Identification Of The Interaction Between DNA N~6-methyladenine Modification And Cey-1 Protein

N~6-methyladenine(6m A)is the most common DNA modification in prokaryotes,and it plays important roles in biological processes such as DNA replication,transcription and cellular defense.With the development of detection technology,6m A has also been found in eukaryotes DNA as a potential epigenetic mark.Caenorhabditis elegans(C.elegans)is a model organism,which is often used as human disease model for research.6m A modificaiton has been proved existed in C.elegans.The whole genome map of C.elegans and distribution of 6m A sites have been drawn,but there are few studies on the binding proteins related to 6m A.In order to understand the 6m A-marked DNA-binding protein in eukaryotes,we used C.elegans as the research model to identified Cey-1 protein by dimethyl quantitative protein assay,and studied its biological functions.This thesis is mainly developed from the following aspects:(1)Methods of isotope labeling can identify and quantify proteins.We chose the stable isotope dimethyl labeling strategy of quantitative proteomics combined with DNA pull-down assay to identify potential binding proteins of 6m A modification in C.elegans.We used 6m A modified ds DNA probe to screen potential binding proteins in vitro,and use formaldehyde to form isotope labels on the peptides.According to this,LC-MS/MS was performed,and the 6m A binding protein Cey-1 had been identified,which provided a worthy reference for 6m A study.(2)Cey-1 is a Cey family protein containing conserved cold shock domain,studies have shown that Cey family proteins have an effect on ribosomes formation.For eukaryotic expression,we constructed a Cey-1 recombinant plasmid containing Flag-tag as eukaryotic expression vector,and transfected it into 293T cells.Combined DNA pull-down assay with Western blot,we proved Cey-1 can directly bind to ds DNA probe with 6m A modification in vitro.This result provides an important theoretical basis for the study of 6m A binding proteins in eukaryotes.(3)We explored the potential biological functions of Cey-1 in C.elegans depthly.Using RNAi technology,we constructed Cey-1 deficient C.elegans mutants successfully.By counting the number of C.elegans progeny of wild-type and Cey-1 deficient mutants,the effect of Cey-1 on the reproduction and survival was explored.In addition,we constructed a Cey-1 involved transcription system,and quantified transcript abundance by frequency of sequences through next-generation sequencing.The results indicated that Cey-1 did not have any effect on transcription efficiency and transcription fidelity.This study broadens our understanding of Cey-1.In summary,this study used the dimethyl quantitative protein analysis method combined with DNA pull-down assay to identify 6m A potential binding protein Cey-1 in C.elegans.Western Blot further verified Cey-1 can directly bind to 6m A modified ds DNA probe in vitro.Statistical experiments on the number of Cey-1-deficient C.elegans indicate that knocking down the Cey-1 will not affect the reproduction and survival of the C.elegans.Through analysis of transcript abundance,Cey-1 will not affect the efficiency and fidelity of in vitro transcription,but upon mitochondrial stress,Cey-1 may participate in the regulation of cross-generational inheritance in another passway.