The Effects Of Cd²⁺, Pb²⁺ And Zn²⁺ On Caenorhabditis Elegans And Using RNAi To Study Several Genes Which Affect Lifespan

Caenorhabditis elegans was used in this paper to study the factors affecting lifespan.We studied the toxic effects of three heavy metals Cd²⁺, Pb²⁺ and Zn²⁺ on C. elegans. The results indicated that: after C. elegans exposed to the three heavy metals 24hs, the toxicity of three heavy metals was Pb²⁺> Cd²⁺>Zn²⁺, LC 50 of three heavy metals was 41774μg·L^-1, 65945μg·L^-1 and 113323μg·L^-1 respectively; after exposed to the three heavy metals 48hs, the toxicity of the three metals was: Pb²⁺>Cd²⁺>Zn²⁺, LC 50 of three heavy metals was 31567μg·L^-1,38679μg·L^-1 and 63423μg·L^-1 respectively; after exposed to the three heavy metals 72hs, the toxicity of the three metals was Cd²⁺≈Pb²⁺>Zn²⁺, LC50 of three heavy metals was 24669μg·L^-1, 24946μg·L^-1 and 40668μg·L^-1 respectively. In the three metals, the toxicity of Cd²⁺ changed most with the time and the toxicity of the Pb²⁺ changed least with the time. The toxicity of Pb²⁺ changed most with the concentration and the toxicity of Zn²⁺ changed least with the concentration.The experiments of concentration effects in combination of two metals presented that: the toxic effect of Cd²⁺ and Zn²⁺ in combination was additional in low concentration and was antagonistic in high concentration; the toxic effect of Pb²⁺ and Zn²⁺ in combination was additional in all concentration in our experiment; the toxic effect of Cd²⁺ and Pb²⁺ was additional in low concentration and synergistic in high concentration.Using additive index method to know the time effects in the combination, we found that: when Cd²⁺ and Zn²⁺ were mixed in 1: 2 in weight, the AI of 24h, 48h and 72 h were minus and the combination effects were antagonistic; when Pb²⁺ and Zn²⁺ were mixed in 1: 2 in weight, the AI of 24h, 48h and 72 were minus and the combination effects were antagonistic; when Cd²⁺ and Pb²⁺ were mixed in 1: 1 in weight, the AI of 24h was positive and the combination effect was synergistic but the AI of 48h and 72 h were minus and the combination effects were antagonistic. Transgenic worm PC72 was used as a biomonitor for the heavy metal contamination which has a high sensitivity. Results indicated: in all metals, the staining ratio changed with the lethal ratio and prolonging the processing time could enhance the staining ratio. We used score method to estimate the effect and found that score changed with the staining ratio. Compared with lethality method, staining method was more quick and sensitive.The staining method also got a good result in the practice of monitoring the pollution of sea water in Xiamen. The staining ratio and the lethal ratio had correlation. The staining method could shorten the experiment time and enhance the sensitivity.We used RNAi to study the genes which could affect the lifespan of C. elegans. Four RNAi vectors: R148.3a, Cnfkb (C. elegans nfkb homologus), sqv-4 and helicase were constructed.The result of R148.3a RNAi showed that lost function of R148.3a could reduce the egg number, shorten the lifespan and decrease the immunity in N2, daf-2(e1370) and daf-16(mu86) mutants.Cnfkb was a homologus of human nfκb light polypeptide gene in C.elegans. The similarity of this gene to nfκb was 31. 09%. Feeding N2 with Cnfκb RNAi bacteria could not change the lifespan but decreased N2 immunity, and the worm easy infected with S. aureus. The ability of cnfkb reduced the immunity depended on daf-16. Lost function of Cnfkb by RNAi couldn't reduce the lifespan of daf-16(mu86) mutant further.Nematodes fed on helicase (RNAi) bacteria laid less eggs and the lifespan of the nematode was 2.9 days shorter than normal nematode. Lost function of helicase by RNAi also made some larvae die at early stage.When the nematodes were fed on sqv-4 (RNAi) bacteria, the number of eggs nematodes laid was reduced dramaticly. Lost function of sqv-4 the worm has the high embryo lethality. 96. 7 % of the eggs couldn't become larva and died in the early stage. At same time the lifespan of the sqv-4 mutant by RNAi was 1.5 day shorter than N2. The lifespan of lin-28(n1119) mutant was 8% longer than N2. The lifespan of lin-4(e912) mutant was 47% shorter than N2. The lifespan of let-7(n2853) mutant is 36% shorter than N2. The double mutant lin-28(n719);lin-4(e912) mutant lived no longer than lin-28(n1119) mutant. This implies that lin-28 works downstream of lin-4.