| Grass carp (Ctenopharyngodon idella) is one of the most impotantfresh aquaculture fishes in China and even the world’s, has a long historyof farming. The diseases, especially the grass carp viral hemorrhagic, areserious problems in freshwater fish breeding process, which is a limitingfactor to fresh water breeding. Up to date, the prevention of such infectionsis still very difficult, traditional methods have their limitations.Use ofmolecular for beeding can promote the sustainable development offreshwater aquaculture, so comprehensive in-depth study of the pathogenpathogenesis and fish immune-related genes. The complement system, as arepresentative of innate immunity, plays a key role in the host defenseagainst infections. C1q is the member of complement classical pathwaytarget recognition protein, involved in innate immunity and adaptiveimmunity, and it plays an important role in the classic complementactivation pathway. In this thesis, the grass carp C1qB, C1qC and C1q-likegenes were cloning and characterized. Analysis of their physicochemicalproperties, amino acid structure, compare their homology, by challengewith grass carp reovirus, study them expression change at different developmental process, different organizations and by GCRV afterinfection in grass carp, and obtained the the results as followed.1. C1qB and C1qC cDNA fragments were cloned from the grass carpby homologous cloning, a578bp fragment of C1q-like gene was obtainedfrom a normalized full-length cDNA library of head kidney tissues of grasscarp. Using RACE PCR to abtain the full-length cDNA of C1qB, C1qC andC1q-like genes were939bp，916bp，1202bp, respectively contain coding243aa (729bp)，244aa (732bp)，248aa (744bp) open reading frame(ORF). The first26,23,22residues for C1qB, C1qC and C1q-like werepredicted to be a signal peptideres respectively.2. BLAST search against the databases to display other similar proteinsequences deduced amino acid sequence shows high similarity. The grasscarpC1qB shares67%amino acid identity with C1qB in zebrafish. Thegrass carp C1qC shares71%amino acid identity with C1qC in zebrafish.The grass carp C1q-like have the highest identity with C1qtnf5in zebrafish.Select the amino acid sequence of the respective genes of different speciesby MEGA5.0software phylogenetic tree was constructed using theneighbor joining (NJ) method and found that grass carp and other teleostclosest relationship, these results support the theory of evolution, reflect theactual geneticrelationship.3. Analysis found that multiple sequence alignment and functional domains showed: The C1qB and C1qC protein contains three conserveddomain structures including an N-terminal signal sequence, a collagendomain and C-terminal globular domain. Four cysteine residues conservedin mammalian and avian C1q molecules were also seen in grass carp C1qBand C1qC, and they were conserved completely among fish and othervertebrates. But amino acid sequences of C1qB and C1qC are not samecompletely, indicating that their function has some differences.TheC1q-like protein contains two important structural part of C1q/TNF familymembers, respectively the C-terminal globular highly homologous tocomplement C1q and the THD domain similar with TNF-α.4. C1qB, C1qC and C1q-like mRNA contents in tissues were detectedon the samples from three unchallenged fish to illustrate the spatialexpression patterns of3genes in grass carp using qRT-PCR. Varioustissues were tested, including blood, gill, fin, skin, muscle, liver, spleen,trunk kidney, intestine, heart, head kidney and brain. Total RNA wasextracted as described above. Testing results showed that,3genes wasdetected in all tissues are expressed, suggesting that they may play a broadrole in grass carp. C1qB, C1qC and C1q-like mRNA were highestexpression in spleen, trunk kidney and head kidney. Fluorescent real-timequantitative RT-PCR was used to examine the expression after challengewith GCRV. Upon challenge with grass carp reovirus, expression of3 genes was significantly up-regulated in most tissues. The immune-relatedtissues expression levels increased significantly in early infected wihGCRV and then down-regulated. In addition, there are also found in theskin, gills, intestines and other mucosal tissues involved in the immuneresponse signs. Analysis of3genes in healthy and challenge grass carpshow that the GCRV can affect the C1q-related genes expression, whichhas a certain impact in grass carp.5. Fluorescent real-time quantitative RT-PCR was used to examine theembryogentic expression patterns. We detected the expression of3genes ineggs, suggesting that they may be matrilineal origin. Grass carp C1qB andC1q-like genes have similar expression profiles, higher expression in theearly embryo development.Taken together the above results, the expression of grass carp C1qB,C1qC and C1q-like are modulated by GCRV infection, suggesting itsimportance in both immune.3genes have already exists in early embryonicdevelopment, suggesting its importance in developmental processes. |
