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Mohammad VATANPARAST,Vahid HOSSEININAVEH 한국곤충학회 2010 Entomological Research Vol.40 No.6
The alfalfa weevil Hypera postica is a serious economic pest in most alfalfa grown in many countries worldwide. Digestive -amylase and pectinase activities of larvae were investigated using general substrates. Midgut extracts from larvae showed an optimum activity for -amylase against starch at acidic pH (pH 5.0). -Amylase from larval midgut was more stable at mildly acidic pH (pH 5–6) than highly acidic and alkaline pH. The enzyme showed its maximum activity at 35°C. -Amylase activity was significantly decreased in the presence of Ca2+, Mg2+ and sodium dodecylsulfate. On the contrary, K+ and Na+ did not significantly affect the enzyme activity. Zymogram analysis revealed the presence of one band of -amylase activity in in-gel assays. Pectinase activity was assayed using agarose plate and colorimetric assays. Optimal pH for pectinase activity in the larval midgut was determined to be pH 5.0. Pectinase enzyme is more stable at pH 4.0–7.0 than highly acidic and alkaline pH. However, the enzyme was more stable at slightly acidic pH (pH 6.0) when incubation time increased. Maximum activity for the enzyme incubated at different temperatures was observed to be 40°C. Optimum pH activity for -amylase and pectinase is not completely consistent with the pH prevailing in the larval midgut. This is the first report of the presence of pectinase activity in H. postica.
Mohammad VATANPARAST,Vahid Hosseininaveh,Mohammad Ghadamyari,Seyede Minoo Sajjadian 한국응용곤충학회 2012 Journal of Asia-Pacific Entomology Vol.15 No.4
The elm leaf beetle, Xanthogaleruca luteola, is a serious pest of elm trees in urban areas. Partial biochemical characterization of pectinases and cellulases was conducted using the larval digestive system of the pest. Midgut extracts from larvae showed optimum activity for pectinase and cellulase against pectin and carboxymethyl cellulose, respectively, under acidic conditions (pH 6). Pectinases and cellulases were respectively more stable under acidic conditions (pH 4–7) and slightly acidic conditions (pH 5–7) than under highly acidic and alkaline conditions. However, the enzymes were more stable in slightly acidic conditions (pH 6) when incubation time was increased. Maximum activity for the pectinases and cellulases incubated at different temperatures was observed at 45 and 50 °C, respectively. Mg2+ remarkably increased pectinase activity,and cellulase activity increased significantly in the presence of Ca2+ and Mg2+. Sodium dodecyl sulfate significantly decreased pectinase and cellulase activity. The Michaelis–Menten constant (KM) and the maximal reaction velocity (Vmax) values for pectinase were 2 mg·mL−1 and 0.017 mmol·min−1·mg−1 protein toward pectin, respectively. Zymogram analyses revealed the presence of one and five bands of pectinase and cellulase activity, respectively, in the larval midgut extract.
Mohammad Vatanparast,김용균 한국응용곤충학회 2019 Journal of Asia-Pacific Entomology Vol.22 No.3
Mating disruption by using sex pheromone is an ecofriendly alternative way to control insect pests. To be effective, large amounts of sex pheromone are needed, leading to a relatively high production cost. To reduce the cost for chemical synthesis of sex pheromone, yeast engineering technology has been devised. This study used a baker's yeast, Saccharomyces cerevisiae, to express genes associated with sex pheromone biosynthesis of the Oriental fruit moth, Grapholita molesta. Compared to other fatty acid biosynthetic pathways, two steps that are unique to pheromone gland of G. molesta are proposed: desaturation at even number catalyzed by desaturase (Gm-DES) and terminal reduction catalyzed by fatty acyl reductase (Gm-FAR). Gm-DES and Gm-FAR were cloned into a yeast expression vector, pYES2.1. They were used to transform S. cerevisiae by a double transfection method. The transformed yeast was induced with 2% galactose to over-express these two exogenous genes. Their expression was confirmed by RT-PCR and western blotting. To facilitate pheromone production, transformed yeasts were supplied with myristic acid during over-expression. Resulting fatty acid composition was analyzed by GC-MS after fatty acid methyl ester derivatization. Control yeast produced mostly saturated fatty acids. However, a single gene (Gm-DES)-transformed yeast produced unsaturated fatty acids at Δ9 such as Z9-tetradecenoic acid (Z9-14:1), palmitoleic acid (Z9-16:1), and oleic acid (Z9-18:1) in addition to saturated fatty acids. The double-transformed yeast produced an additional component, alcohol form of oleic acid (Z9-18:OH). These results suggest that Gm-DES can catalyze desaturation of fatty acids at Δ9 and Gm-FAR can reduce terminal carboxylic acid into alcohol.
Oral delivery of dsRNA using recombinant bacteria against a chymotrypsin gene of Spodoptera exigua
Mohammad Vatanparast,Yonggyun Kim 한국응용곤충학회 2017 한국응용곤충학회 학술대회논문집 Vol.2017 No.04
RNAi (RNA interference) is a tool for silencing of target genes through sequence-specific manner. Spodoptera exigua belongs to Noctuidae family of Lepidoptera and is serious threat to crops of economic importance. One of S. exigua chymotrypsin gene (SeCHY2) was cloned into the L4440 vector to produce sequence specific dsRNAs (double-stranded RNAs). Recombinant L4440 vectors were transformed into Escherichia coli strain HT115 (DE3). Oral delivery of bacterially expressed dsRNA gave significant larval mortality. Quantitative real-time PCR results showed that expression level of target SeCHY2 gene in the larval gut tissue was significantly down-regulated. Pretreatment with an ultra-sonication and heating to disrupt bacterial cell wall/membrane significantly increased the insecticidal activity of the transformed bacteria