Molecular clone and expression of a NAD+-dependent glycerol-3-phosphate dehydrogenase isozyme gene from the halotolerant alga Dunaliella salina
Glycerol is an important osmotically compatible solute in Dunaliella. Glycerol-3-phosphate dehydrogenase (G3PDH) is a key enzyme in the pathway of glycerol synthesis, which converts dihydroxyacetone phosphate (DHAP) to glycerol-3-phosphate. Generally, the glycerol-DHAP cycle pathway, which is driven...
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| description | Glycerol is an important osmotically compatible solute in Dunaliella. Glycerol-3-phosphate dehydrogenase (G3PDH) is a key enzyme in the pathway of glycerol synthesis, which converts dihydroxyacetone phosphate (DHAP) to glycerol-3-phosphate. Generally, the glycerol-DHAP cycle pathway, which is driven by G3PDH, is considered as the rate-limiting enzyme to regulate the glycerol level under osmotic shocks. Considering the peculiarity in osmoregulation, the cDNA of a NAD(+)-dependent G3PDH was isolated from D. salina using RACE and RT-PCR approaches in this study. Results indicated that the length of the cDNA sequence of G3PDH was 2,100 bp encoding a 699 amino acid deduced polypeptide whose computational molecular weight was 76.6 kDa. Conserved domain analysis revealed that the G3PDH protein has two independent functional domains, SerB and G3PDH domains. It was predicted that the G3PDH was a nonsecretory protein and may be located in the chloroplast of D. salina. Phylogenetic analysis demonstrated that the D. salina G3PDH had a closer relationship with the G3PDHs from the Dunaliella genus than with those from other species. In addition, the cDNA was subsequently subcloned in the pET-32a(+) vector and was transformed into E. coli strain BL21 (DE3), a expression protein with 100 kDa was identified, which was consistent with the theoretical value. |
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Glycerol-3-phosphate dehydrogenase (G3PDH) is a key enzyme in the pathway of glycerol synthesis, which converts dihydroxyacetone phosphate (DHAP) to glycerol-3-phosphate. Generally, the glycerol-DHAP cycle pathway, which is driven by G3PDH, is considered as the rate-limiting enzyme to regulate the glycerol level under osmotic shocks. Considering the peculiarity in osmoregulation, the cDNA of a NAD(+)-dependent G3PDH was isolated from D. salina using RACE and RT-PCR approaches in this study. Results indicated that the length of the cDNA sequence of G3PDH was 2,100 bp encoding a 699 amino acid deduced polypeptide whose computational molecular weight was 76.6 kDa. Conserved domain analysis revealed that the G3PDH protein has two independent functional domains, SerB and G3PDH domains. It was predicted that the G3PDH was a nonsecretory protein and may be located in the chloroplast of D. salina. Phylogenetic analysis demonstrated that the D. salina G3PDH had a closer relationship with the G3PDHs from the Dunaliella genus than with those from other species. In addition, the cDNA was subsequently subcloned in the pET-32a(+) vector and was transformed into E. coli strain BL21 (DE3), a expression protein with 100 kDa was identified, which was consistent with the theoretical value.