Cloning And Expression Analysis Of Serine Protease And Its Homolog Genes Of Swimming Crab, Portunus Trituberculatus

The swimming crab Portunus trituberculatus (Decapoda: Brachyura: Portunidae)is one of the important aquaculture species in China. However, with the developmentof intensive culture, various diseases caused by bacteria, viruses and fungi hadfrequently occurred in cultured P. trituberculatus stocks and caused catastrophiceconomic losses to crab aquaculture. Studies on immune defense mechanisms of P.trituberculatus and novel way for disease control are helpful for sustainabledevelopment in crab aquaculture. Serine protease and its homologs which are widelydistributed in invertebrates are involved in innate immunity, including bloodcoagulation, antibacterial peptide synthesis, cell adhesion and melanization reactionand so on, it provides important scientific basis for us to study the importance ofserine protease and its homologs in resisting external microbial process.In our lab, six full-length cDNA sequences of Portunus trituberculatus serineprotease and its homologs were characterized based on analysis of expressed sequencetags from haemocytes and eyestalk cDNA libraries. With the use of gene cloning,real-time PCR, recombinant protein prokaryotic expression and other molecularbiology techniques, we carried out studies on these gene structure, tissue distribution,expression after different types of microbial infection and some genes’s genomesequence and prokaryotic expression, to further discuss the role of them in P.trituberculatus immunity.These genes all possessed a Tryp_SPc domain at the C-terminal. Two groups areseparated according to their protein motif including clip domain or not. One serineprotease (PtSP) and one serine protease homolog (PtSPH1) without clip domainwhich were identified from haemocytes cDNA library formed the first group. Another group including clip domain contains three clip domain serine proteases (PtcSP1,PtcSP2and PtcSP3) and one clip domain serine protease homolog (PtcSPH). Theywere characterized based on analysis of expressed sequence tags from haemocytesand eyestalk cDNA libraries respectively. As with most serine proteases, the thirdcatalytic site Ser (S) was replaced by Gly (G) of PtSPH1, while the second catalyticresidue of PtcSPH Arg (D) replaced by Ala (A).The full length of PtSP was1266bp, including a861bp open reading frame(ORF) encoding a286-amino acid protein, The full length of PtSPH1was1507bpwith an open reading frame of1236bp, encoding a protein of411aa. The full-lengthcDNA sequences of PtcSPs and PtcSPH were1513,1363,1461and1530bp,respectively. PtcSP1contained an open-reading frame (ORF) of1146bp encoding382deduced amino acids. PtcSP2included an ORF of1203bp encoding400aminoacids. PtcSP3consisted an ORF of1248bp encoding415amino acids, while PtcSPHcomprised of an ORF of1137bp encoding379amino acids. Meanwhile, we clonedand analyzed the genome sequences of PtSP and PtSPH1. Six introns presented inPtSP genomic DNA with one uncommon splice site (GG) were discovered at the exon1/intron1boundary region. Four introns with common splice sites were found inPtSPH1genomic DNA.The fuorescent quantitative real-time PCR assay was performed to determine themRNA distribution of serine protease and its homologs in different tissues includinghaemocytes, muscle, hepatopancreas, eyestalk, heart, gill and stomach of Portunustrituberculatus. The results showed that the expression levels were different from eachother. RT-PCR results showed that PtSP mRNA was mainly distributed in haemocytes,gill and eyestalk, whereas PtSPH1transcript was mainly expressed in stomach. ThemRNA transcripts of PtcSPs and PtcSPH could be detected widely in all the examinedtissues with remarkable different expression levels. The PtcSP1mRNA transcriptswere highly expressed in gill, but were less expressed in haemocytes and muscle.PtcSP3transcripts were mainly expressed in haemocytes, with a low level ofexpression in others organs. The relative highest expression level of PtcSPH wasobserved in haemocytes and stomach, but less expressed in gill, eyestalk, heart, hepatopancreas and muscle.To further investigate the roles of seine proteases in innate immunity, thetemporal expression of serine protease genes transcripts in haemocytes of crabschallenged with Vibrio alginolyticus, Micrococcus luteus, and Pichia pastoris weredetermined by quantitative real-time RT-PCR. PtSP showed slight increase during thefirst48h compared to control groups except8h point after Micrococcus luteuschallenge. However, signifcant up-regulation was observed in the expression level ofPtSPH1challenged by Gram-negative bacteria V. alginolyticus, Gram-positivebacteria M. luteus and fungi P. pastoris during the first48h. It indicates that PtSPH1might be more sensitive to microorganism challenges compared with PtSP. Thetemporal expressions of PtcSPs and PtcSPH demonstrated different time-dependentexpression pattern post V. alginolyticus, M. luteus and P. pastoris challenge.Especially, the expression of PtcSPH transcripts showed greater change against V.alginolyticus compared with the other two microorganisms. These fndings suggestthat serine proteases and its homolog containing clip domain play different roles in theantibacterial defense mechanism of Portunus trituberculatus crab.