转Bt基因棉花选择标记基因nptⅡ转录后沉默分析  

Post-transcriptional Silencing of the nptⅡ Gene in Transgenic Bt Cotton

作　　者：晋昕[1,2] 常瞡 司怀军[1] 张宁[1] 吴家和[1,2] 

机构地区：[1]甘肃农业大学生命科学技术学院,兰州730070 [2]中国科学院微生物研究所,北京100101

出　　处：《棉花学报》2015年第5期385-390,共6页Cotton Science

基　　金：棉花生物学国家重点实验室开放课题基金(CB2014B02);国家自然科学基金项目(31471544)

摘　　要：本研究利用病毒诱导基因沉默技术对转Bt基因棉花选择标记基因nptⅡ进行沉默,分析标记基因转录后沉默的可行性。以烟草脆裂病毒载体为骨架构建nptⅡRNAi载体,利用农杆菌浸染棉花子叶,获得nptⅡ干涉的转Bt基因棉花植株,然后涂抹卡那霉素进行检测。结果表明nptⅡ沉默植株叶片出现黄斑症状,RT-PCR和q RT-PCR检测表明叶片、根和茎中nptⅡ基因转录分别被抑制了99.25%、99.05%和98.65%,沉默后期的转录抑制也达到98%。本研究结果为解决已经在环境中释放转基因生物发生意外扩散和不可预料的生物安全事件提供了快速有效的应对方法和新思路。The biosafety of transgenic plants can largely affect their planting area, speed of spread, and acceptance and sale in the market for transgenic products. In this study, we employed virus-induced gene silencing（VIGS） technology to inhibit nptⅡ transcript expression in transgenic Bt cotton. The p YL-156-nptⅡ construct was generated by inserting an nptⅡ gene fragment into the tobacco rattle virus（TRV） vector p YL-156. The plants were transformed with Agrobacterium harboring p YL-156-nptⅡ by injecting cotyledons with a 1-m L no-needle syringe. Two weeks later, 0.5%（mass concentration） kanamycin was applied to the leaves of the silenced plants. The leaves of the silenced plants treated with kanamycin showed necrotic spots, but control leaves grew normally. Based on RT-PCR and q PCR analyses, the transcript level of npt Ⅱ in the leaf, root and stem in the silenced plants was reduced by 99.9%, 92.5% and 98.5%, respectively, compared with control plants; similar nptⅡ transcript levels were observed in silenced plant leaves at 30 and 60 days after injection. Our results demonstrate that foreign genes in transgenic plants, especially selectable marker genes, can be eliminated or inhibited by post-transcriptional gene silencing, which could help to reduce the accidental escape and unpredicted risks of transgenic plants growing in an open environment.

关 键 词：棉花 选择标记基因 病毒诱导的基因沉默 转录后调控 转基因生物安全 

分 类 号：S562[农业科学—作物学]