No abstract

Antimicrobial peptides (AMPs) are important components of the host innate immune response against microbial invasion. In the present study, we report the identification and characterization of a crustin (CrusSp) from the hemocyte of mud crab, Scylla paramamosain using an expressed sequence tag (EST) and rapid amplification cDNA end (RACE) approaches. Analysis of the nucleotide sequence revealed seven different variances of the CrusSp cDNA in mud crab. The open reading frame encodes a protein of 111 amino acids with 21 residues signal sequence. The predicted molecular mass of the mature protein (90 amino acids) is 10.27 kDa with an estimated pI of 8.54. Analysis of the protein domain features indicated typical conserved cysteine residues containing a single whey acidic protein (WAP) domain at the C-terminus. A neighbour-joining tree showed that S. paramamosain crustin is closely related to other crustin homologues, and displays the highest similarity to crustin antimicrobial peptide in shore crab Carcinus maenas. Four exons and three introns were identified within the 999 bp genomic DNA sequence of CrusSp. Tissue distribution analysis showed that CrusSp was highly expressed in hemocytes, gills, intestines and muscle but it was not expressed in hepatopancreas and eyestalks. To gain insight into the in vitro antimicrobial activities of CrusSp, the mature peptide coding region was cloned into E. coli for heterologous expression. The recombinant CrusSp could inhibit the growth of gram-positive bacteria but had no inhibition activity against gram-negative bacteria. These results indicated the involvement of CrusSp in the innate immunity of S. paramamosain.

No abstract

Two cellulase cDNAs (GHF10-Pc1 and GHF10-Pc3) belonging to glycoside hydrolase family 10 (GHF10) were successfully isolated and characterized from stomach tissue of golden apple snail (Pomacea canaliculata), a kind of herbivorous mollusca. Sequencing analysis revealed full-length cDNAs of 1300 and 1277 bp in length, respectively. The open reading frame (ORF) of cellulase cDNA was 1188 and 1191 bp, encoding 395 and 396 amino acid, respectively. Sequence alignment revealed that GHF10-Pc1 and GHF10-Pc3 shared high identity with glycosyl hydrolase family 10 (GHF10) and had an overall similarity of 98 and 82% to those of Ampullaria crossean cellulase EGX. A neighbour-joining tree showed a clear differentiation between each species and also indicated that GHF10-Pc1 and GHF10-Pc3 from P. canaliculata and A. crossean EGX are closely related phylogenetically. The genomic organization of cellulase GHF10-Pc1 and GHF10-Pc3 genes was also investigated. The GHF10-Pc1 and GHF10-Pc3 genes spanned over 4937 and 4512 bp, respectively. Both genes contained 9 exons interrupted by eight introns. The result verified the endogenous origin of the GHF10-Pc1 and GHF10-Pc3 genes. Analysis of RNA by RT-PCR from several ages of P. canaliculata revealed that neither gene was expressed in eggs. GHF10-Pc1 was also expressed in 1- and 10-day-old juvenile snails whereas GHF10-Pc3 was expressed only in 1-day-old juvenile snails. The result showed that two GHF10-Pc transcripts were developmentally expressed.

Major Royal Jelly Protein cDNAs of