Gene Clone And Expression Analysis Of C-type And T-type Lysozyme In Qihe Crucian Carp Carassius Auratus

Qihe crucian carp Carassius auratus has the high nutrition and economic value. In recent years, the high density and intensive farming mode has resulted in the outbreak of fish diseases. Therefore, to improve the immune level is particularly important to control fish disease. Lysozyme as an important nonspecific immune factor, can kill bacteria by hydrolyzing β-1,4-glycosidic linkages of peptidoglycan layer in the bacterial cell wall, plays an important role in fish against the outside invasion of bacteria. In this study, Qihe crucian carp C. auratus, as the experimental fish, comes from the Hebi breed farm in Henan. The c-type and g-type lysozyme of C. auratus were cloned by RT-PCR and RACE methods using the total RNA as templates; the mRNA expression level and lytic activity were detected in various organs and tissues using real time PCR and turbidimetey method respectively; after the injections of A. hydrophila and LPS, the changes of mRNA expression levels and activities of c-type and g-type lysozymes were also analyzed. These all laid the theoretical foundation for the exploring immune defense function and the research of molecular mechanism of the lysozyme of C. auratus.The results showed that c-type lysozyme was 697 bp in full length cDNA with an open reading frame(ORF) of 438 bp encoding 145 amino acids. The theorical isoelectric point(pI) and molecular mass were 8.86 and 1.66 ku respectively. It possesses two conserved catalytic sites(Glu53, Asp69) and eight cysteine residues which can form four disulfied bonds. Moreover, it contained the N-terminal signal peptide as a secreted protein. The g-type lysozyme was 806 bp containing 564 bp ORF encoding 187 amino acid residues, the pI and molecular mass were 9.04 and 2.09 ku, respectively. It possesses three conserved catalytic sites(Glu73, Asp86 and Asp97) and one cysteine residues, but no disulfied bond. It lacks of the N-terminal signal peptide as an intracellular protein.The homology analysis showed that c-type lysozyme of C. auratus exhibited the most high identity(99.3%) with goldfish, and the identity with other fish in cypriniformes were above 80%, however, exhibited the lower homology with other teleost fishes, just about 40%. The g-type lysozyme of C. auratus exhibited about 70% to 80% identity with other fish in cypriniformes, and 50% to 70% with other teleost fishes. The phylogenetic tree indicated that the c-type and g-type lysozymes were clustered into two branches. The c-type lysozymes in fishes were splited into two clusters: one was the fish of cypriniformes, and the another cluster contained other fishes. This result was consisted with the homology analysis result, suggesting that c-type lysozymes of cypriniformes maintain its independence in the evolution history. The g-type lysozyme in fishes became one big cluster.The expression level of c-type and g-type lysozyme indicated the universality and difference in different tissues in C. auratus. The c-type lysozyme was highly expressed in kidney and head kidney(316.7 and188.6 fold to liver), while g-type lysozyme was similar in all tissues. All of the tested tissues exist lysozyme activities in C. auratus, the skin mucous is the highest with the extremely significant difference compared to the other tissues(P<0.01).After intraperitoneal injection of Aeromonas hydrophila and LPS respectively, the changes of mRNA expression levels of c-type and g-type lysozymes showed the upregulated in liver with the extremely significant difference( P<0.01). Moreover, the c-type and g-type lysozyme were in the similar upregulated in gill. In spleen, the c-type lysozyme was upregulated, but the g-type lysozyme was almost no change. However, in kidney and head kidney, the g-type lysozye was upregulated and the c-type lysozyme was no change. The changes of lysozyme activities after intraperitoneal injection of Aeromonas hydrophila and LPS, respectively, were similar to the mRNA expression levels, indicating a trend with the rising in early stage and then declining. The mRNA expression level and enzyme activities of c-type and g-type lysozymes in various tissues after inoculation, indicate that both the two type lysozyme have the response to the pathogens and analogues. It was demonstrated that the c-type and g-type lysozyme play an imprtant role in immune defenses against the bacteria invasion.Conclusions:1. The full length cDNA of c-type and g-type lysozymes in Qihe Crucian carp Carassius auratus were achieved by cloning. The c-type lysozyme was 679 bp, encoding 145 amino acids; the g-type lysozyme was 806 bp, encoding 187 amino acids. Both have the typical structural characteristics of lysozymes.2. The c-type and g-type lysozyme in C. auratus have expressed broadly in various tissues. The c-type lysozyme was highly expressed in kidney and head kidney, and the g-type lysozyme showed little difference. All of the tissues exist lysozyme activities and the skin mucous is the highest.3. After intraperitoneal injection with A.hydrophila and LPS, the mRNA levels and enzyme activities were increased in c-type and g-type lysozyme, indicating that these two lysozymes could defense the bacteria invasion, and play an important role in non-specfic immunity.