Molecular Cloning And Genetic Diversity Analysis Of The Seventh Component Of Complement And Interleukin-1β In Hypophthalmichthys Molitrix

The Hypophthalmichthys molitrix is one of the most important fresh wateraquaculture species in China. But in the breeding process, they are often infected withdisease. The major disease is septicaemia, its pathogen is Aeromonas hydrophilia. Thedisease caused by this pathogen is malignant infection disease and high mortality. Inthis article, we clone the complement C7and interleukin-1β genes, analysis theexpression in different tissues. This study will help to understant the mechanism ofdisease resistance, and also provide a scientific basis for the Hypophthalmichthysmolitrix breeding for disease resistance.Complement activation via the classical, the mannan binding lectin or thealternative pathway leads to the formation of the terminal complement complex(TCC). Complement C7is an important component of the complement system. Itsincorporation into the TCC allows its hydrophilic-amphiphilic transition, whichsubsequently leads to the direct binding of the complex to target cell membranes.Interleukin-1plays an important role in mediating inflammatory response andenhancing the immune response. In the present study, the complement C7andinterleukin-1β genes were cloned in Hypophthalmichthys molitrix.1. Clone and analysis of Hypophthalmichthys molitrix C7: the full-length C7cDNA was isolated from the Hypophthalmichthys molitrix cDNA library, which was2625bp and encoded821amino acids. Hypophthalmichthys molitrix C7, like otherreported teleost C7, had many conserved structural motifs, such as TSP1,LDLRa,MACPF,EGF,TSP1,CCP. Phylogenetic analysis for the seventh component ofcomplement of12Hypophthalmichthys molitrix, we found six mutation sites, five ofwhich were synonymous mutations, another was non-synonymous mutations, andidentifiedsix alleles. RT-PCR analysis demonstrated that the Hypophthalmichthysmolitrix C7mRNA was only detected in the spleen at the normal state, but theexpression in all tissues investigated growth rapidly after they infected Aeromonashydrophilia. With the removal of pathogens, the expression gradually reduced until todisappear. 2. Clone and analysis of Hypophthalmichthys molitrix IL-1β: A conservedfragment of the interleukin-1β was cloned by using degeneracy primers,then we getthe full-length cDNA of interleukin-1β through RACE-PCR. Which was1534bp andcontains an ORF of825bp coding275amino acids. The Hypophthalmichthys molitrixinterleukin-1β contains a357bp5'UTR and a351bp3'UTR with three instabilitymotifs (ATTTA). Homology analysis showed that the predicted amino acid sequenceexhibited highest identity (77%) with Ctenopharyngodon idella. TheHypophthalmichthys molitrix IL-1β contains the classic protein signal of IL-1family:[FCL]-x-S-[ASLV]-x(2)-[PSK]-x(2)-[FYLIV]-[LI]-[SCA]-T-x(7)-[LIVM].RT-PCR analysis demonstrated that the Hypophthalmichthys molitrix IL-1β mRNAwas only detected in the spleen at the normal state, but the expression in all tissuesinvestigated growth rapidly after they infected Aeromonas hydrophilia. With theremoval of pathogens, the expression gradually reduced until to disappear.The result of expression analysis showed that complement C7and interleukin-1βof Hypophthalmichthys molitrix were related genes with the infection of Aeromonashydrophila.