Effects Of HIF-1 On The Expression Of Three Glucose Metabolizing Enzymes And Virus Reproduction And The Functional Analysis Of C-type Lysozyme In Scylla Paramamosain

The mud crab,Scylla paramamosain,is one of the most important commercially cultivated crustaceans along the coast of southern China because of its excellent quality as a food source and high market value.However,with the increase of breeding scale and density,the disease problem has become serious,which has hindered the development of the industry.The lack of dissolved oxygen in water is one of the important causes of disease breakout.The viruses are important pathogens to the crab culture,and their reproduction depend on the energy and substances from the host.In order to understand the mechanism of viral disease in the hypoxic environment,we investigated the effects of HIF-1 on the regulation of three key enzymes(SpLDH,SpPFK and SpFBP)in the glucose metabolism pathway and their effects on virus reproduction in the crab.In addition,the full length of SplyzC was cloned,and studied its tissue distribution and biological function.The specific research results are as follows:(1)The full-length cDNA of SpLDH was 1453 bp with an open reading frame(ORF)of 996 bp,and encoded a polypeptide of 332 amino acids.The full-length cDNA of SpPFK was 3634 bp with an ORF of 2859 bp,and encoded a polypeptide of 953 amino acids.The full-length cDNA of SpFBP was 1442 bp with an ORF of 1005 bp,and encoded a polypeptide of 335 amino acids.Homology analysis showed that the SpLDH,SpPFK and SpFBP are highly similar to arthropods.SpLDH,SpPFK and SpFBP transcripts were detected in all tested tissues and increased after hypoxia compared with normoxia.Significant up-regulation of SpLDH,SpPFK and SpFBP was measured in the gill and hepatopancreas after injection MCDV-1.The silencing of HIF-1 blocked the increase in LDH,PFK and FBP mRNA,and LDH activity,which were induced by hypoxia in gill and muscle tissues.Silencing of HIF-1α blocked the LDH,PFK and FBP expression and LDH activity,along with glucose and lactate concentrations producing after MCDV-1 infection.The above experimental results verify that the pathogenesis of MCDV-1 can be regulated by inducing metabolic changes.(2)The full-length cDNA of SplyzC was 849 bp with an ORF of 669 bp,and encoded a polypeptide of 223 amino acids with a calculated molecular mass of 23.7 kDa and an isoelectric point of 8.90.SplyzC shared conserved active sites with c-type lysozymes from other species,detected in all tested tissues and had higher expression levels in hepatopancreas and gill tissues.The expression of SplyzC was up-regulated in hepatopancreas and gill after infection with V.parahaemolyticus,S.aureus and MCDV-1.The density of bacteria in the hemolymph and the mortality of crabs increased following infection with V.parahaemolyticus after SplyzC expression was silenced by injecting double-strand RNA of SplyzC.The copies of virus in the detected tissues and the mortality of crabs increased following infection with MCDV-1 after SplyzC expression was silenced by injecting double-strand RNA of SplyzC.The recombinant protein of the S.paramamosain c-type lysozyme(rSplyzC)exhibited antibacterial activities against M.lysodeikticus,S.aureus,V.harveyi and V.parahaemolyticus.These results indicate that SplyzC could help eliminate bacteria and virus in S.paramamosain and play an important role in resistance to pathogen infection.In summary,hypoxia stress and MCDV-1 infection can up-regulate the expression of SpLDH,SpPFK and SpFBP.HIF-1 can regulate the expression of SpLDH,SpPFK and SpFBP,and the proliferation of MCDV-1 in the body of the crab,thereby reducing the death caused by the virus infection.SplyzC has a broad spectrum of antibacterial activity,and also has a certain resistance to virus infection.