Coram Tristan E, Pang Edwin C K
RMIT University, School of Applied Sciences, Biotechnology and Environmental Biology, Building 223, Level 1, Plenty Road, Bundoora, Victoria 3083, Australia.
Plant Biotechnol J. 2006 Nov;4(6):647-66. doi: 10.1111/j.1467-7652.2006.00208.x.
Using microarray technology and a set of chickpea (Cicer arietinum L.) unigenes, grasspea (Lathyrus sativus L.) expressed sequence tags (ESTs) and lentil (Lens culinaris Med.) resistance gene analogues, the ascochyta blight (Ascochyta rabiei (Pass.) L.) resistance response was studied in four chickpea genotypes, including resistant, moderately resistant, susceptible and wild relative (Cicer echinospermum L.) genotypes. The experimental system minimized environmental effects and was conducted in reference design, in which samples from mock-inoculated controls acted as reference against post-inoculation samples. Robust data quality was achieved through the use of three biological replicates (including a dye swap), the inclusion of negative controls and strict selection criteria for differentially expressed genes, including a fold change cut-off determined by self-self hybridizations, Student's t-test and multiple testing correction (P < 0.05). Microarray observations were also validated by quantitative reverse transcriptase-polymerase chain reaction (RT-PCR). The time course expression patterns of 756 microarray features resulted in the differential expression of 97 genes in at least one genotype at one time point. k-means clustering grouped the genes into clusters of similar observations for each genotype, and comparisons between A. rabiei-resistant and A. rabiei-susceptible genotypes revealed potential gene 'signatures' predictive of effective A. rabiei resistance. These genes included several pathogenesis-related proteins, SNAKIN2 antimicrobial peptide, proline-rich protein, disease resistance response protein DRRG49-C, environmental stress-inducible protein, leucine-zipper protein, polymorphic antigen membrane protein, Ca-binding protein and several unknown proteins. The potential involvement of these genes and their pathways of induction are discussed. This study represents the first large-scale gene expression profiling in chickpea, and future work will focus on the functional validation of the genes of interest.
利用微阵列技术以及一组鹰嘴豆（Cicer arietinum L.）单基因、草豌豆（Lathyrus sativus L.）表达序列标签（EST）和小扁豆（Lens culinaris Med.）抗性基因类似物，对包括抗性、中度抗性、易感和野生近缘种（Cicer echinospermum L.）基因型在内的四种鹰嘴豆基因型的褐斑病（Ascochyta rabiei (Pass.) L.）抗性反应进行了研究。该实验系统将环境影响降至最低，并采用参照设计进行，其中模拟接种对照的样本用作接种后样本的参照。通过使用三个生物学重复（包括染料交换）、纳入阴性对照以及对差异表达基因采用严格的选择标准，包括通过自身杂交确定的倍数变化阈值、学生t检验和多重检验校正（P < 0.05），实现了稳健的数据质量。微阵列观察结果也通过定量逆转录聚合酶链反应（RT-PCR）进行了验证。756个微阵列特征的时间进程表达模式导致至少一种基因型在一个时间点有97个基因差异表达。k均值聚类将每个基因型的基因分组为具有相似观察结果的簇，对褐斑病抗性和易感基因型之间的比较揭示了预测有效褐斑病抗性的潜在基因“特征”。这些基因包括几种病程相关蛋白、SNAKIN2抗菌肽、富含脯氨酸的蛋白、抗病反应蛋白DRRG49-C、环境胁迫诱导蛋白、亮氨酸拉链蛋白、多态性抗原膜蛋白、钙结合蛋白和几种未知蛋白。讨论了这些基因的潜在参与及其诱导途径。本研究代表了鹰嘴豆首次大规模基因表达谱分析，未来的工作将集中在对感兴趣基因的功能验证上。
