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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 NIb gene of potato Y virus Chinese isolate (PVY-C) replicase was expressed in E. coli and its antiserum was prepared. The NIb gene was frame-shifted by PCR using site-directed mutagenesis - 1, and a frame-coding - NIb gene (UN) plant expression vector was constructed. Tobacco NC89 was transformed by Agrobacterium tumefaciens LBA44 0 4 to obtain 51 regenerated plants. The molecular test results on the regenerated plants showed that the corresponding RNA transcripts of UN gene were detected in the transgenic tobacco, suggesting that the gene has been integrated into the tobacco chromosome. The challenge test revealed that the resistance of transgenic UN tobacco plants (T0 generation) to two PVY lines and tobacco etch virus was the same as that of the full length and deletion of the 381 bp Nib gene tobacco at the 5 ’end (T2 generation). The results of Western blotting showed that the expression level of NIb gene was not detected in transgenic T2 generation tobacco at the level tested in this experiment. The experimental results support the hypothesis that the NIb gene of PVY-C replicase mediates a relatively broad-spectrum resistance at the transcriptional level.