WO2013000279A1 - Gene-auto-excision binary carrier for controlling biosafety of transgenic plant by sexual reproduction - Google Patents

Abstract

Constructed is a new Gene-Auto-Excision Binary System (GAEBS) for controlling the biosafety risk of a plant exogenous gene in sexual reproduction. Mainly using the transcription activating system as a molecular switch to control a recombinase excision system, on one hand, the recombinase gene located between the recombinase recognition sites is maintained silent in the sexual generation of a plant and heredity stability of the exogenous gene introduced by "GM-gene-deletor" technology in the sexual reproduction of the parent plant is realized; and on the other hand, by performing hybridization of parent plants carrying an effector element and an activator element respectively, the effector element and the activator element can be linked to present transcription activating factor activity. At the same time, using a plant tissue specific promoter to promote the expression of the recombinase, tissue specific excision of the exogenous gene is realized and a safe transgenic plant is obtained.

Description

 Automatic deletion of binary vectors by genetically controlling the safety of transgenic plants

Technical field

 The present invention relates to the field of plant genetic engineering, and in particular to methods and systems for the safety control of transgenic plants.

Background technique

 Since the birth of the first GM tobacco in 1983, with the extensive development of genetic engineering technology, a large number of genetically modified plants and their products have emerged, which has set off a new climax of biotechnology development worldwide. According to statistics, by 2009, the global GM crop planting area reached 134 million hectares.

 Although plant transgenic technology has become one of the most important means of increasing food production and reducing environmental pollution in the world. However, since the birth of this technology, people have been arguing constantly. Compared with traditional breeding techniques, the biggest advantage of transgenic technology is that it breaks through the reproductive isolation between species. Under artificial conditions, the target gene is transferred to the recipient plants, and the traits of plants are modified to achieve resistance, such as insect resistance, drought resistance and disease resistance. Wait for the desired purpose of humans to increase crop yields. Biotechnology can be used to introduce genes from bacteria, viruses, animals, humans, distant plants, or even synthetic genes into plant genomes. This situation is almost impossible in nature, so one cannot predict how this transfer of genes will affect the future ecological environment and human health. Therefore, countries around the world (especially Europe) have concerns about the safety of GM technology, and fear that GM plants will cause damage to the ecological environment and even adversely affect human health. As the scope of GMO applications continues to expand, the scientific community and the public are increasingly concerned about the safety of GMOs. This concern has seriously affected the large-scale commercialization of GM crops.

 At present, people's concerns about the biosafety of GM crops are mainly concentrated in two aspects:

 I. Ecological safety: Studies have shown that pollen-mediated gene escape is common between many crops and their wild relatives. Therefore, people are worried that foreign genes such as insect resistance, disease resistance and herbicide resistance in transgenic plants may escape into the natural world, and so-called "super weeds" appear. Certain insect-resistant genes (such as the Bt gene) not only kill pests effectively, but also harm non-target organisms. In addition, genetically modified seeds may also enter other environments by means of vectors such as wind, insects, and birds, which may damage the local ecological balance. All of the above problems can have disastrous consequences for ecological balance and biodiversity.

 2. Food Safety: The public is concerned that insecticidal, antibacterial and allergenic proteins in genetically modified products can be detrimental to human health, causing harm such as chronic food poisoning, cancer or allergic reactions. A recent study found that certain genes of microbes in the human body can be transferred horizontally to the human genome. The foreign genes of genetically modified foods are likely to be transferred to human chromosomes by human microbes, posing a potential hazard to our health and future generations. This potential danger has led to strong opposition from some groups and individuals to GMOs, and even requires the destruction and total resistance of GMOs.

It is one-sided to adopt complete exclusion and resistance to emerging technologies. JH's way is to first fully understand the safety hazards of genetically modified organisms, and at the same time establish an assessment and control system for the safety of genetically modified organisms to ensure the safety of their application. At present, the major developed countries and some developing countries in the world have formulated their own regulations on genetically modified organisms (including plants), which are responsible for evaluating and monitoring their safety. However, this kind of management, evaluation and monitoring takes a long time, which is not only costly but also very difficult. Moreover, the characteristics of agricultural production in China are mainly based on small family economies. Therefore, it is more difficult to achieve complete control of genetically modified seeds, which is not conducive to the commercial development of genetically modified products.

 While strengthening the management of genetically modified organisms, scientists are actively exploring biotechnologies that control the safety risks of genetically modified organisms. The primary goal of eliminating GM safety hazards is to control or eliminate pathways through which biomarkers of foreign genes may pass through pollen and seeds. The main point of this technique is to eliminate the potential safety hazards of these foreign genes before the transgenic plants spread to the environment or their products are consumed after the biological functions of the foreign genes (such as insect, disease, and herbicide resistance) are completed. Biotechnology is favored by scientists all over the world because of its simple operation and low cost. However, most of the existing biotechnologies cannot meet the needs of the safety control of large-scale planting of transgenic crops in ffl, especially the biosafety risks of exogenous genes of hybrid crops such as rice, corn and rape. At present, there is no effective application. Biological control measures.

 There have been many reports on technical research on controlling the safety hazards of genetically modified organisms. These studies have focused on how to control and eliminate the biosafety risks of screening marker genes such as antibiotics. One strategy is to use currently uncontroversial biosafety marker genes such as the green fluorescent protein gene (GFP), the ribitol operon (NL), and the 6-mannose mannose isomerase gene (PMI). This strategy does not address the potential biosafety risks of the target gene (such as disease resistance genes) and promoters. Another strategy is to remove biochemical means from the genome of the transgenic plant after the marker gene such as antibiotics completes its function, thereby eliminating the potential biosafety hazards caused by the transgene. Biotechnology for the removal of foreign genes such as antibiotics is usually considered in two ways: On the one hand, in order to prevent ecological problems caused by transgenic escape, pollen and seed infertility technology, "terminator" technology, chloroplast transgenic technology, etc. have been developed to eliminate The spread of foreign genes through pollen or seeds. On the other hand, it is about the safety of genetically modified foods. The related technologies include: transposon technology, co-transformation technology, and antibiotic-free screening marker gene transformation technology.

 However, the above techniques have the following disadvantages: 1. The technical operation is difficult and cumbersome; 2. The problem of the genetically modified environment and food safety cannot be solved at the same time; 3. Only the deletion of the resistance marker gene is considered, and the target gene may be neglected. Safety issues; Fourth, the removal of foreign genes is inefficient and long-term. Therefore, the existing technologies cannot meet the needs of large-scale planting of GM crop safety control.

In addition, the site-specific recombinase system derived from microorganisms is also a good tool for deleting foreign genes. At present, site-specific recombinase systems that have been widely used on plants include: 1. Cre//o cadaveric system derived from bacteriophage PI {Bacteriophage PI); 2. From yeast Saccharomyces cerevisiae) 2μ plasmid FLP/Fi?r system;

raMxif pSRl plasmid R/3⁄4.

