in Egg2 and Egg6 (P<0.05). This result suggests that SnHsp90 may be involved in the process of correct folding of proteins, yolk synthesis and environmental stress resistance in this stage of S. nudus. The above results have accumulated basic data for further exploration of the developmental mechanism of N. glabrata oocytes.
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