The application of the yeast N-acetyltransferase MPR1 gene and the proline analogue L-azetidine-2-carboxylic acid as a selectable marker system for plant transformation

摘要

The yeast N-acetyltransferase MPR1 gene has previously been shown to confer resistance to the toxic proline analogue azetidine-2-carboxylic acid (A2C) in yeast and transgenic tobacco. Here experiments were carried out to determine if MPR1 and A2C can work as a selectable marker system for plant transformation. The MPR1 gene was inserted into a binary vector under the control of the cauliflower mosaic virus 35S promoter and nopaline synthase terminator, and transformed into tobacco via the Agrobacterium tumefaciens-mediated leaf disc method. A2C was applied in the selection medium to select for putative transformants. PCR analysis showed that 28.4% and 66.7% of the plantlets selected by 250 μM and 300 μM A2C were positive for the MPR1 gene, respectively. Southern and northern blot analysis and enzyme activity assay confirmed the stable gene incorporation, transcription, and translation of the MPR1 transgene in the transgenic plants. The transgene-carrying T1 progeny could be distinguished from the recessive progeny when grown on 400, 450, or 500 μM A2C. Examination of the metabolism of 22 transgenic plants by gas chromatography–mass spectrometry profiling did not reveal any significant changes. In conclusion, the results demonstrate that MPR1/A2C is a safe and efficient selection system that does not involve microbial antibiotic or herbicide resistance genes. Recent studies showed that MPR1 can protect yeast against oxidative stresses by decreasing the accumulation of the proline catabolite Δ1-pyrroline-5-carboxylate (P5C). However, H2O2 treatment resulted in contradictory responses among the five transgenic lines tested. Further experiments are required to assess the response of MPR1 transgenic plants under oxidative stress.