D0406 Molecular cloning and cell-free expression of an endoglucanse belonging to the glycoside hydrolase family GHF45 from the western corn rootworm, Diabrotica virgifera virgifera

Wednesday, December 16, 2009
Hall D, First Floor (Convention Center)
Arnubio Valencia , Plant Science, University of Caldas, Manizales, Colombia
Analiza P. Alves , Insect Resistance Management Science / Trait Characterization & Development, DuPont Pioneer, Johnston, IA
Haichuan Wang , Entomology, University of Nebraska-Lincoln, Lincoln, NE
Blair D. Siegfried , Department of Entomology, University of Nebraska, Lincoln, NE
A novel endogenous endoglucanase (Dvv-EGase I) gene belonging to the glycoside hydrolase family (GHF) 45 was cloned from the western corn rootworm beetle, Diabrotica v. virgifera. cDNA of the Dvv-EGase consisted of 735 bp-open reading frame coding a 245-amino-acid protein. Analysis of the amino acid sequence revealed that Dvv-EGase I has a 54% protein sequence identity when compared with cellulase II, a cellulase belonging to GHF45 from another beetle, Apriona germari. Using neural networks (NN) and hidden Markov models it was possible to know that Dvv-EGase I contains a signal peptide with a most likely cleavage site between position 16 and 17 (AVA-TP). Western blot analyses showed that Dvv-EGase I was expressed in three gut regions, but indicating that the midgut and hindgut are the prime sites for Dvv-EGase I synthesis in D. v. virgifera larvae. In addition, the cDNA encoding Dvv-EGase I was expressed using three Cell-Free systems.

doi: 10.1603/ICE.2016.43396