Fladung M, Deutsch F, Hönicka H, Kumar S
BFH Institute for Forest Genetics and Forest Tree Breeding, Grosshansdorf, Germany.
Plant Biol (Stuttg). 2004 Jan-Feb;6(1):5-11. doi: 10.1055/s-2003-44745.
We have investigated the somatic activity of the maize Activator (Ac) element in haploid and diploid aspen with the objective of developing an efficient transposon-based system for gene isolation in the model tree species Populus. It was shown that Ac is reinserted, frequently into or near coding regions in aspen, and therefore can be used for gene tagging studies. A number of phenotypic variants were also found following transformation of constructs harbouring the rolC gene. Comparative analyses of T-DNA flanking regions of variants and wild type lines indicate that T-DNA insertion has occurred in or near coding regions. However, the frequency of T-DNA insertion into genes is about one half of the frequency of Ac insertion hitting coding sequences. The results obtained give a proof-of-concept for transposon tagging in a tree system. Given the long generation cycles in tree species, gene tagging strategies are practical only to obtain dominant gain-of-function mutants that do not require selfing or test crossing. In order to obtain recessive loss-of-function mutants, we have regenerated haploid lines from immature pollen. These lines were successfully transformed with a construct containing the rolC transgene from Agrobacterium rhizogenes and Ac element from maize. The results indicate that Ac is also active in haploid aspen and hence can be used in general for gene tagging in trees.
我们研究了玉米激活子（Ac）元件在单倍体和二倍体白杨中的体细胞活性，目的是开发一种基于转座子的高效系统，用于在模式树种杨树中分离基因。结果表明，Ac元件经常重新插入到白杨的编码区或其附近，因此可用于基因标签研究。在转化携带rolC基因的构建体后，还发现了许多表型变异体。对变异体和野生型品系的T-DNA侧翼区域进行比较分析表明，T-DNA插入发生在编码区或其附近。然而，T-DNA插入基因的频率约为Ac插入编码序列频率的一半。所获得的结果为树木系统中的转座子标签提供了概念验证。鉴于树种的世代周期长，基因标签策略仅适用于获得不需要自交或测交的显性功能获得型突变体。为了获得隐性功能缺失型突变体，我们从未成熟花粉中再生出了单倍体系。这些品系成功地用含有发根农杆菌rolC转基因和玉米Ac元件的构建体进行了转化。结果表明，Ac在单倍体白杨中也具有活性，因此可普遍用于树木的基因标签研究。
