Sakhno L A, Sytnik E S, Cherep N N, Komarnitskiĭ I K, Kuchuk N V, Klimiuk V I
Tsitol Genet. 2002 Nov-Dec;36(6):3-8.
Transposon mediated insertional mutagenesis is one of the approaches for the unique gene cloning. A wild species of Cruciferae family Orychophragmus violaceus (L.) O.E. Schulz, which is of interest for practical breeding as a donor of improved plant oil, was an object of the investigation. Plasmid construction used in the experiments included selective NPT II gene, reported GUS gene serving as an excision marker, structural BAR gene located within the dSpm element and Spm transposase. The GUS gene of this plasmid had not his own promoter and became functional only after Spm-transposition. Transformed Orychophragmus violaceus (L.) O.E. Schulz. plants were obtained by direct mesophyll protoplast transformation as well as Agrobacterium tumefaciens-mediated root explant transformation. Gene transfer and the transposition event were confirmed by the GUS activity and the PCR analysis. Relative transformation efficiency using protoplasts was 5.8%.
转座子介导的插入诱变是独特基因克隆的方法之一。十字花科野生种诸葛菜(Orychophragmus violaceus (L.) O.E. Schulz)作为改良植物油的供体在实际育种中具有重要意义,是本研究的对象。实验中使用的质粒构建包括选择性NPT II基因、作为切除标记的报告基因GUS、位于dSpm元件内的结构BAR基因和Spm转座酶。该质粒的GUS基因没有自身的启动子,只有在Spm转座后才具有功能。通过直接叶肉原生质体转化以及根癌农杆菌介导的根外植体转化获得了转化的诸葛菜(Orychophragmus violaceus (L.) O.E. Schulz)植株。通过GUS活性和PCR分析证实了基因转移和转座事件。使用原生质体的相对转化效率为5.8%。