Agrobacterium-Mediated Virus-Induced Gene Silencing Assay In Cotton
Agrobacterium-Mediated Virus-Induced Gene Silencing Assay In Cotton
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DOI：
10.3791/2938
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发表时间：
2011-08-01
影响因子：
1.2
通讯作者：
He, Ping
中科院分区：
文献类型：
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作者：
Gao, Xiquan;Britt, Robert C.;He, Ping
陆地棉（Gossypium hirsutum）是世界上最重要的农作物之一。在新品种的分子育种方面已经做出了大量努力。棉花的大规模基因功能分析落后于大多数现代植物物种，这可能是由于其基因组庞大、基因重复和多倍体现象、生长周期长以及对遗传转化的抗性等原因造成的。为了促进棉花的高通量功能遗传学/基因组学研究，我们试图开发快速有效的瞬时检测方法来评估棉花基因的功能。
病毒诱导的基因沉默（VIGS）是一种基于宿主转录后基因沉默（PTGS）开发的强大技术，用于抑制病毒增殖。农杆菌介导的VIGS已成功应用于多种双子叶植物物种，如茄科、拟南芥和豆科植物，以及单子叶植物物种，包括大麦、小麦和玉米，用于各种功能基因组学研究。由于这种快速有效的方法避免了植物转化并克服了功能冗余，它对于像棉花这样不易转化的作物物种的功能基因组学研究特别有吸引力且适用。
在这项研究中，我们报告了农杆菌介导的棉花VIGS系统的详细方案。在几种病毒VIGS载体中，烟草脆裂病毒（TRV）能侵染多种宿主，并且能够在整个植株中强烈传播，同时在宿主上产生轻微症状。为了监测沉默效率，我们克隆了棉花中拟南芥叶绿体改变1基因（AtCLA1）的同源基因GrCLA1，并将其插入到VIGS双元载体pYL156中。CLA1基因参与叶绿体发育，先前的研究表明AtCLA1功能缺失会导致真叶出现白化表型，这为沉默效率提供了一个极好的视觉标记。在农杆菌浸润后大约两周，白化表型开始在真叶上出现，在所有重复实验中沉默效率均为100%。通过逆转录聚合酶链反应（RT - PCR）分析也证实了内源基因表达的沉默。重要的是，在我们测试的所有品种中都能有效地发生沉默，包括德克萨斯州各种商业种植的品种。这种快速有效的农杆菌介导的VIGS检测方法为在棉花全基因组水平上快速大规模分析基因功能提供了一种非常强大的工具。
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.