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      Overexpression of Tisochrysis lutea Akd1 identifies a key cold-induced alkenone desaturase enzyme

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          Abstract

          Alkenones are unusual long-chain neutral lipids that were first identified in oceanic sediments. Currently they are regarded as reliable palaeothermometers, since their unsaturation status changes depending on temperature. These molecules are synthesised by specific haptophyte algae and are stored in the lipid body as the main energy storage molecules. However, the molecular mechanisms that regulate the alkenone biosynthetic pathway, especially the low temperature-dependent desaturation reaction, have not been elucidated. Here, using an alkenone-producing haptophyte alga, Tisochrysis lutea, we show that the alkenone desaturation reaction is catalysed by a newly identified desaturase. We first isolated two candidate desaturase genes and found that one of these genes was drastically upregulated in response to cold stress. Gas chromatographic analysis revealed that the overexpression of this gene, named as Akd1 finally, increased the conversion of di-unsaturated C 37-alkenone to tri-unsaturated molecule by alkenone desaturation, even at a high temperature when endogenous desaturation is efficiently suppressed. We anticipate that the Akd1 gene will be of great help for elucidating more detailed mechanisms of temperature response of alkenone desaturation, and identification of active species contributing alkenone production in metagenomic and/or metatranscriptomic studies in the field of oceanic biogeochemistry.

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          Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method.

          The two most commonly used methods to analyze data from real-time, quantitative PCR experiments are absolute quantification and relative quantification. Absolute quantification determines the input copy number, usually by relating the PCR signal to a standard curve. Relative quantification relates the PCR signal of the target transcript in a treatment group to that of another sample such as an untreated control. The 2(-Delta Delta C(T)) method is a convenient way to analyze the relative changes in gene expression from real-time quantitative PCR experiments. The purpose of this report is to present the derivation, assumptions, and applications of the 2(-Delta Delta C(T)) method. In addition, we present the derivation and applications of two variations of the 2(-Delta Delta C(T)) method that may be useful in the analysis of real-time, quantitative PCR data. Copyright 2001 Elsevier Science (USA).
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            Multiple sequence alignment with the Clustal series of programs.

            R Chenna (2003)
            The Clustal series of programs are widely used in molecular biology for the multiple alignment of both nucleic acid and protein sequences and for preparing phylogenetic trees. The popularity of the programs depends on a number of factors, including not only the accuracy of the results, but also the robustness, portability and user-friendliness of the programs. New features include NEXUS and FASTA format output, printing range numbers and faster tree calculation. Although, Clustal was originally developed to run on a local computer, numerous Web servers have been set up, notably at the EBI (European Bioinformatics Institute) (http://www.ebi.ac.uk/clustalw/).
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              W-IQ-TREE: a fast online phylogenetic tool for maximum likelihood analysis

              This article presents W-IQ-TREE, an intuitive and user-friendly web interface and server for IQ-TREE, an efficient phylogenetic software for maximum likelihood analysis. W-IQ-TREE supports multiple sequence types (DNA, protein, codon, binary and morphology) in common alignment formats and a wide range of evolutionary models including mixture and partition models. W-IQ-TREE performs fast model selection, partition scheme finding, efficient tree reconstruction, ultrafast bootstrapping, branch tests, and tree topology tests. All computations are conducted on a dedicated computer cluster and the users receive the results via URL or email. W-IQ-TREE is available at http://iqtree.cibiv.univie.ac.at. It is free and open to all users and there is no login requirement.
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                Author and article information

                Contributors
                emilhux@biol.tsukuba.ac.jp
                Journal
                Sci Rep
                Sci Rep
                Scientific Reports
                Nature Publishing Group UK (London )
                2045-2322
                25 July 2018
                25 July 2018
                2018
                : 8
                : 11230
                Affiliations
                [1 ]ISNI 0000 0001 2369 4728, GRID grid.20515.33, Faculty of Life and Environmental Sciences, , University of Tsukuba, ; 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8572 Japan
                [2 ]ISNI 0000 0001 2159 3886, GRID grid.412018.e, Present Address: College of Science and Engineering, , Kanto Gakuin University, ; 1-50-1 Mutsuura-higashi, Kanazawa-ku, Yokohama, Kanagawa 236-8501 Japan
                Article
                29482
                10.1038/s41598-018-29482-8
                6060089
                30046151
                6d683a9d-02da-4231-8bb8-0f3c86f5a2c5
                © The Author(s) 2018

                Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

                History
                : 10 April 2018
                : 12 July 2018
                Funding
                Funded by: FundRef https://doi.org/10.13039/501100002241, Japan Science and Technology Agency (JST);
                Award ID: 16010580400
                Award Recipient :
                Funded by: FundRef https://doi.org/10.13039/501100001863, New Energy and Industrial Technology Development Organization (NEDO);
                Award ID: not provided
                Award Recipient :
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