Relatively speaking, the gene deletion technology based on the site-specific recombinase system CxdloxP and has been widely used in biosafety control of transgenic plants in recent years due to its advantages of simple operation and high deletion efficiency. These systems work in a simple manner, and the components that complete the recombination reaction only need to be: a recombinase and a recognition site. ₀ The reaction process: The recombinase recognizes the corresponding specific recognition site in the genome and combines with it. "Holliday"structure; then the nucleophilic attack of the hydroxyl group of the C-terminal tyrosine residue of the recombinase destroys the phosphate bond at both ends of the asymmetric 8 bp sequence in the recognition site; then the recombinase 4 monomers undergo a lipid transfer reaction , will be located between the recognition sites of DNA fragments and One recognition site is deleted, and finally, the genomic DNA is re-ligated to complete the recombination. Since the application of the recombinase system in plants, deletion of marker genes has been obtained in various plants such as tobacco, maize, rice, and Arabidopsis.

 There are two main ways to delete a foreign gene using a site-specific recombinase system:

 1. Trans-acting deletes foreign genes. It is mainly used for the deletion of marker genes. The basic strategy is to construct two plant expression vectors, one of which contains a pair of homologous recognition sites, a marker gene and a gene of interest, and the marker gene is placed between the same recognition sites. Another vector contains a recombinase gene expression element controlled by a constitutive promoter such as CaU5S. When the transgenic plant containing the gene of interest is obtained, the deletion of the marker gene can be carried out in two ways: First, the constitutively expressed recombinant enzyme gene is introduced into the transgenic plant by secondary transformation, and the marker gene located between the recognition sites is deleted. (Russell et al., 1992; Corneille et al., 2001; Marjanac et al., 2008); second, hybridization of a transgenic plant containing the gene of interest to a plant containing the recombinase gene, in a transgenic plant containing the gene of interest Marker gene deletion (Corneille et al., 2001; Arumugam et al., 2007). In this deletion strategy, the recombinase gene and recognition site are located on different chromosomes, and the interaction between the two can be seen as a trans-action. Plants with marker-free genes obtained by secondary transformation or hybridization pathways need to be genetically separated by sexual generation to remove the recombinase gene, which is cumbersome and time-consuming, and cannot be used for marker genes in asexually propagated plants. Deleted.

 2. Site-specific recombinase-mediated gene deletion. CaMV in the strategy of trans-acting delete foreign genes

35S promoter control Cre recombinase overexpression often leads to plant phenotypic abnormalities such as plant growth retardation and leaf yellowing (Coppoolse et al., 2003). Therefore, as a modification of the first method, the recombinase gene, the recognition site sequence and the foreign gene are placed on the same expression vector, and all the foreign genes are located between the recognition sites except the target gene. The expression of the recombinase gene is controlled by an inducible promoter or a tissue-specific promoter, and all foreign genes (including marker genes) other than the gene of interest can be deleted at any stage or at any stage of growth of the transgenic plant. The inducible promoter-controlled recombinase deletion technique has the advantage of controlling the deletion of the foreign gene, that is, when we need to delete, the recombinant enzyme can be artificially induced to delete the foreign gene, and the recombinase gene can be silenced when it is not needed. However, due to its inefficient removal and incompleteness, it is difficult to use in agricultural production to control the biosafety problems that GM may bring. The recombinant enzyme deletion technology controlled by tissue-specific promoters has been widely studied and applied due to its simple operation and high deletion efficiency (Gidoni et al., 2008).

 In 2002, Keenan et al. proposed a technical idea for producing non-GM foods from genetically modified plants (Keenan and

Stemmer, 2002): Place all foreign genes between the / ₀ recognition sites and control the expression of the recombinase gene with a chemically-inducible promoter or a tissue-specific promoter. Under the control of a chemically-inducible promoter, all foreign genes can be deleted at any time to obtain non-transgenic plants. What is even more exciting is: If tissue-specific promoters such as seeds, pollen, and fruits are used to control the expression of recombinase, seeds, pollen, and fruits without any foreign genes can be produced from transgenic plants to solve the transgenic passage. Ecological safety issues caused by pollen and seeds, and food safety issues of genetically modified foods.

On this basis, the experimental researchers have successfully established the "GM-gene-deletor" (Luo et al., 2007) in cooperation with the University of Connecticut. The system consists essentially of a pair of homozygous fusion recognition sites/οχΡ-Γ and a recombinase gene FLP controlled by a tissue (eg pollen or seed) specific promoter. Among them, all foreign genes are placed between the /οχΡ-Γ recognition site sequences. The results of the transgenic tobacco test show that the deletion system has the advantages of high efficiency, accuracy and thoroughness. In transgenic plants using the "GM-gene-dektor" system, pollen or seed-specific promoters control weight The expression of the enzyme, the target gene (such as the normal expression in the vegetative (root, stem, leaf) of the transgenic plant to play its biological function, and all the foreign genes (NP77/, Gi/S and recombinase genes) in the pollen The genome of the seed and the seed is efficiently and completely deleted, and the deletion efficiency is 100%. The idea of "generating non-GMO products with transgenic plants" by Keenan et al. can be realized. After the publication of this technology, it is considered to be "a major revolution".

 Although the "GM-gene-deletor" deletion system has achieved very high deletion efficiency (100%) in transgenic tobacco, and the complete deletion of all foreign genes has been achieved, the system can only be applied to asexual reproduction. In transgenic plants. This is because once the foreign gene is introduced into the plant genome by using this technology, the deletion of the foreign gene is automatically and procedurally carried out along with the developmental process of the plant, resulting in the foreign gene being completely replaced in the transgenic plant To pollen or seed. resection. Therefore, in the progeny of transgenic plants, there will no longer be genes for exogenous purposes such as improving plant resistance and improving crop quality. That is to say, the excellent genes introduced through the "GM-gene-deletor" technology cannot be passed on to the offspring through sexual reproduction, and the farmers cannot enjoy the benefits brought by the genetic modification. Major crops such as corn, rice, wheat and canola are cultivated in the form of sexual reproduction. Therefore, if the "GM-gene-ddetor" system can be applied to plants that are sexually propagated, its importance is self-evident.

 To achieve stable inheritance of a foreign gene introduced by the "GM-gene-deletor" technique in a sexual generation of plants, it is necessary to maintain the recombinase gene located between the recombinase recognition site sequences in the sexual generation of plants. Zuo et al. (2001) used the XVE chemical induction system to control the conditional expression of the recombinant enzyme gene, and realized the stable inheritance of the foreign gene in the progeny of the transgenic plants. It can be used for sexually reproducing plants, but the deletion efficiency is only 29%-60%. , only for the deletion of selectable marker genes, and the use of chemicals can also cause environmental pollution problems. Zhang et al. (2003) introduced recombinase and recognition sites into tobacco, respectively. Although the deletion of genes in transgenic trophozoites and germ cells was achieved, the potential biosafety risks caused by recombinant enzyme genes could not be solved. Although the harm of recombinant enzymes to human health has not been reported, studies have shown that high levels of Cre recombinase protein can cause 100% male sterility in transgenic mice (Schmidt et al., 2000, therefore, using recombinase When deleting the system to control the biosafety hazards of foreign genes, it is necessary to consider the biosafety risks that recombinant enzymes may bring. Therefore, how to use existing biotechnology to ensure that the recombinase deletion system is stably inherited in the sexual reproduction of plants, It is a major technical bottleneck faced by the current application of recombinant enzyme deletion technology to efficiently delete all foreign genes including the recombinase gene when the foreign gene needs to be deleted.

