Identification And Characterization Of AMPK/SIRT/PPAR And Their Distinct Expression Patterns In Response To Air Exposure In The Manila Clam (Ruditapes Philippinarum)

As a typical tidal flat shellfish,clam live in the intertidal zone between the ocean and land transition area.In addition to several hours of air exposure stress per day,complex changes in environmental conditions such as temperature,dissolved oxygen,CO2,salinity and pH are also need to be addressed.Based on the whole genome of the clam,it is important to study the AMPKs,SIRTs and PPARs gene family response mechanisms related to energy metabolism in the clams under air exposure,which was important for revealing the adaptability of the clam in tidal flats.Research on terrestrial life provides provide important reference for the study of marine organisms from aquatic to terrestrial life.In this study,a family of genes related to energy metabolism was obtained from clam genome: AMPKs,PPARs and SIRTs.Through bioinformatics methods,the related information such as gene structure was analyzed.The results showed that the AMPKs of the clams contain three genes,AMPK?,AMPK? and AMPK?.The open reading frames of AMPK?,AMPK? and AMPK? are 1722,837 and 1137 bp,respectively,encoding 595,284 and 392 amino acids,respectively.The relative molecular weight range is predicted to be 30.83-64.55 kDa,and the isoelectric point range is 6.28-8.69.In addition,AMPK? has 21 exons,AMPK? has 8 exons,and AMPK? has 12 exons.Phylogenetic analysis showed that clam and ampullaria gigas spix AMPK alpha/beta/gamma were closely related.The human SIRT gene contains SIRT1-7.According to the database alignment,the SIRT genes identified by the clams are named SIRT1,SIRT2,SIRT4,SIRT6 and SIRT7,respectively.The open reading frames of five genes are 2523,804,978,1623 and 1278 bp,respectively encoding 840,425,325,540 and 267 amino acids,with a predicted relative molecular weight range of 30.29-93.09 kDa,isoelectric point range is 4.56-9.09.In addition,SIRT1,SIRT2,SIRT4,SIRT6,and SIRT7 have 9,13,6,6,and 8 exons,respectively.Phylogenetic analysis showed that the SIRT protein of the clams had higher homology with the scallops and Pacific oysters.The PPAR gene of the clams includes: PPAR?,PPAR? and PPAR?.The open reading frames of PPAR?,PPAR? and PPAR? are 912,1644 and 2124 bp,respectively,encoding 239,547 and 707 amino acids,respectively.The predicted relative molecular weight range is 26.74-80.62 kDa,and the isoelectric point range is 5.68-9.46.In addition,PPAR? has 5 exons,PPAR? has 9 exons,and PPAR? has 11 exons.Phylogenetic analysis indicated that both PPAR? and PPAR? were closely related to scallops,and PPAR? was closely related to Ampullaria gigas spix.In this study,real-time quantitative fluorescent PCR was used to detect the expression of different developmental stages,tissues in clams and three tissues(gonads,intestines and gills)of clams under air exposure.The expression pattern of AMPK was similar in the five tissues,and the expression was highest in the gill,followed by the intestine.After 48 h of air exposure,the expression of AMPK? in the low temperature group was significantly increased at 48h(P<0.05).Overall,air exposure did not promote the expression of AMPK?.After 48 h,the expression patterns of AMPK? and AMPK? were similar.The expression levels of both were significantly increased(P<0.05),indicating that AMPK? and AMPK? can be activated by air exposure,and the expression of AMPK gene is higher than 5°C at 20°C.The abnormal expression pattern of AMPK gene may indicate that energy metabolism in bivalves is highly sensitive to high temperature,which may lead to circulatory or respiratory failure,when AMPK is activated to control ATP consumption and synthesis.Study on SIRT gene family showed that SIRT was expressed in different developmental stages of clams,with the highest expression of SIRT1.SIRT was mainly expressed in gonads,intestines and gills in different tissues.After exposure to air,the expression of SIRT genes increased significantly except SIRT4(P<0.05);after 24 hours of exposure,SIRT1 and SIRT2 in low temperature group were significantly higher than that in normal temperature group(P< 0.05),which may indicate that prolonged air exposure may alter the metabolic pattern of clams dominated by aerobic pathway at low temperature.Overall,SIRT gene family,as a key gene regulating the level of NAD+/NADH in cells,are involved in regulating the redox pathway of energy metabolism in clams during air exposure,thus improving the antioxidant capacity of clams.Studies on PPAR gene family showed that PPAR? was almost not expressed in juvenile clam,and PPAR? and PPAR? were highly expressed in juvenile clam.The results of expression in different tissues showed that the three genes were expressed in different levels in all tissues.The highest expression of PPAR? was found in gills,and the highest expression of PPAR? and PPAR? was found in intestines.The expression of PPAR gene after air exposure showed that the expression of PPAR? was significantly affected by low temperature air exposure.The expression of PPAR? was significantly increased(P < 0.05),and the expression of PPAR? was significantly increased in normal temperature group(P < 0.05).