Plant Physiology and Biotechnology Division, UPASI Tea Research Institute, Valparai, Tamil Nadu, India.
Appl Biochem Biotechnol. 2012 Dec;168(7):1917-27. doi: 10.1007/s12010-012-9907-1. Epub 2012 Oct 12.
Tea (Camellia sinensis (L.) O. Kuntze) is an economically important plant cultivated for its leaves. Infection of Pestalotiopsis theae in leaves causes gray blight disease and enormous loss to the tea industry. We used suppressive subtractive hybridization (SSH) technique to unravel the differential gene expression pattern during gray blight disease development in tea. Complementary DNA from P. theae-infected and uninfected leaves of disease tolerant cultivar UPASI-10 was used as tester and driver populations respectively. Subtraction efficiency was confirmed by comparing abundance of β-actin gene. A total of 377 and 720 clones with insert size >250 bp from forward and reverse library respectively were sequenced and analyzed. Basic Local Alignment Search Tool analysis revealed 17 sequences in forward SSH library have high degree of similarity with disease and hypersensitive response related genes and 20 sequences with hypothetical proteins while in reverse SSH library, 23 sequences have high degree of similarity with disease and stress response-related genes and 15 sequences with hypothetical proteins. Functional analysis indicated unknown (61 and 59 %) or hypothetical functions (23 and 18 %) for most of the differentially regulated genes in forward and reverse SSH library, respectively, while others have important role in different cellular activities. Majority of the upregulated genes are related to hypersensitive response and reactive oxygen species production. Based on these expressed sequence tag data, putative role of differentially expressed genes were discussed in relation to disease. We also demonstrated the efficiency of SSH as a tool in enriching gray blight disease related up- and downregulated genes in tea. The present study revealed that many genes related to disease resistance were suppressed during P. theae infection and enhancing these genes by the application of inducers may impart better disease tolerance to the plants.
茶（Camellia sinensis (L.) O. Kuntze）是一种经济上重要的植物，因其叶子而被栽培。叶部感染拟盘多毛孢（Pestalotiopsis theae）会导致灰斑病，给茶叶产业造成巨大损失。我们利用抑制性消减杂交（SSH）技术来揭示茶树灰斑病发展过程中的差异基因表达模式。来自抗病品种 UPASI-10 的 P. theae 感染和未感染叶片的 cDNA 分别作为测试和驱动群体。通过比较β-肌动蛋白基因的丰度来确认消减效率。正向和反向文库分别获得了大小大于 250bp 的插入片段的 377 和 720 个克隆进行测序和分析。BLAST 分析显示正向 SSH 文库中有 17 个序列与疾病和过敏反应相关基因具有高度相似性，20 个序列与假设蛋白具有高度相似性，而在反向 SSH 文库中，有 23 个序列与疾病和应激反应相关基因具有高度相似性，有 15 个序列与假设蛋白具有高度相似性。功能分析表明正向和反向 SSH 文库中大多数差异调控基因具有未知（61%和 59%）或假设功能（23%和 18%），而其他基因在不同的细胞活动中具有重要作用。大多数上调基因与过敏反应和活性氧产生有关。基于这些表达序列标签数据，讨论了差异表达基因在疾病中的可能作用。我们还证明了 SSH 作为一种工具，可在茶中富集与灰斑病相关的上调和下调基因。本研究表明，在 P. theae 感染期间，许多与抗病性相关的基因受到抑制，通过应用诱导剂增强这些基因可能赋予植物更好的抗病性。
