| The nonsense-mediated mRNA decay (NMD) pathway serves important functions on mRNA quality control in eukaryotic cells. It can specifically identify and degrade aberrant transcripts harbouring a premature translational termination codon (PTC), thereby prevent the production of truncated proteins which may be deleterious to cell. Glutathione-S-transferases (GSTs) are composed of two homologous dimer subunit, which is one of important Phase II antioxidant enzymes, mu subunit of Glutathione-S-transferase (GSTM2) is related to reproductive performance in sows by regulating embryo implantation. Porcine GSTM2gene was found to contain a premature termination codons (PTCs) triggering NMD. GSTM2mutation may be result in pre-or post-natal death in piglets. In order to investigate the GSTM2gene function on embryonic development, we silenced GSTM2to in vitro simulate mutated GSTM2by RNA interference technology, and furthermore analyzed the network related to GSTM2by deep sequencing. The results are as follows:1.3siRNA fragments responsive to GSTM2were designed, synthesized and transfected both into ST cell line and PK cell line by LipofectamineTM2000. The transfection efficiency in PK cell is higher when compared with it in ST cell.2. The expression level of GSTM2mRNA was detected by real-time PCR and the result indicated that both PK and ST transfected cells had a low expression level of GSTM2, whereas former cells presented lower expression level.3. The expression level of GSTM2mRNA was detected at24h,48h,72h after transfection. The result showed that the expression of GSTM2mRNA at24h is lowest, whereas there was a normal expression level of GSTM2in ST cell but in not PK cell at72h.4. The expression level of GSTM2protein was detected by Western Blot. The analysis demonstrated a low expression of GSTM2protein in the cells after trasnfection as compared with the control.5.243differentially expressed genes (q>0.8) were isolated after sequencing between the normal and RNAi ST cell. |
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