| Wolbachia(Wolbachia pipientis)is a kind of intracellular symbiotic bacteria that widely found in insects.They can regulate the reproduction of the host in a variety of ways.One of the most common way is about inducing the hosts to produce cytoplasmic incompatibility(CI).That is to say,after the male infected with Wolbachia mating with the female host which is uninfected or infected with different strains of Wolbachia,the embryo hatching rate of the offspring will be significantly reduced or unable to produce offspring.On the other hand,the male and female hosts infected with the same Wolbachia can produce normal offspring after mating.Although there has been many researches focus on CI so far,the molecular mechanism of CI is still being explored.In recent years,epigenetics has been tightly associated with development,physiology,and pathology.DNA methylation and protein phosphorylation,which plays an important role in post-translational modification,have also become the popular research subjects.Different from many genetic diseases caused by genetic variation,the diseases caused by epigenetic changes are reversible.This may lead to the further investigation of epigenetics,and make it become one of the most prevalent research in biology,medicine and many other fields.The aim of this study is to investigate whether Wolbachia infection can affect DNA methylation and protein phosphorylation in host reproductive system and embryo,using Wolbachia infected and uninfected Drosophila melanogaster as the experiment materials.The relationship between Wolbachia and male fertility will be discussed to provide a new idea for further study on the molecular mechanism of CI production induced by Wolbachia.Also,it will offer us further opinions on the molecular mechanism of Wolbachia affecting epigenetic inheritance in Drosophila.In this study,we first use fluorescence quantitative PCR to detect the effects on the expression level of 6mA demethylase DMAD after Wolbachia infection.The result showed that the relative expression level was significantly up-regulated in Wolbachia infected flies compared with the uninfected one.However,there has no effect on ovary.In addition,the expression level of DMAD was significantly different in various embryos at different stages[TT(the control,with both males and females uninfected),TW(viable,males were uninfected while females were Wolbachia-infected),and WT(lethal CI embryos,with males Wolbachia-infected and females uninfected)].In early CI embryos(0.5h,pre-midblastula transition),DMAD expressionin level was significantly higher than those of the control and TW embryos.In the embryos at midblastula transition(3h),the transcriptional levels of DMAD in TW and CI were extremely higher than that in the control embryos.In 6h embryos(post-midblastula transition),however,DMAD expressionin level was the lowest in CI embryos as compared with the control and TW embryos.These results together suggested that Wolbachia infection might interfere the level of 6mA methylation in the testis of Drosophila,together with the modification of sperm,thus result in abnormal gene expression and the death of early embryos derived from Wolbachia-infected males and uninfected females.In terms of protein phosphorylation,we collected male reproductive system(including testes,accessory gland,spermaduct and ejaculation balls,and the samples are marked as Dmel wMel RS and Dmel T RS,respectively)of 1d Wolbachia infected and uninfected Drosophila melanogaster.Then we add PhosSTOP(Rochee),an inhibitor of phosphatase,and extract the protein,then labeled with iTRAQ and enriched by phosphorylation.After that,the protein was detected by HPLC and MS.The differential phosphorylated proteins in male reproductive system were compared and analyzed.We totally identificated 1583 Phosphorylated peptides,and 1403 Phosphorylated position(phosphoRS probability≥0.75),which located on 871 phosphorylated proteins.It was found that there were significant differences in phosphorylation among 33 peptide segments(the difference multiple≥1.5 or ≤0.667,with P<0.05),27 of which were up-regulated and 6 were down-regulated.From the biological process,most of the differential phosphorylated proteins are related to the reproduction of multicellular organisms(such as spermatogenesis and sperm motility).Some others are related to cell process,catalytic process,regulation of biological process,immune process and so on.Since there has no antibodies corresponding to these proteins,we first detected the corresponding genes of the 33 peptides by qRT-PCR technique.It was found that 11 of the genes were significantly up-regulated and 1 gene was down-regulated because of Wolbachia infection.This suggests that Wolbachia not only leads to differences in phosphorylated proteins in the male reproductive system of the host,but also changes the level of these genes at the transcriptional level.We are preparing antibodies to explore the function of these differential phosphorylated proteins at the following stage.In brief,Wolbachia infection can change the expression level of 6mA and protein phosphorylation in the host.These two modifications all play an in portant role in epigenetics and may lead to changes in the male reproductive system and fertility of Drosophila.This study will provide a new idea for the molecular mechanism of CI induced by Wolbachia in Drosophila melanogaster. |
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