Three isoforms of anti-lipopolysaccharide factor identified from eyestalk cDNA library of swimming crab Portunus trituberculatus

摘要

Anti-lipopolysaccharide factors (ALFs), as the potent antimicrobial peptides, can bind and neutralize lipopolysaccharide (LPS) and exhibit broad spectrum antimicrobial activities. In this study, three isoforms of the ALF homologues (PtesALF1-3) were identified from eyestalk cDNA library of swimming crab Portunus trituberculatus. The full-length cDNA sequences of PtesALF1, 2 and 3 were 1138, 1052 and 1057aabp encoding 92, 108 and 123 amino acids, respectively. PtesALF1-3 contained two conserved cysteine residues and shared high similarity with other reported ALFs. Predicted tertiary structures of PtesALF2 and 3 containing four beta -strands and three alpha -helix were similar to that described in Limulus polyphemus, while PtesALF1 had only one alpha -helix in its spatial structure. Sequence analysis revealed PtesALF1-3 were encoded by the same genomic locus and generated by alternative splicing of the pre-mRNA. Totally 89 SNPs including 18 in coding region and 71 in noncoding region were detected by direct sequencing of 30 genomic samples. The mRNA expression of PtesALF1 and PtesALF1-3 transcripts was mainly detected in haemocytes but showed different expression pattern in other tissues including hepatopancreas, gill, eyestalk and muscle. After challenge with Vibrio alginolyticus, the temporal expression level of PtesALF1-3 transcripts in haemocytes showed a clear time-dependent response expression pattern with two peaks within the experimental period of 32aah, while PtesALF1 was up-regulated only once with obvious decrease at 6aah and significant increase at 24aah. These results suggest that the PtesALF isoforms have different tissue specificity and might provide multiple protective functions against invading bacteria in P. trituberculatus. a-[ordm Three ALF isoforms are identified from eyestalk cDNA library of Portunus trituberculatus. a-[ordm PtesALF1-3 are generated by alternative splicing of the same pre-mRNA. a-[ordm 18 cSNPs are detected from PtesALF by direct sequencing of 30 genomic samples. a-[ordm PtesALF1-3 show different tissue specificity and operating times against pathogens.