Study On The Effects Of Inbreeding On Energy Metabolism Of Portunus Trituberculatus

The swimming crab Portunus trituberculatus(Crustacea: Decapoda: Brachyura)is widely distributed in the coastal waters of Korea,Japan,China,and Southeast Asia,and it is a dominant species in portunid crab fisheries worldwide.P.trituberculatus is an important economic species,and its production in China was 117,772 tons in 2015.However,uncontrolled expansion of the farming scale and unavailable or incomplete natural weed stocks have led to the decline of P.trituberculatus germplasm resources and genetic diversity.Inbreeding of P.trituberculatus is practically unavoidable because of restricted wild seed stock.Inbreeding results in homozygosity,which can increase the chances of the offspring being affected by recessive or deleterious traits and can lead to the depression of key performance traits.This study systematically investigated the possible effects of inbreeding on the key enzymes activities and relative m RNA expression of oxidative phosphorylation and carbohydrate metabolism of P.trituberculatus from different inbred families,in order to reveal the feasible mechanism underlying the depression in key performance traits.The detailed content of the research is as follows: 1.Effects of inbreeding on the oxidative phosphorylation of P.trituberculatus.In order to investigate the effects of inbreeding on the oxidative phosphorylation of P.trituberculatus,we cloned and obtained the c DNA sequence of the key subunit genes of the five Complexes in the respiratory chain of mitochondria by the rapid amplification of c DNA ends(RACE),and then analyzed the genes sequences by bioinformatics technology.The full-length c DNA sequence of core subunit(Ndufv2)genes of the Complex ? was 1005 bp,which contains 705 bp open reading frame(ORF)that encodes 234 amino acids polypeptides,named as pt Ndufv2(Genebank: KY682717).The full length of Complex ? key subunit(SDH)c DNA was 915 bp named as pt SDHC(Genebank: KY406169).It contained an ORF of 540 bp which encoded 179 amino acids.The full-length c DNA sequence of subunit(Cytc1)genes of the Complex ? was 2371 bp,which contains 942 bp open reading frame(ORF)that encodes 313 amino acids polypeptides,named as pt Cytochrome c1(Genebank: KY406171).The full length of Complex ? key subunit(COX6B)c DNA was 1171 bp named as pt COX6B(Genebank: KY406170).It contained an ORF of 318 bp which encoded 105 amino acids.The full-length c DNA sequence of core subunit(F-ATPase?)genes of the Complex ? was 1965 bp,which contains 1053 bp open reading frame(ORF)that encodes 350 amino acids polypeptides,named as pt F-ATPase?(Genebank: KY130458).Homology and phylogenetic analysis revealed that the amino acid sequence of the five Complexes shared a high similarity with close species,and had higher conservation in evolution,which can provide reference for other marine organisms.Then,we investigated the activities and the m RNA expression levels of five Complexes in hepatopancreas and heart mitochondria of P.trituberculatus.The results showed that inbreeding caused a decline in all five Complexes activity and its subunit gene in the hepatopancreas,and a decline in four Complexes eactivity and its subunit gene(except Complx II)in heart,which confirmed that inbreeding gradually reduces the oxidative phosphorylation pathway of P.trituberculatus.2.Effects of inbreeding on the carbohydrate metabolism of P.trituberculatus.The purpose of this section was to investigate the possible effects of inbreeding on the carbohydrate metabolism of P.trituberculatus from different inbred families.To this end,the activities of carbohydrate metabolism-related enzymes(hexokinase [HK],lactate dehydrogenase [LDH],pyruvate kinase [PK],and succinate dehydrogenase [SDH])and glycogen content in P.trituberculatus were measured.The results show that inbreeding weakens the activities of HK in the hepatopancreas and muscle;PK,muscles and serum;and LDH,gills,but enhances the activity of SDH in the muscles and gills.Futhermore,the m RNA expression levels of the carbohydrate metabolism-related genes in the hepatopancreas,muscle,gill,and hemocyte were investigated.The results indicated that the relative m RNA expression levels of the metabolism-related genes decreased as the inbreeding generations increased.The results illustrate that inbreeding gradually reduces carbohydrate metabolism in P.trituberculatus.3.The differences in carbohydrate metabolism between big and small size in same family of P.trituberculatus.In order to analysis the relationship between the differentiation of body weight and carbohydrate metabolism,the key enzymes and its relative m RNA expression levels of the carbohydrate metabolism in big and small individuals from same inbred family were investigated by enzyme assay and RT-PCR.The results show that there were significant differences in carbohydrate metabolism between big(BIs)and small individuals(SIs): HK activity of the BIs in hepatopancreas and muscle of F6,and in the muscle of F10 were significantly higher than that of SIs;PK activity of the BIs in hepatopancreas,muscle,and gill of F6,and in the hepatopancreas and gill of F10 were significantly higher than that of SIs.The results illustrate that the level of glycolysis of BIs is stronger than that of SIs,which suggests that BIs have stronger ability of carbohydrate utilization compare with SIs both in F6 and F10.SDH activity of the BIs in hepatopancreas and gill of F6,and in the gill of F10 were significantly higher than that of SIs.We speculate that the SDH activity of the SIs demonstrates the lower aerobic metabolism level of the SIs.LDH activity of the BIs in hepatopancreas and gill of F6 were significantly higher than that of SIs,which suggests that the higher anaerobic metabolism level of the BIs compared with SIs.The glycogen content of BIs was more than that of SIs,which demonstrates that BIs was able to store more glycogen and maintain higher blood glucose levels.4.The construction of yeast two hybrid c DNA library and the identification of interacting proteins with F-ATPase? of P.trituberculatus.The yeast two hybrid c DNA library of P.trituberculatus was constructed in Clontech Co.,Ltd.The quality parameters of the c DNA library were up to standard,and the c DNA library was available for screening interacting proteins of the key genes.In order to explore the function of the F-ATPase?,we constructed the expression plasmid of bait protein(p GBKT7-Pt ATPase?)by the mehods of In-Fusion Cloning and Yeastmaker Yeast Transformation.After screening with the high defect medium,three frame coding,reverse hybridization,sequencing and bioinformatics analysis,we identified 11 interacting proteins of F-ATPase? by yeast two hybrid system.The interacting proteins are as follows: Elongation Factor 2,Farnesoic acid O-methyltransferase,Probable 60 S ribosomal protein L37-A,Endonuclease-reverse transcriptase-like protein,Dynactin subunit 3-like,Putative cytochrome c oxidase subunit VIb/Cox12,Protein phosphatase 1 regulatory subunit 3C,NADH dehydrogenase [ubiquinone] 1 beta subcomplex subunit 2 mitochondrial,S-methyl-5-thioribose kinase,Masquerade-like protein,and Hypothetical protein.Therefore,this study suggests that the Pt ATPase? played important roles in the oxidativephosphorylation,protein synthesis,glycogen metabolism,mitosis,and in the osmotic pressure regulation of P.trituberculatus.The definition of the interacting proteins is vital to understanding the physiological function of ATPase?,and has potential value for further study on the protein function in ATPase? interactions.