The use of a thermostable β-glucanase gene from Clostridium thermocellum as a reporter gene in plants

Abstract

In order to take advantage of the high thermostability of its product, beta-1,3;1,4-glucanase (lichenase), we used a modified version of the licB gene from Clostridium thermocellum as a reporter gene for the analysis of gene expression in transformed plants. The coding region of the licB gene was truncated at both ends. The truncated enzyme retained its activity and thermostability. The modified gene (m-licB), with and without a plant leader peptide-encoding sequence, was expressed in tobacco plants under control of either the Agrobacterium octopine TR-DNA 2' gene promoter or the promoter of the gene for the small subunit of ribulose-1,5-bisphosphate carboxylase. Expression of licB can be measured quantitatively and accurately, the assay is sensitive and simple enough to be used for analysis of various gene fusion systems or for screening of transformants. The enzyme is very stable and remains active in tissue extracts even after storage for 1 year and survives many thawing-freezing cycles. The lichenase-encoding gene was expressed at high levels in transformed tobacco plants without any apparent detrimental effects on vegetative growth or flowering.

A reporter system for prokaryotic and eukaryotic cells based on the thermostable lichenase from Clostridium thermocellum

“…The lichenase gene previously cloned in a pRT3 vector (Piruzian et al 1998) was modi®ed by deleting a segment at its 3¢ end by PCR using the primers 5¢-GAAAGCATAACACAACAAGCC-3¢ and 5¢-TCGGAGTAGAAGGAGCAATCGT-3¢. The ampli®ed gene, designated as L-licBM2 was inserted into a pT7-blue-T vector (Novagen) to give pTLU.…”

“…Lichenase activity in the lysates was determined as described previously at 65°C using lichenan as substrate (Piruzian et al 1998). Reducing sugars released from the substrate were determined with the dinitrosalicyl reagent according to Wood and Bhat (1988).…”

“…The enzyme was characterized by the zymogram method described earlier (Piruzian et al 1998). The bands of active enzyme appear as clear spots since the dye binds only to unhydrolyzed lichenan.…”

“…We have previously proposed a new reporter system for plants that takes advantage of the thermostability of a lichenase from Clostridium thermocellum (Piruzian et al 1998. Like most bacterial glycosyl hydrolases, lichenase is an one-subunit protein with a number of structural domains: a signal peptide (of 27 amino acids), a catalytic domain (224 residues), a Pro-Thr-box (13 residues), and a`dockerin' domain (5 residues) (Schimming et al 1992).…”

“…Like most bacterial glycosyl hydrolases, lichenase is an one-subunit protein with a number of structural domains: a signal peptide (of 27 amino acids), a catalytic domain (224 residues), a Pro-Thr-box (13 residues), and a`dockerin' domain (5 residues) (Schimming et al 1992). Deletion of the 5¢ and 3¢ ends of the Clostridium thermocellum licB (Piruzian et al 1998) gene coding for b-1,3±1,4-glucanase (lichenase P29716; EC 3.2.1.73) resulted in the truncated licBM2 gene . Due to the removal of 31 N-terminal and 71 C-terminal amino acid residues, the truncated lichenase contains only the catalytic domain sequence and part of the Pro-Thr-box.…”