Midgut Proteases From Mamestra configurata (Lepidoptera: Noctuidae) Larvae: Characterization, cDNA Cloning, and Expressed Sequence Tag Analysis

摘要

The activities of digestive protease within the midgut of Mamestra configurata (bertha armyworm) larve were examined using specific substrates and protease inhibitors. The bulk of the activity was associated with serine proteases comprising trypsin-, chymotrypsin-, and elastase-like enzy6mes. At least 10-15 serine protease isozymes were detected suing one-dimension gelatin gel electrophoresis. Cysteine or aspartic protease activities were not present; however, amino-and carboxypeptidase activities were associated with the migdut extract. Midgut proteases were active in the pH range of 5.0-12.0 with peaks at pH 7.5 and 11.0. In general, the middle region of the midgut exhibited a higher pH (approximately 8.0) than either the posterior or anteiror regions (approximately 7.3-7.7). Moulding larvae possessed a neutral gut pH that was 0.5-1.5 units below that of feeding larvae. Degenerate PCR and expressed sequence tag (EST)-based approaches were used to isolate 30 distinct serine protease encoding cDNAs from a midgut-specific cDNA library including 8 putative trypsin, 9 chymotrypsins, 1 elastase, and 12 whose potential activities could not be determined. cDNAs encoding three amino-and two carboxypeptidases were also identified. Larvae feeding upon artificial diet containing 0.2% soybean trypsin inhibitor experienced a significant delay in development.