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

我们感谢博士。 SP的Dinesh Kumar和尤尔刘TRV - VIGS载体，和Drs。查Kenerley，特里轮车，吉姆斯塔尔提供棉花种子和拜耳作物科学。这项工作是由NSF资助LS和NIH的PH

1。种植棉花幼苗<ol><li>母猪的棉花（陆地棉）各种Fibermax 832 Phytogen 425RF，Phytogen 480WR和Deltapine 90盆（直径7厘米）含地铁混合700（SunGR，Beavile，WA）的种子。 </li><li>盆保持在一个托盘用塑料圆顶覆盖23℃，120μE米-2小号-1光，在增长室12个小时的light/12小时黑暗的光照。 </li><li>删除时，有两个子叶出现圆顶。 </li><li>大约两个星期后，使用两个子叶完全展开VIGS检测的植物。在这个阶?...

<ol>
	<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已被证明是一个瞬时击倒内源性基因的表达，在功能基因组学分析的有力工具。在这项研究中，我们开发利用TRV -基于二进制向量农杆菌介导的VIGS。棉花CLA1（GrCLA1）基因作为一种视觉标记，监察沉默效率。我们一直得到100％的基因沉默的效率，表现出白化​​病的表型出现在所有品种的片真叶测试，大约两个星期后渗透开始。然而，CLA1沉默的棉花植株难以发展成纤维或种子阶段...

<table border="1"><tbody><tr><th>试剂和设备的名称</th><th>公司</th><th>目录/序列号</th><th>评论</th></tr><tr><td>滚子鼓</td><td> GLAS - COL，LLC。 </td><td> 099A TC108 </td><td>农业细菌培养</td></tr><tr><td>孵化器</td><td>谢尔顿制造公司</td><td> 01046209 </td><td>农业细菌培养</td></tr><tr><td>紫外/可见分光光度计</td><td> Beckman Coulter公司</td><td>型号：DU530 </td><td>测量外径</td></tr><tr><td>基因脉冲</td><td> BioRad公司</td><td>型号：编号：1652076; 154BR3880 </td><td>电穿孔</td></tr><tr><td>脉冲控制器</td><td> BioRad公司</td><td>型号：编号：1652098; 232BR4833 </td><td>电穿孔</td></tr><tr><td> Micropulser电穿孔的比色皿</td><td> BioRad公司</td><td> 165-2081 </td><td>电穿孔</td></tr><tr><td> 1 ml注射器</td><td> BD公司</td><td> 30962 </td><td>农业细菌接种</td></tr><tr><td>地铁混合700 </td><td> SUNGR </td><td> SKU＃553001 </td><td>育苗</td></tr><tr><td>兵马俑锅</td><td> TOPlastics </td><td>全球定位系统3001B2 </td><td>育苗</td></tr><tr><td> MES的水合物</td><td> USB </td><td> 18886 </td><td>渗透缓冲区</td></tr><tr><td>乙酰丁香酮</td><td>西格玛</td><td> D134406 </td><td>渗透缓冲区</td></tr></tbody></table>

agrobacterium-mediated virus-induced gene silencing assay in cotton

棉花（ 陆地棉 ）是全球最重要的农作物之一。分子育种新品种已相当大的努力。大规模基因功能分析棉花已经落后于最现代的植物物种，可能是由于其基因组，基因复制和多倍体，生长周期长和遗传转化 1顽抗的大尺寸。为了方便的高通量功能基因/基因棉花的研究，我们尝试发展快速，高效的瞬态分析评估棉花基因的功能。 病毒诱导的基因沉默（VIGS）是一个强大的技术开发基于主机的转录后基因沉默（PTGS）抑制病毒增殖2,3。农杆菌介导的VIGS已被成功地应用在双子叶植物， 如茄科，拟南芥和豆科植物物种的广泛，单子叶植物物种，其中包括大麦，小麦和玉米， 各种功能基因组研究3,4。由于这种快速和有效的方法避免了厂房改造和克服的功能冗余，这是特别有吸引力，适合在作物品种，如棉花不适合转型功能基因组学研究。 在这项研究中，我们报告VIGS在棉花农杆菌介导的系统的详细协议。其中几种病毒的VIGS载体，烟草脆裂病毒（TRV）侵入的主机范围广，能够传遍整个工厂的大力尚未产生症状轻微的hosts5。要监视的沉默效率，GrCLA1 ​​alterados 1基因棉花（AtCLA1），已被克隆，并插入到VIGS二进制向量pYL156。CLA1基因参与叶绿体发展 6，和以往的研究表明，拟南芥 Cloroplastos的同源基因， AtCLA1损失的功能，导致7片真叶白化病的表型，沉默效率提供了一个良好的视觉标记。 农杆菌浸润在大约两个星期后，白化病的表型开始出现片真叶，与100％，在所有实验复制的沉默效率。内源性基因表达的沉默，也证实了RT - PCR分析。值得注意的是，沉默potently可能发生在我们测试的所有品种，包括各种商业化种植的品种在得克萨斯州。这种快速，高效的的农杆菌介导的VIGS检测，提供了一个快速大规模的棉花全基因组水平的基因功能分析非常强大的工具。

