Identification And Regulation Of Chicken Embryo Liver By Transcriptome Sequencing Key Genes And Functional Verification Of Antioxidant Enzyme Activity

During the whole hatching stage,chick embryos rely on external oxygen input to participate in the basic life metabolism and maintain the normal development of the body.However,with the increase of embryo growth,the production of reactive oxygen species will accelerate,and its excessive accumulation will destroy the balance of oxidative stress,causing interference and damage to the body in the later stage.At this time,antioxidant enzymes such as Superoxide dismutase(SOD),Glutathione peroxidase(GSH-Px),and Peroxidase(POD),which exist in the chicken embryo,will produce decomposition reaction to ensure the balance of oxidative stress in the development process.Some studies showed that the types and activities of antioxidant enzymes dispersed in different parts of chicken embryo during the whole incubation process are constantly changing,but the mechanism of regulating antioxidant enzyme activity is not clear,so the main purpose of this study is to make clear the key genes regulating antioxidant enzyme activity.Based on transcriptome sequencing technology,we screened out differentially expressed genes that regulate antioxidant function in chick embryo liver tissues,and a hydrogen peroxide induced oxidative stress model in chicken embryo hepatocytes was also constructed to explore the regulatory mechanism of key genes.1.Detection of antioxidant enzyme activity: it was shown that the activity of SOD and GSH-Px in liver tissue of chicken embryo from 12 days to 18 days had a trend of first decreasing and then increasing,and SOD activity in liver tissue of chicken embryo from 12 days and 16 days was significantly different from GSH-Px activity(P < 0.05).Therefore,the liver tissue of chicken embryo from 12 days to 16 days was chosen as the next transcriptome sequencing object.2.Identification and screening of key genes: by sequencing the liver transcriptome of chicken embryo incubated to 12 and 16 days,216 genes with significant difference expression(including 81 up-regulated genes and 135 down-regulated genes)were found,421 significantly enriched GO terms and 8 significantly enriched pathways were obtained by enrichment analysis.Go enrichment and KEGG pathway analysis showed that GSTA3,GSTA,?-TTP,MGST3,HMOX2,GSTA1,ODC1,MAOA and GST may be the key candidate genes that affect,the activity of antioxidant enzyme during chicken embryo development.3.Functional verification of key genes: chicken embryo liver parenchyma cellswere cultured in vitro,the oxidative stress model of cell growth induced by hydrogen peroxide was used,and vitamin C remission was used as the control group.The results showed that the expression of GSTA3 gene in the treatment group was significantly higher than that in the blank group and the control group(P < 0.05),and GSTA3 gene was associated with the antioxidant regulation.Meanwhile,the treatment with high concentration of vitamin C can effectively alleviate the body damage,1000 ?g/ml vitamin C is better than 750 ?g/ml vitamin C.The new antioxidant genes identified in this study provide a theoretical foundation for further exploring the antioxidant mechanism of chicken embryo liver.