【摘要】 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。更多还原

【Abstract】 Using specific primers designed according to the cloned genome sequence of Potato virus YN (PVYN), 400 bp cDNA fragments were amplified from 3′ end of HC-Pro, CI, NIb and CP genes, respectively. The four cDNA fragments were inserted into binary vector pRCHS contained a intron derived from Chalcone synthase to construct the antisense vectors pRCHS-HC-Pro, pRCHS-CI, pRCHS-NIb and pRCHS-CP, containing intron splicing hpRNA (ihpRNA), respectively. Recombinant binary vectors were introduced into Tobacco NC89 mediated by Agrobacterium tumefaciens and transgenic plants were obtained. The virus resistance tests indicated that the proportion of PVY-resistant transgenic tobaccos with pRCHS-HC-Pro, pRCHS-CI, pRCHS- NIb and pRCHS-CP was 55.34%, 73.69%, 61.54% and 84.21%, respectively. Northern blot revealed an inverse correlation between transgenic transcript accumulation and virus resistance, and siRNA was detected in the presence of disease-resistant plants. It demonstrated that the resistance was RNA mediated.更多还原