Cotton benzoquinone reductase: Up-regulation during early fiber development and heterologous expression and characterization in Pichia pastoris

Benzoquinone reductase (BR; EC 1.6.5.7) is an enzyme which catalyzes the bivalent redox reactions of quinones without the production of free radical intermediates. Using 2D-PAGE comparisons, two proteins were found to be up-regulated in wild-type cotton ovules during the fiber initiation stage but not in the fiberless line SL 1-7-1. These proteins were excised from the gel, partially sequenced and identified to be BR isoforms. PCR was used to amplify both full length coding regions of 609 bp and once cloned, the restriction enzyme HindIII was used to distinguish the clones encoding the BR1 (one site) and BR2 (two sites) isoforms. Both deduced protein sequences had 203 residues which differed at 14 residues. The molecular mass and pIs were similar between the measured protein (2D-PAGE) and the theoretical protein (deduced). Heterologous proteins BR1 and BR2 were produced for further study by ligating the BR1 and BR2 clones in frame into the α-factor secretion sequence in pPICZαA vector and expressed with Pichia pastoris. Both BR1 and BR2 were approximately 26.5 kDa and did enzymatically reduce 2,6-dimethoxybenzoquinone similar to the fungal BR.