Hamzeh Shiva, Motallebi Mostafa, Zamani Mohammad Reza, Moghaddassi Jahromi Zahra
Department of Agricultural Biotechnology, National Institute of Genetic Engineering and Biotechnology, Tehran, Iran.
Iran J Biotechnol. 2015 Sep;13(3):1-9. doi: 10.15171/ijb.1099.
Selectable marker gene (SMG) systems are critical for generation of transgenic crops. Transgenic crop production without using SMG is not economically feasible. However, SMGs are non-essential once an intact transgenic plant has been established. Elimination of SMGs from transgenic crops both increases public acceptance of GM crops and prepares gene stacking possibility for improvement of complex traits. Synthetic inducible promoters provide an efficient and flexible strategy to regulate transgene expression.
This study aimed to construct a transformation vector based on Cre/ recombination system to enhance efficiency of SMG-free transgenic plant production followed by post-excision expression of gene of interest in transgenic plants by a pathogen inducible promoter.
In pG-IPFFDD-cre-gus construct, recombinase and selectable marker gene () cassettes were placed between the two recognition sites in direct orientation. Seed-specific Napin promoter was used for regulation of Cre expression in transgenic seeds. In the construct, flanked sequence containing and recombinase cassettes, located between a pathogen inducible promoter containing FFDD cis-acting elements and β-glucuronidase coding region. The cunstuct was transformed into via -mediated transformation.
The results showed that both and excision occurs in T1 progeny tobacco plants through seed-specific expression. The excisions were confirmed by methods activation of the gene, germination test on kanamycin-containing medium and molecular analysis. Inducibility of expression by FFDD-containing promoter in T1 leaf tissues was confirmed by histochemical staining assay.
The established system is not only an efficient tool for marker gene elimination but also provides possibility for inducible expression of the transgene.
选择标记基因（SMG）系统对于转基因作物的产生至关重要。不使用SMG进行转基因作物生产在经济上不可行。然而，一旦完整的转基因植物建立起来，SMG就不再是必需的。从转基因作物中消除SMG既增加了公众对转基因作物的接受度，又为改善复杂性状的基因堆叠创造了可能性。合成诱导型启动子提供了一种有效且灵活的策略来调控转基因表达。
本研究旨在构建基于Cre/lox重组系统的转化载体，以提高无选择标记转基因植物的生产效率，并在转基因植物中通过病原体诱导型启动子实现目的基因切除后的表达。
在pG-IPFFDD-cre-gus构建体中，重组酶和选择标记基因（）盒以直接方向置于两个lox识别位点之间。种子特异性的Napin启动子用于调控转基因种子中Cre的表达。在该构建体中，包含和重组酶盒的侧翼序列位于含有FFDD顺式作用元件的病原体诱导型启动子和β-葡萄糖醛酸酶编码区之间。该构建体通过农杆菌介导的转化转入。
结果表明，通过种子特异性的表达，T1代烟草植株中发生了和切除。通过基因激活方法、在含卡那霉素培养基上的发芽试验和分子分析证实了切除。通过组织化学染色试验证实了含FFDD启动子在T1叶组织中对表达的诱导性。
所建立的系统不仅是消除标记基因的有效工具，还为转基因的诱导表达提供了可能性。
