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.
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 muM and 300 muM 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 T(1) progeny could be distinguished from the recessive progeny when grown on 400, 450, or 500 muM 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 Delta(1)-pyrroline-5-carboxylate (P5C). However, H(2)O(2) 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.
Tsai
Fei-Yi
FY
Department of Crop Sciences, E. R. Madigan Laboratory, University of Illinois at Urbana-Champaign, 1201 W. Gregory Drive, Urbana, IL 61801, USA.
Zhang
Xing-Hai
XH
Ulanov
Alexander
A
Widholm
Jack M
JM
D000123:Acetyltransferases / Q000235:genetics*; D001383:Azetidinecarboxylic Acid / Q000737:chemistry / Q000494:pharmacology*; D020090:Chromosome Segregation / Q000235:genetics; D003433:Crosses, Genetic; D008401:Gas Chromatography-Mass Spectrometry; D018506:Gene Expression Regulation, Plant / Q000187:drug effects; D005800:Genes, Fungal / Q000235:genetics; D005819:Genetic Markers; D005822:Genetic Vectors; D018745:Genome, Plant / Q000235:genetics; D006861:Hydrogen Peroxide / Q000494:pharmacology; D007612:Kanamycin / Q000494:pharmacology; D055442:Metabolome / Q000235:genetics; D018384:Oxidative Stress / Q000187:drug effects; D030821:Plants, Genetically Modified; D025341:Principal Component Analysis; D011392:Proline / Q000031:analogs & derivatives* / Q000737:chemistry; D017382:Reactive Oxygen Species / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000201:enzymology / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics*; D012641:Selection, Genetic; D014026:Nicotiana / Q000235:genetics*; D014170:Transformation, Genetic / Q000187:drug effects*; D019076:Transgenes / Q000235:genetics
D005819:Genetic Markers; D017382:Reactive Oxygen Species; D029701:Saccharomyces cerevisiae Proteins; D007612:Kanamycin; D001383:Azetidinecarboxylic Acid; D011392:Proline; D006861:Hydrogen Peroxide; D000123:Acetyltransferases; C492447:Mpr1 protein, S cerevisiae