Marion-Poll A, Marin E, Bonnefoy N, Pautot V
Laboratoire de Biologie Cellulaire, INRA, Versailles, France.
Mol Gen Genet. 1993 Apr;238(1-2):209-17. doi: 10.1007/BF00279549.
The maize autonomous transposable element Ac was introduced into haploid Nicotiana plumbaginifolia via Agrobacterium tumefaciens transformation of leaf disks. All the regenerated transformants (R0) were diploid and either homozygous or heterozygous for the hygromycin resistance gene used to select primary transformants. The Ac excision frequency was determined using the phenotypic assay of restoration of neomycin phosphotransferase activity and expression of kanamycin resistance among progeny seedlings. Some of the R0 plants segregated kanamycin-resistant seedlings in selfed progeny at a high frequency (34 to 100%) and contained one or more transposed Ac elements. In the primary transformants Ac transposition probably occurred during plant regeneration or early development. Other R0 transformants segregated kanamycin-resistant plants at a low frequency (< or = 4%). Two transformants of this latter class, containing a unique unexcised Ac element, were chosen for further study in the expectation that their kanamycin resistant progeny would result from independent germinal transposition events. Southern blot analysis of 32 kanamycin-resistant plants (R1 or R2), selected after respectively one or two selfings of these primary transformants, showed that 27 had a transposed Ac at a new location and 5 did not have any Ac element. Transposed Ac copy number varied from one to six and almost all transposition events were independent. Southern analysis of the R2 and R3 progeny of these kanamycin-resistant plants showed that Ac continued to transpose during four generations, and its activity increased with its copy number. The frequency of Ac transposition, from different loci, remained low (< or = 7%) from R0 to R3 generations when only one Ac copy was present.(ABSTRACT TRUNCATED AT 250 WORDS)
通过农杆菌介导叶盘转化法，将玉米自主转座元件Ac导入单倍体烟草。所有再生的转化体（R0）均为二倍体，对于用于筛选初级转化体的潮霉素抗性基因而言，要么是纯合的，要么是杂合的。利用新霉素磷酸转移酶活性恢复的表型分析以及子代幼苗中卡那霉素抗性的表达来确定Ac的切除频率。一些R0植株在自交后代中高频（34%至100%）分离出卡那霉素抗性幼苗，并且含有一个或多个转座的Ac元件。在初级转化体中，Ac转座可能发生在植物再生或早期发育过程中。其他R0转化体以低频（≤4%）分离出卡那霉素抗性植株。选择了后一类中的两个转化体，它们含有一个独特的未切除的Ac元件，期望它们的卡那霉素抗性后代将来自独立的生殖转座事件。对这些初级转化体分别进行一次或两次自交后选出的32株卡那霉素抗性植株（R1或R2）进行Southern杂交分析，结果表明，27株在新位置有一个转座的Ac，5株没有任何Ac元件。转座的Ac拷贝数从1到6不等，几乎所有转座事件都是独立的。对这些卡那霉素抗性植株的R2和R3后代进行Southern分析表明，Ac在四代中持续转座，并且其活性随着拷贝数的增加而增强。当仅存在一个Ac拷贝时，从R0到R3代，来自不同位点的Ac转座频率仍然很低（≤7%）。
