How to remove the traditional marker gene sequence in transgenic plants?

At present, there are four methods to remove marker genes from transgenic plants: transformation, transposon-mediated relocation, homologous recombination and site-specific recombination. The first two methods are to integrate marker genes and exogenous genes in different positions, and they can be separated in subsequent plant hybridization and meiosis. In the latter two methods, the marker gene is deleted by recombination, so the marker gene is removed.

1.*** transformation: The principle of this method is to put the marker gene and foreign gene in two different T-DNA fragments, which can be linked or exist in different DNA molecules.

The transformation method used can be emphasis-mediated: role = italicagrobacteriumemphasis, or particle bombardment. During the transformation, two T-DNA fragments were integrated into different positions in the plant genome. During meiosis, marker genes and foreign genes will be separated. Only 25% of the * * * transformed offspring produced by this method have isolated foreign genes, so it is necessary to screen from a large number of transgenic plants. In addition, this method requires sexual reproduction of plants and is not suitable for the transformation of asexual plants such as potatoes and sugarcane.

Moreover, with gene gun transformation, sometimes two DNA molecules are integrated, and plants with excision marker genes cannot be effectively obtained.

2. Transposon-mediated relocation: This method is based on maize AcDs and SpmdSpm. Transposition in transgenic plants can separate DNA fragments with marker genes from target genes. Through hybridization between transgenic plants and non-transgenic plants and molecular analysis of offspring, offspring plants without marker genes can be obtained. Theoretically, transgenic plants only contain terminal repeats of the target gene and transposable element, and all other DNA used for transformation and transposition, such as T-DNA, marker gene and transposase, are excluded. The transpositional activity of different plants is very different. When the transpositional frequency is very low, a lot of screening work is needed.

3. Homologous recombination: This method is to put the marker gene between repeated sequences and cut the marker gene through homologous recombination of repeated sequences. These repetitive sequences can be any part of the transgenic cassette, such as promoters and termination signals. The frequency of homologous recombination in plants is very low and it takes a long time. Generally, two rounds of continuous screening are needed, and the overall efficiency is very low.

4. Site-specific recombination: At present, at least four recombinant enzyme-mediated site-specific recombination systems have been proved to play a role in plant cells: CRElox system of phage P 1, 2μm plasmid FLPFRT system of beer yeast, RRS system of fermenting yeast rouxxi and Gin recombinant enzyme system of Mu phage. These methods can only be used for plants that propagate through sexual hybridization, not for plants with longer generations (such as trees). Recently, the specific recombination system mediated by λ phage attP site has also been tried to eliminate marker genes in transgenic tobacco. The attP site sequences of two 352bp λ phages were placed on both sides of the marker gene npt, and the marker gene was removed by recombination between them. The system does not need the expression of other proteins, nor does it need the genetic separation step to remove the recombinant enzyme gene, so it may become an effective tool to remove non-target genes, especially for asexual plants.