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

我们目前为农杆菌介导的病毒诱导的基因沉默（VIGS）棉花检测的详细协议。烟草脆裂病毒（TRV）派生的VIGS载体部署GrCLA1​​棉花，Cloroplastos alterados 1基因诱导RNA沉默。白化沉默GrCLA1​​引起的表型观察接种后2个星期内，在苗期。

农杆菌介导的病毒诱导的基因沉默含量在棉花

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

Research

JoVE Journal

Biology

Virus-induced Gene Silencing (VIGS) in Nicotiana benthamiana and Tomato

Virus-induced Gene Silencing VIGS in Nicotiana benthamiana and Tomato

病毒诱导的基因沉默（VIGS）烟草benthamiana和番茄

RNA interference (RNAi) is a highly specific gene-silencing phenomenon triggered by dsRNA1. This silencing mechanism uses two major classes of RNA ...

科研

生物学

Live Cell Calcium Imaging Combined with siRNA Mediated Gene Silencing Identifies Ca2+ Leak Channels in the ER Membrane and their Regulatory Mechanisms

Live Cell Calcium Imaging Combined with siRNA Mediated Gene Silencing Identifies Ca2+ Leak Channels in the ER Membrane and their Regulatory Mechanisms

活细胞钙成像相结合的siRNA介导的基因沉默标识CA 2 +泄漏通道内质网膜和监管机制

In mammalian cells, the endoplasmic reticulum (ER) plays a key role in protein biogenesis as well as in calcium signalling1. The heterotrimeric Sec61 ...

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

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核糖体束缚新生多肽的分离<em&gt;在体外</em&gt;确定沿mRNA的翻译暂停网站

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the ...

Using SecM Arrest Sequence as a Tool to Isolate Ribosome Bound Polypeptides

作为一种工具来隔离核糖体的束缚多肽SECM逮捕序列

Extensive research has provided ample evidences suggesting that protein folding in the cell is a co-translational process1-5. However, the exact pathway ...

Optimization and Utilization of Agrobacterium-mediated Transient Protein Production in Nicotiana

Optimization and Utilization of Agrobacterium-mediated Transient Protein Production in Nicotiana

优化和利用<em&gt;农杆菌</em&gt;介导的瞬态蛋白生产中<em&gt;烟草</em

Agrobacterium-mediated transient protein production in plants is a promising approach to produce vaccine antigens and therapeutic proteins within a short ...

Chitosan/Interfering RNA Nanoparticle Mediated Gene Silencing in Disease Vector Mosquito Larvae

壳聚糖/干扰RNA纳米介导的基因沉默的疾病病媒蚊幼虫

Vector mosquitoes inflict more human suffering than any other organism&#8212;and kill more than one million people each year. The mosquito genome projects ...

Meiotic Spindle Assessment in Mouse Oocytes by siRNA-mediated Silencing

减数分裂纺锤体评估在小鼠卵母细胞的siRNA介导沉默

Errors in chromosome segregation during meiotic division in gametes can lead to aneuploidy that is subsequently transmitted to the embryo upon ...

Silencing the Spark: CRISPR/Cas9 Genome Editing in Weakly Electric Fish

沉默的火花：CRISPR/Cas9基因组编辑在弱电鱼

Electroreception and electrogenesis have changed in the evolutionary history of vertebrates. There is a striking degree of convergence in these ...

Agrobacterium-Mediated Immature Embryo Transformation of Recalcitrant Maize Inbred Lines Using Morphogenic Genes

Agrobacterium-Mediated Immature Embryo Transformation of Recalcitrant Maize Inbred Lines Using Morphogenic Genes

使用形态基因的农业细菌 -顽固玉米近亲繁殖系的介导未成熟胚胎转化

Demonstrated here is a detailed protocol for Agrobacterium-mediated genetic transformation of maize inbred lines using morphogenic genes Baby boom (Bbm) ...

Agrobacterium-Mediated Genetic Transformation, Transgenic Production, and Its Application for the Study of Male Reproductive Development in Rice

Agrobacterium-Mediated Genetic Transformation, Transgenic Production, and Its Application for the Study of Male Reproductive Development in Rice

农业细菌-中生菌遗传转化、转基因生产及其在水稻雄性生殖发育研究中的应用

Male sterility is an important agronomic trait for hybrid seed production that is usually characterized by functional defects in male reproductive ...