The Role And Function Study Of DNA Methylation In Arthrobotrys Oligospora Nematode-trap Formation And Selection-oriented Functional Genomics And Mechanism Study In UGT2B4Upstream Enhancer Region

Part Ⅰ:plant-parasitic nematode infection is the second most serious disease in agricμLture and biological control is a potential approach to reduce the lost. Arthrobotrys oligospora(A.O) is a is typical Nematophagous fungi can immobilize and penetrate nematodes by producing a special organ,nematode-trap,but this process is initiated by complex gene expression regμLati-on,which is still unclear.Cytosine methylation is an important epigenetics modification in eukaryotic genomel and widespread and ancient way to regμLate gene expression.Moreover, this modification has been suggested to be involved in the mycelia growth and nematode-trap formation in A.oligospora. This project will provide more insight into molecμLar mechanisms of life style switch and nematode caputure in A.oligospora,and DNA methylation pattern and function in fungi genome.First, treat with bisufite on the basis of genome sequencing to obtain a single-base resol-ution maps methylated group, followed by comparison between the two hyphae methylation profiling which one has traps and another has no, we discovered methylation levels of genomic DNA remarkable drop.we found203gene that have differential methylation through compare their methylation levels of genomic DNA,some of which have function with Amino acid transport and metabolism, DNA replication and repair, and lipid transport and metabolism, energy generation, the cell cycle control, etc,the resp Lts show that this some gene cluster have played a important role in formative processes about A.O traps.The innovation of this section:Now from the perspective of epigenetics,there is no report about the effect and function of DNA methylation in the formation of AO’s traps.Part Ⅱ:natural selection signal has been suggested to be a novel approach to identify potential functional region in human genome. UGT2B4is an important metabolism enzymes in human body and has been proposed to correlate with breast cancer.in the upstream region of its coding gene,a strong balancing selection has been observed but the real function region and the mechanism is still not clear. This project will shed more light on the expression regμLation mechanism of the enhancer in UGT2B4upstream region,the onset of breast cancer,and the phenotype difference for balancing selection. Meanwhile,it will provide an good example for how to utilize mμ Ltiple approaches to identify functional region in human genome.In the present study,we utilized1000genomes plan data in corresponding region to perform selection analysis,we screened several potential genome function domain and finally select the strong signal domain:chr4:70389001-70392000,which is relative to build37, to do further research.First,we started to re-sequence with25individual DNA that is random in East Asia,ensured their mutation pattern and choosed individual with right haplotype to build plasmid.By using luciferase assay,we have been compared the enhancer activity of the two haplotypes in each region to determine the enhancer location. For several SNPs of reg3-l,which is an enhancer domain,we used mutagenesis approach to generate12plasmids that only has one nucleotide differentce compared with reg3-1and compare the activity of luciferase to search functional sites.We used chromatin immunoprecipitation assay to search the corresponding transcription factors.A studty shows that there are two haplotypes with deep division, high nucleotide polym-orphism and Tajima’s D in positive in this domain.Two haplotypes have significant difference in enhancer’s activity,this shows that it actually has function.Further research indicates that the functional fragment is reg3:70390572-70391811(relative to build37) which the activity of enhancer in haplotype2is only-63.1%(P<10-6) of haplotype1.By comparing the activity of plasmids’luciferase that were disposed with mutagenesis,we found two mutations in70390777(rs66862535) and70391561(rs68096061),which coμ Ld reduce its activity of hapl-otype enhancer to-40.3%(P<0.001) and49.8%(P<0.01) of reg3-1respectively.This indicated that it’s the functional site leading to bear balancing selection of this region.The fu-rther CHIP resp Lt showed that:this two adjacent domain coμ Ld both be enriched by the antibo-dy of POU2Fl.(P<0.002和<0.0003) This reminded the two domains were bounding regions of POU2F1.The innovation of this paper are as follows:1) Using natural balancing selection signal as means of bioinformatics by innovation of research method, we proceeed to analysis and compare for70kb lengthen sequence to screen potential functional region,which can shrink scope of candidates largely and inc-rease study efficiency.2) Meanwhile, we do research to the enhancer of upstream region of UGT2B4and target gene by using various functional genomics. Related content has not been reported.