iTILLING: A Personalized Approach to the Identification of Induced Mutations in Arabidopsis
iTILLING: A Personalized Approach to the Identification of Induced Mutations in Arabidopsis
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DOI：
10.1104/pp.110.159897
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发表时间：
2010-09-01
期刊：
影响因子：
7.4
通讯作者：
Krysan, Patrick J.
中科院分区：
文献类型：
--
作者：
Bush, Susan M.;Krysan, Patrick J.
定向诱导基因组局部突变（TILLING）是一种成熟的用于鉴定在感兴趣基因中携带点突变的植物的方法。一个传统的TILLING项目需要大量的时间和资源投入来建立突变群体和筛选设施。在此，我们描述了一种改良的TILLING程序，它大大减少了进行突变筛选所需的投入。我们开发iTILLING的动机是使单个实验室能够利用特殊的遗传背景快速进行突变筛选。利用iTILLING，从诱变的植物群体中大量收集M2种子，大大减少了管理突变株系所需的劳动力。用于突变筛选的M2幼苗的生长、组织采集以及DNA提取都以96孔板的形式进行。然后利用基因特异性聚合酶链反应产物的高分辨率熔解曲线分析来鉴定突变。携带感兴趣突变的单株植物从96孔生长板转移到土壤中。一名科学家可以在不到4个月的时间内完成一次iTILLING筛选。作为原理验证试验，我们将iTILLING应用于对促分裂原活化蛋白激酶激酶激酶1（mekk1 - 1）突变为纯合且还携带一个MEKK1拯救构建体的拟南芥（Arabidopsis thaliana）植株。我们筛选的目的是鉴定紧密连锁的MEKK2和MEKK3基因座中的突变。我们在MEKK2中获得了5个突变，在MEKK3中获得了7个突变，所有突变都位于mekk1 - 1 T - DNA插入位点的20kb范围内。通过iTILLING过程的重复迭代，可以产生三个或更多串联重复基因的突变。
TILLING (for Targeting Induced Local Lesions IN Genomes) is a well-established method for identifying plants carrying point mutations in genes of interest. A traditional TILLING project requires a significant investment of time and resources to establish the mutant population and screening infrastructure. Here, we describe a modified TILLING procedure that substantially reduces the investment needed to perform mutation screening. Our motivation for developing iTILLING was to make it practical for individual laboratories to rapidly perform mutation screens using specialized genetic backgrounds. With iTILLING, M2 seeds are collected in bulk from the mutagenized population of plants, greatly reducing the labor needed to manage the mutant lines. Growth of the M2 seedlings for mutation screening, tissue collection, and DNA extraction are all performed in 96-well format. Mutations are then identified using high-resolution melt-curve analysis of gene-specific polymerase chain reaction products. Individual plants carrying mutations of interest are transferred from the 96-well growth plates to soil. One scientist can complete an iTILLING screen in less than 4 months. As a proof-of-principle test, we applied iTILLING to Arabidopsis (Arabidopsis thaliana) plants that were homozygous for the mekk1-1 (for MAPK/ERK kinase kinase 1) mutation and also carried a MEKK1 rescue construct. The goal of our screen was to identify mutations in the closely linked MEKK2 and MEKK3 loci. We obtained five mutations in MEKK2 and seven mutations in MEKK3, all located within 20 kb of the mekk1-1 T-DNA insertion. Using repeated iterations of the iTILLING process, mutations in three or more tandemly duplicated genes could be generated.