Cloning,expression And Function Of ALD,GP And ACBP In Shrimp Litopenaeus Vannamei Defending Against WSSV Infection

In recent years,the cultivation of Litopenaeus vannamei as an important breeding species has been seriously restricted by the disease.Related studies have shown that the disease-resistant immune process of shrimp requires enormous amounts of energy,and some energy metabolism-related genes are also involved in this process,which is closely related to the immune response.Therefore,an in-depth understanding of the metabolic pathway and disease-resistant immune process of L.vannamei is of great significance of the development of shrimp culture.In this study,we cloned the glycogen phosphorylase(GP),aldolase(ALD),and acyl-CoA binding protein(ACBP)genes by homologous cloning and RACE.The complete cDNA sequences was used to study the expression profiles in different tissues of shrimp and the expression of three genes in the hepatopancreas and hemocytes of shrimp after WSSV stimulation.In order to further study the function of the genes above,RNA interference(RNAi)was carried out on the three genes,respectively,and then infected with WSSV.The proliferation of WSSV in shrimps and the mortality of shrimps were analyzed after RNAi.Furthermore,the contents changes of metabolic products such as glucose,lactate,pyruvate,glycogen and adenosine triphosphate(ATP)in hepatopancreas and muscle tissues were analyzed.Changes in the expression of immune factors such as crustin,lectin and anti-lipopolysaccharide factors(ALF1,ALF2,ALF3).The main results are as follows:(1)Aldolase(ALD)The LvALD gene has a full length of 1634 bp,a 5’ untranslated region(UTR)of 175 bp,a 3’untranslated region(UTR)of 486 bp,and an open reading frame(ORF)of 1098 bp encoding 365 amino acids.The predicted LvALD protein sequence contained a typical glycolytic domain(15 to 365 aa)and shared 77%-97%identities with ALD from other species.Phylogenetic analysis revealed that LvALD showed the closest relationship with ALD from Fenneropenaeus chinensis.Tissue expression profiles showed that LvALD existed in most examined tissues,with the most predominant expression in muscle,then followed by nerve.WSSV stimulation cause the up-regulation of the LvALD expression at 24 h and reach the peak value in hepatopancreas,then returned to normal level.The transcription level of LvALD gene in hemocytes was shown a similar trend.In the LvALD gene interference experiment,our findings showed that WSSV proliferation and shrimp accumulative mortality increased significantly after LvALD RNAi(p<0.01).The results of relevant important metabolic indicators showed that after WSSV infection,the glucose content in hepatopancreas of LvALD knockdown group decreased significantly(p<0.01).The lactate content decreased significantly at 0 h and 48 h(p<0.01).The pyruvate content increased significantly after WSSV infection(p<0.01).However,the ATP level was inhibited from 0 h to 48 h.In muscle tissue,the glucose content of LvALD knockdown group decreased significantly after 24 hours of WSSV infection(p<0.05).The lactate content decreased significantly at 0 h and 48 h(p<0.01).The pyruvate increased significantly after WSSV infection and reach the highest level at 48 h(p<0.01).However,the ATP level was not change after WSSV infection during LvALD silencing.Moreover,lectin and crustin were inhibited in LvALD silencing shrimp,anti-lipopolysaccharide factor1(ALF1),anti-lipopolysaccharide factor2(ALF2)were induced after WSSV infection.Our results suggested that the LvALD might play a crucial role in shrimp defending against WSSV infection by regulating metabolism and affecting the anti-infectious genes expression.(2)Glycogen phosphorylase(GP)The LvGP gene has a full length of 3242 bp,a 5’ untranslated region(UTR)of 48 bp,a 3’untranslated region(UTR)of 635 bp,and an open reading frame(ORF)of 2559 bp encoding 852 amino acids.The predicted LvGP protein sequence contained a typical phosphorylase domain(113 to 829 aa)and shared 72%-97%identities with GP from other species.Phylogenetic analysis revealed that LvGP showed the closest relationship with GP from Marsupenaeus japonicus.Tissue expression profiles showed that LvGP existed in most examined tissues,with the most predominant expression in brain,then followed by muscle and stomach.LvGP transcripts in hepatopancreas was firstly up regulated but then down regulated after WSSV challenge.The transcription level of LvGP gene in hemocytes was shown a similar trend.In the LvGP gene interference experiment,our findings showed that WSSV proliferation and shrimp accumulative mortality increased significantly after LvGP RNAi(P<0.01).The results of related metabolic indicators showed that after 24 hours of WSSV infection,the content of glycogen,glucose and pyruvate decreased and the concentration of lactate in hepatopancreas decreased significantly in the LvGP knockdown group,and then returned to the control level(p<0.05).More importantly,ATP concentration also increased significantly at 24 h after WSSV injection,but decreased significantly after 48 h(p<0.05).In muscle tissue,the content of glycogen and glucose levels didn’t show significant changes after WSSV infection and the concentration of pyruvate and lactate increased significantly at 24 hpi(p<0.01).Conversely,ATP concentration didn’t show significant change after WSSV infection In addition,LvGP is able to inhibit immune-related genes including lectin and ALF after WSSV infection.However,after injection of WSSV,the expression level of crustin was reduced in the LvGP interference group.Our results suggested that the LvGP might play a crucial role in shrimp defending against WSSV infection by regulating metabolism and affecting the anti-infectious genes expression.(3)Acyl-CoA binding protein(ACBP)The LvACBP gene is 568 bp in length,66 bp in the 5’ untranslated region(UTR),229 bp in the 3’ untranslated region(UTR),and 273 bp in the open reading frame(ORF),encoding 90 amino acids.The predicted LvACBP protein sequence contained a typical acyl-CoA binding domain(4 to 84 aa)and shared 50%to 98%identities with ACBP from other species.Phylogenetic analysis revealed that LvACBP showed the closest relationship with ACBP from Fenneropenaeus chinensis.Tissue expression profiles showed that LvACBP existed in most examined tissues,with the most predominant expression in hepatopancreas,then followed by brain and intestine,and low expression in heart and intestine tissues.After WSSV stimulation,the expression of LvACBP gene in hepatopancreas of L.vannamei was significantly up-regulated at 24 h(p<0.01),and then significantly decreased at 96 h(p<0.01).The expression of LvACBP gene in hemocytes was similar to that in hepatopancreas,and it was significantly up-regulated at 24 h(p<0.05)and it was significantly down-regulated at 96 h(p<0.01).In the LvACBP gene interference experiment,our findings showed that WSSV proliferation and shrimp accumulative mortality increased significantly after LvACBP RNAi(P<0.05).The results of related metabolic indicators showed that in hepatopancreas tissue after 24 h of dsRNA injection,the content of pyruvate decreased significantly(p<0.05),but increased after 24 h of WSSV infection.The concentration of lactate decreased significantly from 0 h to 48 h after WSSV infection in the LvACBP knockdown group(p<0.01).More importantly,triglyceride and total cholesterol concentrations also increased significantly after WSSV injection(p<0.01).In muscle tissue WSSV infection inhibited the producing of the pyruvate,and the lactate producing was reduced after 24 h of dsRNA injection.The concentrations of triglyceride and total cholesterol both significantly increased after WSSV infection.In addition,LvACBP is able to inhibit the expression of lectin,but induce the crustin expression level.Our results indicated that LvACBP might play a crucial role in shrimp defending against WSSV infection by regulating lipid metabolism.