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根据已克隆的马铃薯Y病毒坏死株系(PVYN)的基因组序列设计引物,利用PCR扩增HC-Pro、CI、NIb和CP4个基因的3′端400bpcDNA区段,分别反向插入含有査尔酮合成酶基因(Chalcone synthase,CHS)内含子的双元载体pRCHS中,构建了含内含子的发夹结构RNA(intron splicing hpRNA,ihpRNA)表达载体pRCHS-HC-Pro、pRCHS-CI、pRCHS-NIb和pRCHS-CP。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。病毒抗性检测的结果显示:转pRCHS-HC-Pro、pRCHS-CI、pRCHS-NIb和pRCHS-CP的烟草中,抗性植株的比例分别为55.34%、73.69%、61.54%和84.21%。大分子RNA和siRNA的Northern blot分析表明,目的片段转录产物的积累量与转基因植株的抗病性呈负相关,转基因植株中可检测到siRNA,说明所获得的抗病性是RNA沉默介导的。
Primers were designed according to the genomic sequence of the cloned PVY virus. PCR was used to amplify the 400bp 3’-end cDNA of HC-Pro, CI, NIb and CP4 genes, The intron splicing hpRNA (ihpRNA) expression vector pRCHS-HC-Pro, pRCHS-CI and pRCHS were constructed in the binary vector pRCHS containing the intron of Chalcone synthase (CHS) -NIb and pRCHS-CP. Using Agrobacterium-mediated transformation of tobacco variety NC89, a variety of transgenic plants were obtained. The results of virus resistance test showed that the percentage of resistant plants in transgenic tobacco plants transfected with pRCHS-HC-Pro, pRCHS-CI, pRCHS-NIb and pRCHS-CP were 55.34%, 73.69%, 61.54% and 84.21%, respectively. Northern blot analysis of macromolecular RNA and siRNA showed that the accumulation of the transcripts of the target fragment was negatively correlated with the disease resistance of the transgenic plants, and siRNA could be detected in the transgenic plants, indicating that the acquired disease resistance is mediated by RNA silencing .