Efficient virus-induced gene silencing in plants using a modified geminivirus DNA1 component
Efficient virus-induced gene silencing in plants using a modified geminivirus DNA1 component
复制标题
使用改良的双生病毒 DNA1 成分在植物中进行有效的病毒诱导基因沉默
DOI：
10.1111/j.1467-7652.2008.00395.x
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发表时间：
2009-04-01
影响因子：
13.8
通讯作者：
Zhou, Xueping
中科院分区：
文献类型：
--
作者：
Huang, Changjun;Xie, Yan;Zhou, Xueping
病毒诱导的基因沉默（VIGS）是目前公认的一种功能基因组学应用的强有力的反向遗传学工具。DNA 1是一种与双生病毒科（Geminiviridae）相关的卫星样单链DNA分子，已被证明可以自主复制，但需要辅助病毒进行传播。我们开发了一个VIGS载体的基础上的DNA 1组件的烟草曲茎病毒（TbCSV），一个单分的菜豆花叶病毒，通过插入一个多克隆位点之间的复制相关蛋白的开放阅读框和A-丰富的区域，随后插入的DNA片段的基因沉默。当将宿主基因（硫，Su）或转基因（绿色荧光蛋白，GFP）插入到修饰的DNA 1载体中并与TbCSV共农杆菌接种时，在本氏烟草植物中观察到同源基因的有效沉默。更有趣的是，我们证明了这种修饰的DNA 1可以有效地抑制转基因N.当与番茄黄化曲叶中国病毒（TYLCCNV）（另一种不诱导任何病毒症状的单分菜豆花叶病毒）共接种时，烟草植物中的本塞姆氏病毒或内源Su。烟草属中的基因沉默系统，然后使用TYLCCNV和修饰的DNA 1载体建立番茄和矮牵牛植物。该系统可用于沉默与分生组织和花发育有关的基因。利用改造后的DNA 1载体沉默N.本萨米亚纳NbTOM 1或NbTOM 3的沉默可以将病毒的增殖降低到较低水平，并且同时沉默两个基因可以完全抑制病毒的增殖。先前的研究已经报道，DNA 1与单分和双分菜豆花叶病毒以及短病毒都相关。因此，该载体系统可用于研究、分析和发现多种重要作物中的基因功能。
Virus-induced gene silencing (VIGS) is currently recognized as a powerful reverse genetics tool for application in functional genomics. DNA1, a satellite-like and single-stranded DNA molecule associated with begomoviruses (Family Geminiviridae), has been shown to replicate autonomously but requires the helper virus for its dissemination. We developed a VIGS vector based on the DNA1 component of tobacco curly shoot virus (TbCSV), a monopartite begomovirus, by inserting a multiple cloning site between the replication-associated protein open reading frame and the A-rich region for subsequent insertion of DNA fragments of genes targeted for silencing. When a host gene (sulphur, Su) or transgene (green fluorescent protein, GFP) was inserted into the modified DNA1 vector and co-agroinoculated with TbCSV, efficient silencing of the cognate gene was observed in Nicotiana benthamiana plants. More interestingly, we demonstrated that this modified DNA1 could effectively suppress GFP in transgenic N. benthamiana or endogenous Su in tobacco plants when co-agroinoculated with tomato yellow leaf curl China virus (TYLCCNV), another monopartite begomovirus that does not induce any viral symptoms. A gene-silencing system in Nicotiana spp., Solanum lycopersicum and Petunia hybrida plants was then established using TYLCCNV and the modified DNA1 vector. The system can be used to silence genes involved in meristem and flower development. The modified DNA1 vector was used to silence the AtTOM homologous genes (NbTOM1 and NbTOM3) in N. benthamiana. Silencing of NbTOM1 or NbTOM3 can reduce tobamovirus multiplication to a lower level, and silencing of both genes simultaneously can completely inhibit tobamovirus multiplication. Previous studies have reported that DNA1 is associated with both monopartite and bipartite begomoviruses, as well as curtoviruses. This vector system can therefore be applied for the study, analysis and discovery of gene function in a variety of important crop plants.