Among the existing transgene regulation technologies, transcriptional activation systems have been widely used in functional studies of plant genes. The working principle of this type of system is based on the trans-action principle of gene transcription activation. It is well known that transcription of a cellular initiation gene requires the involvement of a trans-transcriptional activator. A trans-transcriptional activator, such as the yeast transcription factor GAL4, is structurally modular and is often composed of two or more structurally separate, functionally independent domains, among which DNA A binding domain (DNA-BD) and an activation domain (DNA-AD). These two binding domains still function separately when they are separated, but they cannot activate transcription. Only when the separated two are spatially close by appropriate pathways can the full GAL4 transcription factor be re-presented and activated. The downstream promoter of the upstream activating sequence (UAS) allows transcription of genes downstream of the promoter. Moreover, the DNA binding domain and the transcriptional activation domain of different transcription factors are combined to form a hybrid protein, and the protein can still be corresponding The cis-acting element binds to activate transcription of the downstream gene. This cis-reaction is specific, that is, the specific Target promoter only responds to the corresponding DNA binding domain (DNA-BD). Obviously, the expression of the gene controlled by the target promoter and its expression pattern are affected. The regulation of transcriptional activator is controlled and consistent with the expression pattern of the latter, and in the absence of the corresponding transcriptional activator, the downstream gene controlled by the target promoter will remain silent.

 At present, many efficient transcriptional activation systems designed according to this principle have been widely used in plant gene function, gene cloning, enhancer trapping and other research. Among them, the transactivation principle of transcriptional activation system to activate the target gene by sexual hybridization coincides with the characteristics of sexual reproduction plants, that is, the green activation system can be used as a molecular switch to control the recombinase deletion system and maintain the foreign gene. Stable inheritance in the era of sexual reproduction of crops, and then through the hybridization method to achieve the removal of foreign genes in the specific development of plants, solving the existing recombinant enzyme deletion technology, especially the "GM-gene-ddetor" technology can not be used for sexuality The problem of reproductive plant biosecurity control. Summary of the invention

 In view of the above situation, in order to solve the technical problem that the existing gene deletion technology such as "GM-gene-deletor" cannot solve the safety hazard control of the exogenous gene of sexually reproducing plants, the present invention provides an effective control of sexually reproducing plant genetically modified organisms. Safe Gene Automatic Deletion Binary System (GAEBS). The system uses the green-activated system as a molecular switch to control the expression of the recombinase deletion system. On the one hand, the recombinase gene located between the recombinase recognition sites is silenced in the sexual generation of plants, achieving "GM-gene- The deletor "technology-introduced foreign gene is stably inherited in the sexual generation of plants; on the other hand, by means of hybridization, the two interacting elements of the transcriptional activation system are brought together to exhibit transcriptional activator activity while utilizing plants The tissue-specific promoter initiates the expression of the recombinase gene to realize the deletion of the foreign gene, and solves the biosafety potential problem that the foreign gene may bring.

According to an aspect of the present invention, it is an object of the present invention to provide an automatic deletion binary system for the safe control of exogenous gene organisms for sexually reproducing plants, which automatically deletes the binary system by constructing two plant expressions. The vectors, referred to as the first plant expression vector and the second plant expression vector, respectively, are used to control the safety of the exogenous gene organism of the sexually propagated plant. Wherein, the plant expression vector comprises the necessary elements and a plasmid vector carrying the elements, and the element for constructing the first plant expression vector is called an effector element, hereinafter referred to as an Effector element, for constructing a second plant expression vector element called activation Subcomponents, hereinafter referred to as Activator components. Specifically, the Effector element comprises two homologous recombinase specific recognition sites; and the following genes or nucleotides are inserted between the two same recombinase specific recognition sites: Controlling the expression of the foreign gene a promoter, a multiple cloning site for introducing a foreign gene, a recombinase gene, and a target promoter that maintains the recombinase gene silencing (the target promoted Activator element includes two homologous recombinase-specific recognition sites; Inserting the following genes or nucleotides between the two recombinant alcohol-specific recognition sites: a promoter for controlling expression of a foreign gene, a multiple cloning site for introducing a foreign gene, and controlling initiation of a transcriptional activator gene And a transcriptional activator gene that cooperates with a target promoter in the Effector element. The first plant expression vector and the second plant expression vector are constructed by operably inserting the above effector element/activator element into the plasmid vector, by the first plant An expression vector and a second plant expression vector, respectively introducing the Effector element and the Activator element into the plant genome, when carrying the Effector When the element and the plant of the Activator element hybridize as a parent, the target promoter in the Effector element will The transcriptional activator gene in the Activator element combines to activate the activity of the transcriptional activator and activate the promoter activity of the transcriptional activator gene to express the recombinase gene, enabling all exogenous sources including the recombinase gene. The deletion of genes achieves the goal of controlling the safety of exogenous genetic organisms in sexually reproducing plants.

Further, the recombinase deletion system in the automatic deletion binary system for the safety control of sexually reproducing plant transgenic organisms in the present invention may be a natural recombinase system isolated and cloned from animals, plants or microorganisms, or It is an artificially engineered, designed synthetic recombinase system; preferably a CrdloxP system derived from the bacteriophage PI { Bacteriophage PI ) and a Cre ^int / xP system, or a system derived from the yeast Saccha yces cerev / w'ae ) 2 plasmid. The recombinant enzyme is preferably a sequence specific recognition site / α, lox2272, tec5 7 ^ Γ recognition sites and the recombinase gene is selected from FLP, Cre and Cn? '"''Gene. Wherein cre'" ^t group Since the artificially engineered recombinase gene containing a plant-derived intron has a nucleotide sequence as shown in SEQ ID No. 11, wherein the plant-derived intron is pCAMBIA 1305.1 (GenBank accession number: AF354045.1) The intron of the GUS gene on the vector is catalase intron.

 Further, the promoter for controlling the transcription activator gene in the present invention may be a natural promoter isolated and cloned from an animal, a plant or a microorganism, or may be a artificially engineered or designed synthetic promoter; A sub- or tissue-specific promoter, in particular the cauliflower mosaic virus (CaMV) 35S promoter (Ca 5S).

Further, the transcriptional activation system of the present invention may be a natural transcriptional activation system obtained by isolation and cloning in an animal or a microorganism, or an artificially modified, designed and synthesized transcriptional activation system, preferably tTA/TOP10/pTAX, Gal4: VP16/UAS, mGal4: Artificially designed transcriptional activation system such as VP16 UAS or pOp/LhG4. In particular, the target promoter is ρθρ, and the transcriptional activator gene associated therewith is ^G ^ra , which is an improved version of the transcriptional activator gene AC^ with codons having Arabidopsis codon preference.

 According to another aspect of the present invention, there is provided a plant expression vector comprising the above Effector element and an Activator element, namely a first plant expression vector and a second plant expression vector, hereinafter referred to as an Effector plant expression vector and an Activator plant expression vector, respectively. The Effectsor plant expression vector comprises two homologous recombinase specific recognition sites; and the following genes or nucleotides are inserted between the two same recombinase specific recognition sites: controlling the expression of the foreign gene a promoter, a multiple cloning site for introducing a foreign gene, a recombinase gene, and a target promoter that maintains the recombinase gene silencing. The Activator plant expression vector comprises two homologous recombinase specific recognition sites; the following genes or nucleotides are inserted between the two recombinase specific recognition sites: a promoter for controlling expression of a foreign gene a multiple cloning site for introducing a foreign gene, a promoter for controlling a transcriptional activator gene, and a transcriptional activator gene that cooperates with a target promoter in an Fcector element.

