Bhaskar Pudota B, Venkateshwaran Muthusubramanian, Wu Lei, Ané Jean-Michel, Jiang Jiming
Department of Horticulture, University of Wisconsin-Madison, Madison, Wisconsin, United States of America.
PLoS One. 2009 Jun 5;4(6):e5812. doi: 10.1371/journal.pone.0005812.
Potato is the third most important food crop worldwide. However, genetic and genomic research of potato has lagged behind other major crops due to the autopolyploidy and highly heterozygous nature associated with the potato genome. Reliable and technically undemanding techniques are not available for functional gene assays in potato. Here we report the development of a transient gene expression and silencing system in potato. Gene expression or RNAi-based gene silencing constructs were delivered into potato leaf cells using Agrobacterium-mediated infiltration. Agroinfiltration of various gene constructs consistently resulted in potato cell transformation and spread of the transgenic cells around infiltration zones. The efficiency of agroinfiltration was affected by potato genotypes, concentration of Agrobacterium, and plant growth conditions. We demonstrated that the agroinfiltration-based transient gene expression can be used to detect potato proteins in sub-cellular compartments in living cells. We established a double agroinfiltration procedure that allows to test whether a specific gene is associated with potato late blight resistance pathway mediated by the resistance gene RB. This procedure provides a powerful approach for high throughput functional assay for a large number of candidate genes in potato late blight resistance.
马铃薯是全球第三大重要粮食作物。然而，由于马铃薯基因组的同源多倍体性质和高度杂合性，其遗传和基因组研究落后于其他主要作物。目前尚无可靠且技术要求不高的技术用于马铃薯功能基因分析。在此，我们报道了一种马铃薯瞬时基因表达和沉默系统的开发。利用农杆菌介导的浸润法，将基因表达或基于RNA干扰的基因沉默构建体导入马铃薯叶细胞。对各种基因构建体进行农杆菌浸润，均能持续实现马铃薯细胞转化，并使转基因细胞在浸润区域周围扩散。农杆菌浸润效率受马铃薯基因型、农杆菌浓度和植株生长条件的影响。我们证明，基于农杆菌浸润的瞬时基因表达可用于在活细胞的亚细胞区室中检测马铃薯蛋白。我们建立了一种双重农杆菌浸润程序，可用于测试特定基因是否与由抗性基因RB介导的马铃薯晚疫病抗性途径相关。该程序为大量马铃薯晚疫病抗性候选基因的高通量功能分析提供了一种强大的方法。
