Name: agrobacterium-mediated virus-induced gene silencing assay in cotton
Uploaded: 2011-08-20T12:00:00
Description: Watch this Scientific Journal Video about Agrobacterium-Mediated Virus-Induced Gene Silencing Assay In Cotton at JoVE.com

Мы благодарим доктора. SP-Динеш Кумар и Юла Лю для ТРВ-VIGS векторов, и д-ра. Чак Kenerley, Терри Уилер, Джим Старр и Bayer CropScience за предоставление семян хлопчатника. Эта работа финансировалась NSF для LS и NIH для PH

1. Саженцы выращивают хлопок <ol><li> Сейте семена возвышенности хлопка (Gossypium hirsutum) многообразие Fibermax 832, Phytogen 425RF, Phytogen 480WR и Deltapine 90 в горшках (7 см в диаметре), содержащий Metro Mix 700 (SunGR, Beavile, штат Вашингтон). </li><li> Имейте в горшках поднос, покрытый пластиковым куполом при 23 ° С, 120 мкЕ м -2 с -1 ...

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	<li>Chen, Z. J., Scheffler, B. E., Dennis, E., Triplett, B. A., Zhang, T., Guo, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toward+sequencing+cotton+(Gossypium)+genomes.">Toward sequencing cotton (Gossypium) genomes.</a> Plant Physiol. 145, 1303-1310 (2007).</li><li>Dinesh-Kumar, S. P., Anandalakshmi, R., Marathe, R., Schiff, M., Liu, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Virus-induced+gene+silencing.">Virus-induced gene silencing.</a> Methods Mol Biol. 236, 287-294 (2003).</li><li>Hamilton, A. J., Baulcombe, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+species+of+small+antisense+RNA+in+posttranscriptional+gene+silencing+in+plants.">A species of small antisense RNA in posttranscriptional gene silencing in plants.</a> Science. 286, 950-952 (1999).</li><li>Burch-Smith, T. M., Anderson, J. C., Martin, G. B., Dinesh-Kumar, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Applications+and+advantages+of+virus-induced+gene+silencing+for+gene+function+studies+in+plants.">Applications and advantages of virus-induced gene silencing for gene function studies in plants.</a> Plant J. 39, 734-746 (2004).</li><li>Liu, Y., Schiff, M., Dinesh-Kumar, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Virus-induced+gene+silencing+in+tomato.">Virus-induced gene silencing in tomato.</a> Plant J. 31, 777-786 (2002).</li><li>Estevez, J. M., Cantero, A., Romero, C., Kawaide, H., Jimenez, L. F., Kuzuyama, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+the+expression+of+CLA1,+a+gene+that+encodes+the+1-deoxyxylulose+5-phosphate+synthase+of+the+2-C-methyl-D-erythritol-4-phosphate+pathway+in+Arabidopsis.">Analysis of the expression of CLA1, a gene that encodes the 1-deoxyxylulose 5-phosphate synthase of the 2-C-methyl-D-erythritol-4-phosphate pathway in Arabidopsis.</a> Plant Physiol. 124, 95-104 (2000).</li><li>Mandel, M. A., Feldmann, K. A., Herrera-Estrella, L., Rocha-Sosa, M., Leon, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CLA1,+a+novel+gene+required+for+chloroplast+development,+is+highly+conserved+in+evolution.">CLA1, a novel gene required for chloroplast development, is highly conserved in evolution.</a> Plant J. 9, 649-658 (1996).</li><li>Gao, X., Wheeler, T., Li, Z., Kenerley, C., He, P., Shan, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Silencing+GhNDR1+and+GhMKK2+compromised+cotton+resistance+to+Verticillium+wilt.">Silencing GhNDR1 and GhMKK2 compromised cotton resistance to Verticillium wilt.</a> Plant J. 66, 293-305 (2011).</li></ol>

VIGS был доказан, чтобы быть мощным инструментом в области функциональной геномики анализа временно сбить экспрессии эндогенных генов. В этом исследовании, мы разработали агробактериальной VIGS за счет использования TRV основе двоичного вектора. Хлопок CLA1 (GrCLA1) ген был разработан как ?...

