| During the growth and development of chicken embryo,the nutrients that embryo demand only can be provided by which deposited in the egg before egg laying,and there is no organ that can discharge metabolic waste with the outside world,and can only rely on itself to decompose metabolites,such as reactive oxygen species generated during growth and development.During the development of the embryo,the body metabolizes faster,more oxygen is needed to provide energy,and more reactive oxygen species(ROS)are generated at the same time.High levels of reactive oxygen species cause protein,lipid per-oxidation and DNA damage.At this point,as the first defense line of defense against ROS-superoxide dismutase(SOD),glutathione peroxidase(GSH-PX),peroxidase(POD)works together to clear ROS.Studies have shown that in the process of growth and development,there are differences in each organization.However,the identification and functional verification of genes that regulate changes in these antioxidant enzyme activities are still in their infancy.Therefore,in this study,transcriptome sequencing was used to screen the key genes that regulate the activity of different antioxidant enzymes,to clarify the causes of the changes of antioxidant enzymes during the development of chicken embryos from the biological point of view,and to validate the selected key genes,Based on the above research purposes,this study obtained the following three aspects of the test results:1.Identification of antioxidant activity: During different hatching periods of chicken embryos,the activity of SOD was higher than that of GSH-PX and POD,and the highest activity in liver was 145.77 ± 4.37 U / mgprot.Therefore,the activity of SOD in the liver was taken as the screening index,and the liver tissues incubated on the 16 th day and the 20 th day were collected for the follow-up study.2.Transcriptome Sequencing Screening of Key Genes: By sequencing the chicken embryo liver transcriptomes hatched to 16 days and 20 days,57292698-65547680 raw reads were obtained,and 51868410-58937096 clean reads were obtained after filtering,reaching 79% of the reads of the reference genome,the above.The correlation coefficient of gene expression level between samples was greater than 0.97,threshold value p <0.05,foldchange?1,and 1154 differentially expressed genes were screened out,and 1069 significantly enriched GO terms were obtained by differential genomic GO enrichment analysis There were 10 GO terms related to antioxidant enzyme activity.GO enrichment and KEGG pathway analysis indicated that GSTA2,GSTA4,MGAT1,GPX3 and HAO2 are involved in glutathione metabolism and reactive oxygen species production,which are considered as key candidate genes that affect antioxidant enzyme activity.3.Functional validation of key genes: Isolation and culture of chicken embryonic liver parenchymal cells,synthesis of small RNA interference fragment targeting GPX3 gene,transfected cells were collected after 48 h to test the enzyme activity.The results showed that transfection efficiency could reach to 70%.Three small RNA fragments had a certain interference effect on the expression of GPX3 gene.The GPX3-gga-714 fragment had the most obvious interference effect on GPX3 gene,reaching(55.27 ± 0.06)%.In addition,the activity of SOD and CAT increased by(12.85 ± 0.01)U / mgprot and(1.19 ± 0.38)U / mgprot,GSH-PX,POD activity was reduced by(0.52±0.44)U/mgprot and(0.89±0.85)U/mgprot,respectively.In this study,we first studied the anti-oxidase system of chicken embryos during the hatching period by transcriptome sequencing,identified key genes that regulate the activities of antioxidant enzymes during embryonic development and their regulatory mechanisms,providing a theoretical basis for the further study of the growth and development of chicken embryos,it is of great significance for improving the nutritional value and economic benefits of chicken products and egg products?... |
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