转cry1Ac1基因抗虫水稻的培育

Development of Insect-resistant Transgenic Rice with cry1Ac1 Gene

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 the present study, the transgenic rice ＂Xiushui 134＂ with crylAcl was developed by Agrobacterium mediated transformation method. Confirmed by PCR, all the 12 independent transformants were crylA cl-positive plants. Using semi-quantitative reverse transcription PCR （RT-PCR）, the crylA c1 gene expression could be detect- ed in all independent transformants. Using real time quantitative PCR （qPCR） analysis, we found that 3 out of 12 transformants harbored a single copy crylA c I gene. In order to study the integration site, the flanking sequences of these three transformants were amplified by thermal asymmetric interlaced （TAIL）-PCR. The result showed that the T-DNA of one of them inserted into genes interspace on the rice chromosome 2, about 2.4 kb upstream from the Os02G0539500 （Ch.2： 20858661-20860159） gene, and did not disturb other rice functional genes. This trans- formant was used as primary material to self-cross and segregate, and finally we got the homozygous crylAcl lines. The resistance to leaf folder and agronomic traits of the lines were tested in field at natural conditions. The results showed that the homozygous crylA cl rice plants had strong resistance to leaf folder while main agronomic traits were almost unaffected. These results indicate that we have successfully developed homozygous crylAcl lines with highly resistant to leaf folder but main agronomic traits almost the same as controls.