Thuenen-Institute (TI) of Forest Genetics, Sieker Landstrasse 2, 22927, Grosshansdorf, Germany,
Transgenic Res. 2013 Dec;22(6):1167-78. doi: 10.1007/s11248-013-9719-2. Epub 2013 Jun 6.
Stable expression of foreign genes over the entire life span of a plant is important for long-lived organisms such as trees. For transgenic forest trees, very little information is available on long-term transgene expression and genomic stability. Independent transgenic lines obtained directly after transformation are initially screened in respect to T-DNA integration and transgene expression. However, very little consideration has been given to long-term transgene stability in long-lived forest trees. We have investigated possible genome wide changes following T-DNA integration as well as long-term stability of transgene expression in different transgenic lines of hybrid aspen (Populus tremula × Populus tremuloides) that are up to 19 years old. For studies on possible genome wide changes following T-DNA integration, four different independent rolC-transgenic lines were subjected to an extensive AFLP study and compared to the non-transgenic control line. Only minor genomic changes following T-DNA integration could be detected. To study long-term transgene expression, six different independent rolC-transgenic lines produced in 1993 and since that time have been kept continuously under in vitro conditions. In addition, 18 transgenic plants belonging to eight independent rolC-transgenic lines transferred to glasshouse between 1994 and 2004 were chosen to determine the presence and expression of the rolC gene. In all transgenic lines examined, the rolC gene could successfully be amplified by PCR tests. Both, the 19 years old tissue cultures and the up to 18 years old glasshouse-grown trees revealed expression of the rolC transgene, as demonstrated by the rolC-phenotype and/or northern blot experiments confirming long-term transgene expression.
稳定表达外源基因是植物整个生命周期中非常重要的，尤其是对于像树木这样的长寿生物。对于转基因林木来说，关于长期转基因表达和基因组稳定性的信息非常有限。最初，直接从转化中获得的独立转基因系在 T-DNA 整合和转基因表达方面进行筛选。然而，对于长寿的森林树木，很少考虑长期转基因的稳定性。我们已经研究了 T-DNA 整合后可能的全基因组变化，以及不同杂种白杨（Populus tremula × Populus tremuloides）转基因系中长达 19 年的转基因表达的长期稳定性。对于研究 T-DNA 整合后可能的全基因组变化，我们对四个不同的独立 rolC 转基因系进行了广泛的 AFLP 研究，并与非转基因对照系进行了比较。仅检测到 T-DNA 整合后的微小基因组变化。为了研究长期转基因表达，我们对 1993 年产生的六个不同的独立 rolC 转基因系进行了研究，并在那之后一直保持在体外条件下。此外，选择了 18 株属于 1994 年至 2004 年间转移到温室中的八个独立 rolC 转基因系的转基因植物，以确定 rolC 基因的存在和表达。在所检查的所有转基因系中，rolC 基因都可以通过 PCR 测试成功扩增。在所有检测的转基因系中，19 年的组织培养物和长达 18 年的温室生长的树木都表现出 rolC 转基因的表达，这证明了 rolC 表型和/或 northern blot 实验证实了长期的转基因表达。
