Study On Anti-oxidative Effect Of Ngb And Hypoxia Response Mechanism Of Cytoskeleton In C. Elegans

qBrenner first reported C. elegans(Caenorhabditis elegans) as a model organism in 1974. C. elegans has many advantages, and is the preferred model in genetic studies because of short life cycle and lifespan, simple laboratory rearing, characteristics of full development and anatomy. It has completely-sequenced genome and convenient genetic manipulation, such as transgene, gene mutation, RNA interference. Therefore, C. elegans was used in this study to probe the response of Ngb on oxidation injuries and cytoskeleton changes under hypoxia stress.1. Anti-oxidative Mechanism of Ngb in C. elegansNgb is a recently discovered as oxygen binding globin and specifically expressed in the nervous system. Ngb has two kinds of important amino acids: histidine and cysteine, which play important roles in the structure and anti-oxidative function of this protein. To investigate the antioxidant role of Ngb and the underlying mechanism, we used the human Ngb (hNgb) transgenic strain of C. elegans to treat with paraquat (PQ). Results showed that Ngb has antioxidant function. We used co-immunoprecipitation to pull down the potential interacting proteins of Ngb, but had no significant results. Ngb may play antioxidant role through its structure. We also constructed the histidine and cysteine double mutants of Ngb expression vector for microinjection, in order to obtain Ngb mutant transgenic strains and further exploring the oxidative mechanism of Ngb.C. elegans has 33 globins, which is far more than vertebrates. glb-13 is the homolog of hNgb, which has 20% similarity and the same functional domains. To investigate its antioxidant role and the signaling pathway, we constructed the mutant strain by mating the major pathways of hif-1 and glb-13 double mutant strains, then treated with PQ for adult rate and mRNA expression analysis. Results showed that the glb-13(tm2825) mutant strains can not be restored upon PQ induced oxidative damage. The antioxidative effects of the hif-1 and glb-13 double mutant strains were weaker than that of the strain glb-13(tm2825). Therefore, our result indicated that glb-13 is antioxidant and HIF-1 does not influence the antioxidative function of glb-13, which DAF-16 and Nrf-2 might plays important roles in the antioxidant signaling pathway of glb-13. We also constructed the glb-13 promoter of expression vector to observe the expression and distribution of glb-13 tagged EGFP in C. elegans in order to detect the protein level of glb-13. Taken together, the antioxidant of glb-13 also agreed with the antioxidant role of Ngb. It's important for understanding the evolution of Ngb.2. Hypoxia Response Mechanism of Cytoskeleton in C. elegansCytoskeleton, which support the cell structure, is indispensable for cell functions. Cytoskeleton is involved in cell movement, material transport and energy conversion, cell differentiation, signal transduction. The anti-hypoxia function is equally important. To investigate the role in hypoxia, the tropomyosin mutant strain lev-11(x12) was selected. Our results showed that the lev-11(x12) mutant is sensitive to hypoxia, and the LEV-11 protein level increased under hypoxia stress. Therefore, these data indicated that cytoskeleton can recover the damage induced by hypoxia.