May Bruce P, Liu Hong, Vollbrecht Erik, Senior Lynn, Rabinowicz Pablo D, Roh Donna, Pan Xiaokang, Stein Lincoln, Freeling Mike, Alexander Danny, Martienssen Rob
Cold Spring Harbor Laboratory, 1 Bungtown Road, Cold Spring Harbor, NY 11724, USA.
Proc Natl Acad Sci U S A. 2003 Sep 30;100(20):11541-6. doi: 10.1073/pnas.1831119100. Epub 2003 Sep 3.
We describe an efficient system for site-selected transposon mutagenesis in maize. A total of 43,776 F1 plants were generated by using Robertson's Mutator (Mu) pollen parents and self-pollinated to establish a library of transposon-mutagenized seed. The frequency of new seed mutants was between 10-4 and 10-5 per F1 plant. As a service to the maize community, maize-targeted mutagenesis selects insertions in genes of interest from this library by using the PCR. Pedigree, knockout, sequence, phenotype, and other information is stored in a powerful interactive database (maize-targeted mutagenesis database) that enables analysis of the entire population and the handling of knockout requests. By inhibiting Mu activity in most F1 plants, we sought to reduce somatic insertions that may cause false positives selected from pooled tissue. By monitoring the remaining Mu activity in the F2, however, we demonstrate that seed phenotypes depend on it, and false positives occur in lines that appear to lack it. We conclude that more than half of all mutations arising in this population are suppressed on losing Mu activity. These results have implications for epigenetic models of inbreeding and for functional genomics.
我们描述了一种用于玉米中位点特异性转座子诱变的高效系统。使用罗伯逊氏突变体（Mu）花粉亲本产生了总共43776株F1植株，并进行自花授粉以建立一个转座子诱变种子库。每个F1植株产生新种子突变体的频率在10^-4至10^-5之间。作为对玉米研究群体的一项服务，玉米靶向诱变通过聚合酶链反应（PCR）从该文库中选择感兴趣基因中的插入突变。系谱、基因敲除、序列、表型和其他信息存储在一个强大的交互式数据库（玉米靶向诱变数据库）中，该数据库能够对整个群体进行分析并处理基因敲除请求。通过抑制大多数F1植株中的Mu活性，我们试图减少可能导致从混合组织中选出假阳性的体细胞插入。然而，通过监测F2中剩余的Mu活性，我们证明种子表型依赖于它，并且在看似缺乏Mu活性的品系中会出现假阳性。我们得出结论，该群体中产生的所有突变中超过一半在失去Mu活性时受到抑制。这些结果对近亲繁殖的表观遗传模型和功能基因组学具有启示意义。
