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目的:获得具有高水平表达Cry1Ac、Cry1Ig和EPSPS基因并且具有抗虫、抗草甘膦活性的转基因水稻株。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。
OBJECTIVE: To obtain transgenic rice plants with high levels of expression of Cry1Ac, Cry1Ig and EPSPS genes and having insect-resistant and glyphosate-resistant activities. Innovations: Cry1Ig is a newly discovered insecticidal gene of Bacillus thuringiensis (Bt); the use of Cry1Ig in combination with Cry1Ac can delay the pest’s resistance to Bt insecticidal protein. Methods: The expression cassettes of Cry1Ac, Cry1Ig and EPSPS (G10) genes were inserted into the same transfer DNA (T-DNA) in turn. Then the T-DNA was transformed into rice by Agrobacterium-mediated transformation. From the transgenic offspring, the three genes that could express at high level and T (subscript T) were screened by Western blot, Southern blot and TAIL-PCR. Transformants in which a single copy of DNA was inserted and no significant insertion mutation occurred. For the selected transformants, the expression levels of Cry1Ac, Cry1Ig and G10 genes were further analyzed by Western blot. The expression level of Cry1Ac was detected by enzyme-linked immunosorbent assay (ELISA). Using the cotton bollworm, As the object, the anti-insect activity of the transgenic rice was determined; and the glyphosate-resistant activity of the transgenic rice was determined by the glyphosate spraying method. Finally, in the field trials, the basic agronomic traits of transgenic and non-transgenic rice were investigated to see whether the T-DNA insertion had a significant effect on the growth of transgenic rice. Conclusion: In this experiment, GAI-14 with high level of expression of Cry1Ac, Cry1Ig and G10 genes and single copy insertion of T-DNA was obtained without any significant change. GAI-14 has obvious resistance to cotton bollworm, rice stem borer, rice leaf roller and glyphosate. Field experiments showed that GAI-14 had no significant difference in basic agronomic traits with non-transgenic rice.