CDNAcloning And Expression Analysis Of C1qBP And MP1 In Penaeus Monodon

Black tiger shrimp (Penaeus monodon) is an important economic marine aquaculture species.In recent years,black tiger shrimp have broken through the bottleneck that broodstock culture can not be artificial regulated,we have aquired great progress in controling nutritional regulation and eco-based regulation technology of artificial broodstock , but in the breeding process, disease threat and low resistant to disease is still an important reason for the low yield. This urgent need to start from immune system of black tiger shrimp, identify and analyze the immune-related genes, to study the immune regulation mechanism and resistance to disease mechanism, lay the foundation for screening disease resistance genes,and enhancing anti-shrimp ability.In order to study the interaction between host and pathogen, Penaeus monodon infected by Vibrio alginolyticus,expressed before and after the transcriptome database, by NCBI database blast, discovered the complement system complement 1q ligand binding protein (complement component 1 q subcomponent-binding protein, C1qBP) and mitogen-activated protein kinase (mitogen-activated protein kinases, MAPKs) signaling pathway ligand MAPK kinase 1 interacting protein (MAPK kinase 1 interacting protein 1, MP1 ) expressed sequence tags (Expression Sequence Tag, EST). In this study, tiger shrimp immune related genes PmC1qB and PmMap2k1ip1 (MP1) were cloned and analysis with RACE, semi-quantitative analysis, Real time-PCR technique. by stimulation of the pathogen, we analysis the genes expression levels in black tiger shrimp, at the same time we analysis their molecular structure, tissue distribution and responses model to different pathogenic stimulation, at last, we recombinanted gene expression of PmC1qB in vitro and identificated by SDS-PAGE, and discussed function.PmC1qB and PmMapk1ip1 (MP1) are two important proteins, PmC1qB plays an important role in the complement system. While the PmMP1 plays an important role in signal transduction, which do not only regulate immune responses ,but also regulates the development in Penaeus monodon.In this paper, we study gene structure and expression regulation mechanisms under certain condition. The PmC1qBP cDNA was 1303 bp long and consisted of a 5'-untranslated region (UTR) of 104 bp, 3'-UTR of 410 bp, there are two AATAAA in 3'-UTR.PmC1qBP gene obtained a gene accession number.which is HQ 412588, Predicted protein contains 262 amino acids with an estimated molecular mass of 29.07 kDa and a theoretical isoelectric point of 4.74, There are two potential glycosylation sites in sequences, which is Asn31 and Asn127. BLAST homology analysis show that amino acid sequence of PmC1qBP contains a conserved MAM33 superfamily domain, Which can bind to C1q. Homology analysis of the deduced amino acid sequence of the PmC1qBP with other known C1qB sequences by MatGAT software revealed that the PmC1qBP shared 46.6%-82.4% similarity and 25.2%-71.9% identity to the other known PmC1qBP sequences, Comparison of amino acid sequences showed that PmC1qB is more closely related to invertebrate C1qBP. Tissue expression pattern analysis showed that the PmC1qBP mRNA was expressed in all tested tissues, including hepatopancreas, intestine, gill, lymph, nerve, stomach, testeis, hemocytes. There is big difference in some organizations of female and male individuals, the highest expression level is lymphatic in male shrimp, while the highest expression is ovaries in females. LPS, PGN and Vibrio immunization stress test showed, gene expression of PmC1qBP in the hepatopancreas increased significantly at 6 h after LPS and Vibrio challenge, reaching the highest value. There is no significant difference in stimulation group and control group after PGN stimulation. These results maybe implied that the PmC1qBP plays an important roles in the innate immune responses of Penaeus monodon.The PmC1qBP gene was linked into prokaryotic vector PET32a, and the PmC1qBP fusion protein with 29.0 kDa molecular. Fusion protein was successfully expressed in E.coli BL21.And then, we prepared polyclonal antibody by immuning the mice with PmC1qBP. After obtaining polyclonal antibody of PmC1qBP, we tested tissue protein with Western-blot, the result show that PmC1qBP is expressed in sperm, hepatopancreas, intestine. ELISA results that PmC1qBP may bind collagen-like zone of complement component C1q . PmMap2k1ip1 (MP1) plays a key role in the signal transduction pathway of MAPK,which regulate cell proliferation, differentiation, migration and inflammation.MP1 as a regulator in MAPK, has been more and more concerned. The MP1 cDNA was 910 bp long and consisted of a 5'-untranslated region (UTR) of 29 bp, a 3'-UTR of 509 bp, 372 bp ORF and a polyadenylation signal (ATTTA), PmMP1 gene obtained a gene accession number. HQ 339913, Predicted protein contains 123 amino acids with an estimated molecular mass of 13.6 kDa and a theoretical isoelectric point of 6.3,BLAST homology analysis, PmMP1 amino acid sequence contains a conserved MAPKK1-int domain, in other species, MP1 or MAPKsp1, which is located 3 and 123 amino acids. Homology analysis of the deduced amino acid sequence of the PmMP1 with other known MP1 sequences by MatGAT software revealed that PmMP1 has a high conservation. PmMP1 shared 48% and 47.5% identity with N.vitripennis and S. purpuratus respectly. The phylogenetic tree showed that PmMP1 clustered with the marine invertebrates sea urchin. The stimulation expression profile of LPS and PGN shows that, the expression level of MP1 mRNA was significantly up-regulated after PGN or LPS stimulation, The results suggested that the PmMP1 could be involved in the immune responses of Penaeus monodon. However expression analysis results of different developmental stages show that early development in shrimp, PmMP1 is highest expression, which maybe need more mRNA for early life activities and participate in innate immunity during larval development.