Designing, generating anti-fungal gene cassettes and engineering of Citrus cells for transient expression
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Date
2010
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Punjab Agricultural University
Abstract
In the present study two anti-fungal gene constructs were generated comprising CaMV35SChitinase-
NOS and CaMV35S-Glucanase-NOS using 905 bp and 1764bp Open Reading
Frames (ORFs) respectively from the indigenously cloned chitinase (Accession no.
GU187942) and glucanase (Accession no. GU300766) genes. ORFs were PCR amplified
using specifically designed primers containing overhanging restriction sites and ligated into
pBI121 cleaved by corresponding restriction enzymes. The gene constructs were further subcloned
into high copy number plasmids pUC18 and pGEM 9Zf(-) respectively for particle
bombardment of citrus cells for transient expression analysis. The gene constructs were
bombarded individually as well as in combination. PCR analysis demonstrated insertion of
gene construct in 78% of calli bombarded with chitinase gene construct, in 28% of the calli
bombarded with glucanase gene construct and in 9.3% of the calli co-bombarded with both
the gene constructs.
Keywords: Sub-cloning, chitinase, glucanase
Description
Keywords
biotechnology, glucanase