الفهرس 
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Abstract
The present study aimed to investigate the inhibitory effect of partial purified proteinase inhibitor from seeds of soybean Glycine max against the 4th instar larvae of cotton leaf worm Spodoptera littoralis (Bosid.) (Lepidoptera: Noctuidea). This study was divided into two main parts:
The first part of the study was concerned with the purification of proteinase inhibitor from seeds extract of soybean and its inhibitory effect against trypsin and chymotrypsin-like enzyme in midgut extract of 4th larval instar of S. littoralis.
The second part was concerned with the efficiency of purified fractions on serine enzymes activity, larval growth and development after feeding 4th instar larvae on SPI.
1. Purification of proteinase inhibitor and screening of its inhibitory activity.
1.1. Purified fractions of soybean proteinase inhibitor SPI and its inhibitory activity against trypsin-like enzyme from S. littoralis.
Results revealed that, F0-30 (NH4)2SO4 (w/v) saturation was efficient for precipitating the proteinase inhibitor. It gave the highest inhibitory activity against trypsin-like enzyme of 4th instar larvae of S. littoralis; 3.9 ± 0.2 TIU/mg protein. The fold purification obtained from the fraction was 1.56 times that of the crude extract and the recovery percentage was 71.76%.
Followed by F60-90 (NH4)2SO4 which gave 2.7 ± 0.5 TIU/mg protein and F30-60 (NH4)2SO4 showed the lowest trypsin inhibitory activity, 0.5 ± 0.08 TIU/mg protein and lowest fold purification 0.2 times that of crude extract and recovery percentage 12.94%.
1.2. Purified fractions of SPI and its inhibitory activity against chymotrypsin-like enzyme from S. littoralis.
Data indicated that F0-30 (NH4)2SO4 (w/v) saturation was the most effective fraction against chymotrypsin-like enzyme of 4th instar larvae of S. littoralis, it gave the highest inhibitory activity 5.3± 0.2 CIU/mg protein.
Followed by F60-90 (NH4)2SO4 which gave 2.9 ± 0.1 CIU/mg protein with no significant difference with F30-60 (NH4)2SO4 which was 2.7 ± 0.1 CIU/mg protein.
2. The post-ingestion effect of SPI on the activity of midgut Serine proteinases of 4th instar larvae of S. littoralis.
2.1. Determination of total protein content:
Data indicated that there was a significant reduction in the total protein content in the larvae fed on SPI F90% by percentage of inhibition 21%.
On the other hand, larvae fed on both fraction SPI F30% and SPI F60% showed slightly decreas in total protein concentration; it was no significant difference between treated and control.
2.2. In vivo effect of SPI on the activity of midgut trypsin-like enzyme in 4th instar larvae of S. littoralis after feeding 24h:
The results recorded a significant reduction in trypsin-like enzyme activity in larvae fed on SPI F60% with percentage of inhibition 16.5 compared with control followed by SPI F30% which gave 9.17 inhibition percentage.
On the other hand, feeding larvae with SPI F90% showed no significant difference compared with control.
2.3. In vivo effect of SPI on the activity of midgut chymotrypsin-like enzyme in 4th instar larvae of S. littoralis after feeding 24h:
The data revealed a significant reduction in chymotrypsin-like enzyme in larvae fed on SPI F30% with high percentage of inhibition 19.6 compared with control.
The three fractions were significantly difference with the control, but no significant with each other.
3. Effect of bio-feeding assay of SPI on growth and development of the 4th instar larvae of S. littoralis.
3.1. Effect of SPI on growth and development of the 4th instar larvae of S. littoralis:
The results cleared that a significant reduction in larval weight in larvae fed on SPI F30% and SPI F60% with no significant difference between them; larval weight decreased about 2.3 gm compared with control.
This reduction in larval weight began after seven days of feeding on SPI fractions.
There was also, delaying in the pupation two days in larvae fed on both fractions compared with control.
The larval mortality percentage increased in larvae fed on SPI F60% about 20% than the control followed by SPI F30% which caused 16% larval mortality.
3.2. Effects of SPI on pupal weight, pupation %, and adult emergency of 4th larval instar of S. littoralis:
The present work reflected that there was a significant reduction in the pupal weight; the highest reduction was at larvae fed on SPI F60%. There was no significant difference in pupal weight between control larvae and larvae fed on SPI F30%.
There was also, reduction in the pupation rate from 83% to 80% at SPI F60% and SPI F30%, respectively, while in the untreated larvae it recorded 92%.
In addition, the adult emergency delayed 36h in larvae fed on SPI F30% compared with control which began emergency after 10 days of pupation. The adult emergency rate decreased, which only 50% of the larvae fed on SPI F30% emerged and 75% of the larvae fed on SPI F60% emerged.
4. Zymogram analysis of trypsin-like enzyme activity after feeding 4th instar larvae of S. littoralis 24h on SPI:
The results cleared that little difference in the thickness of cleared zones between the control, SPI F30% and SPI F60%. That is mean little inhibition in the trypsin activity in treated larvae with SPI F30% and SPI F60%.
On the other hand, no differences between the thicknesses of clear zones in control and SPI F90%. Therefore, trypsin activity in treated larvae with SPI F90% did not different than control.