| Chinese mitten crab Eriocheir sinensis (Henri Milne Edwards 1854) is one important aquaculture species in China, possessing striking economic and scientific value. The prophenoloxidase activating system (proPO system), playing key roles in the immune defense response of Chinese mitten crab, is the unique immune component of arthropod innate immunity. In the present study, a cDNA library was constructed and sequenced. The resultant ESTs (Expressed Sequence Tags) were analyzed with bioinformatics tools. RACE (rapid amplification of cDNA ends), real time RT-PCR, protein recombinant and RNAi techeniques were adopted to investigate the cDNA characteristic, mRNA expression and molecular function of key genes of E. sinensis proPO system.In the present study, a high quality cDNA library of Chinese mitten crab haemocytes challenged by bateria was constructed with its capacity up to 3.3×106. After random sequencing, 7535 ESTs were obtained and submitted to the GenBank database. Among them, 4593 ESTs having no significant matches to any protein sequences in the public database were identified as novel genes, and the other 2942 were identified as matched genes. Based on the Gene Ontology (GO) classification system, matched genes were classed into 20 GO classifications in terms of molecular functions or 23 GO groups in terms of biological processes. After EST analysis, 32.9% (969 out of 2942) ESTs were identified as immune genes, which were assembled into 221 unigenes. This percentage was significant higher than that of previous reported crustacean cDNA libraries. In addition, antimicrobial peptides (AMPs) taken up 20.1% (195 ESTs) of the 969 ESTs. The high percentage of immune genes 7535 ESTs and the high expression of AMPs indicated that bacteria stimulation was a useful method for improving the proportion of immune genes in cDNA library. The results mentioned above greatly enriched the genomic information of Chinese mitten crab, laid the first step for the elucidation of crab immune system, and for the first time made the full-scale study of the molecular mechanism under various biological processes of crab possible.In the present study, interests were focused on the molecular characteristic, tissue distribution, temproral expression profiles of ten proPO system members of Chinese mitten crab as well as the regulation machnism and evolution trend of E. sinensis proPO system. Ten full-length cDNAs were obtained from E. sinensis based on EST analysis and RACE (rapid amplification of cDNA ends) technique, including the PRSP (EsPRSP), PAP1 (EsPAP1), PAP2 (EsPAP2), PAP3 (EsPAP3), SPH (EsSPH), proPO (EsproPO), PAPII (EsPAPII), serpin (Esserpin), pacifastin (EsPLC), peroxinectin (Esperoxinectin). Blast analysis and multiple sequence alignment indicated that all these genes were members of the proPO system. Furthe study indicated that the CLIP domain of PAPs, the genes of PRSP, pacifastin, proxinectin and proPO could only be identified in arthropod, which might be the molecular basis for the uniqueness of the proPO system in arthropod. Some regulation elements, such as 15-LOX-DICE, K-box and Brd-box, were identified in the 3′UTR of proPO system genes. The PIs of crab proPO system factors presented significant differences. The mRNA transcripts of these genes were detected in the haemocytes, heart, gill, gonad, muscle, and hepatopancreas. The mRNA of EsPAP1, EsPAP2, EsPAPII were predominately expressed in muscle and lower expressed in haemocytes; Mealwhile, the expression of EsPAP3, EsproPO and EsPLC was highest in haemocytes and lower in muscle. It was notable that the expression level of EsPAP3 in haemocytes was 526.35-fold to that in muscle. The existence of regulation elements in the 3′UTR of proPO system genes and the presentce of more than one proPO activating proteases as well as protease inhibitors, the difference of PIs and mRNAs expression in tissue distribution collectively implied that the proPO system of E. sinensis was regulated at least on the transcription, translation, enzyme activation, and signal pathway levels. The temporal expression profiles of proPO system members in the crabs challenged with Listonella anguillarum were obtained with the Realtime PCR technique. Though some genes was found increase the mRNA expression while the others decrease, the expression levels of EsPAP1, EsPAP2, EsPAP3, EsPLC and EsPAPII reached the extremum at same schedule (2h, 12h). This expression characteristic was similar to that of the mRNA expression and specific activity of EsproPO after L. anguillarum challenge, which implied that EsPAP1, EsPAP2, EsPAP3, EsPLC, EsPAPII might cooperate together with EsproPO to play roles in the immune process of Chinese mitten crab. In addition, the expression of EsPAP2, EsPAP3, EsproPO, EsPAPII and EsPLC increased twice after the bacteria challenge. This multiple up-regulation revealed that the proPO system probably functioned in several immune processes during the interaction between the host and bacteria. Using RNAi method, EsPAP3 was further proved to be an effective proPO activating factor in haemocytes, and EsPAP1 played roles in the coagulation of Chinese mitten crab. Taken together, the present study identified the molecular basis for the uniqueness of the proPO system in arthropod, obtained the mRNA expression characteristic of proPO system members of Chinese mitten crab and provided new insights into the regulation machnism of E. sinensis proPO system.
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