Deletion-Based Reverse Genetics in Medicago truncatula
Deletion-Based Reverse Genetics in Medicago truncatula
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DOI：
10.1104/pp.109.142919
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发表时间：
2009-11-01
期刊：
影响因子：
7.4
通讯作者：
Oldroyd, Giles
中科院分区：
文献类型：
--
作者：
Rogers, Christian;Wen, Jiangqi;Oldroyd, Giles
反向遗传学的主要目标是通过筛选大量随机诱变的植物来鉴定靶基因中的无效突变。基于T-DNA和转座子的诱变已被广泛使用，但仅限于转化和组织培养有效的物种。在其他物种中，基于化学诱变的TILLING（用于靶向诱导的基因组局部损伤）提供了一种鉴定单碱基对突变的有效方法，其中只有5%是无效突变。此外，诱导点突变（如基于插入的突变）的效率取决于靶大小。在这里，我们描述了一种替代的反向遗传策略的基础上物理诱导的基因组缺失，独立的目标大小，完全恢复敲除突变体。缺失TILLING（De-TILLING）采用快中子诱变和基于敏感聚合酶链式反应的检测。156，000株蒺藜苜蓿植物被构建为13座塔，每座塔代表12，000 M2植物。De-TILLING策略允许仅使用四个聚合酶链式反应来筛选单个塔。双重筛选和三维合并允许从塔内有效定位突变体。使用这种方法，我们已经证明了使用每个基因五个靶标，从该群体中以29%的比率检测突变体。这种去TILLING反向遗传策略不依赖于组织培养和有效的植物转化，因此适用于任何植物物种。De-TILLING突变体为作物改良提供了优势，因为它们具有相对较少的背景突变并且没有外源DNA。
The primary goal of reverse genetics, the identification of null mutations in targeted genes, is achieved through screening large populations of randomly mutagenized plants. T-DNA and transposon-based mutagenesis has been widely employed but is limited to species in which transformation and tissue culture are efficient. In other species, TILLING (for Targeting Induced Local Lesions IN Genomes), based on chemical mutagenesis, has provided an efficient method for the identification of single base pair mutations, only 5% of which will be null mutations. Furthermore, the efficiency of inducing point mutations, like insertion-based mutations, is dependent on target size. Here, we describe an alternative reverse genetic strategy based on physically induced genomic deletions that, independent of target size, exclusively recovers knockout mutants. Deletion TILLING (De-TILLING) employs fast neutron mutagenesis and a sensitive polymerase chain reaction-based detection. A population of 156,000 Medicago truncatula plants has been structured as 13 towers each representing 12,000 M2 plants. The De-TILLING strategy allows a single tower to be screened using just four polymerase chain reaction reactions. Dual screening and three-dimensional pooling allows efficient location of mutants from within the towers. With this method, we have demonstrated the detection of mutants from this population at a rate of 29% using five targets per gene. This De-TILLING reverse genetic strategy is independent of tissue culture and efficient plant transformation and therefore applicable to any plant species. De-TILLING mutants offer advantages for crop improvement as they possess relatively few background mutations and no exogenous DNA.