Abstract: Objective To clone the prophenoloxidase（proPO）gene from Macrobrachium nipponense, and to analyze the bioinformatics and spatial and temporal expression of the gene. Methods The proPO gene was cloned from haemocytes of Macrobrachium nipponense using RT-PCR and rapid amplification of cDNA ends (RACE) method, and its sequence was analyzed with a biological software. Spatial and temporal expressions of the gene were detected by RT-PCR and Real-time PCR. The immune challenge test was carried out by injecting with 20μl of a Aeromonas hydrophila suspension (5.0×10SUP>9/SUP>/L) into the ventral sinus of prawns. After 3 hours, 6 hours, 12 hours and 24 hours of injection, haemolymph were collected and used for the proPO mRNA expressions of haemocytes and the phenoloxidase (PO) activity of serum. Results The full-length cDNA of hemocyanin was 2428bp and contains a 71bp of 5’-untranslated region (UTR), a 344bp of 3’UTR and a 2013bp of open reading frame (ORF) that encoded a protein of 671 amino acids with a calculated molecular mass of 76.5 kD and pI at 7.31. It was predicted to possess all the expected features of proPO members, including two putative tyrosinase copper-binding motifs with six histidine residues,a proteolytic activation site and a thiol ester-like motif (GCGWPRHM). Deduced amino acid sequence contained three hemocyanin domains. Comparison of amino acid sequences showed that the proPO-deduced amino acid of Macrobrachium nipponense exhibited higher similarity to that of Macrobrachium rosenbergii(93%) and 50% -53% to that of other crustaceans. The gene was expressed highly in the haemocytes, weakly in the hepatopancreas, and negative in the muscle, gill, intestine, mandibular organ and ovarian. Its transcript derived from haemocytes was highest in stage DSUB>0/1/SUB> among the molt cycle. Injection of pathogenic bacteria A. hydrophila resulted in the significant increase of proPO gene expressions and PO activity 6hours after treatment. Conclusion Like other crustaceans, the proPO gene of Macrobrachium nipponense contains two conserved copper-binding sites, and is highly expressed in haemocytes. The levels of proPO transcripts in haemocytes are the highest in molt stage DSUB>0/1/SUB>. Injection of Aeromonas hydrophila results in increase of proPO gene expressions and PO activity, indicating that proPO may act as an important molecule involved in immune defense against Aeromonas hydrop

Key words: Prophenoloxidase（proPO）, Spatial and temporal expression, Rapid amplification of cDNA ends, Real-time RT-PCR, Macrobrachium nipponense