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

ABSTRACT: 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.

ORGANISM(S): Neurospora crassa OR74A

PROVIDER: GSE75293 | GEO | 2016/11/23

SECONDARY ACCESSION(S): PRJNA303140

REPOSITORIES: GEO

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Project description:Chitosan has been widely used in food industry as a weight-loss aid and a cholesterol-lowering agent. Previous studies have shown that chitosan affects metabolic responses and contributes to anti-diabetic, hypocholestermic, and blood glucose-lowering effects; however, the in vivo targeting sites and mechanisms of chitosan remain to be clarified. In this study, we constructed transgenic mice which carried the luciferase genes driven by peroxisome proliferator-activated receptor (PPAR), a key regulator of fatty acid and glucose metabolism. Bioluminescent imaging of PPAR transgenic mice was applied to report the organs that chitosan acted on, and gene expression profiles of chitosan-targeted organs were further analyzed to elucidate the mechanisms of chitosan. Bioluminescent imaging showed that constitutive PPAR activities were detected in brain and gastrointestinal tract. Administration of chitosan significantly activated the PPAR activities in brain and stomach. Microarray analysis of brain and stomach showed that several pathways involved in lipid and glucose metabolism were regulated by chitosan. Moreover, the expression levels of metabolism-associated genes like apolipoprotein B (apoB) and ghrelin genes were down-regulated by chitosan. In conclusion, these findings suggested the feasibility of PPAR bioluminescent imaging-guided transcriptomic analysis on the evaluation of chitosan-affected metabolic responses in vivo. Moreover, we newly identified that downregulated expression of apoB and ghrelin genes were novel mechanisms for chitosan-affected metabolic responses in vivo . Mice (6 to 8 weeks old) were subcutaneously injected saline or 0.2 g/kg chitosan. Chitosan oligosaccharide lactate (MW=4000-6000, >90% deacetylation) was purchased from Sigma-Aldrich (St. Louis, MO, USA) and dissolved in DDW. For rosiglitazone treatment, mice were orally administered 50 mg/kg rosiglitazone. Mice were then imaged for the luciferase activity or sacrificed for microarray analysis at indicated periods.

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

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