Wang Changchun, Cai Xinzhong, Wang Xuemin, Zheng Zhong
Institute of Biotechnology, and Department of Plant Protection, College of Agriculture and Biotechnology, Zhejiang University, 268 Kai Xuan Road, Hangzhou 310029, People's Republic of China.
State Key Laboratory of Plant Physiology and Biochemistry, College of Life Science, Zhejiang University, 268 Kai Xuan Road, Hangzhou 310029, People's Republic of China.
Funct Plant Biol. 2006 May;33(4):347-355. doi: 10.1071/FP05096.
Arabidopsis thaliana (L.) Heynh. is a model plant species in which to study plant gene functions. Recently developed virus-induced gene silencing (VIGS) offers a rapid and high-throughput technique platform for gene function analysis. In this paper we report optimisation of tobacco rattle virus (TRV)-induced gene silencing in Arabidopsis. The parameters potentially affecting the efficiency of VIGS in Arabidopsis were investigated. These included the concentration and pre-incubation of Agrobacterium inocula (agro-inocula), the concentration of acetosyringone included in agro-inocula, the Agrobacterium inoculation (agro-inoculation) method, the ecotypes and the growth stages of Arabidopsis plants for agro-inoculation, and the growth temperature of agro-inoculated plants. The optimised VIGS procedure involves preparing the agro-inocula with OD of 2.0, pre-incubating for 2 h in infiltration buffer containing 200 μm acetosyringone, agro-inoculating by vacuum infiltration, and growth of agro-inoculated plants at 22 -24°C. Following this procedure consistent and highly efficient VIGS was achieved for the genes encoding phytoene desaturase (PDS) and actin in Arabidopsis. The silencing phenotype lasts for at least 6 weeks, and is applicable in at least seven ecotypes, including Col-0, Cvi-0, Sd, Nd-1, Ws-0, Bay-0 and Ler. TRV-induced VIGS was expressed not only in leaves, but also in stems, inflorescences and siliques. However, VIGS was not transmissible through seed to the subsequent generation. The optimised procedure of the TRV-induced gene silencing should facilitate high-throughput functional analysis of genes in Arabidopsis.
拟南芥（Arabidopsis thaliana (L.) Heynh.）是用于研究植物基因功能的模式植物物种。最近开发的病毒诱导基因沉默（VIGS）为基因功能分析提供了一个快速且高通量的技术平台。在本文中，我们报道了烟草脆裂病毒（TRV）诱导拟南芥基因沉默的优化方法。研究了可能影响拟南芥中VIGS效率的参数。这些参数包括农杆菌接种物（农杆菌菌液）的浓度和预培养、农杆菌菌液中乙酰丁香酮的浓度、农杆菌接种（农杆菌注射）方法、用于农杆菌接种的拟南芥植物的生态型和生长阶段，以及农杆菌接种植物的生长温度。优化后的VIGS程序包括制备OD值为2.0的农杆菌菌液，在含有200 μM乙酰丁香酮的浸润缓冲液中预培养2小时，通过真空浸润进行农杆菌接种，以及在22 - 24°C下培养农杆菌接种的植物。按照此程序，在拟南芥中对编码八氢番茄红素去饱和酶（PDS）和肌动蛋白的基因实现了一致且高效的VIGS。沉默表型持续至少6周，并且适用于至少七种生态型，包括Col-0、Cvi-0、Sd、Nd-1、Ws-0、Bay-0和Ler。TRV诱导的VIGS不仅在叶片中表达，也在茎、花序和角果中表达。然而，VIGS不能通过种子传递到下一代。TRV诱导基因沉默的优化程序应有助于对拟南芥中的基因进行高通量功能分析。
