Healy J, Corr C, DeYoung J, Baker B
Department of Plant Pathology, University of California, Berkeley 94720.
Genetics. 1993 Jun;134(2):571-84. doi: 10.1093/genetics/134.2.571.
We have introduced a genetically marked Dissociation transposable element (Dsneo) into tomato. In the presence of Ac transposase, Dsneo excised from an integrated T-DNA and reinserted at numerous new sites in the tomato genome. The marker genes of Dsneo (NPTII) and the T-DNA (HPT) facilitated identification of plants bearing transposon excisions and insertions. To explore the feasibility of gene tagging strategies in tomato using Dsneo, we examined the genomic distribution of Dsneo receptor sites, relative to the location of the donor T-DNA locus. Restriction fragment length polymorphism mapping of transposed Dsneo elements was conducted in two tomato families, derived from independent primary transformants each bearing Dsneo within a T-DNA at a unique position in the genome. Transposition of Dsneo generated clusters of insertions that were positioned on several different tomato chromosomes. Dsneo insertions were often located on the same chromosome as the T-DNA donor site. However, no insertion showed tight linkage to the T-DNA. We consider the frequency and distance of Dsneo transposition observed in tomato to be well suited for transposon mutagenesis. Our study made use of a novel, stable allele of Ac (Ac3) that we discovered in transgenic tomato. We determined that the Ac3 element bears a deletion of the outermost 5 base pairs of the 5'-terminal inverted repeat. Though incapable of transposition itself, Ac3 retained the ability to mobilize Dsneo. We conclude that a dual element system, composed of the stable Ac3 trans-activator in combination with Dsneo, is an effective tool for transposon tagging experiments in tomato.
我们已将一个带有遗传标记的解离转座元件（Dsneo）导入番茄。在Ac转座酶存在的情况下，Dsneo从整合的T-DNA上切除，并重新插入到番茄基因组的众多新位点。Dsneo的标记基因（NPTII）和T-DNA（HPT）有助于鉴定带有转座子切除和插入的植株。为了探索利用Dsneo在番茄中进行基因标签策略的可行性，我们研究了Dsneo受体位点相对于供体T-DNA位点位置的基因组分布。在两个番茄家族中对转座后的Dsneo元件进行了限制性片段长度多态性图谱分析，这两个家族来自独立的初级转化体，每个转化体在基因组的独特位置的T-DNA中都带有Dsneo。Dsneo的转座产生了位于几条不同番茄染色体上的插入簇。Dsneo插入位点常常与T-DNA供体位点在同一条染色体上。然而，没有插入位点与T-DNA表现出紧密连锁。我们认为在番茄中观察到的Dsneo转座频率和距离非常适合用于转座子诱变。我们的研究利用了我们在转基因番茄中发现的一种新型、稳定的Ac等位基因（Ac3）。我们确定Ac3元件在5'端反向重复序列的最外层5个碱基对处有一个缺失。虽然Ac3本身不能转座，但它保留了激活Dsneo转座的能力。我们得出结论，由稳定的Ac3转激活因子与Dsneo组成的双元件系统是番茄中转座子标签实验的有效工具。
