秀丽线虫野生株系和缺氧敏感株系的差异蛋白质组学研究

Proteomic Studies of Wild Type N2 and Hypoxia-sensitivity Strain(ia04) in Caenorhabditis Elegans

目的研究秀丽线虫(Caenorhabditis elegans)野生株系N2和缺氧敏感株系(ia04)的差异蛋白质,为进行线虫在缺氧环境下的应答机制研究奠定基础.方法同步化培养2种株系的线虫,培养至L4期时,常规方法提取总蛋白,采用双向电泳分离和串联质谱分析,搜索Swiss-Prot线虫数据库,鉴定蛋白种类并根据基因本体论(gene ontology,GO)进行分析.结果质谱成功鉴定了其中的25个差异显著蛋白(ratio>2.0,P<0.05),其中N2株系检测到的表达量比ia04株系高的主要是热休克蛋白(HSP-70,HSP-60)、GTP结合蛋白(TAG-210)、乙酰辅酶A脱氢酶中链(GN=T);ia04株系检测到的表达量比N2高的是延伸因子(EFT-3)和铁硫蛋白-1(ISP-1),在ia04株系中,HSP的含量明显低于N2株系,说明这可能是ia04株系对缺氧敏感的原因之一.根据GO注释结果发现,鉴定到的这些差异蛋白与线虫的DNA合成及生长发育的相关性较大.结论蛋白质组学的方法为线虫缺氧的研究提供了有参考价值的数据,不同株系差异蛋白的发现可促进对缺氧应答机制的了解,且可从蛋白质水平揭示重要表型的基因表达机制.

Objective To investigate the different proteins between wild type N2 and hypoxia-sensitivity strain（ia04） and lay the foundation for studying the response mechanism of C.elegans during hypoxia.Methods The worms of two strains were cultured synchronously to L4 stage.Then the total proteins were extracted by using routine method.After that,two-dimensional electrophoresis was performed and the selected protein spots were then identified by MALDI/TOF/TOF tandem mass spectrometry and search the Swiss-Prot database.Go term（Gene Ontology） analysis was also performed.Results Twenty-five significantly different spots were finally identified by MS（ratio2.0,P0.05）.The expression levels of main proteins identified in N2 were higher than those in ie04 strain： heat shock proteins（HSP-70 and HEP-60）,Putative GTP-binding protein（TAG-210）,Probable medium-chain specific acyl-CoA dehydrogenase（GN=T）.The expression levels of main proteins identified in ie04 strain were higher than those in N2 strain： Elongation factor-1 alpha（EFT-3）and Iron-sulfur protein（ISP-1）.The expression level of HSP in ie04 strain was obviously lower than in N2 strain,it may be the one reason why worms of ie04 strain were sensitivity to hypoxia.According to the Go term,most proteins identified were related to the development and DNA synthesis of C.elegans.Conclusions Proteomic research can offer the valuable reference data to study the hypoxia in C.elegans.Different proteins found in different strains may promote the understanding of the hypoxia responsive mechanism and uncover the mechanism of important phenotype gene expression at the protein level.