A stable RNA virus-based vector for citrus trees
A stable RNA virus-based vector for citrus trees
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DOI：
10.1016/j.virol.2007.06.038
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发表时间：
2007-11-10
期刊：
影响因子：
3.7
通讯作者：
Dawson, William O.
中科院分区：
文献类型：
--
作者：
Folimonov, Alexey S.;Folimonova, Svetlana Y.;Dawson, William O.
病毒载体是植物分子生物学和植物基因组学研究的重要工具。许多基于感染草本植物的病毒的载体被用于表达或沉默植物中的基因，以及筛选功能未知的序列。然而，木本植物需要有用的基于病毒的载体，由于系统感染和分析所需的时间较长，因此需要更大的稳定性。我们以绿色荧光蛋白(GFP)为报告基因，研究了利用柑橘衰退病毒(CTV)构建外源基因瞬时表达载体的几种策略。这些策略包括将p13开放阅读框架(ORF)替换为GFP的开放阅读框架(ORF)，构建GFP框架内与主要外壳蛋白(CP)的自我加工融合，或将GFP ORF作为额外基因从由复制的CTV CP sgRNA控制元件(CE)或引入的甜菜黄化病毒的异源CE控制的亚基因组(Sg)mRNA中表达。研究了构建的工程载体在原生质体多代过程中的复制、包裹和GFP表达，以及它们在柑橘植株中感染、移动、表达GFP和维持的能力。基于“添加基因”策略的最成功的载体一直非常稳定，在柑橘树上持续产生GFP荧光超过4年。(C)2007 Elsevier Inc.保留所有权利。
Virus-based vectors are important tools in plant molecular biology and plant genomics. A number of vectors based on viruses that infect herbaceous plants are in use for expression or silencing of genes in plants as well as screening unknown sequences for function. Yet there is a need for useful virus-based vectors for woody plants, which demand much greater stability because of the longer time required for systemic infection and analysis. We examined several strategies to develop a Citrus tristeza virus (CTV)-based vector for transient expression of foreign genes in citrus trees using a green fluorescent protein (GFP) as a reporter. These strategies included substitution of the p13 open reading frame (ORF) by the ORF of GFP, construction of a self-processing fusion of GFP in-frame with the major coat protein (CP), or expression of the GFP ORF as an extra gene from a subgenomic (sg) mRNA controlled either by a duplicated CTV CP sgRNA controller element (CE) or an introduced heterologous CE of Beet yellows virus. Engineered vector constructs were examined for replication, encapsidation, GFP expression during multiple passages in protoplasts, and for their ability to infect, move, express GFP, and be maintained in citrus plants. The most successful vectors based on the 'add-a-gene' strategy have been unusually stable, continuing to produce GFP fluorescence after more than 4 years in citrus trees. (C) 2007 Elsevier Inc. All rights reserved.