Effects Of Porcine PFKM On Cell Metabolism And Mitochondrial Function

Phosphofructokinase-muscle(PFKM)is a key multi-regulatory enzyme which is used to catalyze the irreversible transformation of fructose-6-phosphate into fructose-1,6-diphosphate during glycolytic cycle.PFKM gene regulates glucose fatty acid cycle by affecting the structure and function of mitochondria.In previous study,PFKM protein expression in muscle tissues showed obvious difference in Bama pigs,Large White pigs and Jiangquhai pigs with Isobaric Tags for Relative and Absolute Quantitation(iTRAQ)technology.It is likely that PFKM affects energy metabolism by regulating mitochondria,thereby affecting pig growth and metabolism.In order to study the effect of porcine PFKM gene on cell metabolism and mitochondrial function,3 porcine PFKM gene CRISPR/Cas9 knockout vectors and PFKM overexpression vector were used to prepare PFKM gene knockout cell strain and PFKM gene overexpression cell strain respectively,and the effects on mitochondrial function were measured at the cellular and molecular levels.The main contents of this study include the following 3 parts:Study 1:Construction of porcine PFKM gene knockout vector by CRISPR/Cas9 system and the preparation site-specific mutant cell strain.Three sgRNAs were designed to target the PFKM gene and corresponding knockout vectors pPFKM-KO1,pPFKM-KO2 and pPFKM-KO3 were constructed by CRISPR/Cas9 systems.The plasmids were transfected into PK15 cell lines,purinomycin was used to select and enrich PFKM knockout cell population.The DNA of each cell group was extracted and the knockout region was amplified with PCR method.The knockout efficiency was determined at the DNA level by sequencing.Cell RNA and protein of each group were extracted and the expression level of PFKM was detected by qRT-PCR and Western blot respectively.The results showed that mutations or deletions were detected in the PFKM DNA of each knockout group.Both mRNA and protein expression were reduced,and DNA mutation rate in PFKM-KO2 group was the highest.PFKM-KO2 mRNA and protein expression decrease the most.Compared with the control group,the difference was extremely significant(P<0.01).The PFKM-KO2 plasmid with the highest knockout efficiency was transfected into porcine PK15 cells and screened with puromycin to obtain single cell clones.The DNA of each cell clone was extracted,and the knockout region sequence was amplified with PCR method.The expression level of PFKM mRNA and protein was detected by qRT-PCR and Western blot respectively.The results showed that in the single cell clone obtained by transfection of CRISPR/Cas9 gene knockout plasmid,the mutation in the PFKM gene sequence was selected.Compared with normal cells,the results of qRT-PCR showed that the expression of cell strain PFKM mRNA decreased by 89.41%,the difference was very significant(P<0.01)and the results of Western Blot showed that the expression of PFKM protein in the knockout cell strain decreased by 81.63%,the difference was significant(P<0.05).In brief,the PFKM gene knockout cell strain was successfully acquired.Study 2:Construction of porcine PFKM gene overexpression vector and preparation of overexpression cell strainThe porcine PFKM cDNA gene sequence was retrieved from the GeneBank website,the porcine PFKM gene cDNA fragment was synthesized,and the corresponding overexpression vector was constructed.After the vector was transfected into PK15 cells,the cells expressing GFP protein were selected with flow cytometry method.Single cell clones were selected by limiting dilution method.Genomic DNA of each cell clone was extracted,and the GFP region sequence was amplified by PCR.The integration of overexpression plasmids was detected by electrophoresis and sequence determination.The expression levels of PFKM mRNA and protein were detected by qRT-PCR and Western blot.The results showed that the integration of foreign gene GFP was detected in four cell strains,compared with the normal cells,the expression of PFKM mRNA in four cell strains increased.Among them,the expression of PFKM-OE29 cell strain increased 17.86 times,the difference was extremely significant(P<0.01).Compared with normal cells,the expression of PFKM-OE29 PFKM protein increased by 30%.In brief,the PFKM gene overexpression cell strain was successfully acquired.Study 3:The effect of porcine PFKM gene on cell cycle,apoptosis and mitochondrial functionPorcine PFKM gene knockout cell strain,porcine PFKM gene overexpression cell strain and normal cells were used to detect the cell apoptosis and mitochondrial physical and chemical indexes(ATP,ROS,membrane potential,cell cycle)by flow cytometry,the changes of morphology and structure of mitochondria by transmission electron microscopy.The effects of PFKM gene on cell metabolism and mitochondrial function were detected.Compared with the normal cell group,the cell apoptosis rate and expression of ATP,ROS were reduced in PFKM gene knockout cell strain.The expression of membrane potential was increased and the cell cycle was blocked.For PFKM gene overexpression strain showed that compared with the normal cell group,the apoptosis rate increased,and the expression of ROS and ATP increased.The membrane potential decreased and the cell cycle was blocked.The results of transmission electron microscopy showed that compared with normal cells,the number of mitochondria in PFKM knockout cell strain and PFKM overexpression cell strain increased,and swelling and vacuolation occurred.A PFKM gene knockout cell strain and a PFKM gene overexpression cell strain were prepared.The effects of PFKM gene on cell metabolism and mitochondrial function were examined with flow cytometry,Western blot and transmission electron microscopy method.Knockout of porcine PFKM gene could enhance the cells anti-apoptotic ability to a certain extent and improve the energy metabolism efficiency of cells',but it had certain damage to the structure and function of mitochondria.The overexpression of porcine PFKM gene could reduce the cells' anti-apoptotic ability and cell energy metabolism efficiency.The mitochondrial structure and function were also damaged.In this study,further functional verification will be carried out in the pig population,and new target genes regulating skeletal muscle development will be screened out for molecular identification and breeding of excellent pigs.