Effect of a Chimeric Chitinase on the Larval Stage of the Mediterranean Flour Moth (Ephestia kuehniella Zell.)

Document Type : Research Paper

Authors

1 MSc, National Institute of Genetic Engineering and Biotechnology, Tehran, Iran

2 Professor, National Institute of Genetic Engineering and Biotechnology, Tehran, Iran

3 PhD, National Institute of Genetic Engineering and Biotechnology, Tehran, Iran

4 Assistant Professor, Department of Biological Control Research, Iranian Research Institute of Plant Protection, Tehran, Iran

Abstract

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.

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Volume 10, Issue 2
March 2020
Pages 1-8

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