Optimized cDNA libraries for virus-induced gene silencing (VIGS) using tobacco rattle virus
<p>Abstract</p> <p>Background</p> <p>Virus-induced gene silencing (VIGS) has emerged as a method for performing rapid loss-of-function experiments in plants. Despite its expanding use, the effect of host gene insert length and other properties on silencing efficiency ha...
| Main Authors: | , |
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| Format: | Article |
| Language: | English |
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BMC
2008-01-01
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| Series: | Plant Methods |
| Online Access: | http://www.plantmethods.com/content/4/1/5 |
| _version_ | 1818155447365926912 |
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| author | Page Jonathan E Liu Enwu |
| author_facet | Page Jonathan E Liu Enwu |
| author_sort | Page Jonathan E |
| collection | DOAJ |
| description | <p>Abstract</p> <p>Background</p> <p>Virus-induced gene silencing (VIGS) has emerged as a method for performing rapid loss-of-function experiments in plants. Despite its expanding use, the effect of host gene insert length and other properties on silencing efficiency have not been systematically tested. In this study, we probed the optimal properties of cDNA fragments of the <it>phytoene desaturase </it>(<it>PDS</it>) gene for efficient VIGS in <it>Nicotiana benthamiana </it>using tobacco rattle virus (TRV).</p> <p>Results</p> <p><it>NbPDS </it>inserts of between 192 bp and 1304 bp led to efficient silencing as determined by analysis of leaf chlorophyll a levels. The region of the <it>NbPDS </it>cDNA used for silencing had a small effect on silencing efficiency with 5' and 3' located inserts performing more poorly than those from the middle. Silencing efficiency was reduced by the inclusion of a 24 bp poly(A) or poly(G) homopolymeric region. We developed a method for constructing cDNA libraries for use as a source of VIGS-ready constructs. Library construction involved the synthesis of cDNA on a solid phase support, digestion with RsaI to yield short cDNA fragments lacking poly(A) tails and suppression subtractive hybridization to enrich for differentially expressed transcripts. We constructed two cDNA libraries from methyl-jasmonate treated <it>N. benthamiana </it>roots and obtained 2948 ESTs. Thirty percent of the cDNA inserts were 401–500 bp in length and 99.5% lacked poly(A) tails. To test the efficiency of constructs derived from the VIGS-cDNA libraries, we silenced the nicotine biosynthetic enzyme, putrescine <it>N</it>-methyltransferase (<it>PMT</it>), with ten different VIGS-<it>NbPMT </it>constructs ranging from 122 bp to 517 bp. Leaf nicotine levels were reduced by more than 90% in all plants infected with the <it>NbPMT </it>constructs.</p> <p>Conclusion</p> <p>Based on the silencing of <it>NbPDS </it>and <it>NbPMT</it>, we suggest the following design guidelines for constructs in TRV vectors: (1) Insert lengths should be in the range of ~200 bp to ~1300 bp, (2) they should be positioned in the middle of the cDNA and (3) homopolymeric regions (i.e. poly(A/T) tails) should not be included. Our VIGS-cDNA library method, which incorporates these guidelines to produce sequenced, VIGS-ready cDNAs, will be useful for both fast-forward and reverse genetics experiments in TRV vectors.</p> |
| first_indexed | 2024-12-11T14:42:33Z |
| format | Article |
| id | doaj.art-ef915d6adca649809854df73da7d0d38 |
| institution | Directory Open Access Journal |
| issn | 1746-4811 |
| language | English |
| last_indexed | 2024-12-11T14:42:33Z |
| publishDate | 2008-01-01 |
| publisher | BMC |
| record_format | Article |
| series | Plant Methods |
| spelling | doaj.art-ef915d6adca649809854df73da7d0d382022-12-22T01:01:50ZengBMCPlant Methods1746-48112008-01-0141510.1186/1746-4811-4-5Optimized cDNA libraries for virus-induced gene silencing (VIGS) using tobacco rattle virusPage Jonathan ELiu Enwu<p>Abstract</p> <p>Background</p> <p>Virus-induced gene silencing (VIGS) has emerged as a method for performing rapid loss-of-function experiments in plants. Despite its expanding use, the effect of host gene insert length and other properties on silencing efficiency have not been systematically tested. In this study, we probed the optimal properties of cDNA fragments of the <it>phytoene desaturase </it>(<it>PDS</it>) gene for efficient VIGS in <it>Nicotiana benthamiana </it>using tobacco rattle virus (TRV).</p> <p>Results</p> <p><it>NbPDS </it>inserts of between 192 bp and 1304 bp led to efficient silencing as determined by analysis of leaf chlorophyll a levels. The region of the <it>NbPDS </it>cDNA used for silencing had a small effect on silencing efficiency with 5' and 3' located inserts performing more poorly than those from the middle. Silencing efficiency was reduced by the inclusion of a 24 bp poly(A) or poly(G) homopolymeric region. We developed a method for constructing cDNA libraries for use as a source of VIGS-ready constructs. Library construction involved the synthesis of cDNA on a solid phase support, digestion with RsaI to yield short cDNA fragments lacking poly(A) tails and suppression subtractive hybridization to enrich for differentially expressed transcripts. We constructed two cDNA libraries from methyl-jasmonate treated <it>N. benthamiana </it>roots and obtained 2948 ESTs. Thirty percent of the cDNA inserts were 401–500 bp in length and 99.5% lacked poly(A) tails. To test the efficiency of constructs derived from the VIGS-cDNA libraries, we silenced the nicotine biosynthetic enzyme, putrescine <it>N</it>-methyltransferase (<it>PMT</it>), with ten different VIGS-<it>NbPMT </it>constructs ranging from 122 bp to 517 bp. Leaf nicotine levels were reduced by more than 90% in all plants infected with the <it>NbPMT </it>constructs.</p> <p>Conclusion</p> <p>Based on the silencing of <it>NbPDS </it>and <it>NbPMT</it>, we suggest the following design guidelines for constructs in TRV vectors: (1) Insert lengths should be in the range of ~200 bp to ~1300 bp, (2) they should be positioned in the middle of the cDNA and (3) homopolymeric regions (i.e. poly(A/T) tails) should not be included. Our VIGS-cDNA library method, which incorporates these guidelines to produce sequenced, VIGS-ready cDNAs, will be useful for both fast-forward and reverse genetics experiments in TRV vectors.</p>http://www.plantmethods.com/content/4/1/5 |
| spellingShingle | Page Jonathan E Liu Enwu Optimized cDNA libraries for virus-induced gene silencing (VIGS) using tobacco rattle virus Plant Methods |
| title | Optimized cDNA libraries for virus-induced gene silencing (VIGS) using tobacco rattle virus |
| title_full | Optimized cDNA libraries for virus-induced gene silencing (VIGS) using tobacco rattle virus |
| title_fullStr | Optimized cDNA libraries for virus-induced gene silencing (VIGS) using tobacco rattle virus |
| title_full_unstemmed | Optimized cDNA libraries for virus-induced gene silencing (VIGS) using tobacco rattle virus |
| title_short | Optimized cDNA libraries for virus-induced gene silencing (VIGS) using tobacco rattle virus |
| title_sort | optimized cdna libraries for virus induced gene silencing vigs using tobacco rattle virus |
| url | http://www.plantmethods.com/content/4/1/5 |
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