关键词: 梨小食心虫, 蛋白酶, 最适pH, 蛋白酶抑制剂, 酶活性

Abstract: 【Aim】 Grapholita molesta (Busck) is a fruit pest that causes extremely serious damage. Intestinal protease plays an important role in its growth and development processes. To provide new ideas for using protease inhibitors to control pest insects, the effects of the optimum pH, different inhibitors and activators on the midgut protease activities of G. molesta larvae were studied in this study. 【Methods】 Enzyme fluids were extracted from the midgut of the 3rd instar larvae of G. molesta. Using enzyme specificity substrates, the optimum pH of the midgut proteases of G. molesta larvae was detected in three different buffer solutions (ddH₂O as the CK), the effect of protease inhibitors and activators on the midgut protease activities were determined. At the same time, the effect of the different protease inhibitors (PMSF, TLCK, TPCL and STI) on the midgut protease activities was also determined when G. molesta larvae were fed with them. 【Results】 The optimum pH levels of midgut total protease of G. molesta larvae in the three buffers Tris-HCl, KH₂PO₄/NaOH and glycine/NaOH were 10.5, 11.0 and 11.0, respectively, those of high-alkaline trypsin were 10.5, 11.0 and 11.0, respectively, those of low-alkaline trypsin were 8.5, 9.0 and 9.0, respectively, and those of chymotrypsin were 8.5, 9.0 and 9.5, respectively. Of the five protease inhibitors (DTT, PMSF, TLCK, TPCL and STI), except that TLCK activated the chymotrypsin activity, the other inhibitors inhibited the activities of all proteases; and the higher the inhibitor concentration was, the more significant the inhibitory effects were. The inhibition effects of the inhibitor DTT on the activities of total protease and low-alkaline trypsin were greater than those of the other inhibitors. Of the four protease activators (MgCl₂, CaCl₂, EDTA and EGTA), MgCl₂ inhibited the activities of total protease and chymotrypsin but activated the trypsin activity, CaCl₂ activated the activities of casein enzyme and low-alkaline trypsin but inhibited the activities of high-alkaline trypsin and chymotrypsin, EDTA inhibited the activities of the four proteases, and EGTA inhibited the activities of the other three proteases except high-alkaline trypsin. When G. molesta larvae were fed with different protease inhibitors (PMSF, TLCK, TPCL and STI), all inhibitors could inhibit the activities of the four proteases, with obviously different inhibitory activities at different sampling time points. The inhibitory effects of STI (50 μg/mL) on the activities of the four proteases were greater than those of the other inhibitors; and the higher the inhibitor concentrations were, the more significant the inhibitory effects were. In three experimental treatments with 10, 20 and 50 μg/mL STI, sampling at 4 h post treatment the activities of the four proteases increased, and the increased levels was related to the concentration of STI. The protease activity went to its lowest level in the 20 μg/mL STI experimental treatment at 48 h post treatment, and the 50 μg/mL STI experimental treatment at 60 h post treatment, and the inhibitor STI showed a highly sustainable efficiency. 【Conclusion】 This study indicates that protease inhibitors have certain inhibitory effects on the protease activities in larval midgut of G. molesta, and the inhibitor STI has a great application value in pest control.

Key words: Grapholita molesta, protease, optimum pH, protease inhibitors, enzyme activity