| Cadmium, as one of the most important pollutions among heavy metals, has become one of the hotest research topics in recent years. Cadmium has been widely put into use into manufacturing nickel-cadmium batteries, painting, electroplating and alloy industry, and cause every year there are a large number of cadmium were discharged into the environment through industrial activities, then accumulate and enter the human body through such as fog, sewage, food chain, endanger peoples’ health. At present, the seriously problem of cadmium pollution has been rapidly increasing, poses a great environmental threat to human survival. The study on the biological toxicity of cadmium now have been widely and deeply, researchs reveals that cadmium can cause the rat liver cells and cerebral cortex neurons and HL-60cell apoptosis. Cadmium can cause oxidative damage, interferer ion-channels and protein activity, activate p53or Caspase-3pathway, these two pathways are considered to be the molecular mechanism of apoptosis. In addition, cadmium can also induce epigenetic changes, especially DNA methylation modification, consequently affect the stability of the genome and gene expression. Drosophila Melanogaster as a classical genetic model, has a simple but efficient methylation modify system. DNA methylation is one of the most important aspects of the epigenetic related research, and participate in all the growth and development progress in Drosophila Melanogasters, it can specific silence genes, inhibit mitosis and modify transcription patterns to resisit against stress like Cd, the modifications of DNA methylation pattern may also be one of the important factors in starting apoptosis. In order to uncover the influence on Cadmium affect Drosophila melanogaster DNA methylation and apoptosis related genes, this article set the Canton-S strains as experimental materials, and used three parts to support the topic.Firstly, in order to uncover the gender difference and epigenetic heritability of DNA methylation related genes. Real-time PCR technology was applied to analysis the expression level between genders and generations. This article tested the changes of DNA methylation modification related genes (dDNMT2, dMBD2/3) at the expression of mRNA level, on those flies which were treated in medmiums with different cadmium mass fractions (0,0.9375,1.875,3.75,7.5,15,30,60mg/L), we also tested different tissues as reproduct system, reproduct system removed body cells, and intact flies, extented to the offspring (Fi) flies. Results uncoverd that compared with control group, Cd stress induced the expression of dDNMT2, dMBD2/3mRNA level to arise in parental drosophila tissues like ovary, testis, ovary removed, testis removed, intact female flies, intact male flies, the expression increases with the arise of cadmium exposure concentrations, seems like its on a dose dependent. The reproductive cells is found rise greater than the intact ones, and is more greater than the reproductive removed residue, and the comparison between male and female result in females rise more greater than males(P<0.05), and the change in expression patterns have heritability, can continuely pass to the next generation.Secondly, a sample of half-lethal concentrations (52mg/L) cadmium treated female fly, which is considered with significant genetic diversity, was high-throughput sequenced with a next-generation-sequencing technology named digital gene expression profile (DGE). According to the results, both control group and treatment group have received nearly6million original labels, after removing impurity tags and match those tags,4906113unique match was found in CK group (87.09%), and5118729(90.09%) in TG group. Compare all differently expressed genes there were1443up-regulated genes,527down-regulated genes have been discovered. Further more, target genes had been filtered and screened, focus on DNA methylation related genes and apoptosis related genes, four significantly different expressed genes were found involved in DNA methylation process, not only the DNA methyltransferase gene DNMT2and methyl binding protein gene MBD2/3, which are directly involved in the methylation, there are two indirect genes:Su(var)205, dSETDBl, which control DNA methylation by histone methylation, also been found. In apoptosis pathway, eight significantly different expressed genes had been found, including telomere regulation factor tefu, which increases p53expressions in upstream, or IAP family gene CG17019, apoptosis excute gene Damm, participate in Caspase-3pathways and regulate the apoptosis pathway. These results discovered that the epigenetic related and cell apoptosis related genes are both changes while under cadmium treatment, reveal that these two process may work in a combination way, at least play an important role in resisiting the toxic effect of cadmium. The transcription activity changed by the modifications of DNA methylation pattern in cell apoptosis related genes may be one of the reasons that cell apoptosis occurance in fruit flies, but the proof of the specific contact has to be further studied.Thirdly, the Real-time PCR method were applied not only for the screening of apoptosis related genes, but also for testing and verifing the reliability of the DGE data, and expanding of the experimental conditions to explore the gene expression patterns under different environmental factor changes. This research tested some key genes related to the apoptosis such as tefu, p53, Caspase-3, CG17019, Damm, their expression were examined in female and male fruit flies treated in medmiums which contains different mass fraction of cadmium (0,7.5,15,30,60mg/L). The results show that, compared with the control group, the male and female fruit flies apoptosis related gene were significantly raised, but has its specificity between genetic model, no significant difference were found between genders. CG17019falls after raise, the others remain a dose dependent but have difference in relative abundance against reference gene.In sammary, this research analyzed the effects of transcription activity on DNA methylation genes in cadmium induced fruit flies, found that cadmium increased dDNMT2, dMBD2/3expression, with a dose effect and has the heritability. The result will contribute as a experimental basis in the investigation of epigenetic variation of induced by cadmium, has some practical significance for the study of human epigenetic risks on environmental factors such as cadmium or other heavy metals. The Using of digital gene expression profiles for screening significantly changed apoptosis-related genes in Cadmium treated Drosophila will respond more deeply into the molecular mechanisms of cadmium induced apoptosis, and further provide research support in the investigation of the link between DNA methylation and apoptosis, and it will also contribute in learning more about the possible regulation of the relationship between the occurrence of apoptosis induced by heavy metals and epigenetic modification system, provide new ideas in diagnose diseases through the detoxification of heavy metal. |
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