The CDNA Libraries Construction, Expressed Sequence Tag Analysis And Anti-lipopolysaccharide Factors Of Swimming Crab Portunus Trituberculatus

The swimming crab Portunus trituberculatus (Miers, 1876) is one important aquaculture species in China. However, with the development of intensive culture, various diseases caused by bacteria, viruses and fungi had frequently occurred in cultured P. trituberculatus stocks and caused catastrophic economic losses to crab aquaculture. Studies on immune defense mechanisms of P. trituberculatus and novel way for disease control are helpful for sustainable development in crab aquaculture. Two full-length enriched cDNA libraries were constructed from hemocytes and eyestalk of P. trituberculatus, respectively, and randomly sequenced to collect genomic information and identify genes involved in immune defense response. Seven isoforms of anti-lipopolysaccharide factors of P. trituberculatus were studied by molecular biotechnologies including RACE, RT-PCR, and recombinant protein expression. Their gene structure of cDNA and genomic sequences were determined. Their splicing patterns of pre-mRNAs and functions involved in immune system were also investigated.In the present study, two high quality cDNA libraries of P. trituberculatus were constructed from hemocytes and eyestalk with the capacity up to 8.0×105 and 5.6×105, respectively. After random sequencing in hemocytes library, the assembly of 4452 high quality ESTs created 1066 unigenes consisting of 461 contigs and 605 singletons. In eyestalk library, 4606 high quality ESTs were assembled into 510 unigenes with 301 contigs and 209 singletons. BLAST analysis revealed 660 unigenes from hemocytes library and 365 unigenes from eyestalk library shared high similarity with genes in the public database. A total of 99 unigenes including 64 unigenes in hemocytes library and 35 unigens in eyestalk library were identified to be immune genes. These genes were categorized into six classes, viz. antimicrobial peptides, redox proteins, melanization related proteins, chaperone proteins, clottable proteins and other immune factors. Five immune genes containing multiple protein isoforms were identified and characterized, including anti-lipopolysaccharide factor (PtALF1-7), crustin (PtCrustin1-3), thioredoxin (PtTrx1-2), clip domain serine proteinase (PtcSP1-5) and kazal-type proteinase inhibitor (PtKPI1-4). The results mentioned above will greatly enrich the genomic information of P. trituberculatus and lay the first step for the elucidation of crab immune genes.Seven isoforms of anti-lipopolysaccharide factors of P. trituberculatus (PtALF1-7) were cloned based on the obtained ESTs. The open reading frame (ORF) encoded a polypeptide consisted of a signal peptide and two conserved cysteine residues, which shared high similarity with other reported ALFs. Predicted tertiary structures of PtALF2, PtALF3, PtALF5 and PtALF7 containing fourβ-strands and threeα-helices were similar to that described in other reported ALFs, while PtALF1 had only oneα-helix and PtALF4 had fiveβ-strands in the spatial structure. Sequence analysis revealed PtALF1-3 were encoded by the same genomic locus and generated by alternative splicing of the pre-mRNA. PtALF4-7 were encoded by different genomic loci. Several tandem repeats were found in introns of PtALF4, PtALF5 and PtALF7. Totally 89 SNPs of PtALF1-3 and 128 of PtALF7 were detected by direct sequencing of genomic samples. In healthy P. trituberculatus, PtALF1-7 mRNA could be found in all the tissues detected, however, they showed the different expression pattern. After challenge with Vibrio alginolyticus, the temporal expression level of PtALF1-7 in hemocytes showed a clear time-dependent response expression pattern. These results suggest that the PtALF isoforms are inducible acute-phase and long-lasting effector factors. The protein expression of PtALFs in Escherichia coli BL21 (DE3) was not ideal. The recombinant PtALF5 displayed no antibacterial activity against both Gram-positive and negative bacteria. After adding IPTG, the recombinant PtALF3 and PtALF7 had strong antibacterial activity against E. coli. The results mentioned above show the molecular basis and mRNA expression characteristics of ALFs and their isoforms of P. trituberculatus, and will be helpful in understanding the structures and functions of ALFs.