Long D, Swinburne J, Martin M, Wilson K, Sundberg E, Lee K, Coupland G
Cambridge Laboratory, John Innes Centre, Norwich, UK.
Mol Gen Genet. 1993 Dec;241(5-6):627-36. doi: 10.1007/BF00279905.
The Ac/Ds transposon system of maize shows low activity in Arabidopsis. However, fusion of the CaMV 35S promoter to the transposase gene (35S::TPase) increases the abundance of the single Ac mRNA encoded by Ac and increases the frequency of Ds excision. In the experiments reported here it is examined whether this high excision frequency is associated with efficient re-insertion of the transposon. This was measured by using a Ds that carried a hygromycin resistance gene (HPT) and was inserted within a streptomycin resistance gene (SPT). Excision of Ds therefore gives rise to streptomycin resistance, while hygromycin resistance is associated with the presence of a transposed Ds or with retention of the element at its original location. Self-fertilisation of most individuals heterozygous for Ds and 35S::TPase produced many streptomycin-resistant (strep(r)) progeny, but in many of these families a small proportion of strep(r) seedlings were also resistant to hygromycin (hyg(r)). Nevertheless, 70% of families tested did give rise to at least one strep(r), hyg(r) seedling, and over 90% of these individuals carried a transposed Ds. In contrast, the Ac promoter fusion to the transposase gene (Ac::TPase) produced fewer strep(r)hyg(r) progeny, and only 53% of these carried a transposed Ds. However, a higher proportion of the strep(r) seedlings were also hyg(r) than after activation by 35S::TPase. We also examined the genotype of strep(r), hyg(r) seedlings and demonstrated that after activation by 35S::TPase many of these were homozygous for the transposed Ds, while this did not occur after activation by Ac::TPase. From these and other data we conclude that excisions driven by 35S::TPase usually occur prior to floral development, and that although a low proportion of strep(r) progeny plants inherit a transposed Ds, those that do can be efficiently selected with an antibiotic resistance gene contained within the element. Our data have important implications for transposon tagging strategies in transgenic plants and these are discussed.
玉米的Ac/Ds转座子系统在拟南芥中活性较低。然而，将花椰菜花叶病毒35S启动子与转座酶基因融合（35S::TPase）会增加Ac编码的单个Ac mRNA的丰度，并提高Ds切除的频率。在本文报道的实验中，研究了这种高切除频率是否与转座子的有效重新插入有关。这是通过使用一个携带潮霉素抗性基因（HPT）并插入链霉素抗性基因（SPT）内的Ds来测量的。因此，Ds的切除会产生链霉素抗性，而潮霉素抗性与转座的Ds的存在或元件在其原始位置的保留有关。大多数对Ds和35S::TPase杂合的个体自交产生了许多链霉素抗性（strep(r)）后代，但在许多这些家族中，一小部分strep(r)幼苗也对潮霉素（hyg(r)）有抗性。然而，70%的测试家族确实产生了至少一株strep(r)、hyg(r)幼苗，并且这些个体中超过90%携带了转座的Ds。相比之下，将Ac启动子与转座酶基因融合（Ac::TPase）产生的strep(r)hyg(r)后代较少，并且这些后代中只有53%携带了转座的Ds。然而，与35S::TPase激活后相比，更高比例的strep(r)幼苗也是hyg(r)。我们还检查了strep(r)、hyg(r)幼苗的基因型，并证明在35S::TPase激活后，其中许多对转座的Ds是纯合的，而在Ac::TPase激活后则没有这种情况。从这些和其他数据中我们得出结论，由35S::TPase驱动的切除通常发生在花发育之前，并且尽管一小部分strep(r)后代植物继承了转座的Ds，但那些继承了的可以用元件中包含的抗生素抗性基因有效地筛选出来。我们的数据对转基因植物中转座子标签策略具有重要意义，并对此进行了讨论。
