| Zinc is a kind of essential microelement and it is represented as "spark of life". It plays an importment role in the processes of growth, development, aging, digestion, reproduction of organism, becoming one of a hotspot research in the field of trace element. DNA methylation is the best known epigeneteics process and existing research shows that the change of DNA methylation is the biological events earlier than gene mutation. In higher eukaryotes DNA methylation plays an important role in different biological processes, including regulaion of embryo development, preservation of chromosomal integrity, chromosome X inativation, silencing of gene expression and genomic imprinting. Drosophila is the most important model organism in biologic study and it was demonstrated that genomic methylated DNA is prevalent during the early embryonic development. The overall methylation level in Drosophila is lower than in vertebrates and plants. As in other organisms, whether DNA methylation has a functional role in Drosophila is not known yet and needs more research. Moreover, DNA methylation can be affected by environmental factors and many studies demonstrated that the alteration in DNA-methylation and hence possible changes in gene expression can be transferred to non-exposed generations through epigenetic inheritance.However, the effects of zinc on DNA methylation in the model organism, fruit fly, are seldom reported.The Canton-S D. melanogaster strain was used in all our experiments for all analyses and the AFLP method was used with some modifications. We studied DNA methylation polymorphism during development changes(0～3 h embryos,6～9 h embryos,12～15 h embryos,18～21 h embryos,3rd instar larvae, pupae, males and females adult flies).The effects of zinc treatment were analyzed about the index of lifespan(middle lifespan, mean lifespan, maximum lifespan,90% survival time), reproduction rate of parental and filial flies, and DNA methylation polymorphism during different development stages in Fi flies. The results were as follows:1. High quality genomic DNA from Canton-S was extracted by improved SDS method and several key factors, including EcoR I/Alu I digestion, adapter ligation, pre-amplification, selective amplification were analyzed. The optimized system was obtained as①ligation reaction system:T4 ligase 1 U, A-adapter 50 pmol, E-adapter 5 pmol,12 h for ligation at 4℃.②Pre-amplification reaction was performed in a 25μL volume containing Mg2+ 1.0 mmol/L, dNTPs 0.2 mmol/L, E-00 50 ng, A-00 50 ng,2.5μL of template DNA, Taq DNA polymerase 2 U.③The selective amplification was contained Mg2+ 1.0 mmol/L, dNTPs 0.15 mmol/L,2.0μL of the diluted product, Taq DNA polymerase 1.5 U, E+3 40 ng, A+3 40 ng, and the total volume was 25μL. The clear AFLP fingerprintings have been obtained. By virtue of high stability and good repeatability, this optimized AFLP system can be utilized for the methylation analysis for fruit fly.12 pairs were used for DNA methylation polymorphism which selective primers were screened with the type of distribution, high polymorphism bands and high resolution in 64 pairs of primers.2. Differences in the level of methylation between differently aged Drosophila, however, the Polymorphism Loci are mustered the embryo phase, quotiety of the polymorphism loci is not diversity between the hole embryo phase. The level of DNA methylation is different between male and female fruit fly.3. The propriety concentrations of zinc advanced the parental flies lifespan (middle lifespan, mean lifespan, maximum lifespan,90% survival time) and reproduction capacity of parental fruit fly; high concentration of zinc reduced the lifespan of Drosophila, finally, these organisms are aging and dead aheadly, also, the high concentration of zinc reduced the reproduction number is zero, it demonstrated that eat more zinc is not the better.4. The propriety concentrations of zinc increased the lifespan and egg laying amount of filial flies. It demonstrated that the effect on lifespan and reproduction ability of flies can be extended to F1 generations at least.5. The appropriate concentration of zinc medium (zinc concentrations were 2.5%,5%,10%) were used to culture Drosophila, and its offspring 0～3 h embryos, third instar larvae, pupae, and male Drosophila DNA methylation polymorphic bands ratio did not change significantly; 5～8 h embryo DNA methylation percentage of polymorphicbands in the experimental group F1-1, F1-2, F1-3 was significantly higher than control group; experimental group F1-2 (zinc concentrations was 2.5%) most affected by the zinc in 5～8 h embryos,12～15 h embryos,18～21 h embryos and female flies in the polymorphic DNA methylation band ratios were significantly higher. Appropriate levels of zinc material can increase DNA methylation polymorphism of offspring in Drosophila.Drosophila is the importment model organism in biologic study. We first studied the effects after zinc treatment on DNA methylation polymorphism, lifespan and spawning in Drosophila. These studies will provide experimental data for the DNA methylation research in Drosophila, but also help people to research trace elements and the relationship between DNA methylation and environment factors. |
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