Study On Prophenoloxidase Activating System Related Genes And Hemocyanin Gene From Scylla Paramamosain

Scylla paramamosain is one of the most precious marine crabs aquacultured in China, is prone to infection of various microbes which threats the mud crab industry. In this study, we cloned two genes related to prophenoloxidase activating system and hemocyanin gene from S. paramamosain, and detected the expression profiles in different cases using Quantitative real-time PCR. The main results are as follows:1. Prophenoloxidase activating factors(PPAFs) are a group of clip domain serine proteinase that can convert prophenoloxidase(pro-PO) to the active form of phenoloxidase(PO), causing melanization of pathogens. Here, two full-length PPAF c DNAs from Scylla paramamosain(Sp PPAF1 and Sp PPAF2) were cloned and characterized. The full-length Sp PPAF1 c DNA was 1677 bp in length, including a 5′-untranslated region(UTR) of 52 bp, an open reading frame(ORF) of 1131 bp coding for a polypeptide of 376 amino acids, and a 3′-UTR of 494 bp. The full-length Sp PPAF2 c DNA was 1808 bp in length, including a 5′-UTR of 88 bp, an ORF of 1125 bp coding for a polypeptide of 374 amino acids, and a 3′-UTR of 595 bp. The estimated molecular weight of Sp PPAF1 and Sp PPAF2 was 38.43 and 38.56 k Da with an isoelectric point of 7.54 and 7.14, respectively. Both Sp PPAF1 and Sp PPAF2 proteins consisted of a signal peptide, a characteristic structure of clip domain, and a carboxyl-terminal trypsin-like serine protease domain. Expression analysis by q RT-PCR showed that Sp PPAF1 m RNA was mainly expressed in the gill, testis, and hemocytes, and Sp PPAF2 m RNA was mainly expressed in hemocytes. In addition, Sp PPAF1 and Sp PPAF2 m RNA was expressed in a time-dependent manner after Vibrio parahaemolyticus challenge. The results showed that expression of both Sp PPAF1 and Sp PPAF2 was related to the bacterial challenge but the expression patterns differed. These findings suggest that Sp PPAF is a serine proteinase and may be involved in the pro-PO activation pathway of the crab innate immune system.2. Prophenoloxidase is a copper-dependent enzyme. It catalyzes the oxidation of dopa rapidly to dopaquinone, which in turn polymerize non-enzymetically into insoluble melanin. In this study, full-length pro PO c DNA from Scylla paramamosain(Sppro PO) were cloned and characterized. The full-length Sppro PO c DNA was 2688 bp in length, including a 5’ untranslated region of 40 bp, an open reading frame(ORF) of 2133 bp coding for a polypeptide of 710 amino acids, and a 3’ untranslated region of 515 bp. The estimated molecular weight of Sppro PO was 81.5 k Da with an isoelectric point of 6.01. Expression analysis by q RT-PCR showed that Sppro PO m RNA was mainly expressed in the hemocytes and testis. This study provides useful information regarding the structure and functions of pro PO and will play an important role in immunity and resistance research in Scylla paramamosain.3. Hemocyanin is an important respiratory protein in many arthropod and mollusc species. In this study, four c DNAs encoding distinct hemocyanin from Mud Crab Scylla paramamosain(Sp Hc) were cloned by using EST analysis and rapid amplification of c DNA ends(RACE), named Sp Hc1, Sp Hc2, Sp Hc3 and Sp Hc4. The four full-length c DNA fragments of the Sp Hc covered 2281 bp, 2002 bp, 2184 bp and 2069 bp respectively, and encoded four putative protein of 676,570,672 and 671 amino acids. Quantitative real-time PCR(q RT-PCR) analysis revealed that the Sp Hc were mainly expressed in the hepatopancreas, testis and haemocytes. Sp Hc m RNA expressions during continuous developmental stages in zoeal phases(Z1, Z2, Z3, Z4 and Z5), megalopa and juvenile crab I stages were also detected by q RT-PCR. The expression levels of Sp Hc3 and Sp Hc4 were higher than that of Sp Hc1 and Sp Hc2 during the first six stages, and it declined suddenly during the juvenile stage. After infection with Vibrio parahaemolyticus, the expression of Sp Hc m RNA in the megalopa stage were relative to bacterial challenge but may act in different patterns. These findings suggest that hemocyanin may be potentially involved in the immune responses of the mud crab, and that the role of the four subunits may not be all the same in different tissues and during different developmental stages.