</description><identifier>ISSN: 1932-6203</identifier><identifier>EISSN: 1932-6203</identifier><identifier>DOI: 10.1371/journal.pone.0062287</identifier><identifier>PMID: 23626797</identifier><language>eng</language><publisher>United States: Public Library of Science</publisher><subject>Abiotic stress ; Agriculture ; Algae ; Amino Acid Sequence ; Bioinformatics ; Biology ; Chlorophyta - classification ; Chlorophyta - genetics ; Chlorophyta - metabolism ; Cloning ; Cloning, Molecular ; Computational Biology ; Computer applications ; Dehydrogenase ; Dehydrogenases ; Dihydroxyacetone ; Dihydroxyacetone phosphate ; DNA, Complementary - chemistry ; DNA, Complementary - genetics ; Dunaliella ; Dunaliella salina ; Dunaliella tertiolecta ; E coli ; Enzyme Activation ; Gene Expression ; Genes ; Glycerol ; Glycerol-3-phosphate ; Glycerol-3-phosphate dehydrogenase ; Glycerol-3-Phosphate Dehydrogenase (NAD+) - chemistry ; Glycerol-3-Phosphate Dehydrogenase (NAD+) - genetics ; Glycerol-3-Phosphate Dehydrogenase (NAD+) - isolation & purification ; Glycerol-3-Phosphate Dehydrogenase (NAD+) - metabolism ; Isoenzymes ; Laboratories ; Life sciences ; Manufacturers ; Models, Molecular ; Molecular Sequence Data ; Molecular weight ; NAD ; Osmoregulation ; Phosphates ; Phylogeny ; Polymerase chain reaction ; Protein Conformation ; Proteins ; Recombinant Proteins ; Salinity ; Salinity tolerance ; Sequence Alignment</subject><ispartof>PloS one, 2013-04, Vol.8 (4), p.e62287-e62287</ispartof><rights>2013 Cai et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License: https://creativecommons.org/licenses/by/4.0/ (the “License”), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Notwithstanding the ProQuest Terms and Conditions, you may use this content in accordance with the terms of the License.</rights><rights>2013 Cai et al 2013 Cai et al</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c526t-239487dc61b5609ebbb6d8b142078a563b07a1b392c8a5d8d4e866a2a91d2b0a3</citedby><cites>FETCH-LOGICAL-c526t-239487dc61b5609ebbb6d8b142078a563b07a1b392c8a5d8d4e866a2a91d2b0a3</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC3633914/pdf/$$EPDF$$P50$$Gpubmedcentral$$Hfree_for_read</linktopdf><linktohtml>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC3633914/$$EHTML$$P50$$Gpubmedcentral$$Hfree_for_read</linktohtml><link.rule.ids>230,314,727,780,784,864,885,2102,2928,23866,27924,27925,53791,53793</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/23626797$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><contributor>Cotterill, Sue</contributor><creatorcontrib>Cai, Ma</creatorcontrib><creatorcontrib>He, Li-Hong</creatorcontrib><creatorcontrib>Yu, Tu-Yuan</creatorcontrib><title>Molecular clone and expression of a NAD+-dependent glycerol-3-phosphate dehydrogenase isozyme gene from the halotolerant alga Dunaliella salina</title><title>PloS one</title><addtitle>PLoS One</addtitle><description>Glycerol is an important osmotically compatible solute in Dunaliella. Glycerol-3-phosphate dehydrogenase (G3PDH) is a key enzyme in the pathway of glycerol synthesis, which converts dihydroxyacetone phosphate (DHAP) to glycerol-3-phosphate. Generally, the glycerol-DHAP cycle pathway, which is driven by G3PDH, is considered as the rate-limiting enzyme to regulate the glycerol level under osmotic shocks. Considering the peculiarity in osmoregulation, the cDNA of a NAD(+)-dependent G3PDH was isolated from D. salina using RACE and RT-PCR approaches in this study. Results indicated that the length of the cDNA sequence of G3PDH was 2,100 bp encoding a 699 amino acid deduced polypeptide whose computational molecular weight was 76.6 kDa. Conserved domain analysis revealed that the G3PDH protein has two independent functional domains, SerB and G3PDH domains. It was predicted that the G3PDH was a nonsecretory protein and may be located in the chloroplast of D. salina. Phylogenetic analysis demonstrated that the D. salina G3PDH had a closer relationship with the G3PDHs from the Dunaliella genus than with those from other species. In addition, the cDNA was subsequently subcloned in the pET-32a(+) vector and was transformed into E. coli strain BL21 (DE3), a expression protein with 100 kDa was identified, which was consistent with the theoretical value.