Expression of lignin-degrading enzymes in soils using targeted microarrays. Expression of lignin-degrading enzymes in soils using targeted microarrays

Microarrays have become established tools for describing microbial systems, however the assessment of expression profiles for environmental microbial communities still presents unique challenges. Notably, the concentration of particular transcripts are likely very dilute relative to the pool of total RNA, and PCR-based amplification strategies are vulnerable to amplification biases and the appropriate primer selection. Thus, we apply a signal amplification approach, rather than template amplification, to analyze the expression of selected lignin-degrading enzymes in soil. Controls in the form of known amplicons and cDNA from Phanerochaete chrysosporium were included and mixed with the soil cDNA both before and after the signal amplification in order to assess the dynamic range of the microarray. We demonstrate that restored prairie soil expresses a diverse range of lignin-degrading enzymes following incubation with lignin substrate, while farmed agricultural soil does not. The mixed additions of control cDNA with soil cDNA indicate that the mixed biomass in the soil does interfere with low abundance transcript changes, nevertheless our microarray approach consistently reports the most robust signals. Keywords: comparative analysis, microbial ecology, soil microbial communities Overall design: We used lignin degradation as a model process to demonstrate the use of an oligonucleotide microarray for directly detecting gene expression in soil communities using signal amplification instead of template amplification to avoid the introduction of PCR bias. In the current study, we analyzed mRNA isolated from two distinct soil microbial communities and demonstrate our ability to detect the expression of a small subset of lignin degrading genes following exposure to a lignitic substrate. We also included purified control amplicons and mixed target experiments with pure P. chrysosporium genomic cDNA to determine the level of interference from soil biomass on target hybridization.

基因芯片（microarray）已成为描述微生物系统的成熟工具，但对环境微生物群落的表达谱进行分析仍面临独特挑战。值得注意的是，相较于总RNA池，特定转录本的浓度往往极低；且基于聚合酶链式反应（PCR）的扩增策略易产生扩增偏差，且对引物选择的合理性要求较高。因此，本研究采用信号扩增法而非模板扩增法，对土壤中选定的木质素降解酶的表达情况进行分析。本研究设置了以已知扩增子和黄孢原毛平革菌（Phanerochaete chrysosporium）互补脱氧核糖核酸（cDNA）为形式的对照样本，并在信号扩增前后将其与土壤cDNA混合，以评估基因芯片的动态范围。研究结果显示，经木质素底物孵育后的恢复性草原土壤可表达多种木质素降解酶，而耕作农田土壤则无此现象。对照cDNA与土壤cDNA的混合实验表明，土壤中的混合生物质确实会对低丰度转录本的变化产生干扰，但本研究采用的基因芯片方法仍能稳定输出最强健的信号。关键词：比较分析、微生物生态学、土壤微生物群落。整体实验设计：本研究以木质素降解为模型过程，验证了寡核苷酸基因芯片（oligonucleotide microarray）在土壤群落中直接检测基因表达的应用可行性，该方法采用信号扩增而非模板扩增以规避聚合酶链式反应（PCR）偏差的引入。本研究分析了从两种不同土壤微生物群落中分离得到的信使核糖核酸（mRNA），并证明在暴露于木质素底物后，本方法可检测到一小部分木质素降解基因的表达。此外，本研究设置了纯化的对照扩增子实验，并将纯黄孢原毛平革菌基因组cDNA与样本混合，以探究土壤生物质对靶标杂交的干扰程度。