According to still another aspect of the present invention, another object of the present invention is to provide the use of the above-described automatic gene deletion binary system for preparing a safe transgenic plant. When plants carrying the Effector element and the Activator element, respectively, hybridize as a parent, the target promoter in the Effector element will bind to the transcriptional activator gene in the Activator element, exhibit transcriptional activator activity and activate transcriptional activator The promoter activity of the gene enables expression of the recombinase gene, and the deletion of all exogenous genes including the recombinase gene is achieved, thereby achieving the purpose of controlling the safety of the exogenous gene of the sexually propagated plant. Still another aspect of the present invention provides a method for preparing a safe transgenic plant, firstly constructing an Effector element and an Activator element for automatically deleting a binary system, and inserting a foreign gene to be introduced into a plant genome into an Effector element and/or In the multiple cloning site of the Activator element, the obtained Effector plant expression vector and Activator plant expression vector are respectively transformed into the host plant to obtain the first gene plant and the second transgenic plant, respectively, which are respectively referred to as the Effectsor transgenic plant and the Activator transgene. Plants; Finally, the Effectsor transgenic plants and the Activator transgenic plants were crossed as parents, and the Activator elements and the Effectsor elements from the Effectsor transgenic plants and Activator transgenic plants were respectively closed, and the transcriptional activator activity was re-expressed and the promoters specific for plant tissues were The action turns on the expression of the recombinase gene, and finally the deletion of all exogenous genes including the recombinase gene is achieved, and a safe transgenic plant is obtained.

 The present invention is directed to the defect that the gene deletion technology such as "GM-gene-deletor" cannot maintain the stable inheritance of the foreign gene in the sexual generation of the plant, and creatively proposes the use of a transcription activation system (transactivation system) through extensive research and analysis and preliminary experiments. As a molecularly controlled recombinant enzyme deletion system, a new gene automatic deletion binary system GAEBS was constructed. According to the working principle of GAEBS, based on a large number of screening transcriptional activation systems and recombinase systems, the pOp/LhG4 transcriptional activation system and the Cre/loxp recombinase system were used to construct the GAEBS system. The Activator component and the Effector component in the GAEBS system can be transferred. The expression vector is constructed into a vector commonly used in the field and then transferred into the plant genome. A multiple cloning site sequence for insertion of a foreign functional gene is provided in both the Activator element and the Effector element, and any exogenous functional gene can be inserted therein for genetic improvement of plant genetic engineering.

 The experimental results of the transgenic tobacco in the present invention indicate that the sexual hybridization of the Effectsor transgenic plant and the Activator transgenic plant can efficiently and efficiently all the foreign parents derived from the automatic deletion system of the plant tissue-specific promoter CaMV 35S. The source gene is completely removed from the specific tissues or parts of the hybrid progeny, and the deletion efficiency reaches 100%. Therefore, the present invention successfully constructs the gene automatic deletion binary system GAEBS and the plant expression vector comprising the same, and the system successfully solves the technical problem that the prior art cannot solve the stable inheritance of the foreign gene in the sexual reproduction generation, that is, maintains the technical problem. The recombinase gene located between the recombinase recognition site sequences is silenced in the sexual generation of plants, and the foreign genes introduced by the "GM-gene-deletor" technology are stably inherited in the sexual generation of plants; The sexual promoter initiates the expression of the recombinase in a specific tissue or site to delete the foreign gene, and solves the biosafety problem that the foreign gene may bring, and has obvious beneficial effects. BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a schematic diagram showing the structure of a cloning vector pUC-CaMV 35S-GFP-nos carrying a CaMV 35S-GFP-nos element, wherein CaMV35S and 35S are plant tissue-specific promoters derived from cauliflower mosaic virus; _m GF _er (this The invention is abbreviated as "GFP" as a green fluorescent protein gene; it is a transcription termination sequence of the stalk synthase gene. It is an ampicillin resistance gene for screening of positive clones.

 Figure 2 is a schematic diagram showing the structure of a cloning vector pUC-35S-bar-nos carrying a 35S-bar-nos element.

 Among them, bar is a phosphinthricin acetyltransferase (PAT) gene.

Figure 3 is a schematic diagram showing the structure of a cloning vector pBS-spacer containing a Spacer sequence. The Spacer fragment is a non-functional fragment of approximately 2.4 kb in length from the pCAMBIA4956:ET15 vector. Figure 4 is a flow chart showing the construction of the binary expression vector ρίοχρΰΜΒίοχρ carrying the loxp-CaMV35S:GFP-MCS-35S:bar-loxp element.

 Wherein, ΝΡΤΙΠ is a kanamycin resistance gene; ΝΡΤΙΙ is a neomycin phosphotransferase gene; LB is a T-DNA left border; RB is a T-DNA right border; / w ^ is a cre//oxp recombinase system Identify the sequence of sites. pBIN 19 is a backbone vector for construction of plant expression vectors.

 Figure 5 is a flow chart showing the construction of the binary expression vector plox2272GMNlox2272 containing the lox2272-CaMV35S:GFP-MCS-35S:bar-lox2272 element.

 Among them, Loxp2272 is a mutant recognition site sequence of ίοχρ sequence; nosP is a promoter of the opine synthase gene. Figure 6 is a flow chart showing the construction of the binary vector pZ^ GN-MCS containing loxp-CaMV35S:GUS:NPTII-MCS-loxp.

 Amp-r is an ampicillin resistance gene.

Figure 7 is a flow chart showing the construction of the Effectsor plant expression vector pLOCB containing the pOp:cre' ^m element.

T3A is the polyA sequence of the pea ribulose-1,5-bisphosphate carboxylase small subunit rbcS-3A gene; Ρ ^Λ is the ampicillin resistance gene.

Figure 8 is a flow chart showing the construction of the Effectsor plant expression vector pL22720CN containing the pOp:cre' ^m element.

Figure 9 is a flow diagram showing the construction of the Activator plant expression vector pL35SLhG4 containing the 35S:LhG4 ^ATO element.

 Figure 10 is a test diagram of the Effector transgenic tobacco plants.

Among them, A and B are fluorescence detection maps of GF gene expression in different tissues of transgenic plants. A is the expression of GFT gene in transgenic leaves; B is the expression of GFP gene in transgenic roots. CK is a wild-type regenerated plant tissue. C is the PCR validation result for partial pLOCB transgenic tobacco. Among them, the P lane is the pLOCB plasmid as a template; the 1 -10 lane is the DNA of the pLOCB GFP ⁺ positive plant as a template. D is the PCR verification of pL22720CN transgenic tobacco. Among them, the 11-20 lanes were DNA of pL22720CN GFP+ positive plants. The M lane is a DNA molecule marker; the H lane is H ₂ 0 as a template; and the WT is a DNA of a non-transgenic regenerated plant as a template. The amplified target fragment is a pOp-cre ^int fragment of about 0.8 kb in length. Bar = 3mm.

 Figure 11 is a graph showing the acquisition of Activator transgenic tobacco plants.

