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RESEARCH PRODUCT

Role of nitric oxide synthase (NOS) of Klebsormidium nitens: Identification and characterization of partners

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National audience; Nitric oxide (NO) is an important cellular signaling molecule across kingdoms. During bioticor abiotic stresses, NO burst is detected in both plants and mammals although no sequenceencoding the well described mammalian NO synthase (NOS) is highlighted in plants. Comparedto terrestrial plants, some algae present transcripts encoding the NOS-like enzyme. Amongthem, Klebsormidium nitens the model alga to study the early transition step from aquatic algaeto land plants is found. As mechanisms governing NO synthesis and signaling in green lineageremain unclear, the study of NOS from K. nitens (KnNOS) through (i) the identification ofregulator proteins, (ii) the identification of target proteins, and (iii) their expression levels mightbe an important breakthrough knowledge. To achieve this goal, the identification of proteinpartners will be performed by pull down experiments followed by mass spectrometry analysis.Despite the KnNOS is a challenging protein to produce and purify, we are currently optimizinga promising protocol to obtain recombinant GST-tagged KnNOS protein in Escherichia coli.Since expression level analysis require suitable reference genes for reliable normalization, weselected 13 candidate reference genes based on literature, and performed RT-qPCR to studyexpression stability during 4 abiotic stresses commonly used. Final ranking list of most stablereference genes was obtain based on results from algorithms including, the ΔCt method,geNorm, NormFinder and BestKeeper. This finding opens the way for a deeper characterizationof KnNOS and its protein partners and will facilitate further investigation of gene expressionin K. nitens.