Yang C H, Carroll B, Scofield S, Jones J, Michelmore R
Department of Vegetable Crops, University of California, Davis 95616.
Mol Gen Genet. 1993 Nov;241(3-4):389-98. doi: 10.1007/BF00284692.
The maize transposable element, Activator (Ac), is being used to develop a transposon mutagenesis system in lettuce, Lactuca sativa. In this paper, we describe somatic and germinal transactivation of Ds by chimeric transposase genes in whole plants. Constructs containing either the Ds element or the Ac transposase open reading frame (ORF) were introduced into lettue. The Ds element was located between either the 35S or the Nos promoter and a chimeric spectinomycin resistance gene (which included a transit peptide), preventing expression of spectinomycin resistance. The genomic coding region of the Ac transposase was expressed from the 35S promoter. Crosses were made between 104 independent R1 plants containing Ds and three independent R1 plants expressing transposase. The excision of Ds in F1 progenies was monitored using a phenotypic assay on spectinomycin-containing medium. Green sectors in one-third of the F1 families indicated transactivation of Ds by the transposase at different developmental stages and at different frequencies in lettuce plants. Excision was confirmed using PCR and by Southern analysis. The lack of green sectors in the majority of F1 families suggest that the majority of T-DNA insertion sites are not conducive to excision. In subsequent experiments, the F1 plants containing both Ds and the transposase were grown to maturity and the F2 seeds screened on medium containing spectinomycin. Somatic excision was again observed in several F2 progeny; however, evidence for germinal excision was observed in only one F2 family.
玉米转座因子激活子（Activator，Ac）正被用于构建莴苣（Lactuca sativa）的转座子诱变系统。在本文中，我们描述了在整株植物中嵌合转座酶基因对Ds的体细胞和生殖细胞转激活作用。将含有Ds元件或Ac转座酶开放阅读框（ORF）的构建体导入莴苣。Ds元件位于35S或Nos启动子与嵌合壮观霉素抗性基因（包含一个转运肽）之间，从而阻止壮观霉素抗性的表达。Ac转座酶的基因组编码区由35S启动子表达。在104株含有Ds的独立R1植株与3株表达转座酶的独立R1植株之间进行杂交。通过在含有壮观霉素的培养基上进行表型分析，监测F1代中Ds的切除情况。三分之一的F1家系中出现绿色扇形区域，表明转座酶在莴苣植株的不同发育阶段以不同频率对Ds进行了转激活。通过PCR和Southern分析证实了切除情况。大多数F1家系中没有绿色扇形区域，这表明大多数T-DNA插入位点不利于切除。在后续实验中，将同时含有Ds和转座酶的F1植株培育至成熟，并在含有壮观霉素的培养基上筛选F2种子。在几个F2后代中再次观察到体细胞切除；然而，仅在一个F2家系中观察到生殖细胞切除的证据。