Among them, A and B are histochemical staining analysis of gene expression in different tissues of transgenic plants. A is the result of GUS histochemical staining in transgenic roots, stems and leaves; B is the result of GUS histochemical staining of roots, stems and leaves of non-transgenic plants. C is a PCR verification result graph of pL35SLhG4 transgenic tobacco. Wherein, M lane is a DNA molecule marker; WT is a DNA of a non-transgenic regenerated plant as a template. The P lane is the pL35SLhG4 plasmid as a template; the lanes 1-9 are the DNA of the pL35SLhG4 GUS+ positive plant as a template. The amplified target fragment is a 35S-LhG4 ^ATO fragment of about 1.4 kb in length. Bar = 2mm.

 Figure 12 is a graph showing the efficiency of deletion of foreign genes in the hybrid progeny of the same pL35SLhG4 plant and different pLOCB plants by the GAEBS system.

Separate the deletion efficiency of the GFP and Gf/S genes in the offspring seeds, and then calculate the average deletion efficiency - Average deletion efficiency (%) = (efficiency of deletion of GE gene + efficiency of deletion of Gi/S gene) /2.

 Figure 13 is a graph showing the efficiency of deletion of foreign genes in the hybrid progeny of the same pLOCB plant and different pL35SLhG4 plants by the GAEBS system.

 The deletion efficiency of systematic deletion of G and genes in the offspring seeds was separately calculated, and then the average deletion efficiency was calculated: average deletion efficiency (%) = (efficiency of deletion gene + efficiency of deletion of Gi/S gene) /2.

 Figure 14 is a graph showing the efficiency of deletion of exogenous genes in the hybrid progeny of the pL22720CN plant and the pL35SLhG4 plant by the GAEBS system.

 The deletion efficiency of the G P gene and Gi/S was systematically deleted in the offspring seeds, and then the average deletion efficiency was calculated: the average deletion efficiency (%) = (the efficiency of deleting the gene + the efficiency of deleting the gene) /2.

 Figure 15 is a graph showing the statistical results of the efficiency of deleting the exogenous gene by the automatic deletion of the binary system by different genes.

Error bars Vertical bars=SE. Among them, the GAEBS system showed a significantly higher efficiency in deleting the Activator foreign gene in the progeny of pL222720CNxpL35SLhG4 hybridization than in the _P LOCBxpL35SLhG4 hybrid progeny (p<0.05; t-test). It is indicated that the efficiency of deleting the Activator exogenous gene in the progeny of pLOCBxpL35SLhG4 hybridization is significantly lower than that of deleting the foreign gene of Effector, and the difference is extremely significant (p<0.01; t-test).

 Figure 16 is a PCR analysis of the residual fragments of the system after deletion of the foreign gene in the progeny of the transgenic plants. Wherein, M is a DNA molecular marker; G71 15 is a 2272-1 l x35S-5 hybrid genomic DNA as a template; Π 7413 is a L 174X35S-13 hybrid F, and the genomic DNA is used as a template; f413 is a 35S-4xL13 hybrid F, the genomic DNA is used as a template; L13 is the genomic DNA of the L13 transgenic plant as a template; Π95 is the L79x35S-5 hybrid genomic DNA as a template; fl 316 is the L131 > 35S-6 hybrid genomic DNA as a template; 987 is a L198 genomic DNA hybridized with L198x35S-7 as a template; 35S-4 is a parental genomic DNA of a 35S-4 transgenic plant as a template. Primers for amplification are shown in SEQ ID N0.36 and SEQ ID NO. If the foreign gene is deleted, a specific band of about 800 bp will be amplified, otherwise a band of about 7.3 kb or / and 9.1 kb will be amplified.

 Figure 17 is a PCR analysis diagram of deletion of a foreign gene in a progeny of a transgenic plant.

The M lane is the DNA molecule marker. The 2272-1 1 lane uses the 2272-1 1 maternal genome as a template; the Ω7115 lane uses 2272-11 X35S-5 hybrid F, and the genomic DNA is used as a template; the 35S-5 lane uses the 35S-5 paternal genome as a template. . The L79 lane uses the L79 maternal genome as a template; f795 is a hybrid of L79x35S-5! The generation of genomic DNA is a template; the 35S-5 lane is a template of the 35S-5 paternal genome. The L131 lane uses the L131 maternal genome as a template; the Π316 lane uses the L131 x35S-6 hybrid genomic DNA as a template; and the 35S-6 lane uses the 35S-6 paternal genome as a template. The L198 lane uses the L198 maternal genome as a template; the fl 987 lane uses the L198x35S-7 hybrid generation genomic DNA as a template; and the 35S-7 lane uses the 35S-4 paternal genomic DNA as a template. The primer sequences used to amplify the maternal genome are shown in SEQ ID NO. 40 and SEQ ID NO. 41, and the target fragment is the cre ^int recombinase gene. The primer sequences used for amplification of the paternal genome are shown in SEQ ID N0.42 and SEQ ID NO. 43, and the target fragment is the LhG4 ^ATO transcriptional activator gene. The primers used to amplify the hybrid progeny genome are the above two pairs of primers. It can be seen from the figure that the presence of the parental cre ^int recombinase gene and the parental LhG4 ^ATO transcriptional activator gene was not detected in the progeny of the hybrid. Figure 18 is a graph showing the results of sequencing of residual T-DNA fragments in the hybridization of pL22720CN and pL35SLhG4 parents. The about 0.8 kb residual fragment amplified in Figure 16 was subjected to sequencing analysis. The results showed that all foreign genes were deleted, and finally only one UT12 and xp recognition site sequence and one non-functional fragment on the T-DNA fragment were left on the genome. The thick arrow indicates the direction of the sequence of recognition sites (5'→3'). DETAILED DESCRIPTION OF THE INVENTION

 The present invention will be further described in detail with reference to the accompanying drawings, which are not to be construed as limited by the accompanying claims. range.

 The reagents and drugs in the examples of the present invention are not generally described in the general market, and the material methods are not specifically described. Reference is made to the Guide to Molecular Cloning (Sambrook and Lasel, 2002).

 [Example 1] DNA extraction and PCR amplification of the target fragment sequence

 1. DNA extraction

 Select 0.3-0.5g of young leaves of tobacco, quickly grind it into white powder in liquid nitrogen, transfer to lOmL centrifuge tube, add 3 mL of pre-warmed DNA extraction buffer at 65 °C, and mix gently by shaking. Mix in a 65 ° C water bath for 45 min, mixing 2-3 times. Then add 1 mL of 5 mol/L KAc and ice bath for 20 min. It was extracted once with an equal volume (4 mL) of chloroform:isoamyl alcohol (24:1) (10,000 r/min, centrifuged at 25 ° C for 10 min). Take the supernatant, add 2/3 volume of -20 ° C pre-cooled isopropanol, mix, rest at room temperature for about 30 min, pick up the flocculent precipitate, rinse twice with 75% ethanol, then use anhydrous Rinse with ethanol once. Dry at room temperature and resuspend in 600 L TE. 1 L RNaseA (10 mg/mL) was added and treated at 37 ° C for 1 h to remove RNA from the sample. Re-use phenol (Ph8.0): chloroform: isoamyl alcohol (25:24:1) and chloroform: isoamyl alcohol (24:1) for each extraction (10,000 r / min, centrifugation for 10 min), take Clear, 2.5 times the volume of absolute ethanol precipitated at -20 Torr for more than 30 min. The pellet was collected at 13,000 r/min, centrifuged for 10 min, the supernatant was discarded, and the pellet was rinsed with 75% ethanol. After centrifugation under reduced pressure, the precipitate was finally dissolved in 50-200 μL of TE and stored at -20 °C until use.

