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Sample Set Information

ID TSE1344
Title Rice-Arabidopsis FOX line screening with FT-NIR-based fingerprinting for GC-TOF/MS-based metabolite profiling
Description The full-length cDNA over-expressing (FOX) gene hunting system is useful for genome-wide gain-of-function analysis. The screening of FOX lines requires a high-throughput metabolomic method that can detect a wide range of metabolites. Fourier transform-near-infrared (FT-NIR) spectroscopy in combination with the chemometric approach has been used to analyze metabolite fingerprints. Since FT-NIR spectroscopy can be used to analyze a solid sample without destructive extraction, this technique enables untargeted analysis and high-throughput screening focusing on the alteration of metabolite composition. We performed non-destructive FT-NIR-based fingerprinting to screen seed samples of 3000 rice-Arabidopsis FOX lines; the samples were obtained from transgenic Arabidopsis thaliana lines that overexpressed rice full-length cDNA. Subsequently, the candidate lines exhibiting alteration in their metabolite fingerprints were analyzed by gas chromatography-time-of-flight/mass spectrometry (GC-TOF/MS) in order to assess their metabolite profiles. Finally, multivariate regression using orthogonal projections to latent structures (O2PLS) was used to elucidate the predictive metabolites obtained in FT-NIR analysis by integration of the datasets obtained from FT-NIR and GC-TOF/MS analyses. FT-NIR-based fingerprinting is a technically efficient method in that it facilitates non-destructive analysis in a high-throughput manner. Furthermore, with the integrated analysis used here, we were able to discover unique metabotypes in rice-Arabidopsis FOX lines; thus, this approach is beneficial for investigating the function of rice genes related to metabolism.
Authors Suzuki, M., Kusano, M., Takahashi, H., Nakamura, Y., Hayashi, N., Kobayashi, M., Ichikawa, T., Matsui, M., Hirochika, H. and Saito, K.
Reference Metabolomics, March 2010, Volume 6, Issue 1, pp 137–145

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Sample Information

Title Arabidopsis thaliana
Organism - Scientific Name Arabidopsis thaliana
Organism - ID NCBI taxonomy:3702
Compound - ID
Compound - Source
Preparation For the evaluation of discrimination abilities, seed samples of five various Arabidopsis ecotypes (Col-0, Ws, Ler, Nossen, and C24) were used. Arabidopsis transgenic lines expressing rice full-length cDNA (rice FOX lines) under the control of the CaMV 35S promoter were constructed using the Agrobacterium tumefaciens in planta transformation method (Clough and Bent 1998). We screened seed samples of the T2 generation that did not exhibit any visible phenotypes. A total of 3003 lines (74 batches) were analyzed by FT-NIR spectroscopy. FOX lines in each batch comprised samples that were maximum 51 lines harvested during the same growth period with a cultivation container system (Arasystem, Gent, Belgium). To diminish the environmental effect, we analyzed FOX seeds of each batch separately. Subsequently, genomic DNA of each candidate line exhibiting alteration of metabolite fingerprints was isolated from the corresponding seed sample. Then, rice cDNA was sequenced for the annotation of gene function. For annotation of the inserted rice cDNA, the sequences of the candidate genes were analyzed on the basis of information available in the Knowledge-based Oryza Molecular biological Encyclopedia (KOME) and the Rice Annotation Project (RAP) databases. The re-transformants (five individual transgenic plants for each candidate) were constructed to confirm the reproducibility of the metabotype for each line. The details of the method have been described by Kondou et al (2009). Arabidopsis seeds (Col-0), which were harvested at the same time as the re-transformants, were used as the control.
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