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

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.