A Synthetic cryIC Gene, Encoding a Bacillus thuringiensis δ -endotoxin, Confers Spodoptera resistance in Alfalfa and Tobacco
Spodoptera species, representing widespread polyphagous insect pests, are resistant to Bacillus thuringiensis δ -endotoxins used thus far as insecticides in transgenic plants. Here we describe the chemical synthesis of a cryIC gene by a novel template directed ligation-PCR method. This simple and ec...
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Veröffentlicht in: | Proceedings of the National Academy of Sciences - PNAS 1996-12, Vol.93 (26), p.15012-15017 |
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creator | Strizhov, Nicolai Keller, Menachem Mathur, Jaideep Koncz-Kalman, Zsuzsanna Bosch, Dirk Prudovsky, Evgenia Schell, Jeff Sneh, Baruch Koncz, Csaba Zilberstein, Aviah |
description | Spodoptera species, representing widespread polyphagous insect pests, are resistant to Bacillus thuringiensis δ -endotoxins used thus far as insecticides in transgenic plants. Here we describe the chemical synthesis of a cryIC gene by a novel template directed ligation-PCR method. This simple and economical method to construct large synthetic genes can be used when routine resynthesis of genes is required. Chemically phosphorylated adjacent oligonucleotides of the gene to be synthesized are assembled and ligated on a single-stranded, partially homologous template derived from a wild-type gene (cryIC in our case) by a thermostable Pfu DNA ligase using repeated cycles of melting, annealing, and ligation. The resulting synthetic DNA strands are selectively amplified by PCR with short specific flanking primers that are complementary only to the new synthetic DNA. Optimized expression of the synthetic cryIC gene in alfalfa and tobacco results in the production of 0.01-0.2% of total soluble proteins as CryIC toxin and provides protection against the Egyptian cotton leafworm (Spodoptera littoralis) and the beet armyworm (Spodoptera exigua). To facilitate selection and breeding of Spodoptera-resistant plants, the cryIC gene was linked to a pat gene, conferring resistance to the herbicide BASTA. |
doi_str_mv | 10.1073/pnas.93.26.15012 |
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Here we describe the chemical synthesis of a cryIC gene by a novel template directed ligation-PCR method. This simple and economical method to construct large synthetic genes can be used when routine resynthesis of genes is required. Chemically phosphorylated adjacent oligonucleotides of the gene to be synthesized are assembled and ligated on a single-stranded, partially homologous template derived from a wild-type gene (cryIC in our case) by a thermostable Pfu DNA ligase using repeated cycles of melting, annealing, and ligation. The resulting synthetic DNA strands are selectively amplified by PCR with short specific flanking primers that are complementary only to the new synthetic DNA. Optimized expression of the synthetic cryIC gene in alfalfa and tobacco results in the production of 0.01-0.2% of total soluble proteins as CryIC toxin and provides protection against the Egyptian cotton leafworm (Spodoptera littoralis) and the beet armyworm (Spodoptera exigua). To facilitate selection and breeding of Spodoptera-resistant plants, the cryIC gene was linked to a pat gene, conferring resistance to the herbicide BASTA.</description><identifier>ISSN: 0027-8424</identifier><identifier>EISSN: 1091-6490</identifier><identifier>DOI: 10.1073/pnas.93.26.15012</identifier><identifier>PMID: 8986755</identifier><language>eng</language><publisher>United States: National Academy of Sciences of the United States of America</publisher><subject>Alfalfa ; Amino Acid Sequence ; Animals ; Arabidopsis - physiology ; Bacillus thuringiensis Toxins ; Bacterial Proteins - biosynthesis ; Bacterial Proteins - genetics ; Bacterial Toxins ; Base Sequence ; Biological Sciences ; Centrum voor Plantenveredelings- en Reproduktieonderzoek ; DNA ; DNA Primers ; Endotoxins - biosynthesis ; Endotoxins - genetics ; Genes, Bacterial ; Genes, Synthetic ; Genetic vectors ; Hemolysin Proteins ; Insect larvae ; Leaves ; Medicago sativa - microbiology ; Medicago sativa - physiology ; Molecular Sequence Data ; Moths ; Nicotiana - microbiology ; Nicotiana - physiology ; Oligonucleotides ; Pest Control, Biological ; Plants ; Plants, Genetically Modified ; Plants, Toxic ; Polymerase Chain Reaction ; Recombinant Proteins - biosynthesis ; Spodoptera ; Toxins ; Transgenic plants</subject><ispartof>Proceedings of the National Academy of Sciences - PNAS, 1996-12, Vol.93 (26), p.15012-15017</ispartof><rights>Copyright 1996 National Academy of Sciences</rights><rights>Copyright National Academy of Sciences Dec 24, 1996</rights><rights>Copyright © 1996, The National Academy of Sciences of the USA 1996</rights><rights>Wageningen University & Research</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c545t-4830026077789be88e87378f41faecb8db3c1d08864784e2cbba0feb5990df993</citedby><cites>FETCH-LOGICAL-c545t-4830026077789be88e87378f41faecb8db3c1d08864784e2cbba0feb5990df993</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Uhttp://www.pnas.org/content/93/26.cover.gif</thumbnail><linktopdf>$$Uhttps://www.jstor.org/stable/pdf/40819$$EPDF$$P50$$Gjstor$$H</linktopdf><linktohtml>$$Uhttps://www.jstor.org/stable/40819$$EHTML$$P50$$Gjstor$$H</linktohtml><link.rule.ids>230,314,727,780,784,803,885,27923,27924,53790,53792,58016,58249</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/8986755$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Strizhov, Nicolai</creatorcontrib><creatorcontrib>Keller, Menachem</creatorcontrib><creatorcontrib>Mathur, Jaideep</creatorcontrib><creatorcontrib>Koncz-Kalman, Zsuzsanna</creatorcontrib><creatorcontrib>Bosch, Dirk</creatorcontrib><creatorcontrib>Prudovsky, Evgenia</creatorcontrib><creatorcontrib>Schell, Jeff</creatorcontrib><creatorcontrib>Sneh, Baruch</creatorcontrib><creatorcontrib>Koncz, Csaba</creatorcontrib><creatorcontrib>Zilberstein, Aviah</creatorcontrib><title>A Synthetic cryIC Gene, Encoding a Bacillus thuringiensis δ -endotoxin, Confers Spodoptera resistance in Alfalfa and Tobacco</title><title>Proceedings of the National Academy of Sciences - PNAS</title><addtitle>Proc Natl Acad Sci U S A</addtitle><description>Spodoptera species, representing widespread polyphagous insect pests, are resistant to Bacillus thuringiensis δ -endotoxins used thus far as insecticides in transgenic plants. Here we describe the chemical synthesis of a cryIC gene by a novel template directed ligation-PCR method. This simple and economical method to construct large synthetic genes can be used when routine resynthesis of genes is required. Chemically phosphorylated adjacent oligonucleotides of the gene to be synthesized are assembled and ligated on a single-stranded, partially homologous template derived from a wild-type gene (cryIC in our case) by a thermostable Pfu DNA ligase using repeated cycles of melting, annealing, and ligation. The resulting synthetic DNA strands are selectively amplified by PCR with short specific flanking primers that are complementary only to the new synthetic DNA. Optimized expression of the synthetic cryIC gene in alfalfa and tobacco results in the production of 0.01-0.2% of total soluble proteins as CryIC toxin and provides protection against the Egyptian cotton leafworm (Spodoptera littoralis) and the beet armyworm (Spodoptera exigua). To facilitate selection and breeding of Spodoptera-resistant plants, the cryIC gene was linked to a pat gene, conferring resistance to the herbicide BASTA.</description><subject>Alfalfa</subject><subject>Amino Acid Sequence</subject><subject>Animals</subject><subject>Arabidopsis - physiology</subject><subject>Bacillus thuringiensis Toxins</subject><subject>Bacterial Proteins - biosynthesis</subject><subject>Bacterial Proteins - genetics</subject><subject>Bacterial Toxins</subject><subject>Base Sequence</subject><subject>Biological Sciences</subject><subject>Centrum voor Plantenveredelings- en Reproduktieonderzoek</subject><subject>DNA</subject><subject>DNA Primers</subject><subject>Endotoxins - biosynthesis</subject><subject>Endotoxins - genetics</subject><subject>Genes, Bacterial</subject><subject>Genes, Synthetic</subject><subject>Genetic vectors</subject><subject>Hemolysin Proteins</subject><subject>Insect larvae</subject><subject>Leaves</subject><subject>Medicago sativa - microbiology</subject><subject>Medicago sativa - physiology</subject><subject>Molecular Sequence Data</subject><subject>Moths</subject><subject>Nicotiana - microbiology</subject><subject>Nicotiana - physiology</subject><subject>Oligonucleotides</subject><subject>Pest Control, Biological</subject><subject>Plants</subject><subject>Plants, Genetically Modified</subject><subject>Plants, Toxic</subject><subject>Polymerase Chain Reaction</subject><subject>Recombinant Proteins - biosynthesis</subject><subject>Spodoptera</subject><subject>Toxins</subject><subject>Transgenic plants</subject><issn>0027-8424</issn><issn>1091-6490</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>1996</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNp9kUFv0zAYhi0EGqVwR0gIixOHpTiJE9uIS6nGmDSJw8bZcpwvravMDrbD1gP_it_Bb8JZS8UuyJYsfe_7vPKnF6GXOVnkhJXvB6vCQpSLol7kFcmLR2iWE5FnNRXkMZoRUrCM04I-Rc9C2BJCRMXJCTrhgtesqmbo5xJf7WzcQDQaa7-7WOFzsHCKz6x2rbFrrPAnpU3fjwHHzejTyIANJuDfv3AGtnXR3Rl7ilfOduADvhpc64YIXmEPyReV1YCNxcu-U-liZVt87RqltXuOnqRZgBeHd46-fT67Xn3JLr-eX6yWl5muaBUzysu0SU0YY1w0wDlwVjLe0bxToBveNqXOW8J5TRmnUOimUaSDphKCtJ0Q5Rx92OfeqjXYtAJYaZXXJkinjOxN45XfydvRS9tPzzA2QZbk_szRxz2chjfQarDRq14O3txM0BTwULFmI9fuhyzqkrKEvz3g3n0fIUS5daO3aV1ZpMY443WVTGRv0t6F4KE7xudETk3LqWkpyhQq75tOyOt_v3UEDtUm_c1Bn8i_6sOEd_93yG7s-wh3MVlf7a3bEJ0_einhuSj_AIzBykc</recordid><startdate>19961224</startdate><enddate>19961224</enddate><creator>Strizhov, Nicolai</creator><creator>Keller, Menachem</creator><creator>Mathur, Jaideep</creator><creator>Koncz-Kalman, Zsuzsanna</creator><creator>Bosch, Dirk</creator><creator>Prudovsky, Evgenia</creator><creator>Schell, Jeff</creator><creator>Sneh, Baruch</creator><creator>Koncz, Csaba</creator><creator>Zilberstein, Aviah</creator><general>National Academy of Sciences of the United States of America</general><general>National Acad Sciences</general><general>National Academy of Sciences</general><general>The National Academy of Sciences of the USA</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>7QG</scope><scope>7QL</scope><scope>7QP</scope><scope>7QR</scope><scope>7SN</scope><scope>7SS</scope><scope>7T5</scope><scope>7TK</scope><scope>7TM</scope><scope>7TO</scope><scope>7U9</scope><scope>8FD</scope><scope>C1K</scope><scope>FR3</scope><scope>H94</scope><scope>M7N</scope><scope>P64</scope><scope>RC3</scope><scope>5PM</scope><scope>QVL</scope></search><sort><creationdate>19961224</creationdate><title>A Synthetic cryIC Gene, Encoding a Bacillus thuringiensis δ -endotoxin, Confers Spodoptera resistance in Alfalfa and Tobacco</title><author>Strizhov, Nicolai ; 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Here we describe the chemical synthesis of a cryIC gene by a novel template directed ligation-PCR method. This simple and economical method to construct large synthetic genes can be used when routine resynthesis of genes is required. Chemically phosphorylated adjacent oligonucleotides of the gene to be synthesized are assembled and ligated on a single-stranded, partially homologous template derived from a wild-type gene (cryIC in our case) by a thermostable Pfu DNA ligase using repeated cycles of melting, annealing, and ligation. The resulting synthetic DNA strands are selectively amplified by PCR with short specific flanking primers that are complementary only to the new synthetic DNA. Optimized expression of the synthetic cryIC gene in alfalfa and tobacco results in the production of 0.01-0.2% of total soluble proteins as CryIC toxin and provides protection against the Egyptian cotton leafworm (Spodoptera littoralis) and the beet armyworm (Spodoptera exigua). To facilitate selection and breeding of Spodoptera-resistant plants, the cryIC gene was linked to a pat gene, conferring resistance to the herbicide BASTA.</abstract><cop>United States</cop><pub>National Academy of Sciences of the United States of America</pub><pmid>8986755</pmid><doi>10.1073/pnas.93.26.15012</doi><tpages>6</tpages><oa>free_for_read</oa></addata></record> |
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subjects | Alfalfa Amino Acid Sequence Animals Arabidopsis - physiology Bacillus thuringiensis Toxins Bacterial Proteins - biosynthesis Bacterial Proteins - genetics Bacterial Toxins Base Sequence Biological Sciences Centrum voor Plantenveredelings- en Reproduktieonderzoek DNA DNA Primers Endotoxins - biosynthesis Endotoxins - genetics Genes, Bacterial Genes, Synthetic Genetic vectors Hemolysin Proteins Insect larvae Leaves Medicago sativa - microbiology Medicago sativa - physiology Molecular Sequence Data Moths Nicotiana - microbiology Nicotiana - physiology Oligonucleotides Pest Control, Biological Plants Plants, Genetically Modified Plants, Toxic Polymerase Chain Reaction Recombinant Proteins - biosynthesis Spodoptera Toxins Transgenic plants |
title | A Synthetic cryIC Gene, Encoding a Bacillus thuringiensis δ -endotoxin, Confers Spodoptera resistance in Alfalfa and Tobacco |
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