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

हम डीआरएस के आभारी हैं. सपा दिनेश कुमार और यूल लियू TRV-VIGS वैक्टर के लिए, और डीआरएस. चक Kenerley, टेरी व्हीलर, जिम स्टार और कपास के बीज उपलब्ध कराने के लिए बायर CropScience. इस काम NSF द्वारा पीएच रास और NIH के लिए वित्त पोषित किया गया था

1. कपास seedlings बढ़ने <ol><li> Upland कपास (Gossypium hirsutum) किस्म 832 Fibermax, Phytogen 425RF, Phytogen 480WR और बर्तन (व्यास में 7 सेमी) मेट्रो 700 मिक्स (SunGR, Beavile, WA) युक्त में 90 Deltapine के बीज बोना. </li><li> एक 23 में एक प्लास्टिक के गुंबद के साथ कवर ट्रे में बर्तन रखें डि?...

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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 transiently नीचे अंतर्जात जीन की अभिव्यक्ति दस्तक द्वारा कार्यात्मक जीनोमिक्स विश्लेषण में एक शक्तिशाली उपकरण होना सिद्ध किया गया है. इस अध्ययन में, हम एक द्विआधारी TRV आधारित सदिश का उपयोग करके एक Agrobacterium की म...

<table border="1"><tbody><tr><th> अभिकर्मकों और उपकरणों के नाम </th><th> कंपनी </th><th> कैटेलॉग / सीरियल नंबर </th><th> टिप्पणियाँ </th></tr><tr><td> रोलर ड्रम </td><td> Glas - कर्नल, LLC. </td><td> TC108 099A </td><td> एग्रो - जीवाणु संस्कृति </td></tr><tr><td> अण्डे सेने की मशीन </td><td> शेल्डन विनिर्माण, Inc, </td><td> 01046209 </td><td> एग्रो - जीवाणु संस्कृति </td></tr><tr><td> यूवी / विज़ स्पेक्ट्रोफोटोमीटर </td><td> Beckman कल्टर </td><td> मॉडल: DU530 </td><td> मापने आयुध डिपो </td></tr><tr><td> जीन Pulser </td><td> BioRad </td><td> मॉडल: 1652076, सीरियल: 154BR3880 </td><td> Electroporation </td></tr><tr><td> Pulser नियंत्रक </td><td> BioRad </td><td> मॉडल: 1652098, सीरियल: 232BR4833 </td><td> Electroporation </td></tr><tr><td> Micropulser electroporation क्युवेट </td><td> BioRad </td><td> 165-2081 </td><td> Electroporation </td></tr><tr><td> 1 मिलीलीटर सिरिंज </td><td> बी.डी. बायोसाइंसेज </td><td> 30962 </td><td> कृषि जीवाणु का टीका </td></tr><tr><td> मेट्रो 700 मिक्स </td><td> SUNGR </td><td> 553001 # SKU </td><td> बढ़ते अंकुर </td></tr><tr><td> टेरा Cotta बर्तन </td><td> TOPlastics </td><td> जीपीएस 3001B2 </td><td> बढ़ते अंकुर </td></tr><tr><td> एमईएस monohydrate </td><td> यूएसबी </td><td> 18886 </td><td> घुसपैठ बफर </td></tr><tr><td> Acetosyringone </td><td> सिग्मा </td><td> D134406 </td><td> घुसपैठ बफर </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

हम Agrobacterium - वायरस मध्यस्थता प्रेरित कपास में जीन मुंह बंद परख (VIGS) के लिए विस्तृत प्रोटोकॉल प्रस्तुत करते हैं. तंबाकू खड़खड़ (TRV) वायरस व्युत्पन्न VIGS वैक्टर GrCLA1 ​​कपास, Cloroplastos alterados एक जीन का मुंह बंद आरएनए प्रेरित करने के लिए तैनात किया गया था. सूरजमुखी मनुष्य GrCLA1 ​​मुंह बंद करने के कारण phenotype अंकुर चरण में 2 सप्ताह के भीतर टीकाकरण के बाद मनाया गया.

Agrobacterium की मध्यस्थता वायरस प्रेरित कपास जीन का मुंह बंद परख

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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