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以马铃薯Y病毒坏死株系(PVYN)的外壳蛋白(coat protein,CP)基因3′端50bp片段为hpRNA的茎,以pUC19不同长度的序列为hpRNA的环,构建了茎环比例分别为4∶1、2∶1、1∶1、1∶2、1∶4和1∶8的植物表达载体。利用农杆菌介导法转化烟草品种NC89,获得了多种转基因植株。室内抗病性检测发现:不同茎环比例的hpRNA介导的病毒抗性效率不同;茎环比例为4∶1、2∶1和1∶1时效率较高,抗性植株的比例达60%左右;随着环长度的逐渐增加,抗性植株的比例逐渐降低;当茎环比例为1∶8时,抗性植株的比例仅为9.52%。Southern blot分析结果表明:外源基因已整合于烟草的基因组中,且转基因植株的抗病性与转基因的拷贝数之间无明显的相关性。Northern blot分析结果表明:目的片段转录产物的积累量与植株的抗病性呈负相关,证明所获得的抗病性是RNA介导的。
The 50 bp fragment of the 3 ’end of the coat protein (CP) gene of PVYN was used as the stem of hpRNA, and the pUC19 sequences of different lengths were used as the hpRNA loop to construct stem-loop ratios of 4: 1: 1, 1: 2, 1: 4, and 1: 8 plant expression vectors. Using Agrobacterium-mediated transformation of tobacco variety NC89, a variety of transgenic plants were obtained. The results of in-house resistance test showed that hpRNA-mediated virus resistance efficiency varied with different stem-ring ratios; the stem-loop ratios were higher at 4:1, 2:1 and 1:1, and the percentage of resistant plants was 60% The proportion of resistant plants decreased gradually with the increase of the length of the rings. When the stem-ring ratio was 1: 8, the proportion of resistant plants was only 9.52%. Southern blot analysis showed that the exogenous genes were integrated into the genome of tobacco and there was no significant correlation between the resistance of the transgenic plants and the copy number of the transgenic plants. Northern blot analysis showed that the accumulation of the transcripts of the target fragment was negatively correlated with the disease resistance of the plants, indicating that the acquired disease resistance is RNA-mediated.