摘要
本研究克隆了来源于马铃薯Y病毒坏死株系(PVYN)外壳蛋白(CP)基因(PVYNCP)5′端和3′端的反向重复cDNA序列,并构建了植物表达载体pROKII-5′IR和pROKII-3′IR,利用农杆菌介导的叶盘法转化烟草NC89,分别获得166和126株转基因烟草。抗病性试验表明,转化PVYNCP基因3′端茎50 bp(环50 bp)hp RNA(hairpin RNA)的烟草抗病率达69%,而转化PVYNCP基因5′端相同片段长度的hpRNA的烟草却全部发病。Southern blot结果表明,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介导的抗病性。研究结果证明PVYNCP基因的3′端比5′端更能有效地诱发基因沉默,并且hpRNA茎部的长度可减少到50 bp。该结果为探索利用CP基因的最短长度和有效部位的基因片段来培育多抗病毒转基因植物提供了依据。
Two inverted repeats cDNA fragments derived from 5＇ and 3＇ ends of PVY^N CP gene were cloned respectively, and the corresponding expression vectors pROKII-5＇ IR and pROKII-3＇ IR were constructed. Recombinant binary vectors were then introduced into tobacco （NC89） plants via Agrobacterium tumefaciens-mediated transformation. 166 and 126 transgenic plants transformed with pROKII-5＇ IR and pROKII-3＇ IR were obtained, respectively. Resistance tests indicated that plants transformed with pROKII-3＇ IR were highly resistant to PVY infection, and the proportion of disease resistant transgenic plants was 69%, while plants transformed with pROKII-5＇ IR were susceptible to PVY^N infection. Southern blot results indicated that there were no obvious correlation between the copy numbers of transgene and resistance. Northern blot results revealed an inverse correlation between transgenic transcript accumulation and virus resistance, it demonstrating that the resistance was RNA mediated. The results demonstrated that the hpRNA （hairpin RNA） with 50 bp-stem derived from the 3＇ end of the CP gene is sufficient to induce RNA-mediated gene silencing. This information is useful in production of transgenic plants which are resistant to multi-virus infections.
出处
《植物病理学报》
CAS
CSCD
北大核心
2007年第1期69-76,共8页
Acta Phytopathologica Sinica
基金
国家自然科学基金资助项目(30471139
30500327)
