Cloning And Function Of CMDH,E1 And CS In Shrimp Litopenaeus Vannamei Defending Against WSSV

In recent years,The development of Litopenaeus vannamei aquaculture industry has been suffered by serious infectious disease.Previous studies have shown that shrimp metabolism showed a close relation with immunity,and metabolic enzymes might involved in metabolism and immune response.Therefore,the further study about the mechanism of shrimp pathogen infection and immune metabolism reaction of shrimp have important significance for the healthy and sustainable development of shrimp.This study cloned the full length cDNA sequence of the metabolism-related gene cytosolic malate dehydrogenase(cMDH),the pyruvate dehydrogenase complex 1 alpha subunit(E1),and the citrate synthase(CS)gene in shrimp L.vannamei.Moreover,their molecular structure mechanism,their expression levels in different tissues of shimp and their expression in hepatopancreas and haemolymph after WSSV stimulation were studied.Then,each gene was knocked down by RNAi technology and infected with WSSV virus.At the same time,the proliferation of the virus,the mortality rate of shrimp and the enzyme activities of malate dehydrogenase and lactate dehydrogenase,as well as the changes of glucose level,lactic acid level and ATP level were also detected.The effects of cMDH,E1 and CS in shrimp against WSSV infection were analyzed from three levels:gene,enzyme activity and metabolism physiology.The main results are as follows:(1)The full length cDNA of the cMDH gene of L.vannamei was 1096 bp,including a 35 bps of 5-untranslated region(UTR),a 61 bps of 3-UTR with a poly(A)tail and an open reading frame(ORF)of 999 bp encoding a polypeptide of 332 amino acid residues,named LvcMDH.The predicted molecular weight of LvcMDH was 35.81kDa,and isoelectric point(pI)was 5.87.Tissue expression revealed that the most predominant expression of LvcMDH was in muscle and very weak in digestive tract and haemolymph.LvcMDH expression levels increased significantly in hepatopancreas and haemolymph After WSSV injection.Knockdown of LvcMDH resulted in a less WSSV proliferation and a lower mortality of WSSV infected shrimp.Simultaneously,compared with the control group,MDH and LDH activity,lactate content,and ATP content decreased,whereas glucose content increased in the WSSV infected shrimp.These findings suggested that LvcMDH might play a positive role in WSSV proliferation in shrimp by through carbohydrate metabolism.(2)The full length cDNA of the El gene of L.vannamei was 1761 bp,including a 5’-UTR 85 by,3’-UTR 473 bp with a poly(A)tail and an ORF of 1203 bp encoding a polypeptide of 400 aa,named LvE1.The predicted molecular weight of LvE1 was 44.25kDa,and pI was 8.05.Tissue expression revealed that the most predominant expression of LvEl was in muscle and skin,but very weak in hepatopancreas and haemolymph.LvE1 expression levels increased significantly in hepatopancreas and haemolymph After WSSV injection.Knockdown of LvE1 resulted in a more WSSV proliferation of WSSV infected shrimp at 24 h,but mortality changes are not significant.Simultaneously,compared with the control group,MDH and LDH activity,lactate content,and ATP content increased,whereas glucose content decreased in the WSSV infected shrimp;At 48 h,these changes were not significant,but the expression of Adenosine 5’-monophosphate(AMP)-activated protein kinase(AMPK)and mitogen-activated protein kinases p38(p38)in the shrimps was increased at 48 h and the pathways may be activated.These findings suggested that LvE1 might play a positive role in resistant WSSV proliferation in shrimp by through carbohydrate metabolism.(3)The full length cDNA of the CS gene of Lvannamei was 2408 bp,including a 5’-UTR 84 bp,3’-UTR 902 bp with a poly(A)tail and ORF of 1422 bp encoding a polypeptide of 473 aa,named LvCS.The predicted molecular weight of LvCS was 52.02kDa,and pI was 6.48.Tissue expression revealed that the most predominant expression of LvCS was in muscle and heart,but very weak in eyes handle and nerves.LvCS expression levels increased significantly in hepatopancreas and haemolymph After WSSV injection.Knockdown of LvCS resulted in a more WSSV proliferation and a higher mortality of WSSV infected shrimp at 24 h.Simultaneously,compared with the control group,MDH and LDH activity,lactate content,and ATP content increased,whereas glucose content decreased in the WSSV infected shrimp;At 48 h,these changes were not significant,but the expression of AMPK/p38 in the shrimps was increased at 48 h and the pathways may be activated.These findings suggested that LvCS might play a positive role in resistant WSSV proliferation in shrimp by through carbohydrate metabolism.