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为探究靶序列位置对RNA介导的病毒抗性产生的影响,利用聚合酶链式反应(polymerase chain reaction,PCR)技术扩增马铃薯Y病毒(Potato virus Y,PVY)复制酶基因(nuclear inclusion b,NIb)不同位置的cDNA区段,反向插入双元载体pROKII中,构建了发夹RNA(hairpin RNA,hpR-NA)结构的植物表达载体。将构建的植物表达载体采用冻融法转入农杆菌LBA4404,叶盘法转化烟草NC89,获得转基因植株。攻毒试验表明:PVYNIb基因不同位置cDNA区段介导的对PVY的抗性存在显著差异;3′端1/2处和中间位置的序列可介导高水平的病毒抗性,抗性植株的比例在50%以上,而5′端、5′端1/2处和3′端的序列介导的抗性效率较低,抗性植株的比例仅为10%~30%。Northern杂交显示:抗病植株中RNA的积累量明显低于同类型的感病植株,抗性与RNA积累量呈负相关;抗病转基因植株中有siRNA存在,表明病毒抗性是由RNA介导的。
In order to explore the effect of target position on the RNA-mediated virus resistance, a polymerase chain reaction (PCR) technique was used to amplify the nucleobase B gene of the potato virus Y (PVY) , NIb) were inserted into the binary vector pROKII in the reverse direction to construct a plant expression vector with the hairpin RNA (hpR-NA) structure. The constructed plant expression vector was transformed into Agrobacterium LBA4404 by freeze-thaw method, and transformed into tobacco NC89 by leaf disc method to obtain transgenic plants. The challenge experiments showed that there were significant differences in resistance to PVY mediated by cDNA segments of different positions of PVYNIb gene. The sequences at 1/2 and 3 ’of the 3’ end could mediate high level of virus resistance, The proportion is more than 50%, while the sequence-mediated resistance at the 5 ’end, the 5’ end, the 1/2 end and the 3 ’end is less efficient, and the proportion of resistant plants is only 10% -30%. Northern blotting showed that the accumulation of RNA in resistant plants was significantly lower than that in susceptible plants, and there was a negative correlation between the resistance and the accumulation of RNA. The presence of siRNA in the transgenic plants showed that the resistance of the virus was mediated by RNA of.