Molecular cloning and expression analysis of a zeta-class glutathione S-transferase gene in sugarcane

Abstract

Glutathione S-transferases (GSTs) play an important role in stress tolerance in plants. This is the first report of cloning and characterization of a zeta-class GST gene in sugarcane (GenBank Accession number: GQ246461). Sequence analysis showed that the cDNA sequence of Sc-GST gene was 829 bp, contained a 621 bp open reading frame (ORF), the 5’ untranslated region (UTR) of 65 bp and 3’UTR of 143 bp, plus the typical AATAA region and poly (A) tail. It encoded the 206 amino acid residues with a molecular mass of 23.1 KD and isoelectric point of 6.10. Protein domain prediction and multiple sequence alignment demonstrated that the conserved domain in Sc-GST at N-terminus was SSCXXRXRIA, while that at C-terminus was quebec platelet disorder (QPD), both of which were specific for zeta-type GST in eukaryotes. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis and enzyme activity assay indicated that the prokaryotic expression product was a fusion protein with a molecular weight of about 30 KD, which also possessed GST enzyme activity. It was revealed in real-time quantitative polymerase chain reaction (qPCR) analysis that the Sc-GST gene had induced expression under H₂O₂ and Ustilago scitaminea stresses, while it was inhibited and then induced by salicylic acid (SA) stress, suggesting that it is a type of stress-tolerant gene playing a certain role in sugarcane resistance response.

Key words: Saccharum officinarum, glutathione S-transferase, homology, prokaryotic expression, real-time quantitative PCR.