Gene expression profiling of rice seedling with 2-azahypoxanthine . Oryza sativa Japonica Group

Rings or arcs of fungus-stimulated plant growth occur often on the floor of woodlands which are commonly called “fairy rings”. We purified a plant growth-stimulating compound, 2-azahypoxanthine (AHX), from the fairy ring-forming fungus Lepista sordida, and the detection of AHX in the fungus-infected soil near the growth-stimulated turfgrass roots. The growth-promoting activity of AHX towards rice was further analyzed by oligo DNA microarrays. Overall design: Immediately after germination, rice seedlings were treated with AHX and incubated for 2 weeks. Total RNA was isolated from the seedlings using an RNeasy plant mini kit (Qiagen). cDNA was synthesized to provide 400 ng of total RNA (Agilent Technologies). This cDNA was used as a template to synthesize cRNA. The cRNA was labeled with Cyanine-3 (Cy3) CTP or Cyanine-5 (Cy5) CTP. Cy3-labeled cRNA was mixed with the same amount of Cy5-labeled cRNA and the mixture was used for hybridization. After hybridization for 17 h at 65°C, the slides were washed and scanned with an Agilent Microarray Scanner. Genes showing a signal value below 300 in the Cy3 channel of the control were excluded from the analysis. Feature extraction and image analysis software (version A.6.1.1; Agilent Technologies) were used to locate and delineate each spot in the array and to integrate each spot’s intensity, filtering, and normalization. We attempted dye swap experiments to estimate the error of technical and dye labelling. Three-condition experiment, control vs. compound-treated rice (0.05 and 0.2 mM). Biological replicates: control, 0.05 mM AHX-treated, 0.2 mM AHX-treated rice independently grown and harvested. One replicate per array.

真菌诱导植物生长形成的环状或弧状结构常出现在林地地表，这类结构通常被称为“仙环病（fairy rings）”。我们从形成仙环病的真菌紫蜡蘑（Lepista sordida）中纯化得到一种促植物生长化合物2-氮杂次黄嘌呤（2-azahypoxanthine，AHX），并在生长受促的草坪草根际真菌感染土壤中检测到了AHX的存在。我们进一步通过寡聚DNA微阵列（oligo DNA microarray）分析了AHX对水稻的促生长活性。
实验整体设计：水稻种子萌发后立即用AHX处理，随后培养2周。使用RNeasy植物微量试剂盒（Qiagen，凯杰公司）从幼苗中提取总RNA。以400 ng总RNA为模板合成互补DNA（cDNA），相关实验由安捷伦科技（Agilent Technologies）完成。将该cDNA作为模板合成互补RNA（cRNA）。使用氰基3（Cyanine-3，Cy3）CTP或氰基5（Cyanine-5，Cy5）CTP对cRNA进行荧光标记。将等量的Cy3标记cRNA与Cy5标记cRNA混合，用于后续杂交实验。于65℃杂交17小时后，清洗芯片玻片并使用安捷伦微阵列扫描仪（Agilent Microarray Scanner）进行扫描。将对照组Cy3通道信号值低于300的基因排除在后续分析之外。使用特征提取与图像分析软件（版本A.6.1.1，安捷伦科技（Agilent Technologies））对芯片上的每个斑点进行定位、勾勒，并整合各斑点的信号强度，完成过滤与归一化处理。我们设置了染料互换实验，以评估技术操作与染料标记过程带来的误差。本实验包含三个处理组：对照组与两种浓度（0.05 mM、0.2 mM）AHX处理的水稻组。生物学重复：对照组、0.05 mM AHX处理组及0.2 mM AHX处理组的水稻均独立培养并收获，每张芯片对应一个生物学重复。