MGST1 Gene Regulates Chicken Embryo Liver Cells Molecular Mechanism Research

Peroxidase(POD),superoxide dismutase(SOD)and glutathione peroxidase(GSH-Px)form the first antioxidant defense line,which works together to resist the damage caused by reactive oxygen species.In the process of embryo development,these enzymes from the dormant state into active state,to provide protection for the normal growth of chick embryo development.In different incubation stages,the activities of different tissues and organs are also quite different.Based on the key gene microsomal glutathione transferase 1(MGST1),which was screened by our research group in the early stage,we interfered with the target gene by RNA and identified the differentially expressed genes between the interference group(GR)and the blank group(KB)by transcriptome sequencing technology,in order to find out the molecule of MGST1 gene regulating the activity of antioxidant enzymes.The experimental results are as follows.(1)Optimizing the conditions of primary hepatic parenchymal cells of chicken embryo: The chicken embryo eggs hatched for 12-20 days were selected as the experimental raw materials.The isolation and culture conditions of primary cultured chicken embryo hepatic parenchymal cells were explored from the selection of embryonic age,the concentration and digestion time of trypsinase digestion solution,the type and concentration of fetal bovine serum,and the growth factor.Finally,chicken embryo hepatic parenchymal cells were selected.The optimum conditions for culturing primary hepatic parenchymal cells of chicken embryos were as follows: embryo age 16 days,concentration of trypsin digestion solution 0.25%,EDTA,digestion time 12 minutes,imported fetal bovine serum 2%,and growth factor added in culture medium.(2)Transfection test of primary hepatic parenchymal cells of chicken embryos: The siRNA interference fragment of MGST1 gene was successfully constructed and transfected into primary hepatic parenchymal cells of chicken embryos in vitro using lip2000 transfection reagent.The results showed that the transfection efficiency was over 70%,and the transfection efficiency was good,which could meet the requirements of subsequent experiments.At the same time,the five siRNA interference fragments constructed could inhibit MGST1 gene expression in primary hepatic parenchymal cells of chicken embryos to some extent.Among them,MGST1-gga-142 interference fragment had the highest inhibition efficiency on MGST1 gene in primary hepatic parenchymal cells of chicken embryos,reaching 60.33%,and the interference effect was obvious(P < 0.01).(3)Screening differentially expressed genes by transcriptome sequencing: Chicken embryo primary hepatic parenchymal cells interfered with MGST1-gga-142 with the highest interference efficiency were used as interference group(GR)and blank group(KB)for transcriptome sequencing.A total of 1064 differentially expressed genes were screened by sequencing,of which 429 differentially expressed genes were up-regulated and 635 differentially expressed genes were down-regulated.Seven key genes,including MGST2,NQO1,GPX2,GPX3,HAO1,APOA4 and APOA1,were found to be involved in the regulation of antioxidant activity of MGST1.The MGST1 gene regulates the activity of antioxidant enzymes by regulating the seven different genes.The results provide a theoretical basis for the subsequent identification of the metabolic pathway of antioxidant enzymes.