</description><subject>Abiotic stress</subject><subject>Agriculture</subject><subject>Algae</subject><subject>Amino Acid Sequence</subject><subject>Bioinformatics</subject><subject>Biology</subject><subject>Chlorophyta - classification</subject><subject>Chlorophyta - genetics</subject><subject>Chlorophyta - metabolism</subject><subject>Cloning</subject><subject>Cloning, Molecular</subject><subject>Computational Biology</subject><subject>Computer applications</subject><subject>Dehydrogenase</subject><subject>Dehydrogenases</subject><subject>Dihydroxyacetone</subject><subject>Dihydroxyacetone phosphate</subject><subject>DNA, Complementary - chemistry</subject><subject>DNA, Complementary - genetics</subject><subject>Dunaliella</subject><subject>Dunaliella salina</subject><subject>Dunaliella tertiolecta</subject><subject>E coli</subject><subject>Enzyme Activation</subject><subject>Gene Expression</subject><subject>Genes</subject><subject>Glycerol</subject><subject>Glycerol-3-phosphate</subject><subject>Glycerol-3-phosphate dehydrogenase</subject><subject>Glycerol-3-Phosphate Dehydrogenase (NAD+) - chemistry</subject><subject>Glycerol-3-Phosphate Dehydrogenase (NAD+) - genetics</subject><subject>Glycerol-3-Phosphate Dehydrogenase (NAD+) - isolation & purification</subject><subject>Glycerol-3-Phosphate Dehydrogenase (NAD+) - metabolism</subject><subject>Isoenzymes</subject><subject>Laboratories</subject><subject>Life sciences</subject><subject>Manufacturers</subject><subject>Models, Molecular</subject><subject>Molecular Sequence Data</subject><subject>Molecular weight</subject><subject>NAD</subject><subject>Osmoregulation</subject><subject>Phosphates</subject><subject>Phylogeny</subject><subject>Polymerase chain reaction</subject><subject>Protein Conformation</subject><subject>Proteins</subject><subject>Recombinant Proteins</subject><subject>Salinity</subject><subject>Salinity tolerance</subject><subject>Sequence Alignment</subject><issn>1932-6203</issn><issn>1932-6203</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2013</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><sourceid>DOA</sourceid><recordid>eNptUttu1DAQjRAVLYU_QGCJFySUxbc4zgtS1XKpVOAFnq1xPLvJyhsHO0EsP8Ev4-1uq7biyTPjM2fmjE5RvGB0wUTN3q3DHAfwizEMuKBUca7rR8UJawQvFafi8Z34uHia0prSSmilnhTHXCiu6qY-Kf5-CR7b2UMkrc9MBAZH8PcYMaU-DCQsCZCvZxdvS4cjDg6Hiaz8tsUYfCnKsQtp7GBC4rDbuhhWOEBC0qfwZ7tBklMkyxg2ZOqQdODDlOdFyCzgV0Au5iyhR--BpBwM8Kw4WoJP-PzwnhY_Pn74fv65vPr26fL87KpsK66mkotG6tq1itlK0QattcppyySntYZKCUtrYFY0vM2p005iVg4cGua4pSBOi1d73tGHZA63TIYJqapGNlpkxOUe4QKszRj7DcStCdCb60KIKwNx6luPZinrtqoUoJZCSg1acLS15VUlLNQOM9f7w7TZbtC1-YoR_D3S-z9D35lV-GWEEqJhMhO8ORDE8HPGNJlNn9rd3QYM8_XeNa-0kk2Gvn4A_b86uUe1MaQUcXm7DKNm56-bLrPzlzn4K7e9vCvktunGUOIfigPQyQ</recordid><startdate>20130423</startdate><enddate>20130423</enddate><creator>Cai, Ma</creator><creator>He, Li-Hong</creator><creator>Yu, Tu-Yuan</creator><general>Public Library of Science</general><general>Public Library of Science (PLoS)</general><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>3V.