Molecular cloning and characterization of serum amyloid A gene and the analysis of its immune function in Macrobrachium nipponense

In order to study the function of serum amyloid A (SAA) in the immune defense of Macrobrachium nipponense, the full-length cDNA sequence of the SAA gene in M. nipponense, named MnSAA, was cloned using rapid amplification of cDNA ends (RACE) method. The expression levels of MnSAA in different tissues and different development stages of M. nipponense, and the expression profile of MnSAA in hemocytes and hepatopancreas of M. nipponense after Micrococcus lysoleikticus, Aeromonas hydrophila and white spot syndrome virus (WSSV) infections were also examined by quantitative real-time PCR (QPCR). At the same time, the expression changes of activator protein-1 (AP-1) and class B scavenger receptor (CD36) genes in hepatopancreas and cumulative mortality of prawn after MnSAA gene silencing plus A. hydrophila infection were further investigated using RNA interference (RNAi) technology. The results showed that the full-length cDNA of MnSAA was 649 bp, including a 21 bp 5′-untranslated region (UTR), a 232 bp 3′UTR, and a 369 bp open reading frame (ORF) encoding a deduced protein with 131 amino acids. Sequence and phylogenetic analyses revealed that MnSAA belongs to acute-serum amyloid A (A-SAA) and had the closest relationship with the invertebrate Crassostrea hongkongensis. mRNA expression analysis revealed that MnSAA gene was ubiquitously expressed in various tissues and growth stages of M. nipponense, and abundantly expressed in the hepatopancreas and adult prawn stage. Pathogen infection experiment showed that the expressions of MnSAA in hemocytes and hepatopancreas were all significantly increased compared with the control group after M. lysoleikticus, A. hydrophilahe and WSSV infections for 12-72 h. The RNAi experiment showed that after MnSAA silencing, AP-1 and CD36 gene expressions in hepatopancreas of M. nipponense were all significantly decreased compared with the control group after 12-72 h and 6-72 h, respectively, of A. hydrophila infection, and the cumulative mortality of prawn was increased compared with the control group after A. hydrophila infection. These results suggested that MnSAA is involved in the activity of anti-pathogen infection in M. nipponense and plays an important role in the immune defense system of M. nipponense.