转二价核酶基因马铃薯及抗病性研究

 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。

Transgenic potato transformed with divalent ribozyme gene for resistance to Potato leaf roll virus,PLRV

A binary vector of plasmids pROKⅡ/DR containing the divalent ribozyme gene was constructed and transferred into Agrobacterium tumefaciens LBA4404 by tri-parental conjugation to obtain virus-resistant transgenic potato plants.The leaf disks of potato cultivar Desiree were used for gene transformation mediated by Agrobacterium.The gene integration in the regenerated progenies was identified by PCR and Southern-blot.The results revealed that 14.5% of the regenerated plants were transformed by the ribozyme gene.The gene was existed stably in asexual propagated potato plants.Expression of the ribozyme (DR) gene at RNA level was also confirmed by RT-PCR analysis.Detection of the transgenic plants resistant to Potato leafroll virus (PLRV) infection by aphid transmission was carried out in greenhouse and netroom trials.The phenotypes of diseased transgenic potato lines were different.Among them infected by PLRV,the second progenies of asexual propagation of L5,L7,L8 and J-1 lines showed relative high level of resistance.In netroom prevented from aphides,plot test of the transgenic potato lines uninfected by the virus showed the different yields,in which the line L5 and J-1 with high resistance to PLRV were yielded similarly to that of the non-transgene control of potato cultivar Desiree.With these results,it becomes possible to develop potato varieties resistant to PLRV infection by transformation of potato plants with ribozyme genes.