Expression of a novel yeast gene that detoxifies the proline analog azetidine-2-carboxylate confers resistance during tobacco seed germination, callus and shoot formation
Expression of a novel yeast gene that detoxifies the proline analog azetidine-2-carboxylate confers resistance during tobacco seed germination, callus and shoot formation
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DOI：
10.1007/s00299-003-0741-3
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发表时间：
2004-03-01
影响因子：
6.2
通讯作者：
Widholm, JM
中科院分区：
文献类型：
--
作者：
Zhang, XH;Takagi, H;Widholm, JM
在酿酒酵母(Sigma1278b)中发现了一种新的乙酰转移酶(MPr1)，它能特异性地解毒酵母细胞中的一种脯氨酸类似物--L-氮杂乙氨酸-2-羧酸(A2C)[M.Shichiri等人。(2001年)生物化学杂志276：41998-42002]。我们研究了酵母Mpr1基因在植物系统中是否具有类似的功能，以及它的表达是否可以赋予对Pro类似物的抗性。将Mpr1基因由两个不同的构成启动子驱动的编码序列，分别带有或不带有与传统的NOS终止子相邻的Mpr1基因的5‘和3’非编码序列，将其转化到烟草中。Xanthi)植物通过根癌农杆菌侵染。酵母5‘和3’非编码序列的存在似乎增加了Mpr1基因在转基因植物中表达的可能性。卡那霉素筛选的Mpr1活性较高的转基因植株生长正常，其后代表达了能利用A2C、氮杂环丁酸和4-羟基-L-脯氨酸为底物的乙酰基转移酶活性。在叶片组织培养和种子萌发过程中表现出对A2C的抗性，但对其他两种类似物没有表现出抗性。A2C对野生型植物的毒性可被添加的Pro逆转，表明A2C具有类似于Pro的作用。我们的研究证实，Mpr1在烟草中的功能类似于在酵母中的功能，以解毒有毒的Pro类似物A2C，因此它可能被用作一种新的植物转化的选择标记。然而，我们试图利用Mpr1作为根癌农杆菌介导的烟草转化的有效选择标记基因，但没有成功。
A novel acetyltransferase (Mpr1) found in Saccharomyces cerevisiae (strain Sigma1278b) has been shown to specifically detoxify a proline analog, L-azetidine-2-carboxylic acid (A2C) in yeast cells [M. Shichiri et al. (2001) J Biol Chem 276: 41998-42002]. We investigated whether the yeast MPR1 gene would function similarly in a plant system and if its expression could confer resistance to proline analogs. The MPR1 gene coding sequence driven by two different constitutive promoters, with or without the 5'- and 3'-noncoding sequence from the MPR1 gene adjacent to the conventional NOS terminator, was transformed into tobacco (Nicotiana tabacum L. cv. Xanthi) plants via Agrobacterium tumefaciens infection. The presence of the yeast 5'- and 3'-noncoding sequences appeared to increase the likelihood of MPR1 gene expression in the transgenic plants. The kanamycin-selected transgenic plants with a high level of Mpr1 activity grew normally, and their progeny expressed acetyltransferase activity that could utilize A2C, azetidine-3-carboxylic acid and 4-hydroxy-L-proline as substrates. Resistance to A2C, but not to the other two analogs, was exhibited during leaf tissue culture and seed germination. The A2C toxicity to the wild-type plants was reversed by the addition of proline, suggesting that A2C acts as a proline analog. Our studies confirm that MPR1 can function in a similar fashion in tobacco as in yeast to detoxify the toxic proline analog A2C, so it could potentially be used as a new selectable marker for plant transformation. However, our attempts to utilize MPR1 as an efficient selectable marker gene for the A. tumefaciens-mediated transformation of tobacco were unsuccessful.