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首页> 外文期刊>BMC Biotechnology >Somatic cell selection for chlorsulfuron-resistant mutants in potato: identification of point mutations in the acetohydroxyacid synthase gene
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Somatic cell selection for chlorsulfuron-resistant mutants in potato: identification of point mutations in the acetohydroxyacid synthase gene

机译：马铃薯中耐氯磺隆突变体的体细胞选择：乙酰羟酸合酶基因中点突变的鉴定

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BackgroundSomatic cell selection in plants allows the recovery of spontaneous mutants from cell cultures. When coupled with the regeneration of plants it allows an effective approach for the recovery of novel traits in plants. This study undertook somatic cell selection in the potato ( Solanum tuberosum L.) cultivar ‘Iwa’ using the sulfonylurea herbicide, chlorsulfuron, as a positive selection agent. ResultsFollowing 5?days’ exposure of potato cell suspension cultures to 20?μg/l chlorsulfuron, rescue selection recovered rare potato cell colonies at a frequency of approximately one event in 2.7?×?105 of plated cells. Plants that were regenerated from these cell colonies retained resistance to chlorsulfuron and two variants were confirmed to have different independent point mutations in the acetohydroxyacid synthase ( AHAS ) gene. One point mutation involved a transition of cytosine for thymine, which substituted the equivalent of Pro-197 to Ser-197 in the AHAS enzyme. The second point mutation involved a transversion of thymine to adenine, changing the equivalent of Trp-574 to Arg-574. The two independent point mutations recovered were assembled into a chimeric gene and binary vector for Agrobacterium -mediated transformation of wild-type ‘Iwa’ potato. This confirmed that the mutations in the AHAS gene conferred chlorsulfuron resistance in the resulting transgenic plants. ConclusionsSomatic cell selection in potato using the sulfonylurea herbicide, chlorsulfuron, recovered resistant variants attributed to mutational events in the AHAS gene. The mutant AHAS genes recovered are therefore good candidates as selectable marker genes for intragenic transformation of potato.
机译：背景技术植物中的体细胞选择允许从细胞培养物中回收自发突变体。当与植物的再生结合时，它为恢复植物中的新性状提供了一种有效的方法。这项研究使用磺酰脲类除草剂氯磺隆作为阳性选择剂，对马铃薯（Iwa）品种'Iwa'进行了体细胞选择。结果在将马铃薯细胞悬浮培养物暴露于20？μg/ l氯磺隆中5天后，抢救选择以2.7？×？10 5 铺板细胞中的大约一个事件的频率恢复了稀有马铃薯细胞集落。 。从这些细胞集落再生的植物保留了对氯磺隆的抗性，并且证实了两个变体在乙酰羟酸合酶（AHAS）基因中具有不同的独立点突变。一个点突变涉及胞嘧啶向胸腺嘧啶的转变，其在AHAS酶中将Pro-197的等价物替换为Ser-197。第二点突变涉及将胸腺嘧啶转化为腺嘌呤，从而将Trp-574的等价物改变为Arg-574。回收的两个独立的点突变被组装成嵌合基因和二元载体，用于农杆菌介导的野生型“ Iwa”马铃薯的转化。这证实了AHAS基因中的突变赋予了所得转基因植物抗氯磺隆的能力。结论使用磺酰脲类除草剂氯磺隆在马铃薯中进行体细胞选择，可回收归因于AHAS基因突变事件的抗性变异体。因此，回收的突变型AHAS基因是马铃薯内源转化的可选择标记基因的良好候选者。

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