 2. PCR amplification of the sequence of the target fragment

1 OxEx PCR buffer (Mg ²⁺ free) 2.5 μΐ,

 2.5 mmol/L dNTPs 2 μL

25 mmol/L MgCl ₂ 2 μL

 Upstream primer (5 μπιοΙ/L) 1 μL

 Downstream primer (5 μιηοΙ/L) 1 μL

 Ex Taq DNA Polymerase 1 U

 DNA about 20 ng

 25 amplification system

 The amplification procedure was: 94 ° C, 5 min; 94 ° C, 30 sec, 56 ° C, 30 sec, 72 ° C, l ~ 4 min, 35 cycles; 2 ° C extension for 10 min.

3. DNA fragment recovery, ligation, transformation of E. coli DH5a Under a UV lamp, cut the agarose gel block containing the desired fragment with a clean blade. Refer to the instruction manual of the kit (Roche) for the recovery method. The recovered fragments were quantified by electrophoresis on an agarose gel.

 The establishment of the restriction enzyme system and the reaction conditions were carried out in accordance with the instructions of the Roche restriction endonuclease kit. The recovered fragment, or the amplified fragment obtained by amplification, was cloned into pUCm-T (Shanghai Sarigon) or pGEM-T/pGEM-T Easy (Promega) vector according to the ligase kit instructions. The reaction system is as follows:

 10xT4 DNA Ligation Buffer 1

 Vector DNA fragment 1 (50ng)

 Exogenous ligation product DNA fragment 1

 Τ4 DNA ligase 1

 Replenishing 10 connection system with double distilled water

 The molar ratio of the vector DNA fragment to the exogenous ligation product DNA fragment was 1:1.

 Connect at 16 °C for 2-8h. The ligation product was then transformed into E. coli DH3⁄4 competent cells and cultured at 37 F.

 [Example 2] Construction of binary expression vector

 1. Acquisition of pOp/LhG4 transcriptional activation system

 1.1 Cloning of the ρθρ target promoter

 A pair of primers (shown as SEQ ID NO. 1 and SEQ ID NO. 2) were designed based on the pH-pOp(A) (Moore et al., 2006) plasmid on the O promoter sequence, and the 5' end was amplified by PCR. And the ρθρ promoter containing the H «dIII and Xbal endonuclease recognition site sequences at the 3' end, respectively, and T was cloned into the pGEM-T-easy vector to obtain the pGEM-t-pOp vector. Sequencing confirmed.

 1.2 Cloning of transcriptional activator genes

According to the pBIN-LR-LhGR ² (Craft et al., 2005; Samalova et al., 2005), a pair of primers (as shown in SEQ ID N0.3 and SEQ ID NO. 4) were designed on the plasmid (?). The Z/zG^ ¹⁷⁰ gene containing the sequence of the 3' end and the 3' end, respectively, was amplified by PCR and cloned into the pUCm-T vector to obtain the pUCm-t-LhG4 ^ATO vector. Sequencing confirmed.

 2. Artificial synthesis of the ere recombinase gene containing plant-derived introns

The intron (catalase intron) derived from the gene on the pCAMBIAl 305.1 vector (GenBank accession number: AF354045.1) was inserted into the ere recombinase gene encoding Glnl44/Vall45 acid according to the conserved sequence of the intron boundary in the plant genome. Between the codons of the residues. For this, primer pairs c-5F/c-5R (as shown in SEQ ID N0.5 and SEQ ID NO. 6), c-3F/c-3R (such as SEQ ID NO. 7 and SEQ ID NO. 8) and int-L/int-R (as shown in SEQ ID NO. 9 and SEQ ID NO. 10) were amplified to obtain the base sequence cre-5, 3 of the 5' end of the c recombinase gene of 432 bp. 'End 589 bp long base sequence cre-3 and 190 bp long catalase intron. Then, cre-5 intron and blunt end ligation reaction, and then this reaction solution as a template, with 05? / ^ 1-1 primers to give about 600 _1¾) size cre-5 + int fragments. The fragment was ligated to cre-3, and a 1.2 kb cr^" gene fragment was amplified by c-5F/c-3R. Finally, a 1.2 kb long ere'" fragment was recovered and sequenced after cloning. The Cre recombinase gene containing the catalase intron intron between bases 432 and 433 was obtained and designated cre ⁱⁿ ' as shown in SEQ ID NO. 3. Construction of binary expression vector p/oxpGMB/ox carrying loxp-CaMV35S:GFP-MCS-35S:bar-loxp First, a CaMV35S-GFP-nos expression element containing a GFP reporter gene was constructed. A pair of primers (shown as SEQ ID N0.12 and SEQ ID NO. 13) were designed according to the sequence of the Ca 35S promoter upstream of the GUS gene on the vector of pCAMBIA 1305.1 (GenBank accession number: AF354045.1), PCR amplified, and A Bg!ll endonuclease recognition site was introduced at the 3' end of the C3⁄4 35S promoter. Specific primers (shown as SEQ ID N0.14 and SEQ ID NO. 15) were designed according to the sequence of the mGFP-er gene (abbreviated as "GFP" in the present invention) on the vector of pCAMBIA4956: ETl 5 (Johnson et al., 2005). The Bamlll and Nhe\enzyme recognition sites were added to the 5' ends of the two primers; and the sequence of Deutsch bp downstream of the GFP gene (including the GFP termination sequence) was designed, and the primer nos-up/nos-down was designed (such as SEQ ID). NO. 16 and SEQ ID NO. 17), and a Spel enzyme recognition site was introduced at the 5' end of the former. The above target fragment was PCR-amplified, and T-cloning was applied to pUCm-T (Shanghai Shenggong Biotechnology Co., Ltd.) vector to obtain pUCm-T-CaMV35S, pUCm-T-GFP and pUCm-T-nos intermediate vectors, respectively. Sequencing confirmed. On this basis, GFP was excised from pUCm-T-GFP using BamHVNhel, and S/7eI/H/wiin was excised from pUCm-T-nos, both of which were digested with 3⁄4/II/H/mim. The pUCm-T-CaMV35S vector was ligated to obtain pUC-CaMV35S-GFP-nos, which contained the CaMV35S-GFP-nos expression element (as shown in Figure 1). spare.

 Next, a 35S-bar-nos expression element containing a herbicide resistant gene bar was constructed. First, use Hz'mmi/EcoRI

The 35S-GUS-nos element was excised from pBI 121 (GenBank accession number: AF485783.1) and ligated into the pUCm-T vector which was subjected to the same digestion to obtain pUC-35S-GUS-nos vector. Then, a pair of primers (as shown in SEQ ID NO. 18 and SEQ ID NO. 19) were designed according to the sequence of the herbicide resistance gene bar on the vector pFGC5941 (Genbank accession number: AY31090U), and 5' and 3' were obtained by PCR amplification. The bar genes with Spe\ and d, respectively, were T-cloning onto the pUCm-T vector and confirmed by sequencing. The bar gene was excised from the vector by S/?d/d, and ligated into the pUC-35S-GUS-nos vector treated with Xbal/Sacl enzyme to obtain a pUC-35S-bar-nos vector. Then, the vector was digested with Sacl enzyme, and the sticky ends were filled in, and the vector was cyclized to eliminate the 3⁄4d endonuclease recognition site between bar and nos (as shown in Fig. 2). spare.

