At present, the process of DNA transfer in plant transgenic technology (that is, the transformation of foreign DNA fragments into the plant genome) is performed at the tissue level. Only the genome of a part of cells in the transformed plant tissues is integrated into the exogenous DNA, so at the same time There are also untransformed cells. The transformed cells are usually screened from untransformed cells using a marker gene. Most commonly used marker genes are some resistance genes to antibiotics or herbicides. When it is used to co-transform a plant cell with a gene of interest, the plant cell can be provided with the ability to grow on a medium containing an antibiotic or a herbicide, and untransformed cells cannot grow on such a medium, so the surviving cell Only transformed cells. However, the marker gene does not participate in the trait improvement of the plant, and its presence in the plant after the screening is superfluous. This extra gene will have some negative effects. First, in the process of screening for transformed cells, untransformed cells with a large amount of growth inhibition will impede the absorption of nutrients by the transformed cells, and may also secrete some toxic substances to inhibit the growth of transformed cells. Second, when gene stacking is performed using the re-transformation method, each transgene insertion needs to be screened for transformation; however, only a few of the marker genes that can be used for plant transgene selection cannot satisfy the need for retransformation. The requirement for multiple marker genes so that the marker gene can only be deleted from the plant genome after one transformation so that the same marker gene is used in the next transformation. However, the most interesting aspect of the marker gene is its potential "flow" - the level of gene transfer and the possible biosafety problem of gene escape: the antibiotic resistance gene as a marker gene once flowed to harm human health Microorganisms will provide them with resistance, and the herbicide-resistance genes, if they flow into weeds that threaten the growth of food crops, will give them herbicide resistance and become "super weeds." Therefore, selectable marker-free (SMF) has become a new topic in the research of transgenic plants. According to different principles, the method of rejecting marker genes can be divided into two categories: separation culling and recombination culling. In the former, the marker gene and the gene of interest were inserted into the non-linkage sites of the plant genome, respectively, and the separation of the marker gene from the concerned gene was achieved by meiosis to achieve the purpose of removing the marker gene. In the latter case, the marker gene is constructed in the DNA cutting unit, and the cutting gene is recognized by the cutting enzyme (recombinase or transposase) and the cutting unit is removed, thereby removing the marker gene from the plant genome. This article explains these two methods separately. 1 Isolation of Marker Gene 1.1 Principle In the transformation of Agrobacterium tumefaciens T-DNA as a gene transfer vector, the marker gene and the gene of interest are constructed in a T-DNA unit in a closely linked manner, so the two are always common Integration into the plant genome makes it difficult to separate from each other by free or cross-interchange of genes in meiotic cells. To achieve separation of the marker gene and the gene of interest after transformation, the two types of genes can be designed in two T-DNA units, respectively, so that they are inserted separately rather than co-integrated into the plant genome (Komari et al., 1996). Since the insertion of T-DNA is random, the inserted marker gene and the gene of interest may be located on different chromosomes, ie, at non-linkage sites, or at positions far apart on the same chromosome; after meiosis, The marker gene and the gene of interest can be separated so that a transgenic plant without the marker gene can be obtained in the next generation. 1.2 Types According to the type of Agrobacterium used, the type of Ti plasmid, and the positional relationship between the two T-DNAs on the Ti plasmid, the separation and deletion of marker genes can be divided into five types. (1) The two T-DNAs constructing the marker gene and the gene of interest are located on two Ti plasmids, and the plant cells are co-transformed with two Agrobacterium tumefaciens strains. (2) The two T-DNAs constructing the marker gene and the gene of interest are located on two Ti plasmids, and the plant cells are transformed with a soil Agrobacterium strain. (3) The two T-DNAs constructing the marker gene and the gene of interest are located on the same Ti plasmid, and the marker gene and the gene of interest are separated by a large fragment of virulence genes. Two sets of T-DNA border sequences (left, LB and right, RB) are used, where one set defines the marker genes and the other sets the genes of interest. (4) The two T-DNAs constructing the marker gene and the gene of interest are closely arranged on the same Ti plasmid. (5) Design the marker gene and the gene of interest in a T-DNA unit with double right border (DRB). The author et al. (Lu et al., 2001) constructed a binary vector of DRB (Fig. 1) to obtain SMF transgenic rice with S5 gene of interest in the 5th gene fragment of rice dentate dwarf virus genome. Southern blot Tests have shown that 64% of Jarrah rice produces SMF progeny, whereas Chinese rice variety Xiushui is 36%. SMF progeny contain less than 50% of the plants containing the gene of interest. 