Sequencing and characterization of the citrus weevil, Diaprepes abbreviatus, trypsin cDNA - Effect of Aedes trypsin modulating oostatic factor on trypsin biosynthesis

Trypsin mRNA from the citrus weevil. Diaprepes abbreviatus, was reverse tra nscribed and amplified by PCR. A cDNA species of 513 bp was cloned and sequ enced. The 3' and 5' ends of the gene (262 bp and 237 bp, respectively) wer e amplified by rapid amplification of cDNA ends, cloned and sequenced. The deduced sequence of the trypsin cDNA (860 bp) encodes for 250 amino acids i ncluding 11 amino acids of activation and signal peptides and exhibited 16. 8% identity to trypsin genes of selected Lepidoptera and Diptera. A three-d imensional model of Diaprepes trypsin contained two domains of beta-barrel sheets as has been found in Drosophila and Neobellieria. The catalytic acti ve site is composed of the canonical triad of His41, Asp92 and Ser185 and a specificity pocket occupied by Asp179 with maximal activity at pH 10.4. So uthern blot analysis indicated that at least two copies of the gene are enc oded by Diaprepes midgut. Northern blot analysis detected a single RNA band below 1.35 kb at different larval ages (28-100 days old). The message incr eased with age and was most abundant at 100 days. Trypsin activity, on the other hand, reached a peak at 50 days and fell rapidly afterwards indicatin g that the trypsin message is probably regulated translationally. Feeding o f soybean trypsin inhibitor and Aedes aegypti trypsin modulating oostatic f actor affected trypsin activity and trypsin biosynthesis, respectively. The se results indicate that Diaprepes regulates trypsin biosynthesis with a tr ypsin modulating oostatic factor-like signal.