</scope><scope>7QG</scope><scope>7QL</scope><scope>7QO</scope><scope>7RV</scope><scope>7SN</scope><scope>7SS</scope><scope>7T5</scope><scope>7TG</scope><scope>7TM</scope><scope>7U9</scope><scope>7X2</scope><scope>7X7</scope><scope>7XB</scope><scope>88E</scope><scope>8AO</scope><scope>8C1</scope><scope>8FD</scope><scope>8FE</scope><scope>8FG</scope><scope>8FH</scope><scope>8FI</scope><scope>8FJ</scope><scope>8FK</scope><scope>ABJCF</scope><scope>ABUWG</scope><scope>AFKRA</scope><scope>ARAPS</scope><scope>ATCPS</scope><scope>AZQEC</scope><scope>BBNVY</scope><scope>BENPR</scope><scope>BGLVJ</scope><scope>BHPHI</scope><scope>C1K</scope><scope>CCPQU</scope><scope>D1I</scope><scope>DWQXO</scope><scope>FR3</scope><scope>FYUFA</scope><scope>GHDGH</scope><scope>GNUQQ</scope><scope>H94</scope><scope>HCIFZ</scope><scope>K9.</scope><scope>KB.</scope><scope>KB0</scope><scope>KL.</scope><scope>L6V</scope><scope>LK8</scope><scope>M0K</scope><scope>M0S</scope><scope>M1P</scope><scope>M7N</scope><scope>M7P</scope><scope>M7S</scope><scope>NAPCQ</scope><scope>P5Z</scope><scope>P62</scope><scope>P64</scope><scope>PATMY</scope><scope>PDBOC</scope><scope>PIMPY</scope><scope>PQEST</scope><scope>PQQKQ</scope><scope>PQUKI</scope><scope>PRINS</scope><scope>PTHSS</scope><scope>PYCSY</scope><scope>RC3</scope><scope>7X8</scope><scope>5PM</scope><scope>DOA</scope></search><sort><creationdate>20130423</creationdate><title>Molecular clone and expression of a NAD+-dependent glycerol-3-phosphate dehydrogenase isozyme gene from the halotolerant alga Dunaliella salina</title><author>Cai, Ma ; He, Li-Hong ; Yu, Tu-Yuan</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c526t-239487dc61b5609ebbb6d8b142078a563b07a1b392c8a5d8d4e866a2a91d2b0a3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2013</creationdate><topic>Abiotic stress</topic><topic>Agriculture</topic><topic>Algae</topic><topic>Amino Acid Sequence</topic><topic>Bioinformatics</topic><topic>Biology</topic><topic>Chlorophyta - classification</topic><topic>Chlorophyta - genetics</topic><topic>Chlorophyta - metabolism</topic><topic>Cloning</topic><topic>Cloning, Molecular</topic><topic>Computational Biology</topic><topic>Computer applications</topic><topic>Dehydrogenase</topic><topic>Dehydrogenases</topic><topic>Dihydroxyacetone</topic><topic>Dihydroxyacetone phosphate</topic><topic>DNA, Complementary - chemistry</topic><topic>DNA, Complementary - genetics</topic><topic>Dunaliella</topic><topic>Dunaliella salina</topic><topic>Dunaliella tertiolecta</topic><topic>E coli</topic><topic>Enzyme Activation</topic><topic>Gene Expression</topic><topic>Genes</topic><topic>Glycerol</topic><topic>Glycerol-3-phosphate</topic><topic>Glycerol-3-phosphate dehydrogenase</topic><topic>Glycerol-3-Phosphate Dehydrogenase (NAD+) - chemistry</topic><topic>Glycerol-3-Phosphate Dehydrogenase (NAD+) - genetics</topic><topic>Glycerol-3-Phosphate Dehydrogenase (NAD+) - isolation & purification</topic><topic>Glycerol-3-Phosphate Dehydrogenase (NAD+) - 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Academic</collection><collection>PubMed Central (Full Participant titles)</collection><collection>DOAJ Directory of Open Access Journals</collection><jtitle>PloS one</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Cai, Ma</au><au>He, Li-Hong</au><au>Yu, Tu-Yuan</au><au>Cotterill, Sue</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Molecular clone and expression of a NAD+-dependent glycerol-3-phosphate dehydrogenase isozyme gene from the halotolerant alga Dunaliella salina</atitle><jtitle>PloS one</jtitle><addtitle>PLoS One</addtitle><date>2013-04-23</date><risdate>2013</risdate><volume>8</volume><issue>4</issue><spage>e62287</spage><epage>e62287</epage><pages>e62287-e62287</pages><issn>1932-6203</issn><eissn>1932-6203</eissn><abstract>Glycerol is an important osmotically compatible solute in Dunaliella. Glycerol-3-phosphate dehydrogenase (G3PDH) is a key enzyme in the pathway of glycerol