 Then, the construction of the pBS-spacer vector. A 2.4 kb spacer sequence (non-functional sequence) was excised from the pCAMBIA4956:ET15 vector by HindlWEcoRI, and inserted into the pBluescript SK (pBS) (Genbank accession number: X52328.1) vector treated with the same enzyme to obtain pBS-spacer. Carrier, spare (as shown in Figure 3).

Finally, a pair of primers (shown as SEQ ID NO. 20 and SEQ ID NO. 21) were designed based on the hxp recognition site sequence and the CaMV35S-GFP-nos element sequence, wherein the 5' end of primer 1 contains a 34 bp long loxp. The 3' end is matched to the 5' end of the CaMV 35S promoter, and the primer 2 is complementary to the 3' end of the nos. The loxp-CaMV35S-GFP-nos element was amplified by PCR using the pUC-CaMV35S-GFP-nos plasmid as a template. Similarly, using the pUC-35S-bar-nos plasmid as a template, a pair of primers (as shown in SEQ ID N0.22 and SEQ ID NO. 23) was used to amplify the loxp-35S-bar-nos element. The recognition site hxp of Cre recombinase was added to the 5' end of both fragments. The above fragment was T-cloning onto the pUCm-T vector and confirmed by sequencing. First, the loxp-CaMV35S-GFP-nos element was excised from the intermediate vector pUC-loxp-CaMV35S-GFP-nos by SacVKpnl, and ligated into the same enzyme-treated pBIN 19 (Genbank accession number: U09365.1) vector to obtain pLoxp. - GFP intermediate vector. Then, the hol/Xbal enzyme will take the loxp-35S-bar-nos component from The pUC-loxp-35S-bar-nos vector was excised and ligated with the SaWNhel digested pLoxp-GFP vector to generate the p ocpGBZrap intermediate vector. Finally, ^ZwI/ /wI digested the pBS-spacer vector, and filled the Qwl sticky end, and recovered the spacer fragment. At the same time, S lAY^I cut the p£ox GB ox ? vector and ligated with the spacer fragment to produce a plant expression vector. p xpGMWoxp. Among them, a 2.4 kb spacer sequence was inserted between the GFP and Bar expression elements, and a multiple cloning site was included at both ends of the spacer sequence. Thus, all exogenous gene fragments, including the multiple cloning site MCS, are located between the recognition site sequences (as shown in Figure 4).

 4. Construction of binary expression vector containing plox2272GMNlox2272 containing lox2272-CaMV35S:GFP-MCS-35S:bar-lox2272 element

 Similarly, a pair of primers (shown as SEQ ID N0.24 and SEQ ID NO. 25) were designed based on the Ιοχ2272 recognition site sequence and the CaMV35S-GFP-nos element sequence, pUC-CaMV35S-GFP-nos plasmid was used as a template, PCR The lox2272-CaMV35S-GFP-nos element was amplified. Similarly, the IBIN2272-nosP-NPTII-nos element was amplified using a pair of primers (shown as SEQ ID NO. 26 and SEQ ID NO. 27) using the pBIN 19 plant expression vector as a template. The recognition site sequence of Cre Recombinase 10x2272 was added to the 5' end of both fragments. The above fragment was T-donated onto the pUCm-T vector and confirmed by sequencing. The pUC-lox2272 (-nosP-NPTII-nos vector was digested with Xhol/Xbal, and the lox2272-nosP-NPTII-nos fragment was recovered and ligated with the Savon Nhe\treated pBIN 19 vector to obtain the plox2272-NPTII intermediate vector. Then, Sacl/Xbal The vector was recovered by restriction enzyme digestion. At the same time, Sacl/Kpnl digested the pUC-lox2272-CaMV35S-GFP-nos vector, recovered the lox2272-CaMV35S-GFP-nos fragment, and KpnVXbal digested the pBS-spacer, and recovered the spacer fragment, which was recovered. The three fragments were ligated to obtain the plant expression vector p/o 2272GMN/o 2272. Similarly, all exogenous gene fragments including the multiple cloning site MCS were located between the Ιοχ22Ί2 recognition site sequences (as shown in Figure 5). .

 5. Construction of the binary vector p£ox/?GN-MCS containing loxp-CaMV35S:GUS:NPTII-MCS-loxp in order to add the /ox/7 recognition site upstream of the 2xCaU5S promoter, according to the pCAMBIA1305.1 vector The l^CaMV 35S promoter sequence was designed to design a pair of primers (as shown in SEQ ID N0.28 and SEQ ID NO. 2), and the pCAMBIA1305.1 plasmid was used as a template to obtain a loxp-2xCaMV35S element by PCR amplification. Similarly, a pair of primers were designed with the sequence of the pBIN 19 vector (as shown in SEQ ID NO. 30 and SEQ ID NO. 31, and the pBIN 19 plasmid was used as a template to PCR-amplify the nos-loxp element. Thus, The loxp recognition site sequence was added to the 3' end of the nos sequence. T-cloning onto the pGEM-T-easy vector to obtain the pGEM-T-nos-loxp intermediate vector. Sequencing confirmed. Sad/EcoRI digestion pGEM-t- The nos-loxp intermediate vector, the nos-loxp element was recovered and ligated into the pBIN19 vector treated with the same enzyme to produce the pBIN-loxp-nos intermediate vector. Secondly, the GUS:NPTII-nos element was extracted from pSackiss-35S-GUS with BamHVSan enzyme. : NPTII-nos vector was ligated and ligated with pGEM small loxp-35S vector treated with 3⁄4/ΠΑ3⁄4/Ι enzyme to obtain pUC-loxp-35S-GUS: NPTn-nos cloning vector. Finally, HiVjdlll/zd enzyme was used. The vector was digested, and the loxp-35S-GUS:NPTII-nos fragment was recovered and ligated with the same enzyme-treated pBIN-loxp-nos vector to obtain a p/^xpGN-MCS vector. In this vector, GUS, ΝΡΤΠ and The cloning loci MCS are located between a pair of co-directional recognition locus sequences (Figure 6)).

[Example 3] Construction of plant expression vector 1. Construction of an Effector plant expression vector containing pOp re ^int elements

To construct an Effector plant expression vector, the pOp-cre ^int- T3A expression element was first constructed. A pair of primers (shown as SEQ ID N0.32 and SEQ ID NO. 33) were amplified from the pER8 vector to obtain the terminator sequence T3A, which introduced the JiTwI endonuclease recognition site at the 5' end, T-cloning to pUCm- On the T vector, a pUC-T-T3A vector was produced. Sequencing confirmed. Then, Sacl/Xbal cuts the ρθρ promoter from the pGEM-T-pOp vector, and the Τ3Α termination sequence is excised from the pUC Τ3 A vector, and XbaVSdl cre ^mt gene from pUC-T-Cre ^{The int} vector was excised and the three fragments were ligated into the SacVBglll-digested pUC vector to obtain a _P UC-pOp-Cre ^int -T3A vector containing the pOp-Cre ^int -T3A expression element.

