马铃薯Y病毒NIb复制酶基因在转录水平介导的相对广谱抗病性

RNA-mediated Relatively Broad Resistance of Potato Y Virus NIb Replicase Gene

在大肠杆菌中表达了马铃薯Y病毒中国分离物 (PVY -C)复制酶NIb基因 ,并制备了其抗血清。利用PCR定点突变方法使 NIb 基因移码 - 1位 ,构建了移码 - 1位 NIb 基因 (UN)的植物表达载体。通过土壤农杆菌(AgrobacteriumtumefaciensLBA44 0 4)介导转化烟草NC89,获得 5 1株再生植株。对再生植株的分子检测结果表明 ,转基因烟草中检测到UN基因相应的RNA转录产物 ,推测该基因已经整合到烟草染色体中。攻毒试验发现转UN 基因烟草 (T0 代 )对两个PVY株系和烟草蚀纹病毒的抗病性与转全长、缺失 5′端 381bpNIb 基因烟草 (T2 代 )的相同。Western印迹试验结果表明 ,在本试验检测水平上 ,在转基因T2 代烟草中未检测到NIb基因的表达产物。实验结果支持PVY -C复制酶NIb基因在转录水平上介导相对广谱抗病性的假说。

The nuclear inclusion b (NIb) replicase gene of a Chinese isolate of potato Y potyvirus was expressed in E. coli, and the rabbit antibody against replicase was prepared. In order to elucidate the resistant mechanism by PVY NIb gene, -1 frameshift NIb gene was obtained by use of site-mutating PCR. This -1 frameshift NIb gene (UN) was constructed into binary vector pBin438, and then transformed into tobacco NC 89 mediated by Agrobacterium tumefaciens LBA4404. Northern blot results showed that the RNA transcript of UN gene was determined in the regenerated tobacco lines. Of 51 independently transformed lines expressing UN gene in T 0 regeneration, twenty-five were resistant to either one of the two different strains of PVY or tobacco etch potyvirus, of which resistance appeared to be the same as that of transgenic lines expressing full-length or deleted 381bp from 5′ terminus of NIb gene in T 2 generation. Despite the high resistance against potyvirus, the expressed product of NIb gene in transgenic plants was not detected by Western blotting in our experimental level. Therefore, these results support the proposal that PVY-C NIb replicase gene-mediated resistance is both at transcription and relatively broad level.