synthesis, which converts dihydroxyacetone phosphate (DHAP) to glycerol-3-phosphate. Generally, the glycerol-DHAP cycle pathway, which is driven by G3PDH, is considered as the rate-limiting enzyme to regulate the glycerol level under osmotic shocks. Considering the peculiarity in osmoregulation, the cDNA of a NAD(+)-dependent G3PDH was isolated from D. salina using RACE and RT-PCR approaches in this study. Results indicated that the length of the cDNA sequence of G3PDH was 2,100 bp encoding a 699 amino acid deduced polypeptide whose computational molecular weight was 76.6 kDa. Conserved domain analysis revealed that the G3PDH protein has two independent functional domains, SerB and G3PDH domains. It was predicted that the G3PDH was a nonsecretory protein and may be located in the chloroplast of D. salina. Phylogenetic analysis demonstrated that the D. salina G3PDH had a closer relationship with the G3PDHs from the Dunaliella genus than with those from other species. In addition, the cDNA was subsequently subcloned in the pET-32a(+) vector and was transformed into E. coli strain BL21 (DE3), a expression protein with 100 kDa was identified, which was consistent with the theoretical value.</abstract><cop>United States</cop><pub>Public Library of Science</pub><pmid>23626797</pmid><doi>10.1371/journal.pone.0062287</doi><oa>free_for_read</oa></addata></record> |
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| subjects | Abiotic stress Agriculture Algae Amino Acid Sequence Bioinformatics Biology Chlorophyta - classification Chlorophyta - genetics Chlorophyta - metabolism Cloning Cloning, Molecular Computational Biology Computer applications Dehydrogenase Dehydrogenases Dihydroxyacetone Dihydroxyacetone phosphate DNA, Complementary - chemistry DNA, Complementary - genetics Dunaliella Dunaliella salina Dunaliella tertiolecta E coli Enzyme Activation Gene Expression Genes Glycerol Glycerol-3-phosphate Glycerol-3-phosphate dehydrogenase Glycerol-3-Phosphate Dehydrogenase (NAD+) - chemistry Glycerol-3-Phosphate Dehydrogenase (NAD+) - genetics Glycerol-3-Phosphate Dehydrogenase (NAD+) - isolation & purification Glycerol-3-Phosphate Dehydrogenase (NAD+) - metabolism Isoenzymes Laboratories Life sciences Manufacturers Models, Molecular Molecular Sequence Data Molecular weight NAD Osmoregulation Phosphates Phylogeny Polymerase chain reaction Protein Conformation Proteins Recombinant Proteins Salinity Salinity tolerance Sequence Alignment |
| title | Molecular clone and expression of a NAD+-dependent glycerol-3-phosphate dehydrogenase isozyme gene from the halotolerant alga Dunaliella salina |
| url | https://sfx.bib-bvb.de/sfx_tum?ctx_ver=Z39.88-2004&ctx_enc=info:ofi/enc:UTF-8&ctx_tim=2024-12-21T08%3A01%3A20IST&url_ver=Z39.88-2004&url_ctx_fmt=infofi/fmt:kev:mtx:ctx&rfr_id=info:sid/primo.exlibrisgroup.com:primo3-Article-proquest_plos_&rft_val_fmt=info:ofi/fmt:kev:mtx:journal&rft.genre=article&rft.atitle=Molecular%20clone%20and%20expression%20of%20a%20NAD+-dependent%20glycerol-3-phosphate%20dehydrogenase%20isozyme%20gene%20from%20the%20halotolerant%20alga%20Dunaliella%20salina&rft.jtitle=PloS%20one&rft.au=Cai,%20Ma&rft.date=2013-04-23&rft.volume=8&rft.issue=4&rft.spage=e62287&rft.epage=e62287&rft.pages=e62287-e62287&rft.issn=1932-6203&rft.eissn=1932-6203&rft_id=info:doi/10.1371/journal.pone.0062287&rft_dat=%3Cproquest_plos_%3E2956612431%3C/proquest_plos_%3E%3Curl%3E%3C/url%3E&disable_directlink=true&sfx.directlink=off&sfx.report_link=0&rft_id=info:oai/&rft_pqid=1346594983&rft_id=info:pmid/23626797&rft_doaj_id=oai_doaj_org_article_f47c556ae843448a832eb7b2553ba7de&rfr_iscdi=true |