CMV抑制PVY^N CP基因沉默及其介导的抗病性

Cucumber Mosaic Virus Suppresses PVY^NCP Gene Silencing and Breaks Transgenic Resistance to PVY^N in Tobacco

以前曾报道用RNA介导的抗病毒策略,获得了高度抗病的表达马铃薯Y病毒坏死株系外壳蛋白基因(PVYNCP)的转基因烟草,并对T1、T2代转基因植株进行了遗传和抗病性分析。此次以T3代转基因植株为试验材料,在筛选高度抗病植株并证明其抗病性是基于转基因沉默的基础上,采用Northern杂交的方法,证明CMV侵染抑制了转基因植株中PVYNCP基因的沉默,而且CMV对PVYNCP基因沉默的抑制部位是发生在接种后的新生叶上,接种叶及其下部叶片中PVYNCP基因沉默则未受到影响。采用ELISA方法对CMV+PVYN复合接种的转基因植株进行PVYN检测,结果表明,接种叶及下部叶没有检测到PVYN,植株叶片对PVYN表现为抗病。而在CMV接种后植株新生叶中则检测出了高滴度的PVYN,植株叶片对PVYN表现为感病。该文报道了在表达PVYNCP基因的RNA介导抗性转基因植株中,异源病毒侵染抑制了转基因的沉默,并导致转基因植株的抗病性丧失。

The RNA-mediated virus resistant transgenic tobacco lines, in which the resistant mechanism was similar to post-transcriptional gene silencing (PTGS),were attained in our previous study.The genetics and resistance analyses of T1 and T2 progeny transgenic tabacco lines were also studied. Here, using T3 progeny transgenic tobacco in which the expression of untranslatable PVYN CP gene is silenced, we demonstrated that cucumber mosaic virus (CMV) was able to suppress PVYN CP gene silencing and the associated potato virus Y(PVY) resistance in tobacco. Northern blot analysis indicated that CMV infection led to reversal of PVYN CF gene silencing in the renascent upper leaves of the transgenic lines, as revealed by the elevation of transgene mRNA accumulation , but transgene mRNA levels in inoculated and down non-inoculated leaves were unaffected, with very low steady-state levels. ELISA assay at 35 days post inoculation showed that the PVYN was not detected in the inoculated leaf and the down non-inoculated leaves of the transgenic lines after inoculated with PVYN + CMV, while the renascent upper leaves of the transgenic lines became susceptible to infection by PVYN(OD405 values showed no difference from that of non-transgenic plants inoculated with PVYN + CMV). In this study, we demonstrated that the RNA-mediated virus resistance can be overcome via infection by a heterologous virus that suppresses gene silencing,making the plant susceptible to the virus that it was engineered to resist.