Department of Plant Sciences, University of Tennessee, 2431 Joe Johnson Dr, 252 Ellington Plant Sciences Bldg, Knoxville, TN 37996-4561, USA.
Plant Cell Rep. 2011 Mar;30(3):325-34. doi: 10.1007/s00299-010-0961-2. Epub 2010 Dec 8.
Functional analysis of candidate transgenes for insect resistance in stably transformed plants is a time-consuming task that can take months to achieve in even the fastest of plant models. In this study, a rapid screening technique is described, which employs candidate transgene transient expression using agroinfiltration in Nicotiana benthamiana combined with a simple insect bioassay. Using this system the known insecticidal protein Cry1Ac is demonstrated to effectively control Helicoverpa zea. Insects fed tissue with synthesized GFP (green fluorescent protein) as a positive control were shown to have enhanced growth and development. Additionally, a Brassica oleracea proteinase inhibitor (BoPI), a less characterized insect resistance candidate, demonstrated effectiveness to decrease the growth and development of H. zea at high levels of transient expression. Bioassays performed on stable transformants showed that BoPI had a low level of insect resistance at the more typical levels of gene transcription found in stably transformed plants. This agroinfiltration-insect bioassay procedure can give a rapid assessment of insect resistance significantly decreasing the time needed for evaluation of candidate genes.
在稳定转化的植物中，对候选抗虫基因进行功能分析是一项耗时的任务，即使在最快的植物模型中，也需要数月时间才能完成。本研究描述了一种快速筛选技术，该技术采用农杆菌介导的瞬时表达候选转基因，结合简单的昆虫生物测定。利用该系统，已知的杀虫蛋白 Cry1Ac 被证明可有效控制棉铃虫。用合成 GFP（绿色荧光蛋白）作为阳性对照的昆虫喂养组织，显示出增强的生长和发育。此外，一种拟南芥蛋白酶抑制剂（BoPI），一种特征不那么明显的抗虫候选基因，在高水平瞬时表达时表现出降低棉铃虫生长和发育的有效性。对稳定转化体进行的生物测定表明，BoPI 在稳定转化植物中发现的典型基因转录水平下，具有较低的抗虫水平。这种农杆菌瞬时表达-昆虫生物测定程序可以快速评估抗虫性，显著缩短候选基因评估所需的时间。
