Simultaneous silencing of multiple genes in the apple scab fungus, Venturia inaequalis, by expression of RNA with chimeric inverted repeats
Simultaneous silencing of multiple genes in the apple scab fungus, Venturia inaequalis, by expression of RNA with chimeric inverted repeats
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DOI：
10.1016/j.fgb.2004.06.006
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发表时间：
2004-10-01
影响因子：
3
通讯作者：
Plummer, KM
中科院分区：
文献类型：
--
作者：
Fitzgerald, A;van Kan, JAL;Plummer, KM
RNA介导的基因沉默已经在植物、动物和最近在丝状真菌中得到证实。在这里，我们报告高频率，RNA介导的基因沉默在苹果黑星病真菌，黑星菌。绿色荧光蛋白（GFP）转基因在表达GFP的大肠杆菌中沉默。参与黑色素生物合成的内源基因三羟基萘还原酶（THN）也被沉默。这两个基因的沉默导致了明显的体外表型。通过农杆菌转移的GFP或THN基因使用发夹构建体实现了高频率的基因沉默（分别为71%和61%）。THN沉默的转化体表现出独特的浅棕色表型，并保持感染苹果的能力。重要的是来自单个嵌合反向重复发夹构建体的两个基因的同时沉默。这两个基因与此构建体的沉默发生在所有转化体的51%的频率。GFP基因沉默的所有125个集落也对THN沉默。由于THN和GFP沉默的转化体具有容易检测的表型，因此所述基因可用作基因沉默的标记。同时，多个基因沉默，利用这样的标记基因，将使高通量筛选功能基因组学的发展。当与在体外没有明显表型的沉默基因连接时，这种嵌合技术将特别有价值。(C)2004年爱思唯尔公司All rights reserved.
RNA-mediated gene silencing has been demonstrated in plants, animals, and more recently in filamentous fungi. Here, we report high frequency, RNA-mediated gene silencing in the apple scab fungus, Venturia inaequalis. The green fluorescent protein (GFP) transgene was silenced in a GFP-expressing transformant. An endogenous gene, trihydroxynaphthalene reductase (THN), involved in melanin biosynthesis, was also silenced. Silencing of these two genes resulted in obvious phenotypes in vitro. High frequency gene silencing was achieved using hairpin constructs for the GFP or the THN genes transferred by Agrobacterium (71 and 61%, respectively). THN-silenced transformants exhibited a distinctive light brown phenotype and maintained the ability to infect apple. Of significance was the simultaneous silencing of the two genes from a single chimeric, inverted repeat hairpin construct. Silencing of both genes with this construct occurred at a frequency of 51% of all the transformants. All 125 colonies silenced for the GFP gene were also silenced for THN. As THN and GFP silenced transformants have readily detectable phenotypes, the genes have utility as markers for gene silencing. Simultaneous, multiple gene silencing, utilising such marker genes, will enable the development of high through-put screening for functional genomics. This chimeric technology will be particularly valuable when linked with silenced genes that have no obvious phenotype in vitro. (C) 2004 Elsevier Inc. All rights reserved.