stable and efficient nuclear transformation system for the diatom Chaetoceros gracilis
Chaetoceros gracilis belongs to the centric diatoms, and has recently been used in basic research on photosynthesis. In addition, it has been commercially used in fisheries and is also attracting interest as a feedstock for biofuels production and biorefinery. In this study, we developed an efficien...
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| Veröffentlicht in: | Photosynthesis research 2015-02, Vol.123 (2), p.203-211 |
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| creator | Ifuku, Kentaro Yan, Dongyi Miyahara, Mado Inoue-Kashino, Natsuko Yamamoto, Yoshiharu Y Kashino, Yasuhiro |
| description | Chaetoceros gracilis belongs to the centric diatoms, and has recently been used in basic research on photosynthesis. In addition, it has been commercially used in fisheries and is also attracting interest as a feedstock for biofuels production and biorefinery. In this study, we developed an efficient genetic transformation system for C. gracilis. The diatom cells were transformed via multi-pulse electroporation using plasmids containing various promoters to drive expression of the nourseothricin acetyltransferase gene (nat) as a selectable marker. The transformation efficiency reached ~400 positive transgenic clones per 10⁸recipient cells, which is the first example of successful transformation with electroporation in a centric diatom species. We further produced two expression vectors: the vector pCgLhcr5p contains the light-dependent promoter of a fucoxanthin chlorophyll a/c binding protein gene and the vector pCgNRp contains the inducible promoter of a nitrate reductase gene to drive the expression of introduced genes. In both vectors, an acetyl-CoA acetyltransferase promoter drives nat gene expression for antibiotic selection. Stable integration and expression of reporter genes, such as the firefly luciferase and green fluorescent protein Azami–Green genes, were observed in transformed C. gracilis cells. This efficient and stable transformation system for C. gracilis will enable both functional analysis of diatom-specific genes and strain improvement for further biotechnological applications. |
| doi_str_mv | 10.1007/s11120-014-0048-y |
| format | Article |
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In addition, it has been commercially used in fisheries and is also attracting interest as a feedstock for biofuels production and biorefinery. In this study, we developed an efficient genetic transformation system for C. gracilis. The diatom cells were transformed via multi-pulse electroporation using plasmids containing various promoters to drive expression of the nourseothricin acetyltransferase gene (nat) as a selectable marker. The transformation efficiency reached ~400 positive transgenic clones per 10⁸recipient cells, which is the first example of successful transformation with electroporation in a centric diatom species. We further produced two expression vectors: the vector pCgLhcr5p contains the light-dependent promoter of a fucoxanthin chlorophyll a/c binding protein gene and the vector pCgNRp contains the inducible promoter of a nitrate reductase gene to drive the expression of introduced genes. In both vectors, an acetyl-CoA acetyltransferase promoter drives nat gene expression for antibiotic selection. Stable integration and expression of reporter genes, such as the firefly luciferase and green fluorescent protein Azami–Green genes, were observed in transformed C. gracilis cells. This efficient and stable transformation system for C. gracilis will enable both functional analysis of diatom-specific genes and strain improvement for further biotechnological applications.