1.3 Analysis and design The marker genes and genes of interest in the same T-DNA unit are always integrated into the plant genome because they are located in the same transfer unit. At this time, the function of the marker gene is manifested at two levels: the marker function of the gene integration event at the gene level and the screening function of the transformed cell at the cell level. When the marker gene and the gene of interest are located in two T-DNAs, the co-integration relationship between them does not exist, so the marker gene does not have the marker function for the integration event of the concerned gene, and it is in the entire transformation process. The role of this is only reflected in the screening of transformed cells. Therefore, the first step is to remove the marker gene from the T0 plants with the marker gene (ie, which can grow on the selective medium), and screen the plants that contain the gene of interest at the same time, that is, the co-transformation between the screening marker gene and the gene of interest. Plant. In summary, there are two central events in the separation of marker genes-co-transformation and isolation of marker genes and genes of interest; corresponding to two screening processes--screening of T0 co-transformed plants and T1 generation of non-selective marker plants. The screening; therefore there are two measured ratios - the co-conversion rate of the TO generation and the separation rate of the T1 generation. 2 Recombinant knockout of marker genes is different from marker gene knockout. Recombinant deletion eliminates the need to insert marker genes and genes of interest into two T-DNAs, and they are still constructed in a T-DNA in a tightly linked manner. However, inserting the recognition sequence of the DNA cutting system at both ends of the marker gene constitutes a clipping unit, which is trimmed and removed by the action of the enzyme that acts as a cutter. That is to say, the DNA recombination system is composed of two parts: the cutting unit defined by the recognition sequence and the enzyme that plays the role of cutting. After the marker gene completes the screening of the transformed cells, it is removed from the plant genome. There are two core issues in this tailoring process: the first is the control of the timing of the cropping, ie the cropping should be done after the marker gene has been screened; the second is the detection of the cropping event, ie the cropping event occurs only in partially transformed cells. The selectable marker gene has been trimmed of the transformed cells removed from the plant genome. Before discussing these two core issues, a brief introduction was made to the DNA cutting system that has been used for crop gene deletion. 2.1 DNA cutting system The cutting system used to remove marker genes from transgenic plants is essentially some DNA recombination systems, including two kinds of site-specific recombination systems and transposition systems. Both of these systems consist of a cropping unit, a marker gene (defined by the target sequence of a short DNA clipping enzyme at the left and right ends) and a cutting enzyme. Under the cutting action of the enzyme, DNA undergoes intrachromosomal recombination between two adjacent recognition sites, and the DNA fragment located between the recognition sites is cut off from the chromosome; the difference is that the DNA is cut out from the transposition system. The DNA fragment may be re-inserted at another site on the chromosome, and the clipped DNA fragment is lost in the site-specific recombination system. Site-specific recombination systems: Site-specific recombination systems that have been found to be effective in plants include the Cre/loxP system of E. coli bacteriophage P1 (Odell et al., 1990), the R/rs system of Zygosaccharomyces rouxii ( Sugita et al., 2000), the Flp/frt system of Saccharomyces cerevisiae (Lloyd and Davis, 1994, Seibler et al., 1998) and the Xis/attP system of E. coli phage lambda (Better et al., 1983). Among them, Cre/loxP is a more commonly used system and has been well applied in rice research (Tran et al., 1999). McCormac et al. (1999) developed