摘要
人工ta-siRNA基因沉默技术具有同时表达多个ta-siRNA和产生ta-siRNA位置固定等特点,在精确性和高效性等方面有明显优势。本研究以马铃薯Y病毒(potato virus Y,PVY)外壳蛋白(coat protein,CP)为靶基因,选取不同位置的3个siRNA为靶序列,烟草TAS3a作为ta-siRNA表达的骨架,分别构建植物表达载体PVY-CP5'、PVY-CPM和PVY-CP3',并利用叶盘法转化烟草NC89。瞬时表达检测结果显示,3个syn-tasiRNA植物表达载体均能在植物体内成功表达siRNA;抗病性鉴定发现,转PVY-CP5'、PVY-CPM和PVY-CP3'的转基因株系中抗病株系的比率分别为45. 08%、27. 21%、61. 90%;Northern杂交分析显示,目的片段转录产物(总RNA)的积累量与植株的抗病性呈负相关,转基因植株中均有siRNA存在,表明植株的病毒抗性是由RNA介导的。
Synthetic trans-siRNA has the characteristics of simultaneous expression of multiple ta-siRNA and fixed location,which has obvious advantages in accuracy and efficiency.In this study,synthetic trans-siRNA constructs based on3regions of the CP gene of PVY were designed by using TAS3a of tobacco as ta-siRNA expression skeleton.The plant expression vectors dubbed PVY-CP5',PVY-CPM and PVY-CP3'were transformed into tobacco NC89by leaf disc method.The siRNAs were detected in all expression vectors in transient assay,the ratio of disease-resistant lines in transgenic lines of PVY-CP5',PVY-CPM and PVY-CP3'were45.08%,27.21%and61.90%,respectively.Northern blot analysis revealed an inverse correlation between accumulation of target transcripts(total RNA)and plant disease resistance,siRNAs were present in all selected resistant and susceptible transgenic plants.The results proved this resistance was RNA-mediated.
作者
吴斌
姜珊珊
张眉
徐德坤
辛志梅
王升吉
Wu Bin;Jiang Shanshan;Zhang Mei;Xu Dekun;Xin Zhimei;Wang Shengji(Institute of Plant Protection,Shangdong Academy of Agricultural Sciences,Jinan 250100,China;Plant Protection Station of Linyi City,Linyi 276000,China)
出处
《山东农业科学》
2018年第12期86-90,共5页
Shandong Agricultural Sciences
基金
山东省农业科学院青年科研基金项目(2015YQN26)
山东省农业科学院农业科技创新工程项目(CXGC2016A09-监测预警)
