Expression array for Trichoderma atroviride in mycoparasitic interaction with Rhizoctonia solani

A self-designed Trichoderma high density oligonuclotide (HDO) microarray (Roche-NimbleGen, Inc., Madison, WI, USA) was constructed in a similar way than a previous Trichoderma HDO microarray (Samolski et al., 2009). The microarray was composed of 392,779 60-mer probes designed against 13,443 EST-derived transcripts (Trichochip-1) and the genomes of T. atroviride (11,100 genes) and T. virens (11,643 genes). The Trichochip-1 ESTs were obtained from 28 cDNA libraries from eight different species (representing the biodiversity of this genus: T. harzianum, T. atroviride, T. asperellum, T. viride, T. longibrachiatum, T. virens, T. stromaticum and T. aggresivum) under a wide range of growth conditions, including biocontrol-related conditions and different nutritional situations (Vizcaíno et al., 2006). The Trichochip1 EST database was generated in the TrichoEST project funded by the EU (QLK3-CT-2002-02032) T. atroviride P1 mycelium grown (approximately for 24h) at 25ºC on a cellophane sheet on PDA (Difco) plates before contact (at a distance of 5 mm) a R. solani colony grown under identical conditions in the same plate was compared with T. atroviride P1 mycelium after contact (5 mm) the above R. solani colony (mycoparasitic interaction). RNAs from both conditions were extracted and the corresponding cDNAs were use to hybridize by triplicate the Trichoderma HDO microarray.

本研究自主设计的木霉（Trichoderma）高密度寡核苷酸（high density oligonucleotide, HDO）微阵列购自美国威斯康星州麦迪逊市罗氏-尼姆布基因公司（Roche-NimbleGen, Inc., Madison, WI, USA），其构建方法与此前报道的一款木霉HDO微阵列（Samolski等，2009）高度相似。该微阵列共包含392779条60聚体探针，设计靶点涵盖13443条表达序列标签（expressed sequence tag, EST）来源的转录本（即Trichochip-1），以及深绿木霉（T. atroviride）的11100个基因与绿木霉（T. virens）的11643个基因。Trichochip-1的EST序列来源于覆盖该属生物多样性的8个不同木霉物种的28个cDNA文库，涉及物种包括哈茨木霉（T. harzianum）、深绿木霉（T. atroviride）、棘孢木霉（T. asperellum）、绿色木霉（T. viride）、长枝木霉（T. longibrachiatum）、绿木霉（T. virens）、子座木霉（T. stromaticum）及侵袭木霉（T. aggresivum），所有文库均构建于多种生长条件下，包括生防相关条件与不同营养状态（Vizcaíno等，2006）。Trichochip-1的EST数据库由欧盟资助的TrichoEST项目（项目编号QLK3-CT-2002-02032）构建完成。本实验设置两组对照样本：第一组为在PDA（Difco）平板的玻璃纸上于25℃培养约24小时的深绿木霉P1菌丝体，此时其与同一平板上以相同条件培养的立枯丝核菌（R. solani）菌落相距5mm，尚未发生接触；第二组为深绿木霉P1菌丝体与上述立枯丝核菌菌落发生5mm接触（即菌寄生相互作用阶段）后的样本。分别提取两组样本的总RNA，反转录得到对应cDNA后，以三次生物学重复的方式与木霉HDO微阵列进行杂交实验。