Agrobacterium की मध्यस्थता वायरस प्रेरित कपास जीन का मुंह बंद परख
Summary
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.
Abstract
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.
Protocol
Discussion
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…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
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
Materials
| Name of the reagents and equipments | Company | Catalogue/serial number | Comments |
|---|---|---|---|
| Roller drum | Glas-Col, LLC. | 099A TC108 | Agro-bacterium culture |
| Incubator | Sheldon Manufacturing, Inc. | 01046209 | Agro-bacterium culture |
| UV/Vis spectrophotometer | Beckman Coulter | Model: DU530 | Measuring OD |
| Gene Pulser | BioRad | Model: 1652076; Serial: 154BR3880 | Electroporation |
| Pulser Controller | BioRad | Model: 1652098; Serial: 232BR4833 | Electroporation |
| Micropulser Electroporation cuvette | BioRad | 165-2081 | Electroporation |
| 1 ml Syringe | BD Biosciences | 30962 | Inoculation of agro-bacterium |
| Metro Mix 700 | SUNGR | SKU# 553001 | Growing seedling |
| Terra Cotta pot | T.O.Plastics | GPS 3001B2 | Growing seedling |
| MES monohydrate | USB | 18886 | Infiltration buffer |
| Acetosyringone | Sigma | D134406 | Infiltration buffer |
Riferimenti
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