</description><identifier>ISSN: 0166-8595</identifier><identifier>EISSN: 1573-5079</identifier><identifier>DOI: 10.1007/s11120-014-0048-y</identifier><identifier>PMID: 25297896</identifier><language>eng</language><publisher>Dordrecht: Springer-Verlag</publisher><subject>acetyl-CoA acetyltransferase ; Algae ; Analysis ; antibiotics ; Bacillariophyceae ; Bacillariophycidae ; binding proteins ; Biochemistry ; biofuels ; Biomass energy ; Biomedical and Life Sciences ; biorefining ; Chaetoceros gracilis ; Chlorophyll ; clones ; Diatoms - drug effects ; Diatoms - genetics ; Diatoms - physiology ; Drug Resistance, Microbial - genetics ; Electroporation ; Emerging Techniques ; Enzymes ; feedstocks ; fisheries ; Gene expression ; Gene Expression Regulation ; Genetic engineering ; genetic transformation ; Genetic Vectors ; genetically modified organisms ; green fluorescent protein ; Life Sciences ; Luciferase ; nitrate reductase ; Photosynthesis ; Phytochemistry ; Plant Genetics and Genomics ; Plant Physiology ; Plant Sciences ; plasmids ; Plasmids - genetics ; Protein binding ; reporter genes ; Streptothricins - pharmacology ; Transformation, Genetic</subject><ispartof>Photosynthesis research, 2015-02, Vol.123 (2), p.203-211</ispartof><rights>Springer Science+Business Media Dordrecht 2014</rights><rights>COPYRIGHT 2015 Springer</rights><rights>Springer Science+Business Media Dordrecht 2015</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c572t-adef01cf55c0f30317e5ac2817fa68360764526d007d59086ea9194f7b4d7cd93</citedby><cites>FETCH-LOGICAL-c572t-adef01cf55c0f30317e5ac2817fa68360764526d007d59086ea9194f7b4d7cd93</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://link.springer.com/content/pdf/10.1007/s11120-014-0048-y$$EPDF$$P50$$Gspringer$$H</linktopdf><linktohtml>$$Uhttps://link.springer.com/10.1007/s11120-014-0048-y$$EHTML$$P50$$Gspringer$$H</linktohtml><link.rule.ids>314,780,784,27924,27925,41488,42557,51319</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/25297896$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Ifuku, Kentaro</creatorcontrib><creatorcontrib>Yan, Dongyi</creatorcontrib><creatorcontrib>Miyahara, Mado</creatorcontrib><creatorcontrib>Inoue-Kashino, Natsuko</creatorcontrib><creatorcontrib>Yamamoto, Yoshiharu Y</creatorcontrib><creatorcontrib>Kashino, Yasuhiro</creatorcontrib><title>stable and efficient nuclear transformation system for the diatom Chaetoceros gracilis</title><title>Photosynthesis research</title><addtitle>Photosynth Res</addtitle><addtitle>Photosynth Res</addtitle><description>Chaetoceros gracilis belongs to the centric diatoms, and has recently been used in basic research on photosynthesis. In addition, it has been commercially used in fisheries and is also attracting interest as a feedstock for biofuels production and biorefinery. In this study, we developed an efficient genetic transformation system for C. gracilis. The diatom cells were transformed via multi-pulse electroporation using plasmids containing various promoters to drive expression of the nourseothricin acetyltransferase gene (nat) as a selectable marker. The transformation efficiency reached ~400 positive transgenic clones per 10⁸recipient cells, which is the first example of successful transformation with electroporation in a centric diatom species. We further produced two expression vectors: the vector pCgLhcr5p contains the light-dependent promoter of a fucoxanthin chlorophyll a/c binding protein gene and the vector pCgNRp contains the inducible promoter of a nitrate reductase gene to drive the expression of introduced genes. In both vectors, an acetyl-CoA acetyltransferase promoter drives nat gene expression for antibiotic selection. Stable integration and expression of reporter genes, such as the firefly luciferase and green fluorescent protein Azami–Green genes, were observed in transformed C. gracilis cells. This efficient and stable transformation system for C. gracilis will enable both functional analysis of diatom-specific genes and strain improvement for further biotechnological applications.