Agrobacterium-mediated transformation of the filamentous fungus Aspergillus awamori
Many transformation methods have been developed to introduce DNA into filamentous fungi. One of these methods is Agrobacterium -mediated transformation (AMT). Here, we describe an efficient protocol for AMT of Aspergillus awamori . This protocol has been used to determine the function of Agrobacteri...
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| Veröffentlicht in: | Nature protocols 2008-10, Vol.3 (10), p.1671-1678 |
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| creator | Michielse, Caroline B J Hooykaas, Paul J J J van den Hondel, Cees A M J Ram, Arthur F |
| description | Many transformation methods have been developed to introduce DNA into filamentous fungi. One of these methods is
Agrobacterium
-mediated transformation (AMT). Here, we describe an efficient protocol for AMT of
Aspergillus awamori
. This protocol has been used to determine the function of
Agrobacterium
virulence genes during AMT, to identify factors influencing transformation frequencies, to generate insertional mutants and to generate
A. awamori
gene knockout transformants. This protocol in not only applicable to
A. awamori
, but can be used as a more general guideline for AMT of other filamentous fungi. Conidiospores are incubated with induced
Agrobacterium
, and, after a cocultivation and selection period, hygromycin-resistant transformants are obtained with a frequency of 200–250 transformants per 1 × 10
6
conidiospores. Using this protocol, transformants can be obtained within 10–12 d. |
| doi_str_mv | 10.1038/nprot.2008.154 |
| format | Article |
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Agrobacterium
-mediated transformation (AMT). Here, we describe an efficient protocol for AMT of
Aspergillus awamori
. This protocol has been used to determine the function of
Agrobacterium
virulence genes during AMT, to identify factors influencing transformation frequencies, to generate insertional mutants and to generate
A. awamori
gene knockout transformants. This protocol in not only applicable to
A. awamori
, but can be used as a more general guideline for AMT of other filamentous fungi. Conidiospores are incubated with induced
Agrobacterium
, and, after a cocultivation and selection period, hygromycin-resistant transformants are obtained with a frequency of 200–250 transformants per 1 × 10
6
conidiospores. Using this protocol, transformants can be obtained within 10–12 d.</description><identifier>ISSN: 1754-2189</identifier><identifier>EISSN: 1750-2799</identifier><identifier>DOI: 10.1038/nprot.2008.154</identifier><identifier>PMID: 18833205</identifier><language>eng</language><publisher>London: Nature Publishing Group UK</publisher><subject>Agrobacterium tumefaciens ; Analytical Chemistry ; Aspergillus ; Aspergillus - genetics ; Bacterial transformation ; Biological Techniques ; Biology ; Biomedical and Life Sciences ; Computational Biology/Bioinformatics ; Deoxyribonucleic acid ; DNA ; DNA, Fungal - genetics ; Enzymes ; Fungi ; Gene Expression Regulation, Fungal ; Genes ; Genes, Fungal - genetics ; Genetic aspects ; Genetic Engineering - methods ; Genetic Markers ; Genomics ; Kanamycin ; Life Sciences ; Methods ; Microarrays ; Organic Chemistry ; Plant molecular genetics ; Plasmids ; protocol ; Rhizobium - metabolism ; Transformation, Genetic - physiology ; Virulence</subject><ispartof>Nature protocols, 2008-10, Vol.3 (10), p.1671-1678</ispartof><rights>Springer Nature Limited 2008</rights><rights>COPYRIGHT 2008 Nature Publishing Group</rights><rights>Copyright Nature Publishing Group Oct 2008</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c564t-faceb7c81f0964d0db9ca807fcb170a9cce77def35bd3845862cd7f3fc564e483</citedby><cites>FETCH-LOGICAL-c564t-faceb7c81f0964d0db9ca807fcb170a9cce77def35bd3845862cd7f3fc564e483</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://link.springer.com/content/pdf/10.1038/nprot.2008.154$$EPDF$$P50$$Gspringer$$H</linktopdf><linktohtml>$$Uhttps://link.springer.com/10.1038/nprot.2008.154$$EHTML$$P50$$Gspringer$$H</linktohtml><link.rule.ids>314,776,780,27901,27902,41464,42533,51294</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/18833205$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Michielse, Caroline B</creatorcontrib><creatorcontrib>J Hooykaas, Paul J</creatorcontrib><creatorcontrib>J J van den Hondel, Cees A M</creatorcontrib><creatorcontrib>J Ram, Arthur F</creatorcontrib><title>Agrobacterium-mediated transformation of the filamentous fungus Aspergillus awamori</title><title>Nature protocols</title><addtitle>Nat Protoc</addtitle><addtitle>Nat Protoc</addtitle><description>Many transformation methods have been developed to introduce DNA into filamentous fungi. One of these methods is
Agrobacterium
-mediated transformation (AMT). Here, we describe an efficient protocol for AMT of
Aspergillus awamori
