Barley stripe mosaic virus-induced gene silencing in a monocot plant
Barley stripe mosaic virus-induced gene silencing in a monocot plant
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DOI：
10.1046/j.1365-313x.2002.01291.x
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发表时间：
2002-05-01
期刊：
影响因子：
7.2
通讯作者：
Pogue, GP
中科院分区：
文献类型：
--
作者：
Holzberg, S;Brosio, P;Pogue, GP
内源植物基因的RNA沉默可以通过病毒介导的同源基因片段的瞬时表达来实现。这种强大的反向遗传方法被称为病毒诱导基因沉默(VIGS)，只在双子叶植物中得到证实，在那里它已经成为功能基因组学的重要工具。大麦条纹花叶病毒(BSMV)是一种三重正义RNA病毒，可侵染大麦、燕麦、小麦和玉米等多种农业上重要的单子叶植物。为了在单子叶宿主中展示VIGS，我们修改了BSMV，以正义或反义方向表达Gammab基因下游不可翻译的外源插入。植烯去饱和酶(PDS)是合成类胡萝卜素所必需的，类胡萝卜素是保护叶绿素免受光漂白的化合物。从大麦中扩增出的部分PDS基因与水稻、玉米和烟草中的PDS基因的同源性分别为90%、88%和74%。大麦感染表达大麦、水稻或玉米PDS片段的BSMV后，光漂白和积累植酸烯(PDS的底物)的方式与PDS的化学抑制剂去甲氟拉松处理相似。相反，感染野生型BSMV或表达N.benthamiana PDS或反义绿色荧光蛋白(GFP)的BSMV的大麦不会光漂白或积累植酸。因此，内源PDS的BSMV沉默是同源依赖的。外壳蛋白的缺失增强了BSMV沉默PDS的能力。这是第一次在单子叶植物中发现VIGS，表明BSMV可以用于单子叶植物的功能基因组学和RNA沉默机制的研究。
RNA silencing of endogenous plant genes can be achieved by virus-mediated, transient expression of homologous gene fragments. This powerful, reverse genetic approach, known as virus-induced gene silencing (VIGS), has been demonstrated only in dicot plant species, where it has become an important tool for functional genomics. Barley stripe mosaic virus (BSMV) is a tripartite, positive-sense RNA virus that infects many agriculturally important monocot species including barley, oats, wheat and maize. To demonstrate VIGS in a monocot host, we modified BSMV to express untranslatable foreign inserts downstream of the gammab gene, in either sense or antisense orientations. Phytoene desaturase (PDS) is required for synthesizing carotenoids, compounds that protect chlorophyll from photo-bleaching. A partial PDS cDNA amplified from barley was 90, 88 and 74% identical to PDS cDNAs from rice, maize and Nicotiana benthamiana, respectively. Barley infected with BSMV expressing barley, rice or maize PDS fragments became photo-bleached and accumulated phytoene (the substrate for PDS) in a manner similar to plants treated with the chemical inhibitor of PDS, norflurazon. In contrast, barley infected with wild-type BSMV, or BSMV expressing either N. benthamiana PDS or antisense green fluorescent protein (GFP), did not photo-bleach or accumulate phytoene. Thus BSMV silencing of the endogenous PDS was homology-dependent. Deletion of the coat protein enhanced the ability of BSMV to silence PDS. This is the first demonstration of VIGS in a monocot, and suggests that BSMV can be used for functional genomics and studies of RNA-silencing mechanisms in monocot plant species.