利用RNA介导的抗病性获得高度抗马铃薯Y病毒的转基因烟草  

RNA-MEDIATED VIRAL RESISTANCE AGAINST POTATO VIRUS Y (PVY) IN TRANSGENIC TOBACCO PLANTS

作　　者：郭兴启[1] 吕士恩[2] 朱常香[2] 宋云枝[2] 孟祥兵[2] 郑成超[2] 温孚江[2] 

机构地区：[1]浙江大学农业与生物技术学院,杭州310029 [2]山东农业大学植物遗传工程实验室,泰安271018

出　　处：《植物病理学报》2001年第4期348-356,共9页Acta Phytopathologica Sinica

基　　金：国家自然科学基金资助 ( 3 9970 4 85 )

摘　　要：以马铃薯 Y病毒坏死株系 (PVYN)的 RNA为模板 ,应用反转录 -聚合酶链式反应 (RT- PCR)方法 ,扩增出长度为 80 1bp的非翻译的马铃薯 Y病毒外壳蛋白基因。将扩增的片段克隆到 p BSK的Bam HI和 Kpn I之间并进行了序列测定。用 Bam HI和 Kpn I从重组克隆载体上切下该基因并插入到质粒 p ROKII内得到植物表达载体 p PVYCP。通过根癌农杆菌 (LBA4 40 4 )介导的方法转化烟草NC89,经卡那霉素抗性筛选、PCR和 Southern blot检测 ,获得 82株转基因植株。 Northern blot和Western blot分析表明 ,转基因植株只在 RNA水平上得到了表达。抗病性试验表明转基因植株之间抗性水平存在着差异 ,其中有 7株是对 PVYN高度抗病性的植株。转基因植株抗病特异性试验初步表明 ,对 PVYN 表现高度抗病的植株对 PVYO 也具有高度抗病性。According to the published nucleotide sequence of potato virus Y necrosis strain (PVY N), the untranslatable PVY N coat protein (CP) gene was synthesized by reverse transcription polymerase chain reaction (RT PCR), and constructed into cloning vector pBSK. Sequence and restriction enzyme analyses indicated that the untranslatable CP gene was the same as expected. The untranslatable PVY N CP gene was introduced into tobacco (NC89) plants via Agrobacterium tumefaciens mediated transformation, transformed tissue was selected in the presence of Kanamycin sulfate. Regenerated plants were screened by PCR, and 82 transgenic plants were obtained. Southern blot analysis confirmed that the foreign gene had been integra ted into the tobacco genome. Northern blot and Western blot analysis demonstrated that the transgene was expressed only at the RNA level. Resistant test indicated that the resistant le vels of the transgenic plants against PVY N varied among different transgenic plants, of which, seven lines were highly resistant to PVY N infection, no matter if the sap (extracted from PVY N infected leaf) or viral RNA was used as inoculum. Further experiment demonstrated that the three lines were also highly resistant to PVY O infection.

关 键 词：RNA介导 病毒抗性 马铃薯Y病毒 转基因烟草 

分 类 号：S572.03[农业科学—烟草工业] S188[农业科学—作物学]