A selection system for transgenic Arabidopsis thaliana using potassium thiocyanate as the selective agent and AtHOL1 as the selective marker
A selection system for transgenic Arabidopsis thaliana using potassium thiocyanate as the selective agent and AtHOL1 as the selective marker
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DOI：
10.5511/plantbiotechnology.26.341
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发表时间：
2009-01-01
影响因子：
1.6
通讯作者：
Nakamura, Tatsuo
中科院分区：
文献类型：
--
作者：
Midorikawa, Keiko;Nagatoshi, Yukari;Nakamura, Tatsuo
对使用细菌抗生素抗性基因作为选择标记的担忧被认为是公众对转基因作物产生抗性的因素之一。我们先前已经表明，拟南芥AtHOL 1蛋白具有高S-腺苷-L-甲硫氨酸依赖性甲基转移酶活性的硫氰酸根离子（NCS-），这是有毒的植物超过一定浓度。与我们关于AtHOL 1作为选择标记的可用性的预期相反，我们在正常生长条件下在含有各种浓度的硫氰酸钾的1/2 MS琼脂培养基上对AtHOL 1过表达拟南芥的初始试验筛选是不成功的。为了探索AtHOL 1在拟南芥转化中应用的可能性，进一步考察了AtHOL 1过表达拟南芥的筛选条件。我们发现，当种子在含有3.0- 5.0mM硫氰酸钾的培养基上萌发并在7天黑暗随后12小时光照/12小时黑暗循环的条件下生长3-7天时，可以筛选转基因幼苗。还进行了基因组中的转基因插入和AtHOL 1 mRNA在筛选的幼苗中的积累的分析。在本文报道的条件下筛选的幼苗既未观察到逃逸幼苗也未观察到生长改变。
Concern about the use of bacterial antibiotic resistance genes as selectable markers is considered to be one of the factors in public resistance towards genetically modified crops. We had previously shown that the Arabidopsis AtHOL1 protein had high S-adenosyl-L-methionine-dependent methyltransferase activity toward the thiocyanate ion (NCS-), which is toxic to plants beyond certain concentrations. Contrary to our expectations regarding the usability of AtHOL1 as a selectable marker, our initial trial screenings for AtHOL1-overexpressing Arabidopsis on 1/2 MS agar medium containing various concentrations of potassium thiocyanate under normal growth conditions had been unsuccessful. In order to explore the possibility of using AtHOL1 in Arabidopsis transformation, the screening conditions for AtHOL1-overexpressing Arabidopsis were further examined. We found that the transgenic seedlings Could be screened when the seeds were germinated and grown under conditions of 7-day darkness followed by a 12-h light/12-h dark cycle for 3-7 days oil a medium containing 3.0-5.0 mM potassium thiocyanate. Analyses of transgene insertions into the genomes and AtHOL1 mRNA accumulation in the screened seedlings were also performed. Neither escaped seedlings nor altered growth was observed with the seedlings screened under the conditions reported here.