Plant Pathology Laboratory, Faculty of Agriculture, Iwate University, Ueda 3-18-8, Morioka 020-8550, Japan.
Plant Methods. 2011 Jun 10;7(1):15. doi: 10.1186/1746-4811-7-15.
Virus-induced gene silencing (VIGS) is an effective technology for the analysis of gene functions in plants. Though there are many reports on virus vectors for VIGS in plants, no VIGS vectors available for Rosaceae fruit trees were reported so far. We present an effective VIGS system in apple, pear, and Japanese pear using Apple latent spherical virus (ALSV) vectors.
Inoculation of ALSV vectors carrying a partial sequence of endogenous genes from apple [ribulose-1, 5-bisphosphate carboxylase small subunit (rbcS), alpha subunit of chloroplast chaperonin (CPN60a), elongation factor 1 alpha (EF-1a), or actin] to the cotyledons of seeds by a particle bombardment induced highly uniform knock-down phenotypes of each gene on the true leaves of seedlings from 2~3 weeks after inoculation. These silencing phenotypes continued for several months. Northern blot and RT-PCR analyses of leaves infected with ALSV containing a fragment of rbcS gene showed that the levels of rbcS-mRNA drastically decreased in the infected apple and pear leaves, and, in reverse, rbcS-siRNAs were generated in the infected leaves. In addition, some of apple seedlings inoculated with ALSV vector carrying a partial sequence of a TERMINAL FLOWER 1 gene of apple (MdTFL1) showed precocious flowering which is expected as a knock-down phenotype of the silencing of MdTFL1 gene.
The ALSV-based VIGS system developed have provides a valuable new addition to the tool box for functional genomics in apple, pear, and Japanese pear.
病毒诱导的基因沉默（VIGS）是一种分析植物基因功能的有效技术。虽然有许多关于植物中 VIGS 载体的报道，但迄今为止尚未报道用于蔷薇科果树的 VIGS 载体。我们使用苹果潜隐病毒（ALSV）载体在苹果、梨和日本梨中展示了一种有效的 VIGS 系统。
通过粒子轰击将携带苹果内源基因部分序列（核酮糖-1,5-二磷酸羧化酶小亚基（rbcS）、叶绿体伴侣蛋白 60a 的α亚基（CPN60a）、延伸因子 1α（EF-1a）或肌动蛋白）的 ALSV 载体接种到种子子叶中，可在接种后 2~3 周从幼苗的真叶上诱导出每个基因的高度均匀的敲低表型。这些沉默表型持续了几个月。用含有 rbcS 基因片段的 ALSV 感染叶片的 Northern blot 和 RT-PCR 分析表明，rbcS-mRNA 在感染的苹果和梨叶片中的水平急剧下降，并且在感染的叶片中产生了 rbcS-siRNAs。此外，一些接种了携带苹果 MdTFL1 基因部分序列的 ALSV 载体的苹果幼苗表现出早熟开花，这预计是 MdTFL1 基因沉默的敲低表型。
开发的基于 ALSV 的 VIGS 系统为苹果、梨和日本梨的功能基因组学提供了一个有价值的新工具。
