Activation tagging in aspen using an inducible two component Ac/Ds-enhancer element system

以玉米Ac/DS二元转座系统为基础，提出了一种适用于山杨杂交种Esch5‘的激活标记方法。所提出的方法是基于我们早先在玉米转座元件Ac的遗传转移及其在杂交和纯杨品系中的功能分析所获得的结果。结果表明，Ac元件在杨树中是活跃的，并在基因组的其他区域以高频方式重新整合。然而，由于部分营养阶段较长，将Ac和D放在一起的两元转座子标记系统在树木上是不可行的。为了克服这一障碍，提出了一个可诱导的二元件Ac/ATDS元件系统，在两个独立的转化步骤之后诱导激活标记突变体。结合35S增强子四聚体和位于ATDS元件两端附近的两个向外的CaMV 35S启动子，可以提高位于元件新整合位点附近的基因的表达。作为ATDS转座的选择标记，考虑同时敲除表型标记(RolC基因)和阴性选择标记基因(TMS)的表达。因此，可以很容易地在初级转基因株系中筛选到转座。

Based on the Ac/Ds two element transposition system from maize an activation tagging approach is suggested for the hybrid aspen (Populus tremula x tremuloides) line ¿Esch5¿. The proposed approach is based on results obtained from our earlier work on the genetic transfer of the maize transposable element Ac and its functional analysis in hybrid and pure aspen lines. It was shown that the Ac element is active in aspen and reintegrates elsewhere in genomic regions in high frequency. However, a two element transposon tagging system where Ac and Ds are put together in crosses is not feasible in trees due to the in part long vegetative phases. To overcome this barrier, an inducible two element Ac/ATDs element system is suggested to induce activation tagged variants following two independent transformation steps. In combination with a 35S enhancer tetramer and outward facing two CaMV 35S promoter located near both ends of the ATDs element, expression of genes can be elevated which are located adjacent to the new integration site of the element. As selective marker for ATDs transposition, both knocking-out the expression of a phenotypic marker (rolC gene) and a negative selection marker gene (tms) are considered. Thus, the transposition can easily be screened in primary transgenic lines.