Transcriptome Analysis And Functional Gene Mining Of Evolutionary Malformation Caused By Nonylphenol In C Auratus Red Var.

Nonylphenol(NP)is an environmental endocrine disruptor widely present in water,which affects growth and development of aquatic organisms.In order to investigate mechanisms of non-phenol-induced developmental malformation of C auratus red var.,fish embryos were placed in NPs.The morphological changes in C auratus red var.embryos under NP stress were observed.Transcriptome sequencing was performed with NCK(neuroblast stage embryos in control group),SCK(21 somite stage in control group),NNP(neural stage that exposed to 5μmol/L NP)and SNP(21 somite stage that exposed to 5μmol/L NP)samples.Candidate genes affecting embryonic development were selected.Their full-length coding sequences were obtained by homologous cloning and RACE technology;The expression specificity of candidate genes were characterized by qRT-PCR.The methylation levels of candidate gene were detected.The results identified malformation of the crucian spine and thrombosis in the embryos under NP stress.Our RNA-seq yielded 89 166 unigenes,pairwise comparison between NCK,SCK,NNP and SNP groups discovered a total of 13 554 differentially expressed genes.25 differentially expressed genes were validated by qRT-PCR,indicating that the sequencing results are reliable.Pick out candidate genes Sox8 and GATA1,the Sox8 cDNA sequence was 2 163 bp in length,encoding 391 amino acids,while the GATA1 gene cDNA sequence was 2 730 bp long,encoding 443 amino acids.Our qRT-PCR analysis showed that Sox8 had the highest expression in the brain,and the expression levels in liver and pituitary were extremely low.GATA1 expression was highest in the testis,with extremely low expression in the liver and ovary.During pharyngeal sac stage,Sox8 expression in embryos was most sensitive to NP treatment.Expression of GATA1 gene in embryos was most sensitive to NP treatment in the somite stage.The 3μmol/L was the most effective concentration for Sox8 and GATA1.Our methylation analysis found that the expression of GATA1 in the control group was significantly positively correlated with the methylation levels in its 5’ UTR region;while methylation levels of GATA1 gene in NP treatment groups was higher than those in the control group.