. This protocol has been used to determine the function of
Agrobacterium
virulence genes during AMT, to identify factors influencing transformation frequencies, to generate insertional mutants and to generate
A. awamori
gene knockout transformants. This protocol in not only applicable to
A. awamori
, but can be used as a more general guideline for AMT of other filamentous fungi. Conidiospores are incubated with induced
Agrobacterium
, and, after a cocultivation and selection period, hygromycin-resistant transformants are obtained with a frequency of 200–250 transformants per 1 × 10
6
conidiospores. Using this protocol, transformants can be obtained within 10–12 d.</description><subject>Agrobacterium tumefaciens</subject><subject>Analytical Chemistry</subject><subject>Aspergillus</subject><subject>Aspergillus - genetics</subject><subject>Bacterial transformation</subject><subject>Biological Techniques</subject><subject>Biology</subject><subject>Biomedical and Life Sciences</subject><subject>Computational Biology/Bioinformatics</subject><subject>Deoxyribonucleic acid</subject><subject>DNA</subject><subject>DNA, Fungal - genetics</subject><subject>Enzymes</subject><subject>Fungi</subject><subject>Gene Expression Regulation, Fungal</subject><subject>Genes</subject><subject>Genes, Fungal - genetics</subject><subject>Genetic aspects</subject><subject>Genetic Engineering - methods</subject><subject>Genetic Markers</subject><subject>Genomics</subject><subject>Kanamycin</subject><subject>Life Sciences</subject><subject>Methods</subject><subject>Microarrays</subject><subject>Organic Chemistry</subject><subject>Plant molecular genetics</subject><subject>Plasmids</subject><subject>protocol</subject><subject>Rhizobium - metabolism</subject><subject>Transformation, Genetic - physiology</subject><subject>Virulence</subject><issn>1754-2189</issn><issn>1750-2799</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2008</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><sourceid>BENPR</sourceid><recordid>eNp1kt1rFDEUxYMotq6--igDQkHKbJPJzCR5HBY_CkXBVnwMmczNmDKTrEkG9b832y60Kyt5uBfyO5ebk4PQa4LXBFN-4bbBp3WFMV-Tpn6CTglrcFkxIZ7e9XVZES5O0IsYbzGuGW3Zc3RCOKe0ws0puu7G4HulEwS7zOUMg1UJhiIF5aLxYVbJeld4U6QfUBg7qRlc8ksszOLGXLq4hTDaacq9-qVmH-xL9MyoKcKrfV2hbx_e32w-lVdfPl5uuqtSN22dSqM09ExzYrBo6wEPvdCKY2Z0TxhWQmtgbABDm36gvG54W-mBGWp2cqg5XaGz-7nZg58LxCRnGzVMk3KQN5StaGlFOcng23_AW78El3eTBNeENrTF-IEa1QTSOuOzCXo3UnZEYC5wk11bofURKp8BZqu9g-wRHAreHQgyk-B3GtUSo7y8_nrInv-f7W6-bz4fXUUHH2MAI7fBzir8ya-Su3TIu3TIXTpkTkcWvNn7sPT5px_wfRwycHEPxHzlRgiPjTo68i-c7MS-</recordid><startdate>20081001</startdate><enddate>20081001</enddate><creator>Michielse, Caroline B</creator><creator>J Hooykaas, Paul J</creator><creator>J J van den Hondel, Cees A M</creator><creator>J Ram, Arthur F</creator><general>Nature Publishing Group UK</general><general>Nature Publishing Group</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>ATWCN</scope><scope>ISR</scope><scope>3V.</scope><scope>7QG</scope><scope>7T5</scope><scope>7T7</scope><scope>7TM</scope><scope>7X7</scope><scope>7XB</scope><scope>88E</scope><scope>8FD</scope><scope>8FE</scope><scope>8FH</scope><scope>8FI</scope><scope>8FJ</scope><scope>8FK</scope><scope>ABUWG</scope><scope>AEUYN</scope><scope>AFKRA</scope><scope>ATCPS</scope><scope>AZQEC</scope><scope>BBNVY</scope><scope>BENPR</scope><scope>BHPHI</scope><scope>C1K</scope><scope>CCPQU</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>LK8</scope><scope>M0S</scope><scope>M1P</scope><scope>M7N</scope><scope>M7P</scope><scope>P64</scope><scope>PATMY</scope><scope>PQEST</scope><scope>PQQKQ</scope><scope>PQUKI</scope><scope>PYCSY</scope><scope>RC3</scope><scope>7X8</scope></search><sort><creationdate>20081001</creationdate><title>Agrobacterium-mediated transformation of the filamentous fungus Aspergillus awamori</title><author>Michielse, Caroline B ; J Hooykaas, Paul J ; J J van den Hondel, Cees A M ; J Ram, Arthur F</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c564t-faceb7c81f0964d0db9ca807fcb170a9cce77def35bd3845862cd7f3fc564e483</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2008</creationdate><topic>Agrobacterium tumefaciens</topic><topic>Analytical Chemistry</topic><topic>Aspergillus</topic><topic>Aspergillus - genetics</topic><topic>Bacterial transformation</topic><topic>Biological Techniques</topic><topic>Biology</topic><topic>Biomedical and Life Sciences</topic><topic>Computational Biology/Bioinformatics</topic><topic>Deoxyribonucleic acid</topic><topic>DNA</topic><topic>DNA, Fungal - genetics</topic><topic>Enzymes</topic><topic>Fungi</topic><topic>Gene Expression Regulation, Fungal</topic><topic>Genes</topic><topic>Genes, Fungal - genetics</topic><topic>Genetic aspects</topic><topic>Genetic Engineering - methods</topic><topic>Genetic Markers</topic><topic>Genomics</topic><topic>Kanamycin</topic><topic>Life Sciences</topic><topic>Methods</topic><topic>Microarrays</topic><topic>Organic Chemistry</topic><topic>Plant