A Ligation-Independent cloning tobacco rattle virus vector for high-throughput virus-induced gene silencing identifies roles for NbMADS4-1 and -2 in floral development
A Ligation-Independent cloning tobacco rattle virus vector for high-throughput virus-induced gene silencing identifies roles for NbMADS4-1 and -2 in floral development
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DOI：
10.1104/pp.107.107391
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发表时间：
2007-12-01
期刊：
影响因子：
7.4
通讯作者：
Dinesh-Kumar, Savithramma P.
中科院分区：
文献类型：
--
作者：
Dong, Yiyu;Burch-Smith, Tessa M.;Dinesh-Kumar, Savithramma P.
病毒诱导的基因沉默（VIGS）是一种广泛使用的，强大的反向遗传学技术。来源于烟草脆裂病毒（TRV）的VIGS载体是最流行的VIGS载体之一。我们已经开发了一种TRV RNA 2载体，该载体允许通过连接非依赖性克隆（LIC）插入基因沉默片段。这种新载体与以前的载体相比具有几个优点，特别是对于涉及分析大量序列的应用，因为以100%的效率获得含有所需插入片段的TRV-LIC载体。重要的是，该载体允许沉默片段的高通量克隆，而不使用重组所需的昂贵的酶，如基于GATEWAY的载体的情况。我们在此TRV-LIC背景下基于400个番茄（Solanum lycopersicum）表达的序列标签产生沉默载体的集合。我们已经使用该载体来鉴定SlMADS 1及其本氏烟草同源物NbMADS 4 -1和NbMADS 4 - 2在开花中的作用。我们发现NbMADS 4 -1和-2在花发育中起非冗余作用，并且任何一个基因的沉默都会导致器官身份的丧失。这种TRV-LIC载体应该是植物群落的宝贵资源。
Virus-induced gene silencing (VIGS) is a widely used, powerful technique for reverse genetics. VIGS vectors derived from the Tobacco rattle virus (TRV) are among the most popular for VIGS. We have developed a TRV RNA2 vector that allows the insertion of gene silencing fragments by ligation-independent cloning (LIC). This new vector has several advantages over previous vectors, particularly for applications involving the analysis of large numbers of sequences, since TRV-LIC vectors containing the desired insert are obtained with 100% efficiency. Importantly, this vector allows the high-throughput cloning of silencing fragments without the use of costly enzymes required for recombination, as is the case with GATEWAY-based vectors. We generated a collection of silencing vectors based on 400 tomato ( Solanum lycopersicum) expressed sequence tags in this TRV-LIC background. We have used this vector to identify roles for SlMADS1 and its Nicotiana benthamiana homologs, NbMADS4-1 and -2 in flowering. We find that NbMADS4-1 and -2 act nonredundantly in floral development and silencing of either gene results in loss of organ identity. This TRV-LIC vector should be a valuable resource to the plant community.