Molecular Cloning, Expression, and Polyclonal Antibody Production of a Novel Flavin-Containing Monooxygenase from Thellungiella halophila.

A novel flavin-containing monooxygenase (FMO)-like gene, designated as TsFMO, was cloned from Thellungiella halophila by reverse-transcriptase polymerase chain reaction (RT-PCR) coupled with rapid amplification of cDNA ends approach. The cDNA sequence of TsFMO had a complete open reading frame encoding a putative protein of 461 amino acids, which contained the conserved domains of FMOs. Sequence analysis showed that it had highest similarity with Arabidopsis FMOGS-OX1. RT-PCR analysis revealed that TsFMO was constitutively expressed in all examined tissues including the roots and rosette leaves of the seedlings and mature plants, bolts, and open flowers. Real-time PCR suggested that the gene was quickly and significantly upregulated in the leaves exposed to NaCl stress, which showed that TsFMO might be regulated with the redox state of plant cell. TsFMO was found to complement and rescue the retardant growth to dithiothreitol of yeast fmo mutant strain. In addition, TsFMO was expressed in Escherichia coli and the recombinant protein was purified to produce polyclonal antibody. Western blot with T. halophila extracts showed that the polyclonal antibody was effective and specific. [ABSTRACT FROM AUTHOR]