<table border="1"><tbody><tr><th> Название реагентов и оборудования </th><th> Компания </th><th> Каталог / серийный номер </th><th> Комментарии </th></tr><tr><td> Каток </td><td> Glas-Col, LLC. </td><td> 099A TC108 </td><td> Агро-бактерии культуры </td></tr><tr><td> Инкубатор </td><td> Sheldon Manufacturing, Inc </td><td> 01046209 </td><td> Агро-бактерии культуры </td></tr><tr><td> UV / Vis спектрофотометр </td><td> Beckman Coulter </td><td> Модель: DU530 </td><td> Измерение ОП </td></tr><tr><td> Гена Pulser </td><td> BioRad </td><td> Модель: 1652076; Последовательный: 154BR3880 </td><td> Электропорация </td></tr><tr><td> Генератор импульсов контроллера </td><td> BioRad </td><td> Модель: 1652098; Последовательный: 232BR4833 </td><td> Электропорация </td></tr><tr><td> Micropulser Электропорация кювет </td><td> BioRad </td><td> 165-2081 </td><td> Электропорация </td></tr><tr><td> 1 мл шприцев </td><td> BD Biosciences </td><td> 30962 </td><td> Прививка агропромышленного бактерии </td></tr><tr><td> Metro Mix 700 </td><td> SUNGR </td><td> SKU # 553001 </td><td> Рост рассады </td></tr><tr><td> Стихаря горшок </td><td> TOPlastics </td><td> GPS-3001B2 </td><td> Рост рассады </td></tr><tr><td> МЧС моногидрат </td><td> USB </td><td> 18886 </td><td> Проникновение буфер </td></tr><tr><td> Acetosyringone </td><td> Сигма </td><td> D134406 </td><td> Проникновение буфер </td></tr></tbody></table>

agrobacterium-mediated virus-induced gene silencing assay in cotton

Cotton (Gossypium hirsutum) is one of the most important crops worldwide. Considerable efforts have been made on molecular breeding of new varieties. The large-scale gene functional analysis in cotton has been lagged behind most of the modern plant species, likely due to its large size of genome, gene duplication and polyploidy, long growth cycle and recalcitrance to genetic transformation1. To facilitate high throughput functional genetic/genomic study in cotton, we attempt to develop rapid and efficient transient assays to assess cotton gene functions.
Virus-Induced Gene Silencing (VIGS) is a powerful technique that was developed based on the host Post-Transcriptional Gene Silencing (PTGS) to repress viral proliferation2,3. Agrobacterium-mediated VIGS has been successfully applied in a wide range of dicots species such as Solanaceae, Arabidopsis and legume species, and monocots species including barley, wheat and maize, for various functional genomic studies3,4. As this rapid and efficient approach avoids plant transformation and overcomes functional redundancy, it is particularly attractive and suitable for functional genomic study in crop species like cotton not amenable for transformation.
In this study, we report the detailed protocol of Agrobacterium-mediated VIGS system in cotton. Among the several viral VIGS vectors, the tobacco rattle virus (TRV) invades a wide range of hosts and is able to spread vigorously throughout the entire plant yet produce mild symptoms on the hosts5. To monitor the silencing efficiency, GrCLA1, a homolog gene of Arabidopsis Cloroplastos alterados 1 gene (AtCLA1) in cotton, has been cloned and inserted into the VIGS binary vector pYL156. CLA1 gene is involved in chloroplast development6, and previous studies have shown that loss-of-function of AtCLA1 resulted in an albino phenotype on true leaves7, providing an excellent visual marker for silencing efficiency. At approximately two weeks post Agrobacterium infiltration, the albino phenotype started to appear on the true leaves, with 100% silencing efficiency in all replicated experiments. The silencing of endogenous gene expression was also confirmed by RT-PCR analysis. Significantly, silencing could potently occur in all the cultivars we tested, including various commercially grown varieties in Texas. This rapid and efficient Agrobacterium-mediated VIGS assay provides a very powerful tool for rapid large-scale analysis of gene functions at genome-wide level in cotton.

Watch this Scientific Journal Video about Agrobacterium-Mediated Virus-Induced Gene Silencing Assay In Cotton at JoVE.com

Agrobacterium-Mediated Virus-Induced Gene Silencing Assay In Cotton

Мы представляем подробный протокол для агробактериальной вирус-индуцированных генов (VIGS) анализа в хлопка. Вирус табачной погремушкой (ТРВ)-производные векторов VIGS были развернуты, чтобы побудить глушителей РНК хлопка GrCLA1, Cloroplastos alterados один ген. Альбиноса фенотип вызванных глушителей GrCLA1 ​​наблюдался на всходов в течение 2 недель после прививки.

Агробактериальной вирус-индуцированного Пробирной Отключение гена В Хлопок

Cotton (Gossypium hirsutum) is one of the most important crops worldwide. Considerable efforts have been made on molecular breeding of new varieties.  The ...

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Biology

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