MOLECULAR-CLONING AND SEQUENCING OF TRYPSIN CDNAS FROM PENAEUS-VANNAMEI (CRUSTACEA, DECAPODA) - USE IN ASSESSING GENE-EXPRESSION DURING THEMOLT CYCLE

Trypsin is the most abundant protease in Crustacea. This enzyme was pu rified from the digestive gland of Penaeus vannamei, revealing three m ajor isoforms (molecular weights 31-32 kDa) and several minor componen ts, Five cDNAs encoding five isoforms of trypsin were detected by two successive screenings of an amplified cDNA library from the digestive gland of P. vannamei. The longest isolated and sequenced cDNA encoded a preproenzyme of 255 amino acids containing a putative precursor pept ide of 14 residues and a highly hydrophobic signal sequence of 14 amin o acids, Amino acid sequence alignments revealed a high degree of iden tity between the trypsin from P. vannamei and that from crayfish (74%) and an equal level of sequence similarity to that from mammals and in sects (approximate to 40). Dot blot hybridization and subsequent analy sis of the variation in trypsin-specific activities revealed that mRNA expression is at a maximum during early premoult (D-1), declining sha rply in late premoult (D-2-D-3). The specific activity of trypsin also followed this pattern, suggesting the regulation of trypsin biosynthe sis is, at least in part, transcriptional. The characterization of try psin cDNA from P. vannamei provides the first description of a putativ e zymogen sequence in a crustacean species, enabling us to elucidate t he regulatory mechanism of trypsin synthesis in these important marine organisms. Copyright (C) 1996 Elsevier Science Ltd.