Molecular Mechanism Of Hox Gene-mediated Teratogenicity Of Chlorpyrifos On Xenopus Tropicalis Embryos

The organophosphorus insecticide chlorpyrifos is highly toxic to aquatic organisms and is frequently detected in water bodies.In the early stage,most amphibians lived in farmland and nearby water bodies,and pesticides were one of the main reasons for the decline of amphibian populations.In response to the stress of external pollutants,amphibians can respond to environmental changes during early development by changing their morphology,behavior,and physiological activities.Among them,apparent modification has a regulatory effect on the body's phenotype,and histone methylation modification is one of the main ways of apparent modification.This thesis takes Xenopus tropicalis as the research object,by studying the teratogenic effects of chlorpyrifos,screening malformation-related genes,and clarifying the differential expression of teratogenic-related genes,and the correlation between histone methylation modifications and related genes.Transcriptome sequencing,Western blotting and chromatin immunoprecipitation studies were performed on malformed embryo samples to clarify the relationship between histone methylation modifications and malformation-related genes annotated by transcriptomics.Lay the foundation for further elucidating the molecular mechanism of chlorpyrifos teratogenicity.The main findings are as follows:1)We used three different concentrations of CPF: 0.05 mg/L,0.20 mg/L,and 0.80 mg/L to treat Xenopus tropicalis embryos at the NF10-12 stage.The results of the 96 h FETAX test showed that as the concentration increased,the death rate Increased,Xenopus embryo mortality has a significant dose-effect relationship,the mortality rate in the highest concentration group reached 25.83%;chlorpyrifos treatment group had different degrees of teratogenic effects,as the concentration increased,the number and severity of abnormalities Increasingly,the teratogenic rate in the 0.8mg/L concentration group is as high as 88.24%,showing a significant dose-effect relationship.The four main abnormal phenotypes in abnormal embryos are notochord curve(22.53%),tail curve(18.77%),pericardial/pericardial cavity edema(15.02%),craniofacial edema(11.26%),of which notochord curve and tail curve.The phenotypic proportion accounted for 41.30%.2)RNA-seq was performed on malformed embryo samples.The sequencing results showed that most of the Hox genes were up-regulated(P-value<0.05,FC>1).28 annotated Hox genes were analyzed,and we verified 6 by real-time fluorescence quantitative PCR.Hox genes with the same expression are:Hoxa3,Hoxa9,Hoxa11,Hoxa13,Hoxc5,Hoxd4,and their expressions are significantly up-regulated(*P<0.05,**P<0.01);3)Western blotting showed that histone H3K4me3 expression was down-regulated in tropical Xenopus malformation embryo tissues,indicating that it was involved in the occurrence and development of embryo malformations.The activity of H3K4me3 specific methyltransferase was further analyzed and regulation was found.The expression of Mll1 of methyltransferase is enhanced,and the expression of LSD1 that regulates demethyltransferase is reduced.This change does not correspond to the modification level of H3K4me3;4)The chromatin immunoprecipitation test(ChIP)found that the binding level of H3K4me3 and Hox genes induced by chlorpyrifos decreased(*P<0.05),that is,the chlorpyrifos induced marker H3K4me3 on the promoter region of the Hox gene decreased,and the H3K4me3 modification level was compared with 6 Hox There is a positive correlation between gene binding levels(Hoxc5(r=0.601,p=0.039)and Hoxd4 have a significant correlation r=0.614,p=0.034),which proves that Hox gene expression is regulated by H3K4me3 modification.The results of this paper show that chlorpyrifos induces significant differences in the expression of6 Hox genes,which may be related to histone H3K4 trimethylation modification.Therefore,histone H3K4me3 modification and its methyltransferase-induced dysregulation of Hox gene expression may be a risk factor for teratogenicity of chlorpyrifos,which requires further research and verification.This thesis is the first attempt to compare and study the histone methylation modification of malformed embryos after 96 h of exposure to chlorpyrifos,and provide a reference for studying the histone methylation modification and DNA methylation of the teratogenic susceptibility genes of embryos induced by chlorpyrifos.Theory and technical support.