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本研究以粳稻“秀水134”为受体材料,通过根癌农杆菌介导的遗传转化方法导入抗虫基因cry1Ac1,获得12个独立转基因株系。经过PCR确认转基因植株均为cry1Ac1阳性植株。半定量RT-PCR实验表明,目的基因在所有转化体中都能够表达。用荧光定量PCR分析拷贝数,结果表明有3个转化体携带单拷贝目的基因。通过TAIL-PCR方法对单拷贝转化体基因插入位点分析,发现其中一个转化体的目的基因插入到基因间区,位于第2染色体Os02G0539500(Chr.2:20858661~20860159)基因上游约2.4 kb处,未对受体材料的已知功能基因造成破坏。通过此单拷贝转化体自交后,从后代群体中获得纯系并进行田间农艺性状及抗虫性调查,结果显示此纯系抗虫效果佳,并且没有造成主要农艺性状的明显改变。研究结果表明,我们已经获得cry1Ac1基因在水稻植株中可以稳定表达,并且主要农艺性状没有明显改变的高抗虫水稻转基因材料。
In this study, japonica rice “Xiushui 134” was used as the recipient material, and the insect-resistant gene cry1Ac1 was introduced into Agrobacterium tumefaciens-mediated genetic transformation to obtain 12 independent transgenic lines. PCR confirmed the transgenic plants are cry1Ac1-positive plants. Semi-quantitative RT-PCR experiments showed that the target gene can be expressed in all transformants. The copy number was analyzed by fluorescence quantitative PCR and the results showed that three transformants carried a single copy of the gene of interest. By TAIL-PCR analysis of single copy transformant gene insertion sites, we found that one of the transformants inserted into the intergenic region, located at about 2.4 kb upstream of the gene on chromosome 2 Os02G0539500 (Chr.2: 20858661 ~ 20860159) , Did not cause damage to the known functional genes of the receptor material. After selfing of this single copy transformant, pure lines were obtained from the progeny population and field agronomic traits and insect resistance surveys were conducted. The results showed that the pure lines had good insect resistance and did not cause significant changes in the main agronomic traits. The results showed that we have obtained the highly insect-resistant rice transgenic material with stable expression of cry1Ac1 gene in rice plants and no significant change of major agronomic traits.