Construct design for efficient, effective and high-throughput gene silencing in plants
Construct design for efficient, effective and high-throughput gene silencing in plants
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DOI：
10.1046/j.1365-313x.2001.01105.x
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发表时间：
2001-09-01
期刊：
影响因子：
7.2
通讯作者：
Waterhouse, PM
中科院分区：
文献类型：
--
作者：
Wesley, SV;Helliwell, CA;Waterhouse, PM
使用反义或共抑制结构对植物基因进行转录后沉默通常仅导致一小部分沉默个体。最近的研究表明，编码自互补“发夹”RNA (hpRNA) 的构建体具有有效沉默基因的潜力。在这项研究中，我们从显示沉默的独立转基因植物的比例和沉默的程度方面检查了有效基因沉默的设计规则。使用含有 98 至 853 nt 有义/反义臂的 hpRNA 构建体可在多种植物物种中产生有效的沉默，并且在这些构建体中包含内含子具有持续增强的效果。含有内含子的构建体（ihpRNA）通常使 90-100% 的独立转基因植物表现出沉默。这些构建体的沉默程度比使用共抑制或反义构建体获得的沉默程度大得多。我们制作了一种通用载体 pHANNIBAL，它可以将感兴趣基因的简单单一 PCR 产物轻松转化为高效的 ihpRNA 沉默构建体。我们还创建了一种高通量载体 pHELLSGATE，它应该有助于使用体外重组酶系统克隆基因库或大量确定的基因，例如 EST 集合中的基因。该系统可能会像RNA一样促进植物基因功能的大规模测定和发现！被用于检查秀丽隐杆线虫的基因功能。
Post-transcriptional silencing of plant genes using anti-sense or co-suppression constructs usually results in only a modest proportion of silenced individuals. Recent work has demonstrated the potential for constructs encoding self-complementary 'hairpin' RNA (hpRNA) to efficiently silence genes. In this study we examine design rules for efficient gene silencing, in terms of both the proportion of independent transgenic plants showing silencing, and the degree of silencing. Using hpRNA constructs containing sense/anti-sense arms ranging from 98 to 853 nt gave efficient silencing in a wide range of plant species, and inclusion of an intron in these constructs had a consistently enhancing effect. Intron-containing constructs (ihpRNA) generally gave 90-100% of independent transgenic plants showing silencing. The degree of silencing with these constructs was much greater than that obtained using either co-suppression or anti-sense constructs. We have made a generic vector, pHANNIBAL, that allows a simple, single PCR product from a gene of interest to be easily converted into a highly effective ihpRNA silencing construct. We have also created a high-throughput vector, pHELLSGATE, that should facilitate the cloning of gene libraries or large numbers of defined genes, such as those in EST collections, using an in vitro recombinase system. This system may facilitate the large-scale determination and discovery of plant gene functions in the same way as RNA! is being used to examine gene function in Caenorhabditis elegans.