Prins M, de Haan P, Luyten R, van Veller M, van Grinsven M Q, Goldbach R
Department of Virology, Wageningen Agricultural University, The Netherlands.
Mol Plant Microbe Interact. 1995 Jan-Feb;8(1):85-91. doi: 10.1094/mpmi-8-0085.
Transgenic tobacco plants have been obtained expressing nucleoprotein (N) gene sequences of three different tospoviruses known to affect vegetable crops: tomato spotted wilt virus (TSWV), tomato chlorotic spot virus (TCSV), and groundnut ringspot virus (GRSV). The chimeric plant transformation vector used comprised the three viral N gene sequences, each with a copy of the CaMV 35S promoter and the nos terminator. Despite the high levels of homology between the different N gene sequences (74-82%) and the presence of repeated promoter and terminator sequences in this construct, unrearranged copies of this triple N gene construct were stably maintained in both Escherichia coli and Agrobacterium tumefaciens plasmids used during the cloning process, as well as in several generations of transgenic tobacco plants. A transgenic tobacco line was obtained that exhibited high levels of resistance to all three tospoviruses, showing the possibility of producing transgenic plants with a broad resistance to tospoviruses by introducing tandemly cloned viral N gene sequences. DNA analysis of this transgenic plant line shows that the multivirus resistance trait is confined to a single genetic locus, which is very convenient for further breeding purposes.
已经获得了表达三种已知影响蔬菜作物的不同番茄斑萎病毒属病毒核蛋白（N）基因序列的转基因烟草植株，这三种病毒分别是番茄斑萎病毒（TSWV）、番茄褪绿斑病毒（TCSV）和花生环斑病毒（GRSV）。所使用的嵌合植物转化载体包含这三种病毒的N基因序列，每个序列都带有一个CaMV 35S启动子拷贝和nos终止子。尽管不同N基因序列之间具有高度同源性（74 - 82%），并且该构建体中存在重复的启动子和终止子序列，但在克隆过程中使用的大肠杆菌和根癌农杆菌质粒中，以及在几代转基因烟草植株中，这种三重N基因构建体的未重排拷贝都得到了稳定维持。获得了一个对所有三种番茄斑萎病毒属病毒都表现出高度抗性的转基因烟草株系，这表明通过引入串联克隆的病毒N基因序列来培育对番茄斑萎病毒属病毒具有广泛抗性的转基因植物是有可能的。对该转基因株系的DNA分析表明，多病毒抗性性状局限于单个基因座，这对于进一步的育种目的非常方便。
