Effect Of Acetylation On The Activity Of The Enzyme PK Related With Energy Metabolism In Alates Of The Termite Reticulitermes Chinensis Snyder

Swarming behavior has important ecological significances for the distribution,spread and reproduction of termites.However,the level of energy supply is an important factor influencing the swarming behavior of termites.The previous studies mainly focused on climatological and biological characteristics involved in swarming behavior.Until now,the effect of protein acetylation modification on energy metabolism has not been reported during swarming behavior of termites.Here,we took the termite Reticulitermes chinensis Snyder as an experimental material to study the effect of acetylation on the activity of pyruvate kinase(PK)that is a key ratelimiting enzyme of glycolysis.Our results will help to understand the mechanism of energy supply during termite swarming,and can provide a new idea about developing energy inhibitors of termite swarming.The main results are as follows: 1.Cloning and expression of PK gene involved in energy metabolismWe cloned a fragment of PK gene with 2,325 bp nucleotide sequences in alates of R.chinensis,containing a coding region of 1,776 bp,and encoding a protein of 591 amino acid with a predicted molecular weight of 64 KDa and theoretical isoelectric point of 7.50.The sequence homology analysis indicated that our PK gene had the highest homology with PK gene from Zootermpsis nevadensis.The qRT-PCR results showed that the expression level of PK gene in throax was significantly higher than that in head and abdomen(p < 0.001).Meanwhile,the expression level of PK gene in abdomen was also significantly higher than that in head(p < 0.01).2.Construction of PK eukaryotic expression vector and its expression in Sf9 cellsThe optimized PK gene was obtained by the method of total gene synthesis,and the recombinant eukaryotic expression vector with green fluorescent markers(pIEx-1-EGFP-PK)was constructed.The recombinant plasmid pIEx-1-EGFP was transfected into Sf9 cells,and the optimum expression time was 72 h.Green fluorescence was observed in the host cells under the inverted fluorescence microscope after 72 h of the pIEx-1-EGFP-PK transfection.About 2,500 bp fusion fragment was detected by RTPCR,and about 100 KDa specific protein band was identified by western blot.These results suggested that PK protein can be heterogeneously expressed in Sf9 cells.3.Screening and validation of the PK acetylation siteThe three acetylated enzymes related with energy metabolism during the glycolysis pathway were screened from the quantitative modification proteomic data,including the acetylation site K160 of pyruvate kinase(PK)that was down-regulated in dealates of R.chinensis(multiple = 0.824).The 478~480 bases encoding 160 amino acid were respectively mutated from AAG(Lysine,K)to CAG(Glutamine,Q)and AGG(Arginine,R)by the method of site-directed mutagenesis.Two mutant plasmids,including PK-K160 Q and PK-K160 R mutants,were constructed.The two mutants and wild-type PK were transfected into Sf9 cells.The results of western blot showed that the acetylation level of wild-type PK was higher than that of the two mutants,suggesting that PK could be acetylated and its acetylation site was K160.4.Effect of deacetylase inhibitors on the PK activity in alates of R.chinensisThe PK activity(p < 0.01),ATP content(p < 0.01)and the pyruvate content(p < 0.05)of alates were significantly decreased after treated by deacetylase inhibitor TSA for 3 d,whereas the expressions of PK gene in alates were significantly increased(p < 0.05).The alates exhibited significantly increased PK activity(p < 0.01),significantly increased ATP content(p < 0.05),marginally significantly decreased expression of PK gene(p = 0.0625),and marginally significantly increased pyruvate content(p = 0.0520)after treated by TSA for 5 d.In addition,alates exhibited significantly increased PK activity(p < 0.01),significantly increased ATP content(p < 0.05),significantly decreased pyruvate content(p < 0.05),and significantly decreased PK gene expression(p < 0.05)after treated by deacetylase inhibitor NAM for 3 d.The alates exhibited significantly increased PK activity(p < 0.05)after treated by NAM for 5 d,but the ATP content(p = 0.1227),PK gene expression(p = 0.3125)and pyruvate content(p = 0.5176)did not significantly change.Meanwhile,both TSA and NAM had no significant effect on the survival rate of alates(p = 0.3341).