a useful dual carrier system based on this system. Transposition system: The transposable system for marker gene knockout of transgenic plants that has been reported so far is the Ac/ds system derived from corn (Hehl and Baker, 1990). As mentioned above, the DNA fragments cut out in the transposition system may be re-inserted into another site on the chromosome. If the old and new two sites are distributed on two chromosomes, the marker gene can be focused on by meiosis. Genes are separated, but if the two sites are located on the same chromosome, crossover is also required. Therefore, both the marker gene and the gene of interest can be constructed in the cutting unit, but the transgenic plants without the marker gene can only be screened in the next generation. 2.2 The deletion of the control marker gene at the time of clipping should be performed after the transformation screening. Therefore, usually the cutting unit and its immediately adjacent gene of interest are inserted into the plant genome, and then the enzyme gene for cutting is introduced; at the marker gene After removal with the cutting unit, the cutting-off enzyme gene inserted in the plant genome is meiotically separated from the gene of interest, so that transgenic plants having only the gene of interest can be obtained in the offspring. There are two methods of introducing the cropping enzyme gene into the genome of a transgenic plant into which the marker gene has been inserted: retransformation and cross-pollination. The retransformation is the screening of the transformed genes of the T0 generation marker genes and the transformation of the enzyme genes. Therefore, the marker gene can be deleted from the plant genome in the T0 generation; however, the later introduced cutting enzyme gene must be separated from the target gene by meiosis in the T1 generation to obtain the transgenic plant with only the concerned gene. Cross-pollination is introduced into the cutting enzyme gene through the T0 generation of sperm cells. Therefore, the marker gene can only be removed in the T1 generation. The cleavage enzyme gene can only be separated from the concerned gene in the T2 generation. In addition, the cut-off rates of the two methods of introducing the cutting enzyme gene are quite different. For example, the cutting rate of the re-introduced Cre recombinase is significantly higher than that of the cross-pollination, and is stable at 90% to 95%, which may be different from There is a difference in the expression of Cre proteins in cells. The above method of introducing the two components of the cutting system into the plant genome, respectively, ensures that the marker gene is removed after the transformation and screening, but it is time consuming and consumes labor and materials. If the marker gene and the tailored enzyme gene are closely arrayed in a cutting unit and inserted into the plant genome, the transgene containing only the concerned gene can be screened out in the TO generation for both the marker gene and the tailored enzyme gene. Plants. However, when the marker gene and the cutting enzyme gene are integrated into the plant genome, the expression of the cutting enzyme gene may cause the marker gene to be cut out prematurely. Therefore, the tailoring enzyme gene should be placed under the control of the inducible promoter. The marker gene was cut off after completing the transformation screening. Zuo et al. (2001) used beta-estradiol to induce Cre gene expression in transgenic Arabidopsis thaliana. Eight copies of the Lex A operator sequence were fused upstream of the Cre gene's CaMV 35S promoter, allowing the Cre gene to be expressed under the induction of β-estradiol. Using the GFP gene as a reporter gene, the cutting unit containing the marker gene, the Cre gene, and the transactivation gene of XVE was inserted between the promoter and the coding region of GFP. Therefore, the GFP gene can be expressed only after the cutting unit is removed. . The transformed tissue was first selected on the selective medium and no GFP expression was detected at this time. The transformed tissue was then transferred to the induction medium containing β-estradiol. As a result, GFP was detected in all the transformed lines. expression. This shows that β-estradiol inducible expression system can strictly control and efficiently induce the expression of the cutting enzyme gene. In another experiment, the R recombinase in the R/rs system achieved induced expression under the control of the Safener-induced GST-II-27 promoter. 