Generation of double-virus-resistant marker-free transgenic potato plants
Generation of double-virus-resistant marker-free transgenic potato plants
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DOI：
10.1016/j.pnsc.2008.08.005
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发表时间：
2009-05-10
影响因子：
4.7
通讯作者：
Zhang, Weifeng
中科院分区：
文献类型：
--
作者：
Bai, Yunfeng;Guo, Zhihua;Zhang, Weifeng
病毒在自然界中广泛分布，在马铃薯生产中引起严重病害和产量损失。dsRNA产生基因的转移可以通过特异性靶向同源病毒RNA来赋予高水平的病毒抗性。在这项研究中，我们构建了一个嵌合基因的无标记表达载体，该嵌合基因来自马铃薯X病毒（PVX）的外壳蛋白序列和马铃薯Y病毒（PVY）的核包含蛋白序列的分子内dsRNA的形式。然后将该嵌合基因导入马铃薯cv。利用农杆菌介导法对紫花白进行遗传转化。通过RT-PCR和DAS-ELISA检测，获得了抗PVX和PVY的无标记基因转基因植株。北方印迹分析表明，转基因衍生的mRNA被切割成短的干扰RNA（siRNA），并且病毒抗性是由RNA沉默介导的。这项研究的一个重要方面是，转基因病毒序列不被翻译，实际的RNA转录物被切割，这可能限制了环境风险，如转基因与外来病毒的转衣壳和重组。此外，由于转基因植物中标记基因的缺失，可以避免标记基因带来的生物安全风险。(C)2009年国家自然科学基金、中国科学院。由Elsevier Limited和Science in China Press出版。All rights reserved.
Viruses are very widespread in nature, and they cause severe diseases and yield losses in potato production. The transfer of the dsRNA-producing gene could confer a high level virus resistance by specific targeting of cognate viral RNA. In this study, we constructed a marker-free expression vector of a chimeric gene derived from the coat protein sequence of Potato virus X (PVX) and the nuclear inclusion protein sequence of Potato virus Y (PVY) in the form of an intramolecular dsRNA. Then this chimeric gene was introduced into potato cv. Zihuabai, a popular variety in China, via Agrobacterium tumefaciens-mediated transformation. Marker gene-free transgenic plants resistant to both PVX and PVY were obtained and confirmed by RT-PCR and DAS-ELISA detection. Northern blot analysis showed that transgene-derived mRNA was cleaved into short interfering RNAs (siRNAs), and that the virus resistance was mediated by RNA silencing. One important aspect of the study is that the transgenic viral sequence is not translated and the actual RNA transcript is cleaved, which possibly limit the environmental risks, such as transcapsidation and recombination of the transgene with an incoming virus. In addition, the biosafety risk resulting from marker genes can be avoided because of the absence of marker genes in transgenic plants. (C) 2009 National Natural Science Foundation of China and Chinese Academy of Sciences. Published by Elsevier Limited and Science in China Press. All rights reserved.