</description><subject>acetyl-CoA acetyltransferase</subject><subject>Algae</subject><subject>Analysis</subject><subject>antibiotics</subject><subject>Bacillariophyceae</subject><subject>Bacillariophycidae</subject><subject>binding proteins</subject><subject>Biochemistry</subject><subject>biofuels</subject><subject>Biomass energy</subject><subject>Biomedical and Life Sciences</subject><subject>biorefining</subject><subject>Chaetoceros gracilis</subject><subject>Chlorophyll</subject><subject>clones</subject><subject>Diatoms - drug effects</subject><subject>Diatoms - genetics</subject><subject>Diatoms - physiology</subject><subject>Drug Resistance, Microbial - genetics</subject><subject>Electroporation</subject><subject>Emerging Techniques</subject><subject>Enzymes</subject><subject>feedstocks</subject><subject>fisheries</subject><subject>Gene expression</subject><subject>Gene Expression Regulation</subject><subject>Genetic engineering</subject><subject>genetic transformation</subject><subject>Genetic Vectors</subject><subject>genetically modified organisms</subject><subject>green fluorescent protein</subject><subject>Life Sciences</subject><subject>Luciferase</subject><subject>nitrate reductase</subject><subject>Photosynthesis</subject><subject>Phytochemistry</subject><subject>Plant Genetics and Genomics</subject><subject>Plant Physiology</subject><subject>Plant Sciences</subject><subject>plasmids</subject><subject>Plasmids - genetics</subject><subject>Protein binding</subject><subject>reporter genes</subject><subject>Streptothricins - pharmacology</subject><subject>Transformation, Genetic</subject><issn>0166-8595</issn><issn>1573-5079</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2015</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><sourceid>ABUWG</sourceid><sourceid>AFKRA</sourceid><sourceid>AZQEC</sourceid><sourceid>BENPR</sourceid><sourceid>CCPQU</sourceid><sourceid>DWQXO</sourceid><sourceid>GNUQQ</sourceid><recordid>eNqNkcFrFTEQxoMo9rX6B3jRBS962DrJbjbZY3loLRQEa72GvOxkm7Kb1CQLff-9eWwV60Ekh8DM7xvmm4-QVxROKYD4kCilDGqgbQ3Qynr_hGwoF03NQfRPyQZo19WS9_yIHKd0CwCyo81zcsQ464Xsuw35nrLeTVhpP1RorTMOfa78YibUscpR-2RDnHV2wVdpnzLOVSlU-Qarwekc5mp7ozEHgzGkaozauMmlF-SZ1VPClw__Cbn-9PHb9nN9-eX8Ynt2WRsuWK71gBaosZwbsA00VCDXhkkqrO5k04HoWs66oZgdeF-2R93TvrVi1w7CDH1zQt6tc-9i-LFgymp2yeA0aY9hSaocAFroO8r-A-WspcClLOjbv9DbsERfjBSqFVxSkLxQpys16gmV8zaUc5nyBpydCR6tK_WzFgRjUrKD4P0jQWEy3udRLympi6uvj1m6sqacNUW06i66Wce9oqAO4as1fFXCV4fw1b5oXj-svexmHH4rfqVdALYCqbT8iPEPX_-Y-mYVWR2UHqNL6vqKAeUADBpgovkJ6XHBKg</recordid><startdate>20150201</startdate><enddate>20150201</enddate><creator>Ifuku, Kentaro</creator><creator>Yan, Dongyi</creator><creator>Miyahara, Mado</creator><creator>Inoue-Kashino, Natsuko</creator><creator>Yamamoto, Yoshiharu Y</creator><creator>Kashino, Yasuhiro</creator><general>Springer-Verlag</general><general>Springer Netherlands</general><general>Springer</general><general>Springer Nature B.V</general><scope>FBQ</scope><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>ISR</scope><scope>3V.</scope><scope>7QP</scope><scope>7X7</scope><scope>7XB</scope><scope>88A</scope><scope>88E</scope><scope>88I</scope><scope>8AO</scope><scope>8FE</scope><scope>8FH</scope><scope>8FI</scope><scope>8FJ</scope><scope>8FK</scope><scope>ABUWG</scope><scope>AFKRA</scope><scope>AZQEC</scope><scope>BBNVY</scope><scope>BENPR</scope><scope>BHPHI</scope><scope>CCPQU</scope><scope>DWQXO</scope><scope>FYUFA</scope><scope>GHDGH</scope><scope>GNUQQ</scope><scope>HCIFZ</scope><scope>K9.</scope><scope>LK8</scope><scope>M0S</scope><scope>M1P</scope><scope>M2P</scope><scope>M7N</scope><scope>M7P</scope><scope>PQEST</scope><scope>PQQKQ</scope><scope>PQUKI</scope><scope>PRINS</scope><scope>Q9U</scope><scope>7X8</scope><scope>F1W</scope><scope>H95</scope><scope>L.G</scope></search><sort><creationdate>20150201</creationdate><title>stable and efficient nuclear transformation system for the diatom Chaetoceros gracilis</title><author>Ifuku, Kentaro ; Yan, Dongyi ; Miyahara, Mado ; Inoue-Kashino, Natsuko ; Yamamoto, Yoshiharu Y ; Kashino, Yasuhiro</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c572t-adef01cf55c0f30317e5ac2817fa68360764526d007d59086ea9194f7b4d7cd93</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2015</creationdate><topic>acetyl-CoA acetyltransferase</topic><topic>Algae</topic><topic>Analysis</topic><topic>antibiotics</topic><topic>Bacillariophyceae</topic><topic>Bacillariophycidae</topic><topic>binding proteins</topic><topic>Biochemistry</topic><topic>biofuels</topic><topic>Biomass energy</topic><topic>Biomedical and Life Sciences</topic><topic>biorefining</topic><topic>Chaetoceros gracilis</topic><topic>Chlorophyll</topic><topic>clones</topic><topic>Diatoms - drug effects</topic><topic>Diatoms - genetics</topic><topic>Diatoms - physiology</topic><topic>Drug Resistance, Microbial - genetics</topic><topic>Electroporation</topic><topic>Emerging Techniques</topic><topic>Enzymes</topic><topic>feedstocks</topic><topic>fisheries</topic><topic>Gene expression</topic><topic>Gene Expression Regulation</topic><topic>Genetic engineering</topic><topic>genetic transformation</topic><topic>Genetic Vectors</topic><topic>genetically modified organisms</topic><topic>green fluorescent protein</topic><topic>Life Sciences</topic><topic>Luciferase</topic><topic>nitrate reductase</topic><topic>Photosynthesis</topic><topic>Phytochemistry</topic><topic>Plant Genetics and Genomics</topic><topic>Plant Physiology</topic><topic>Plant Sciences</topic><topic>plasmids</topic><topic>Plasmids - genetics</topic><topic>Protein binding</topic><topic>reporter genes</topic><topic>Streptothricins - pharmacology</topic><topic>Transformation, Genetic</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Ifuku, Kentaro</creatorcontrib><creatorcontrib>Yan, Dongyi</creatorcontrib><creatorcontrib>Miyahara, Mado</creatorcontrib><creatorcontrib>Inoue-Kashino, Natsuko</creatorcontrib><creatorcontrib>Yamamoto, Yoshiharu Y</creatorcontrib><creatorcontrib>Kashino, Yasuhiro</creatorcontrib><collection>AGRIS</collection><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>Gale In Context: Science</collection><collection>ProQuest Central (Corporate)</collection><collection>Calcium & Calcified Tissue Abstracts</collection><collection>Health & Medical Collection</collection><collection>ProQuest Central (purchase pre-March 2016)</collection><collection>Biology Database (Alumni Edition)</collection><collection>Medical Database (Alumni Edition)</collection><collection>Science Database (Alumni Edition)</collection><collection>ProQuest Pharma Collection</collection><collection>ProQuest SciTech Collection</collection><collection>ProQuest Natural Science Collection</collection><collection>Hospital Premium Collection</collection><collection>Hospital Premium Collection (Alumni Edition)</collection><collection>ProQuest Central (Alumni) (purchase pre-March 2016)</collection><collection>ProQuest Central (Alumni Edition)</collection><collection>ProQuest Central UK/Ireland</collection><collection>ProQuest Central Essentials</collection><collection>Biological Science Collection</collection><collection>ProQuest Central</collection><collection>Natural Science Collection</collection><collection>ProQuest One Community College</collection><collection>ProQuest Central Korea</collection><collection>Health Research Premium Collection</collection><collection>Health Research Premium Collection (Alumni)</collection><collection>ProQuest Central Student</collection><collection>SciTech Premium Collection</collection><collection>ProQuest Health & Medical Complete (Alumni)</collection><collection>ProQuest Biological Science Collection</collection><collection>Health & Medical Collection (Alumni Edition)</collection><collection>Medical Database</collection><collection>Science Database</collection><collection>Algology Mycology and Protozoology Abstracts (Microbiology C)</collection><collection>Biological Science Database</collection><collection>ProQuest One Academic Eastern Edition (DO NOT USE)</collection><collection>ProQuest One Academic</collection><collection>ProQuest