2.3. Screening for Clipped Cells The β-estradiol-induced Cre/loxP system described above had only about 29% to 66% of the T0 generation of germ cell cells that had a cutting event. Therefore, the tissue after induction cutting is actually a chimera, and it is necessary to screen out the cropped cells. This can be done by negative selection marker genes. The so-called negative selectable marker gene means that its product can be toxic to cells under certain conditions, so that cells bearing a negative selectable marker gene cannot survive under such conditions. For example, the cytosine deaminase (cod A) gene from Escherichia coli is a conditional lethal dominant gene that can convert non-toxic 5-fluorocytosine to 5-fluorouracil, which is thymidine. The precursor molecule of the irreversible inhibitor of acid synthase, 5-fluorodeoxyguanosine-phosphate, results in the inability of the cell to synthesize DNA due to the lack of deoxythymidine triphosphate. By constructing such a negative selection marker gene in the cutting unit, only the cells that have been knocked out of the plant genome by the negative selection marker gene and the marker gene and the cutting enzyme motif can survive. The cod A gene has been used for the screening of transgenic tobaccos without marker genes (Gleave et al., 1999; Corneille et al., 2001). In addition to negative selectable marker genes, a class of genes that promotes the reproduction and differentiation of plant cells can also be used to screen for tailored cells. This type of dominant gene promotes the growth of plant cells, but also causes the plant to form an abnormal morphological structure. Such as A. The prenyltransferase gene of tumefaciens PO22 can catalyze the synthesis of cytokinin, promote the proliferation of transformed cells and differentiate to form adventitious shoots different from normal shoots; Ebinuma et al (1997) And Endo et al. (2002) constructed a new plant vector system for MAT (multi-auto-transformation), which contains a chimeric ipt gene inserted in the transposon AC, and the ipt gene can be used as a marker for transformation. gene. The MAT carrier system can greatly shorten the breeding time and is therefore particularly useful for woody plants. From A. The rol gene of rhizogenes NIAESl724 promotes the proliferation of the "hairy root" of transformed tissues by increasing the sensitivity of auxin. These abnormal morphological features can be used as screening markers for transformed cells instead of screening for non-transformed cells with selective agents such as antibiotics or herbicides. Further, if such a growth-promoting gene is constructed in a cutting unit, when it is removed from the plant genome by the action of a clipping enzyme, the transgenic tissue will form a normal morphological structure, and this type of growth-promoting gene acts as a tailoring. Remove the screening tag. For example, with the elimination of the ipt gene, the apical dominance and rooting ability will be restored, and morphologically normal seedlings will appear in the indeterminate pods formed after transformation and screening, and the latter is the transgenic tissue with the marker gene removed. Thus, the growth-promoting gene of plants can be used as both a screening marker for transformation (abnormal morphology) and a screening marker (normal form) for cropping, and at the same time assuming two screenings required for the cultivation of transgenic plants without marker genes. Features. The core of marker gene knockout methods for transgenic plants is the separation of marker genes from the genes of interest. In the excision-elimination method, the marker gene and the gene of interest are constructed in two T-DNAs, respectively, and thus have been "artificially isolated" before transforming the plant tissue; in the trimming culling method, the marker gene and the gene of interest are closely linked. Constructed in a T-DNA, the marker gene was trimmed from the plant genome by a tailoring system and therefore was isolated from the gene of interest in the transformed plant tissue. Since the marker gene and the gene of interest have been separated before the transformation of the plant tissue in the separation and deletion method, the marker function of the marker gene insertion is lost with the release of the co-integration relationship, and the corresponding effective conversion rate is also reduced. From this point of view, the crop culling method has advantages. In the crop culling method, the control of cropping time and the screening of cropped cells are the key issues, which can be solved by the improved design of the cutting unit: the cutting enzyme gene is designed downstream of the inducible promoter to control the time of cropping; Screen cells with a negative selection gene or a special plant growth promotion gene screen. Note: (1) References: Slightly omitted; those who need to contact E-mail or visit the Library of China National Rice Research Institute; (2) Source: Journal of Plant Physiology and Molecular Biology, 2004, Volume 30, Number 2 (3) Authors: Key Laboratory of Proteomics, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences.
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