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Journal of Zhejiang University SCIENCE B
ISSN 1673-1581(Print), 1862-1783(Online), Monthly
2015 Vol.16 No.10 P.824-831
Generation of insect-resistant and glyphosate-tolerant rice by introduction of a T-DNA containing two Bt insecticidal genes and an EPSPS gene
Abstract: Insect resistance and glyphosate tolerance have been two of the most important traits in the genetic improvement of various crops. In this study, two Bacillus thuringiensis (Bt) insecticidal genes, Cry1Ac and Cry1Ig, and a modified glyphosate-tolerant 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene (G10) were combined into a single transferred DNA (T-DNA) fragment and introduced into rice by Agrobacterium-mediated transformation. A transgenic line with single-copy T-DNA insertion named GAI-14 was found to be highly resistant to striped stem borer and rice leaf roller, and tolerant to glyphosate. Analysis of T-DNA border sequence suggested that the transgenes were inserted at the chromosome 3 and appeared to have not interrupted any known or putative genes. A field trial observed no significant difference in the basic agronomic traits between GAI-14 and the recipient rice.
Key words: Bt gene stacking, Bt resistance management, EPSPS, Transgenic rice
创新点：Cry1Ig为新发现的苏云金杆菌（Bt）杀虫基因；Cry1Ig与Cry1Ac共同使用有利于延缓害虫产生对Bt杀虫蛋白抗性。
方法：将Cry1Ac、Cry1Ig和EPSPS（G10）基因的表达框依次插入到同一转移DNA（T-DNA）中，再将此T-DNA利用农杆菌介导法转化到水稻中。从转基因后代中，利用蛋白质印迹法（Western blot）、DNA印迹法（Southern blot）和热不对称交错聚合酶链式反应（TAIL-PCR）技术，筛选出能够高水平表达上述三个基因以及T-DNA单拷贝插入并且未发生明显插入突变的转化株。对于筛选到的转化株，利用Western blot进一步分析Cry1Ac、Cry1Ig和G10基因的表达水平；使用酶联免疫吸附测定（ELISA）检测Cry1Ac的表达量；使用棉铃虫、二化螟和稻纵卷叶螟为对象，测定转基因水稻的抗虫活性；使用草甘膦喷施法，测定转基因水稻的抗草甘膦活性。最后，在大田试验中，考察转基因水稻与非转基因水稻的基本农艺性状，观察T-DNA的插入是否对转基因水稻的生长产生明显的影响。
结论：本实验最终获得具有高水平表达Cry1Ac、Cry1Ig和G10基因以及T-DNA单拷贝插入并且未引起明显变化的转化株GAI-14。GAI-14对棉铃虫、二化螟、稻纵卷叶螟以及草甘膦均具有明显的抗性。田间试验表明GAI-14在基本农艺性状上与非转基因水稻无明显差异。
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DOI:
10.1631/jzus.B1500056
CLC number:
Q182
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On-line Access:
2015-10-03
Received:
2015-03-09
Revision Accepted:
2015-06-09
Crosschecked:
2015-09-13