International Union for the Study of Social Insects (IUSSI2018), August 5-10, 2018 in Guarujá, Brazil.

only days left!
Find us: Ft

From Heterotermes tenuis (Rhinotermitidae) caste transcriptomes to alpha-amylase heterologous expression

Author(s):
Matheus Pedrino Gonçalves, Matheus Pedrino Gonçalves , Ana Paula Timóteo Ferreira , Ana Maria Costa-Leonardo , Caio César de Melo Freite , Anderson Ferreira da Cunha , Francis Morais Franco Nunes
Institution(s):
Programa de Pós-Graduação em Genética Evolutiva e Biologia Molecular (PPGGEv), Universidade Federal de São Carlos (UFSCar), Brasil; Programa de Pós-Graduação em Genética Evolutiva e Biologia Molecular (PPGGEv), Universidade Federal de São Carlos (UFSCar), São Carlos, Brasil ; Programa de Pós-Graduação em Genética Evolutiva e Biologia Molecular (PPGGEv), Universidade Federal de São Carlos (UFSCar), São Carlos, Brasil ; Instituto de Biociências, Universidade Estadual Paulista (UNESP), Rio Claro, Brasil ; Departamento de Genética e Evolução, Universidade Federal de São Carlos, São Carlos, Brasil ; Departamento de Genética e Evolução, Universidade Federal de São Carlos, São Carlos, Brasil ; Departamento de Genética e Evolução, Universidade Federal de São Carlos, São Carlos, Brasil
Termites are economically and ecologically relevant insects and may serve as a model organism in biotechnological studies. We explored transcriptome data from Heterotermes tenuis (Rhinotermitidae) workers and soldiers, searching for potential biotechnological targets for industrial applications and/or pest control. By means of RT-PCR, we validated gene models assembled from the RNA-Seq data of both castes. From the set of validated genes, we selected the gene encoding the an alpha-amylase enzyme for downstream analysis and assays. We successfully assembled and amplified (by PCR) the complete alpha-amylase coding sequence and inserted it into a pET-15b expression vector. An E. coli Rosetta (DE3) chassis was transformed with a recombinant construct. Heterologous production of H. tenuis alpha-amylase was soluble, observed at 30°C and 37°C of induction at different concentrations of IPTG (0.1 mM, 0.4 mM and 1 mM), and confirmed by Western Blot analysis (using 6X His-Tag monoclonal antibody for detection). The predicted molecular weight of α-amylase is ~ 67.3 kDa, similar to that observed in gel electrophoresis: ~ 69.5 kDa. However, production was low and unsatisfactory purification products were recovered after nickel affinity chromatography. Even with some technical problems, the present study represents the first applied steps toward identification and expression of Brazilian termite genes with biotechnological potential, opening avenues of investigations as well as developing and improving biotechnological methods and protocols.
Back