Expression of Rhizobium pydA-pydB fusion gene in Nicotiana tabacum confers resistance to the toxic aromatic compound 3-hydroxy-4-pyridone

摘要

The toxic aromatic compound 3-hydroxy-4-pyridone (HP) is an intermediate in both synthesis and degradation of mimosine, which is produced by the tree legume Leucaena leucocephala. The L. leucocephala root-nodule symbiont Rhizobium TAL1145 contains a dioxygenase (pydA) and a hydrolase (pydB) gene that produce enzymes necessary for the degradation of HP. In order to coordinately express both genes in plant tissues under a single promoter, three different pydA-pydB fusion constructs (G0, G3, and G7) with varying glycine linkers between the two genes were developed. Prior to transferring the fusion constructs into L. leucocephala, which is highly recalcitrant to genetic transformation, we tested the expression and activity of the hybrid proteins in Nicotiana tabacum, a model plant system that can be easily transformed and analyzed. Seven independent transgenic tobacco lines were generated by Agrobacterium-mediated transformation, and stable integration and expression of pydA-pydB in these transgenic lines were confirmed by polymerase chain reaction (PCR), reverse transcriptase PCR (RT-PCR) and Western analysis. Only one of the fusion constructs, G3, containing a 9-nucleotide linker between pydA and pydB, provided significant levels of resistance to 3 mM HP, indicating that the hybrid protein produced by this fusion construct could degrade HP. Additional key words metabolic pathway engineering - Leucaena leucocephala - Rhizobium TAL1145 - mimosine - hybrid protein