University of Stuttgart, Department of Molecular Biology and Plant Virology, Pfaffenwaldring 57, D-70550 Stuttgart, Germany.
J Virol Methods. 2010 Oct;169(1):129-37. doi: 10.1016/j.jviromet.2010.07.010. Epub 2010 Jul 16.
The bipartite Abutilon mosaic virus (AbMV) was engineered as a versatile silencing vector in which the coat protein gene of DNA A was deleted and replaced by sequences of interest. Plants transgenic for the dimeric AbMV DNA B component were used as test hosts to minimize the risk of unintended release of the recombinant DNA. The vector construct was stable genetically upon systemic infection and, in common with the parental virus, the vector remained phloem-limited. For virus-induced gene silencing (VIGS), a phytoene desaturase gene fragment was isolated from Nicotiana benthamiana (NbPDS) and inserted into the vector. After agroinfection, phytoene desaturase silencing was triggered efficiently in all leaf tissues without interference by viral symptoms. In order to facilitate further the use of the system, a technique for cell-free construction of recombinants was established using rolling circle amplification and biolistic inoculation of DNA B-transgenic plants. This novel procedure provides a convenient and safe way for delivering VIGS constructs for functional genomics.
二分体苘麻嵌纹病毒（AbMV）被设计成一种多功能的沉默载体，其中 DNA A 的外壳蛋白基因被删除，并被感兴趣的序列所取代。转基因植物的二聚体 AbMV DNA B 成分被用作测试宿主，以最大程度地降低重组 DNA 意外释放的风险。载体构建体在系统感染后在遗传上是稳定的，并且与亲本病毒一样，载体仍然局限于韧皮部。对于病毒诱导的基因沉默（VIGS），从 Nicotiana benthamiana（NbPDS）中分离出类胡萝卜素脱饱和酶基因片段并插入载体中。农杆菌感染后，在所有叶片组织中有效地触发了类胡萝卜素脱饱和酶沉默，而没有病毒症状的干扰。为了进一步促进该系统的使用，建立了一种使用滚环扩增和生物弹道接种 DNA B-转基因植物的无细胞构建重组体的技术。这种新方法为功能基因组学提供了一种方便和安全的传递 VIGS 构建体的方法。
