秀丽线虫野生株系N2缺氧的荧光定量蛋白质组学研究

Quantitative proteomic (2D-DIGE) studies of wild type Caenorhabditis elegans strain N2 after hypoxia

目的研究秀丽线虫野生株系N2缺氧时的差异蛋白质,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N2株系的线虫,培养至L4期时物理缺氧10 h(0.2%O2)后,提取总蛋白,采用双向荧光差异凝胶电泳(DIGE)分离和串联质谱分析,搜索线虫数据库,鉴定蛋白种类并进行分析。并采用突变株系(prdx-2,prdx-3)进行了4、6、8、10、12 h的缺氧实验研究。结果质谱成功鉴定了其中的5个与对照有显著差异的蛋白质(ratio>2.0,P<0.05),包括2个上调(PRDX-2、PRDX-3)和3个(RPL-7、H28O16.1、VHA-12)下调的蛋白。突变株系的缺氧存活率实验进一步验证了蛋白质组学的结果,也显示了PRDX-2和PRDX-3在抗缺氧中起重要作用。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,缺氧10 h后,线虫中变化的蛋白质与能量、翻译和抗氧化有关,为进一步研究缺氧时的应答机制奠定了基础。

Objective To investigate the different proteins in wild type Caenorhabditis elegans （ C. elegans） N2 after hypoxia and lay the foundation for studying the response mechanism of C. elegans during hypoxia. Methods The worms of N2 were cultured synchronously to IA stage and treated 10 h hypoxia （0.2% oxygen）. Then the total proteins were extracted and two-dimensional difference gel electrophoresis （2D-DIGE） was performed and the selected protein spots were then identified by MALDI/TOF/TOF tandem mass spectrometry and search the database. Two mutant strains （prdx-2, prdx-3） were incubated for 4, 6, 8, 10 and 12 h in sealed hypoxia chamber （0.2% oxygen）. The survival rate was calculated. Results Five significantly different sports were finally identified by MS （ ratio 〉 2.0,P 〈 0.05）. Two proteins were significantly upregulated （ PRDX-2, PRDX-3 ）, and three were down- regulated upon hypoxic stress（ RPL-7, H28016.1 ,VHA-12）. The mutant strain of （prdx-2, prdx-3） also showed a hypoxia-sensitive phenotype in subsequent analyses, confirming the proteomic findings and showed that PRDX-2, PRDX-3 play an important role in anti-hypoxia. Conclusion Proteomic research can offer the valuable reference data to study the hypoxia in C. elegans. Changed proteins found after 10 h hypoxia are related to the energy, translation and anti-oxidant, which may lay the foundation of understanding the hypoxia responsive mechanism.