Two Kinds Of C-type Lectins Response To Bacterial Infection And Ammonia Nitrogen,Salinity Stress In Tiger Shrimp(Penaeus Monodon)

The black tiger shrimp（Penaeus monodon）,as the one of aquatic products of coastal areas of China,has an important role in the development of aquatic economic system of China.Due to the deteriorating water environment and the rampancy of bacteria and virus,the breeding situation of black tiger shrimp is more and more serious.Prawns,as invertebrates,mainly rely on the innate immune system to resist external threats and maintain organism health.Lectin is an important member of the superfamily of pattern recognition receptor（PRR）,and the C-type lectin has various functions such as recognizing pathogen,inducing microorganism agglutination,opsonization and phagocytosis.They can recognize the pathogen-associated molecular patterns（PAMP）exposed on the surface of pathogenic microorganisms so that eliminate the pathogen,which is the most typical immunization route.In addition,we also explore the expression of CTL gene in tiger under the ammonia nitrogen and low salt stress,which can help us understand the immune protection mechanism of shrimp in responsed to environmental stress.The specific experimental results are described as follows:1.Identification of novel lectin gene in the black tiger shrimpThe EST sequence of C-type lectins were acqurired from hepatopancreas transcriptome of the black tiger shrimp Penaeus monodon.The full-length of these lectins which named PmCL1 and Pm Cl2 was obtained by PCR,RACE,TA clone and other biological technologies.The relevant information of the CTLs sequence has been deposited in Genbank database,and the number of CTLs was MK076295 and MK599468,respectively.RACE revealed a complete c DNA sequence of PmCL1 of861 bp,including a 5′-untranslated region（5′-UTR）of 74 bp,an ORF of 495 bp,and a292 bp 3′-UTR with a poly（A）tail.The ORF encoded a 164 amino acid polypeptide with a deduced molecular（without a signal peptide）weight of 16.17 k Da and a theoretical isoelectric point of 4.42.The full length of PmCL2 revaled by RACE comprised a 5′-untranslated region（5′-UTR）of 165 bp,an ORF of 720 bp,and a 146bp 3′-UTR with a poly（A）tail.The ORF encoded a 239 amino acid polypeptide with a deduced molecular（without a signal peptide）weight of 24.95 k Da and a theoretical isoelectric point of 4.75.The structure of PmCL1 and PmCL2 was similar,which including a singal and a CRD domain.They all have a typical carbohydrate-binding motif EPS and six highly conserved cysteine residues.The phylogenetic tree showed that PmCL1 cluster and form a monophyletic clade,and PmCL2 close with the branch including C-type lectin 3 from M.rosenbergii,lectin 3 from E.modestus.Multiple sequence alignment analysis of PmCL1 and PmCL2 against the CTLs from various species revealed different amino acid compositions.2.Expression pattern analysis of PmCL1 and PmCL2 in the black tiger shrimpThe expression pattern of PmCL1 and PmCL2 were detected by q RT-PCR using EF1ɑas a control in various tissues.The results showed that the expression quantity of PmCL1 and PmCL2 was the highest in the hepatopancreas.In addition,the transcription in intestine,gill and stomach were also at a high level,but hardly dected in testis and ovary.Under the salinity stress,PmCL1 transcripts fluctuated in hepatopancreas,with a slight increase at 3 h and reduction at 96 h.For intestinal tissue,PmCL1 expression was down-regulated at 3 h and up-regulated at 6 h,but was still lower than at 0 h.For the expression of PmCL2,both in hepatopancreas and tissue,it was decreased under the ammonia nitrogen stress.In hepatopancreas,PmCL2 transcripts increased from 3h to 72h.In contrast,PmCL2 transcripts was the tendency of fluctuations to rise from 6h to 48h in tissue.The result showed that the tendency of PmCL1 m RNA expression in two group had some similarities that decline under the ammonia nitrogen except increase in intestine of SC group.For hepatopancreas,the transcripts were increased immediately at 3 h and were subsequently reduced during 96h both in SC group and LC group.For intestine,the expression of PmCL1 was up-regulated at 3h and stayed on a high expression until 96h in SC group.The expression quantity variation trend of PmCL2 in LC group was similar to that in SC group,which reduced at 3h with difference but no significance.For intestine,PmCL2 transcripts in SC group increased significantly at 3h and stayed on a high expression until 72h.In LC group,the expression of PmCL2 also increased significantly at 3h,then reduced and stayed on a low expression until 96h.Under the bacteria challenge,the expression of PmCL1and PmCL2 was more sensitive to Gram-negative bacteria compared to Gram-positive bacteria.PmCL1 transcripts increased and got the highest value at 6h in gill and at 12h in hepatopancreas after injected by V.anguillarum.When challenged by V.harveyi,the expression quantity of PmCL1 got the highest value at 24h in gill.For PmCL2,the expression of which came to the highest at 3h in hepatopancreas and 6h in gill after challenged by V.anguillarum.However,when challenged by V.harveyi,the expression came to the highest at 6h in hepatopancreas and 3h in gill,respectively.3.Research on the function of recombinant protein of PmCL1 and PmCL2The recombinant proteins of PmCL1 and PmCL2 were obtained by prokaryotic expression.The predicted molecular weight of r PmCL1 and r PmCL2 was 35.6k D and40.0k D,respectively.We used Ni-NTA His bind Resin which could catch the recombinant protein with His-tag to obtain purified protein.In order to test the binding activity and function against bacteria infection of recombinant protein,we performed enzyme-linked immunosorbent assay（ELISA）,microbial agglutination assay and bacteria clearence assay.The ELISA results showed that the binding of r PmCL1 to PGN was strong and dose-dependent manner,with a high affinity evident at 50–100μg/m L.At the same time,r PmCL1 could also bind LPS but not LTA.In addition,the direct binding assay to carbohydrates revealed r PmCL1binding affinity that was similar for D-glucose andα-lactose,and marginally stronger to D-mannose,followed by galactosamine and trehalose.Similarly,the binding of r PmCL2 to PGN and LPS was dose-dependent manner.However,r PmCL2 weakly bound to LTA.Almost all bacteria in the field of vision were agglutinated after incubation with r PmCL1 and Ca²⁺.Agglutination was markedly inhibited after incubation of r PmCL1in the PBS-Ca²⁺buffer plus EDTA.The similar inhibition effect was also observed in the presence of PGN.Thus,r PmCL1 could enhance the elimination of V.harveyi significantly in tiger shrimp.The result of knocking down the expression of PmCL1 and PmCL2 by RNA interference increased the mortality of shrimps compared to control when injected by V.harveyi.The mortality of shrimp in PBS group did not increased till 12h post-injection.The mortality of shrimp in ds GFP group increased slowly and came to the value a little over 60%.However,the mortality of shrimp in ds PmCL1 and ds PmCL2group increased significantly from 3h to 12h and still kept the growing trend until 24h,while 20 percent higher than the control group.Based on the above experimental results,this project adopted a variety of technologies to have a certain understanding of the new C-type lectin PmCL1 and PmCL2 which involved in innate immunity and responsed to environmental stress in the black tiger shrimp.The analysis of expression pattern under bacteria stimulation and immunity of its recombinant protein could lay a theoretical foundation for the study on improving the immune and disease resistance ability of shrimp.In addition,the response of PmCL1 and PmCL2 to salinity and ammonia nitrogen stress could enrich the functional study of CTLs in the black tiger shrimp.