Cloning Expression And Function Analysis Of The PHKG1P And HKG2 Genes In Guizhou Congjiang Pig

Phosphorylation kinase?PHK?is a key regulatory enzyme in the metabolic process of glycogen metabolism,including four subunits ? ??????,of which ? is a catalytic subunit,and is affected by Phosphorylase kinase ?1?PHKG1?and Phosphorylase kinase ?2?PHKG2?genes.PHKG1 and PHKG2 act together to provide energy for muscle contraction and maintain blood glucose balance.In this experiment,Guizhou Congjiang pigs were used as research objects.The functions of PHKG1 and PHKG2 genes were studied at the molecular and cellular levels using techniques such as molecular cloning,RNAi,and cell culture.The main findings are as follows:?1?Using qRT-PCR to detect the expression of PHKG1 and PHKG2 genes in different tissues of Large white pigs and Congjiang pigs,it was found that PHKG1 gene has the highest expression levels in the longissimus dorsi muscle of Large white pigs and Congjiang pigs,and the expression level is lower in heart,liver,spleen,kidney and other visceral tissues.The expression level of PHKG1 is significant difference in lung tissue from Large white pig and Congjiang pig?P<0.01?;the expression level of PHKG2 gene in the tissues was spleen> lung> large intestine> small intestine> fat> liver> kidney> stomach> heart> longissimus dorsi.There was no significant difference in the expression of all tissues from Large white pigs and Congjiang pigs?P>0.05?.?2?The sequences of the PHKG1 and PHKG2 gene coding regions from Congjiang pig and Large white pig were successfully cloned.There were 4 base mutations in the PHKG1 gene sequence compared with those in the Gen Bank,and there was a mutation in the T945 C gene in comparison with the Large white pig.There is no difference in the encoded amino acid sequence,which is a synonymous mutation.The sequences of PHKG2 from Congjaing pig,Large white pig,Gen Bank pig?NM₀01166317?and Bama pig?KJ 186785?were compared and found that Large white pigs,Congjiang pigs and Bama pig share G236 A,C431T,A726 G,A807G,A816 G,and A867 G mutations.The presence of A614 G mutation site from Congjiang pig caused the 205 th glutamic acid to become alanine.Bioinformatics analysis revealed that both PHKG1 and PHKG2 proteins are transmembrane hydrophilic non-secretory proteins.PHKG1 and PHKG2 are highlyhomologous,and the encoded protein structure also has certain similarities and may have similar biological functions.?3?An overexpression vector p EGFP-N3-PHKG1 and two sh RNAi vectors p LVX-Sh RNA-puro-PHKG1-1,p LVX-Sh RNA-puro-PHKG1-2,and a negative control sequence p LVX-Sh RNA-puro-PHKG1-NC were designed for the PHKG1 gene.For PHKG2 gene,designed an overexpression vector p EGFP-N3-PHKG2 and four sh RNAi vectors p GPU6/GFP/Neo-PHKG2-1,p GPU6/GFP/Neo-PHKG2-2,p GPU6/GFP/Neo-PHKG2-3,p GPU6/GFP/Neo-PHKG2-4 and a negative control sequence p GPU6/GFP/Neo-PHKG2-NC.These vectors were successfully constructed after preliminary double digestion and sequencing.The constructed overexpression vector and RNAi vector were transfected into C2C12 cell line and Congjiang pig kidney cells to detect the expression level of PHKG1 and PHKG2 genes.The results showed that overexpression of PHKG1 gene can significantly increase the expression of PHKG1,PHKG2,PGAM2?Phosphoglycease mutase?genes,and reduce the level of glycogen in the cells;Interference with PHKG1 gene revealed significant downregulation of PHKG1,PGAM2,GYS1(glycogen synthase 1?muscle?genes after transfection with p LVX-Sh RNA-puro-PHKG1-1.After overexpression and interference of the PHKG2 gene,the expressions of the glycogen phosphorylase-related genes PYGM?glycogen phosphorase,,muscle form?,PGAM2,GYS1 were up-regulated and down-regulated,respectively,and the intracellular glycogen content was decreased and increased.The biological functions of PHKG1 and PHKG2 genes were studied from both positive and negative aspects of overexpression and RNAi,indicating that these two genes have regulatory functions in the metabolic process of glycogen metabolism in pigs.