One Academic UKI Edition</collection><collection>ProQuest Central China</collection><collection>ProQuest Central Basic</collection><collection>MEDLINE - Academic</collection><collection>ASFA: Aquatic Sciences and Fisheries Abstracts</collection><collection>Aquatic Science & Fisheries Abstracts (ASFA) 1: Biological Sciences & Living Resources</collection><collection>Aquatic Science & Fisheries Abstracts (ASFA) Professional</collection><jtitle>Photosynthesis research</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Ifuku, Kentaro</au><au>Yan, Dongyi</au><au>Miyahara, Mado</au><au>Inoue-Kashino, Natsuko</au><au>Yamamoto, Yoshiharu Y</au><au>Kashino, Yasuhiro</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>stable and efficient nuclear transformation system for the diatom Chaetoceros gracilis</atitle><jtitle>Photosynthesis research</jtitle><stitle>Photosynth Res</stitle><addtitle>Photosynth Res</addtitle><date>2015-02-01</date><risdate>2015</risdate><volume>123</volume><issue>2</issue><spage>203</spage><epage>211</epage><pages>203-211</pages><issn>0166-8595</issn><eissn>1573-5079</eissn><abstract>Chaetoceros gracilis belongs to the centric diatoms, and has recently been used in basic research on photosynthesis. In addition, it has been commercially used in fisheries and is also attracting interest as a feedstock for biofuels production and biorefinery. In this study, we developed an efficient genetic transformation system for C. gracilis. The diatom cells were transformed via multi-pulse electroporation using plasmids containing various promoters to drive expression of the nourseothricin acetyltransferase gene (nat) as a selectable marker. The transformation efficiency reached ~400 positive transgenic clones per 10⁸recipient cells, which is the first example of successful transformation with electroporation in a centric diatom species. We further produced two expression vectors: the vector pCgLhcr5p contains the light-dependent promoter of a fucoxanthin chlorophyll a/c binding protein gene and the vector pCgNRp contains the inducible promoter of a nitrate reductase gene to drive the expression of introduced genes. In both vectors, an acetyl-CoA acetyltransferase promoter drives nat gene expression for antibiotic selection. Stable integration and expression of reporter genes, such as the firefly luciferase and green fluorescent protein Azami–Green genes, were observed in transformed C. gracilis cells. This efficient and stable transformation system for C. gracilis will enable both functional analysis of diatom-specific genes and strain improvement for further biotechnological applications.</abstract><cop>Dordrecht</cop><pub>Springer-Verlag</pub><pmid>25297896</pmid><doi>10.1007/s11120-014-0048-y</doi><tpages>9</tpages></addata></record> |
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| ispartof | Photosynthesis research, 2015-02, Vol.123 (2), p.203-211 |
| issn | 0166-8595 1573-5079 |
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| subjects | acetyl-CoA acetyltransferase Algae Analysis antibiotics Bacillariophyceae Bacillariophycidae binding proteins Biochemistry biofuels Biomass energy Biomedical and Life Sciences biorefining Chaetoceros gracilis Chlorophyll clones Diatoms - drug effects Diatoms - genetics Diatoms - physiology Drug Resistance, Microbial - genetics Electroporation Emerging Techniques Enzymes feedstocks fisheries Gene expression Gene Expression Regulation Genetic engineering genetic transformation Genetic Vectors genetically modified organisms green fluorescent protein Life Sciences Luciferase nitrate reductase Photosynthesis Phytochemistry Plant Genetics and Genomics Plant Physiology Plant Sciences plasmids Plasmids - genetics Protein binding reporter genes Streptothricins - pharmacology Transformation, Genetic |
| title | stable and efficient nuclear transformation system for the diatom Chaetoceros gracilis |
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