Department of Crop Sciences, E. R. Madigan Laboratory, University of Illinois at Urbana-Champaign, 1201 W. Gregory Drive, Urbana, IL 61801, USA.
J Exp Bot. 2010 Jun;61(10):2561-73. doi: 10.1093/jxb/erq086. Epub 2010 Apr 28.
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.
酵母 N-乙酰转移酶 MPR1 基因先前已被证明能赋予酵母和转基因烟草对有毒脯氨酸类似物氮杂环丁烷-2-羧酸（A2C）的抗性。在这里，进行了实验以确定 MPR1 和 A2C 是否可以作为植物转化的可选择标记系统。MPR1 基因被插入到一个二元载体中，受花椰菜花叶病毒 35S 启动子和胭脂碱合成酶终止子的控制，并通过根癌农杆菌介导的叶盘法转化为烟草。A2C 被应用于选择培养基中以选择假定的转化体。PCR 分析显示，在 250 μM 和 300 μM A2C 选择的植株中，有 28.4%和 66.7%为 MPR1 基因阳性。Southern 和 Northern 印迹分析和酶活性测定证实了 MPR1 转基因在转基因植物中的稳定基因整合、转录和翻译。当在 400、450 或 500 μM A2C 上生长时，携带该基因的 T(1)代可以与隐性后代区分开来。通过气相色谱-质谱分析对 22 个转基因植物的代谢进行检查未发现任何显著变化。总之，结果表明 MPR1/A2C 是一种安全有效的选择系统，不涉及微生物抗生素或除草剂抗性基因。最近的研究表明，MPR1 可以通过减少脯氨酸分解产物 Delta(1)-吡咯啉-5-羧酸（P5C）的积累来保护酵母免受氧化应激。然而，在测试的五个转基因系中，H(2)O(2)处理导致了相反的反应。需要进一步的实验来评估 MPR1 转基因植物在氧化应激下的反应。