molecular genetics</topic><topic>Plasmids</topic><topic>protocol</topic><topic>Rhizobium - metabolism</topic><topic>Transformation, Genetic - physiology</topic><topic>Virulence</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Michielse, Caroline B</creatorcontrib><creatorcontrib>J Hooykaas, Paul J</creatorcontrib><creatorcontrib>J J van den Hondel, Cees A M</creatorcontrib><creatorcontrib>J Ram, Arthur F</creatorcontrib><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: Middle School</collection><collection>Gale In Context: Science</collection><collection>ProQuest Central (Corporate)</collection><collection>Animal Behavior Abstracts</collection><collection>Immunology Abstracts</collection><collection>Industrial and Applied Microbiology Abstracts (Microbiology A)</collection><collection>Nucleic Acids Abstracts</collection><collection>Health & Medical Collection</collection><collection>ProQuest Central (purchase pre-March 2016)</collection><collection>Medical Database (Alumni Edition)</collection><collection>Technology Research Database</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 One Sustainability</collection><collection>ProQuest Central UK/Ireland</collection><collection>Agricultural & Environmental Science Collection</collection><collection>ProQuest Central Essentials</collection><collection>Biological Science Collection</collection><collection>ProQuest Central</collection><collection>Natural Science Collection</collection><collection>Environmental Sciences and Pollution Management</collection><collection>ProQuest One Community College</collection><collection>ProQuest Central Korea</collection><collection>Engineering Research Database</collection><collection>Health Research Premium Collection</collection><collection>Health Research Premium Collection (Alumni)</collection><collection>ProQuest Central Student</collection><collection>AIDS and Cancer Research Abstracts</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>Algology Mycology and Protozoology Abstracts (Microbiology C)</collection><collection>Biological Science Database</collection><collection>Biotechnology and BioEngineering Abstracts</collection><collection>Environmental 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>Environmental Science Collection</collection><collection>Genetics Abstracts</collection><collection>MEDLINE - Academic</collection><jtitle>Nature protocols</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Michielse, Caroline B</au><au>J Hooykaas, Paul J</au><au>J J van den Hondel, Cees A M</au><au>J Ram, Arthur F</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Agrobacterium-mediated transformation of the filamentous fungus Aspergillus awamori</atitle><jtitle>Nature protocols</jtitle><stitle>Nat Protoc</stitle><addtitle>Nat Protoc</addtitle><date>2008-10-01</date><risdate>2008</risdate><volume>3</volume><issue>10</issue><spage>1671</spage><epage>1678</epage><pages>1671-1678</pages><issn>1754-2189</issn><eissn>1750-2799</eissn><abstract>Many transformation methods have been developed to introduce DNA into filamentous fungi. One of these methods is
Agrobacterium
-mediated transformation (AMT). Here, we describe an efficient protocol for AMT of
Aspergillus awamori
. This protocol has been used to determine the function of
Agrobacterium
virulence genes during AMT, to identify factors influencing transformation frequencies, to generate insertional mutants and to generate
A. awamori
gene knockout transformants. This protocol in not only applicable to
A. awamori
, but can be used as a more general guideline for AMT of other filamentous fungi. Conidiospores are incubated with induced
Agrobacterium
, and, after a cocultivation and selection period, hygromycin-resistant transformants are obtained with a frequency of 200–250 transformants per 1 × 10
6
conidiospores. Using this protocol, transformants can be obtained within 10–12 d.</abstract><cop>London</cop><pub>Nature Publishing Group UK</pub><pmid>18833205</pmid><doi>10.1038/nprot.2008.154</doi><tpages>8</tpages></addata></record> |
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| issn | 1754-2189 1750-2799 |
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| subjects | Agrobacterium tumefaciens Analytical Chemistry Aspergillus Aspergillus - genetics Bacterial transformation Biological Techniques Biology Biomedical and Life Sciences Computational Biology/Bioinformatics Deoxyribonucleic acid DNA DNA, Fungal - genetics Enzymes Fungi Gene Expression Regulation, Fungal Genes Genes, Fungal - genetics Genetic aspects Genetic Engineering - methods Genetic Markers Genomics Kanamycin Life Sciences Methods Microarrays Organic Chemistry Plant molecular genetics Plasmids protocol Rhizobium - metabolism Transformation, Genetic - physiology Virulence |
| title | Agrobacterium-mediated transformation of the filamentous fungus Aspergillus awamori |
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