نوع مقاله : مقاله پژوهشی
نویسندگان
1 کارشناسی ارشد، بخش بیوتکنولوژی گیاهی، پژوهشگاه ملی مهندسی ژنتیک و زیست فناوری، تهران، ایران
2 استاد، بخش یوتکنولوژی گیاهی، پژوهشگاه ملی مهندسی ژنتیک و زیست فناوری، تهران، ایران
3 دکترا، بخش یوتکنولوژی گیاهی، پژوهشگاه ملی مهندسی ژنتیک و زیست فناوری، تهران، ایران
4 کارشناسی ارشد، بخش یوتکنولوژی گیاهی، پژوهشگاه ملی مهندسی ژنتیک و زیست فناوری، تهران، ایران
5 استادیار، بخش تحقیقات کنترل بیولوژیک، موسسه تحقیقات گیاه‌پزشکی کشور، تهران، ایران
چکیده
کلیدواژه‌ها
موضوعات
عنوان مقاله [English]
نویسندگان [English]
In this research, the effect of chitin binding domain was investigated on the enhancement of chitinase activity Chit42 enzyme tested on the last instar larvae of Ephestia kuehniella Zell. Chimeric chitinase containing chitin binding domain from Trichoderma atroviride 18-10 chitinase and Chit42 were cloned in pET 26b(+) and over expressed in Escherichia coli. The expressed protein was observed and verified by SDS-PAGE method. Hydrolytic activity of the chimeric and native enzymes was measured on the dry powder of 10 last instar larvae of E. kuehniella. The effect of 3 different concentrations of each enzyme including 30, 45 and 60μg/ml was evaluated on the mortality of 30 E. kuehneilla last instar larvae (each 10 larvae as 1 replication) at 28ºC, 50% RH and 16:8h L:D. Larvae were treated by protein solution lacking enzymes in control. After 24h, the number of dead larvae was counted in treatments and control. Mortality was calculated by Abbott’s formula. The chimeric chitinase and native enzymes activity on the larval chitin (substrate) was calculated to be 15.47 and 9.16U/ml, respectively. Bioassay tests demonstrated that the chimeric chitinase and Chit42 enzymes imposed 65 and 47% mortality to E. kuehniella larvae, respectively. Finally, microscopic observation demonstrated higher amount of chitin degradation in treated larvae with chimeric chitinase compared with those treated by native Chit42.
کلیدواژه‌ها [English]
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