Neurospora crassa transcriptomics reveals oxidative stress and plasma membrane homeostasis biology genes as key targets in response to chitosan. Neurospora crassa OR74A

Purpose: Evaluate and analyze the transcriptional response of N. crassa germinating conidia and determined the main gene functions related with the exposure to chitosan. Identification of main chitosan gene tragets in N. crassa. Methods: From samples in contact with chitosan for 4, 8 and 16h total RNA was isolated from material using TRIzol reagent. Total RNA was washed and then resuspended. For first strand cDNA synthesis, the fragmented poly (A+) RNA was incubated with random hexamers. Superscript II (200 U; Invitrogen) was added to the first cDNA strand synthesis. End-repair was performed by adding T4 DNA ligase buffer T4 DNA polymerase, Klenow DNA polymerase, T4 PNK. Standard Illumina adapters (FC) were ligated to the cDNA fragments using 2× DNA ligase buffer, 1 µL of adapter oligo mix and DNA ligase. The sample was purified in a 2% low-melting point agarose gel, and a slice of gel containing 200-bp fragments was removed and the DNA purified. A aliquot of purified cDNA library was amplified by PCR using the pfx DNA polymerase with genomic primers 1.1 and 2.1 (Illumina). All libraries were sequenced on a HiSeq 2000 platform. Results: A total of 523 N. crassa genes were differentially expressed (log2foldchange ≥ 2; p-value < 0.05) in response to chitosan. Of these, 55.6% (291 genes) were down-regulated and 45.3% (237 genes) up-regulated. This time-course experiment showed a progressive reduction in the number of genes whose expression increased upon exposure to chitosan (142 induced genes at 4h, 119 at 8h and 45 at 16h). In contrast, exposure to chitosan resulted in an increase in the number of genes whose expression levels decreased over time (79, 93 and 207 genes down-regulated at 4, 8 and 16h, respectively). A subset of 22 genes was differentially expressed consistently (log2foldchange ≥ 2; p-value < 0.05) throughout the whole time-course. Expression of 10 N. crassa genes representative of functional categories that were differentially expressed by exposure to chitosan were selected to validate our RNA-seq analysis. Gene expression was evaluated by qRT-PCR following an 8h exposure to chitosan. These genes were involved in the main functions related with the chitosan response. All genes analyzed by qRT-PCR showed an expression pattern consistent with that derived from RNA-seq data analysis. Conclusions: This work provides the first study of the gene expression response of a filamentous fungus (N. crassa) to chitosan. Transcriptomics revealed oxidoreductase activity, membrane homeostasis and microtubule organization as main gene functions differentially expressed. Overall design: Transcriptomic response of N. crassa to sub-lethal concentration of chitosan in a time course experiment (4, 8 and 16h)

研究目的：评估并分析粗糙脉孢菌（N. crassa）萌发分生孢子的转录响应，明确其暴露于壳聚糖（chitosan）时相关的核心基因功能，并鉴定粗糙脉孢菌中壳聚糖作用的主要基因靶点。实验方法：取与壳聚糖分别孵育4、8、16小时的样本，使用TRIzol试剂（TRIzol reagent）提取总RNA，经洗涤后重悬。进行第一链cDNA合成时，将片段化的poly(A)+ RNA与随机六聚体引物共孵育，随后加入Superscript II反转录酶（200 U；Invitrogen公司）完成第一链cDNA合成。通过添加T4 DNA连接酶缓冲液、T4 DNA聚合酶、Klenow DNA聚合酶及T4多核苷酸激酶（T4 PNK）进行末端修复。使用2×DNA连接酶缓冲液、1 μL接头寡核苷酸混合物及DNA连接酶，将标准Illumina接头（FC）连接至cDNA片段。将样本在2%低熔点琼脂糖凝胶中纯化，切取包含200 bp片段的凝胶条带并回收DNA。取等分试样的纯化cDNA文库，使用pfx DNA聚合酶结合Illumina的基因组引物1.1和2.1进行聚合酶链式反应（PCR）扩增。所有文库均在HiSeq 2000测序平台上完成测序。实验结果：共有523个粗糙脉孢菌基因在壳聚糖处理下呈现显著差异表达（log₂倍数变化≥2；P值<0.05）。其中55.6%（291个基因）为下调表达，45.3%（237个基因）为上调表达。本次时间梯度实验显示，随壳聚糖暴露时间延长，上调表达基因的数量逐渐减少：4小时时诱导表达基因为142个，8小时为119个，16小时仅为45个。与之相反，下调表达基因的数量随时间推移逐渐增加：4、8、16小时分别有79、93和207个基因下调。另有22个基因在整个时间进程中均呈现稳定的差异表达（log₂倍数变化≥2；P值<0.05）。选取10个代表壳聚糖响应相关功能类别的粗糙脉孢菌差异表达基因，通过定量实时聚合酶链式反应（qRT-PCR）验证RNA测序（RNA-seq）分析结果：将样本与壳聚糖孵育8小时后，采用qRT-PCR检测基因表达水平，这些基因均参与壳聚糖响应的核心生物学过程。所有经qRT-PCR分析的基因，其表达模式均与RNA-seq数据分析结果一致。研究结论：本研究首次解析了丝状真菌粗糙脉孢菌对壳聚糖的基因表达响应。转录组学分析显示，氧化还原酶活性、膜稳态及微管组织是差异表达基因富集的核心基因功能。实验设计：本研究以亚致死浓度的壳聚糖处理粗糙脉孢菌，通过4、8、16小时的时间梯度实验分析其转录组响应。