embryo-microinjection-knockout-mutant-identification-crisprcas9

Embryo Microinjection and Knockout Mutant Identification of CRISPR/Cas9 Genome-Edited <em>Helicoverpa Armigera</em> (H&#252;bner)

The cotton bollworm, Helicoverpa armigera, is one of the most destructive pests in the world. A combination of molecular genetics, physiology, functional ...

China University of Mining and Technology

Chinese Academy of Agricultural Sciences

Trehalase is an indispensable component of insect hemolymph that plays important role in energy metabolism and stress resistance. In this study, we cloned and expressed the gene encoding soluble trehalase (HaTreh-1) of Helicoverpa armigera (cotton bollworm) and characterized the enzyme. HaTreh-1 had a full-length open reading frame encoding a protein of 571 amino acids. Sequence comparison indicated that HaTreh-1 was similar to some known insect trehalases. Two essential active sites (D321 and E519) and three essential residues (R168, R221, and R286) were conserved in HaTreh-1. The recombinant trehalase was expressed in Escherichia coli and purified by nickel exchange chromatography. Molecular weight of the recombinant protein was about 71 kDa, and the optimum HaTreh-1 enzyme activity is at 55°C with pH 6.0. Enzymatic assays showed a Km value of 72.8 mmol/liter and a Vmax value of 0.608 mmol/(liter·min). Inhibition assays in vitro indicated that castanospermine, a polyhydroxylated alkaloid, was an effective competitive inhibitor of trehalase with a Ki value of 6.7 μmol/liter. The inhibitor action of castanospermine was linked to its modification effect on trehalase structure. The circular dichroism spectrum showed that the percentage of α-helix increased under the presence of castanospermine. Results of our study will aid in developing effective trehalase inhibitors for controlling H. armigera in the future.

Gene Cloning, Prokaryotic Expression, and Biochemical Characterization of a Soluble Trehalase in Helicoverpa armigera Hübner (Lepidoptera: Noctuidae).

The purpose of this study was to evaluate the effects of three toxic β-glucosides, phlorizin, santonin, and amygdalin, on carbohydrate metabolism in the cotton bollworm, Helicoverpa armigera (Hübner), when diets mixed with β-glucosides were fed to third-instar larvae. The growth of the larvae was significantly inhibited by exposure to santonin after 96 hr but not obviously affected by phlorizin and amygdalin. The midgut trehalase activities were only 51.7%, 32%, and 42.5% of that of the control after treatment with phlorizin, santonin and amygdalin at 2 mg/ml, respectively. In the hemolymph and fat body, the amount of trehalose decreased in all cases. However, the effects of santonin on the alteration of the glycogen and glucose levels as well as the activities of glycogen phosphorylase, were different than those of the other two β-glucosides. It appears that the three β-glucosides have different influences on the carbohydrate metabolism of cotton bollworm.

Dong Ai

School of Chemical and Environmental Engineering

China University of Mining and Technology

Embryo Microinjection and Knockout Mutant Identification of CRISPR/Cas9 Genome-Edited Helicoverpa Armigera (Hübner)

Dong Ai

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}School of Chemical and Environmental Engineering

China University of Mining and Technology

Embryo Microinjection and Knockout Mutant Identification of CRISPR/Cas9 Genome-Edited Helicoverpa Armigera (Hübner)

School of Chemical and Environmental Engineering