结论:开发了一种用于大豆叶片的稳健的农业浸润介导的瞬时基因表达方法。植物基因型,发育阶段和叶龄,表面活性剂,和农杆菌培养条件对于成功的农业渗透很重要。本氏烟草的土壤渗透已成为植物生物技术和合成生物学的主要瞬时测定法,以测试双子叶中基因构建体的性能。虽然有效,然而,通常期望直接在作物物种中测定转基因构建体。为此,我们为大豆(大豆)创新了一种非常强大的农业渗透方法,世界上种植最广泛的双子叶植物。发现几个因素与成功的大豆叶片农业渗透有关,包括基因型,表面活性剂,发育阶段,和农杆菌菌株和培养基。我们优化的方案涉及具有适当表达载体的多步农杆菌培养过程,SilwetL-77作为表面活性剂,在VC或V1生长阶段选择完全扩张的叶子,和在-85kPa下5分钟的真空,然后是黑暗的温育期,然后将植物恢复到正常的生长条件。使用此方法,两系大豆幼叶-V17-0799DT,和TN16-5004-是GUS的高表达者,两个共表达的荧光蛋白基因,和RUBY记者产品,betalain.这项工作不仅代表了大豆生物技术的新研究工具,但也指出了指导其他作物物种农业渗透优化的关键参数。我们推测叶片发育阶段可能是成功入渗的最关键因素。
CONCLUSIONS: A robust
agroinfiltration-mediated transient gene expression method for soybean leaves was developed. Plant genotype, developmental stage and leaf age, surfactant, and Agrobacterium culture conditions are important for successful agroinfiltration.
Agroinfiltration of Nicotiana benthamiana has emerged as a workhorse transient assay for plant biotechnology and synthetic biology to test the performance of gene constructs in dicot leaves. While effective, it is nonetheless often desirable to assay transgene constructs directly in crop species. To that end, we innovated a substantially robust
agroinfiltration method for Glycine max (soybean), the most widely grown dicot crop plant in the world. Several factors were found to be relevant to successful soybean leaf
agroinfiltration, including genotype, surfactant, developmental stage, and Agrobacterium strain and culture medium. Our optimized protocol involved a multi-step Agrobacterium culturing process with appropriate expression vectors, Silwet L-77 as the surfactant, selection of fully expanded leaves in the VC or V1 stage of growth, and 5 min of vacuum at - 85 kPa followed by a dark incubation period before plants were returned to normal growth conditions. Using this method, young soybean leaves of two lines-V17-0799DT, and TN16-5004-were high expressors for GUS, two co-expressed fluorescent protein genes, and the RUBY reporter product, betalain. This work not only represents a new research tool for soybean biotechnology, but also indicates critical parameters for guiding agroinfiltration optimization for other crop species. We speculate that leaf developmental stage might be the most critical factor for successful
agroinfiltration.