College of Food Science and Nutritional Engineering, China Agricultural University, Beijing, China.
BMC Plant Biol. 2010 Oct 28;10:234. doi: 10.1186/1471-2229-10-234.
Downy mildew (DM), caused by pathogen Plasmopara viticola (PV) is the single most damaging disease of grapes (Vitis L.) worldwide. However, the mechanisms of the disease development in grapes are poorly understood. A method for estimating gene expression levels using Solexa sequencing of Type I restriction-endonuclease-generated cDNA fragments was used for deep sequencing the transcriptomes resulting from PV infected leaves of Vitis amurensis Rupr. cv. Zuoshan-1. Our goal is to identify genes that are involved in resistance to grape DM disease.
Approximately 8.5 million (M) 21-nt cDNA tags were sequenced in the cDNA library derived from PV pathogen-infected leaves, and about 7.5 M were sequenced from the cDNA library constructed from the control leaves. When annotated, a total of 15,249 putative genes were identified from the Solexa sequencing tags for the infection (INF) library and 14,549 for the control (CON) library. Comparative analysis between these two cDNA libraries showed about 0.9% of the unique tags increased by at least five-fold, and about 0.6% of the unique tags decreased more than five-fold in infected leaves, while 98.5% of the unique tags showed less than five-fold difference between the two samples. The expression levels of 12 differentially expressed genes were confirmed by Real-time RT-PCR and the trends observed agreed well with the Solexa expression profiles, although the degree of change was lower in amplitude. After pathway enrichment analysis, a set of significantly enriched pathways were identified for the differentially expressed genes (DEGs), which associated with ribosome structure, photosynthesis, amino acid and sugar metabolism.
This study presented a series of candidate genes and pathways that may contribute to DM resistance in grapes, and illustrated that the Solexa-based tag-sequencing approach was a powerful tool for gene expression comparison between control and treated samples.
由病原菌葡萄钩丝壳菌（Plasmopara viticola，PV）引起的霜霉病是全球范围内对葡萄（Vitis L.）危害最大的单一病害。然而，葡萄霜霉病发病机制尚不清楚。本研究采用基于 Type I 限制性内切酶产生的 cDNA 片段的 Solexa 测序方法对感病的山葡萄（Vitis amurensis Rupr. cv. Zuoshan-1）叶片进行转录组深度测序，以估计基因表达水平。我们的目标是鉴定与葡萄霜霉病抗性相关的基因。
从 PV 病原菌感染叶片的 cDNA 文库中测序了约 850 万个（M）21-nt cDNA 标签，从对照叶片构建的 cDNA 文库中测序了约 750 万个。经注释，从感染（INF）文库和对照（CON）文库的 Solexa 测序标签中共鉴定出 15249 个假定基因。对这两个 cDNA 文库进行比较分析发现，至少增加 5 倍的独特标签约占 0.9%，而感染叶片中至少减少 5 倍的独特标签约占 0.6%，两个样本之间 98.5%的独特标签差异小于 5 倍。通过实时 RT-PCR 验证了 12 个差异表达基因的表达水平，观察到的趋势与 Solexa 表达谱非常吻合，尽管变化幅度较低。经通路富集分析，确定了一组与核糖体结构、光合作用、氨基酸和糖代谢相关的差异表达基因（DEGs）的显著富集通路。
本研究提出了一系列可能有助于葡萄抵抗霜霉病的候选基因和通路，并表明基于 Solexa 的标签测序方法是比较对照和处理样本基因表达的有力工具。
