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用克隆的特异性切割马铃薯卷叶病毒(Potato leaf roll virus,PLRV)复制酶基因负链RNA的二价核酶基因,构建植物表达载体pROKⅡ/DR,经土壤农杆菌(Agrobacterium tumefaciens)介导叶盘法转化马铃薯外植体,获得再生植株。PCR和Southern-blot检测,证明目的基因已成功地导入马铃薯再生植株,其转化率约为14.5%,并能够在无性繁殖后代植株中稳定存在。RT-PCR检测表明,再生马铃薯植株中的二价核酶基因可以转录表达。经病毒接种的转基因马铃薯株系L5、L7、L8和J-1的无性繁殖后代在继发感染中仍表现出较高的抗病性,为最终获得抗PLRV马铃薯新品系打下了基础。
The bivalent ribozyme gene of negative strand RNA of Potato leaf roll virus (PLRV) replicase gene was cloned, and the plant expression vector pROKⅡ / DR was constructed. Agrobacterium tumefaciens mediated leaf Potato explants were transformed into regenerated plants. PCR and Southern-blot. The results showed that the target gene was successfully introduced into potato regenerated plants, and its transformation rate was about 14.5%. It could stably exist in the plants of clonal progenies. The RT-PCR results showed that the bivalent ribozyme gene in regenerated potato plants could be transcribed. The cloned offspring of the virus-inoculated transgenic potato lines L5, L7, L8 and J-1 still showed higher disease resistance in the secondary infection, which laid the foundation for finally obtaining new potato lines resistant to PLRV.