Finally, the pOp-Cre ^int -T3A expression elements were excised with Hi"dll leg ol and ligated into the HindUl/Xhol digested p xpGMB!oxp and p/ox2272GMN/ox2272 plant expression vectors, respectively, to generate the corresponding Effector plants. The expression vectors pLOCB and pL22720CN (Fig. 7, 8).

2. Construction of Activator plant expression vector pL35SLhG4 containing 35S:LhG4 ^ATO element

First, the LhG4 ^ATO- T3A element was constructed, and the ZJJG^^ gene was excised from the pUCm-t-LhG4 ^ATO vector by wHIA3⁄4/I. XhoVKpnl used the T3A (pea 3A) transcription termination sequence from the pER8 vector (Genbank accession number: AF309825.2) The upper cut was ligated into the amHI/ /wI enzyme-treated pBS vector to obtain pBS-LhG4 ^ATO- T3A, which contained the LhG4 ^ATO- T3A element. Then, the element was excised with Kpnl/Baml, San/B imHl was cleaved from the pSackiss-35S-mcs-nos vector, and the 35S promoter was ligated, and the J55 "promoter and LhG4 ^ATO- T3A elements were ligated together with the SaWKpnl enzyme. In the treated p£ojf/7GN-MCS vector, a pL35SLhG4 plant expression vector (shown in Figure 9) was obtained.

 [Example 4] Transformation of expression vector

 1. The constructed plant expression vector plasmid was introduced into Agrobacterium EHA105 by electroporation.

 The above vector was introduced into Agrobacterium EHA105 by electroporation using the Bio-RAD MicroPulser User's Manual.

 2. The Effector and Activator components of the GAEBS system are integrated into the tobacco genome

 Genetic transformation of tobacco by Agrobacterium tumefaciens-mediated methods

 Table 1: Agrobacterium tumefaciens-mediated medium for tobacco genetic transformation

 Medium name

Basic medium (MSB) MSB (MS inorganic salt + B5 organic)

Seed germination culture (MSB0) MSB+30g/L glucose +7.5 g/L agar, pH 5.8

Co-culture (MSB1) MSB+2.0mg/L 6-BA+0.5mg/L IAA+6g/L Qiongyue, pH5.8 Screening medium (MSB2) MSB+2.0mg/L 6-BA+0.5 Mg/L IAA +200mg/L Cef+ 50mg/L

 Kan+6g/L agar, pH 5.8

Inducing medium (MSB3) MSB+200mg/L Cef+ 50mg/L Kan+6g/L agar, pH5.8 Rooting medium (MSB4) MSB+200mg/L Cef+6g/L agar, pH5.8

MS: Murashige & Skoog, 1962 B5: Gamborg, 1986

 The above expression vector is introduced into tobacco by a method of Agrobacterium-mediated leaf disc. The specific method is as follows:

 Tobacco seeds were sterilized with 1% sodium hypochlorite and germinated on solid medium MSBc under the conditions of 25 ° C, 16 hr light / 8 hr dark photoperiod. A sterile sterile seedling that grows about one month later can be used as a transformed explant.

Select robust blade, cut into about clean table leaf discs 0.5cmx0.5 _C m, moist standby. The toothpick picks up the Agrobacterium single colony cultured on the YEB plate, activates the liquid culture, and then transfers it to a flask to grow to OD _{6 ( )} = 0.5. The cut leaf discs were immersed in the Agrobacterium liquid suspension resuspended in MSB liquid basic medium, and immersed for 10-20 min. The bacterial liquid was decanted, and the excess bacterial liquid on the surface of the leaf disc was aspirated with a sterile absorbent paper, and the leaf disc was placed on the co-culture medium MSB!, and cultured for 24 days in the dark. After the completion of the co-cultivation, the leaf discs were subjected to differentiation culture in the screening medium MSB ₂ for 2 weeks under the conditions of 25 ° C, 16 hr light / 8 hr dark photoperiod. After the regenerating green callus appeared, transfer to the bud medium MSB _{3 to} promote bud production. When the resistant seedlings grow to 3-4 cm in length, they are cut into rooting medium MSB ₄ to induce rooting. When the roots of the resistant seedlings grow to 2-3 cm long, the medium is washed, and the hydroponic seedlings are 2-3 days, transplanted into nutrient mash, and grown in a greenhouse. The obtained transgenic tobacco was not significantly different in phenotype and growth development from the wild type control.

 [Example 5] Screening of transgenic plants

 1. Effector's acquisition of transgenic tobacco plants

 Since the Effector vector contains the green fluorescent protein GF reporter gene, the expression of GF in the leaves of plants and other tissues was detected by fluorescence stereomicroscope in tobacco regenerated plants screened by PPT (pLOCB) and Km (pL22720CN). Happening. GFP green fluorescence identification of transgenic tobacco is described in Haseloff (Haseloff, 1999). The leaves of the resistant seedlings were harvested and the green fluorescence signal of the tobacco (the excitation peak was 488 nm and the emission peak was 520 nm) was observed under an Olympus SZX-ILLB2-200 stereofluorescence imaging system (R) and photographed. As shown in Fig. 10A, B, the green fluorescent signal was detected in both the leaves and roots of the transgenic tobacco, while the control did not detect any green fluorescent signal and only showed the red fluorescent signal of chlorophyll. Among them, the fluorescence of the control plant (CK) root was significantly weakened. Based on the above observations, the effector transgenic plants were screened by the uniform green fluorescent signal of the resistant seedling leaves.

For GFP-positive plants, the integration of foreign genes was verified by PCR. As shown in Fig. 10C-D, specific fragments of about 0.8 kb pOp-Cre ^int were detected in all transgenic tobaccos displaying green fluorescent signals, indicating that these GFP-positive plants were indeed transgenic plants (Fig. 10, C, D). Show). The primers used are shown in the sequences as SEQ ID N0.34 and SEQ ID NO.

 2. Acquisition of Activator transgenic tobacco plants

 Because the Activator vector contains the Gf/S reporter gene. Therefore, the resistant seedlings were first stained by GUS histochemical staining. For the method of GUS tissue staining, see the method of Jefferson et al. (1987). The main process is as follows:

The transgenic plant material (leaf, stem, root, etc.) was cut into thin slices, placed in a freshly prepared X-Gluc staining solution, and incubated at 37 ° C for 12 h. After sufficient staining, it was destained 2 to 3 times with 75% (v/v) ethanol until the control material (wild type plant) was white. And under the stereoscopic observation and photography. As shown in Fig. 11A, B, the roots, stems and leaves of the transgenic plants showed strong GUS blue, while the non-transgenic plants showed no blue color. Then, PCR was used to further identify the integration of foreign genes in transgenic plants with GUS staining in blue. The results showed that all of the transgenic plants with GUS staining were blue. A specific band of 1.4 kb 35S-LhG4 ^{ATO was} detected (shown as C in Figure 11). The primers used are shown in the sequences as SEQ ID N0.36 and SEQ ID NO.

 [Example 6] Analysis of genetic stability of foreign genes in sexual generation of Effector transgenic plants

An appropriate amount of To seeds was added to a 150 ml flask, and then about 30 mL of tap water was added, and the mixture was shaken at 26 rpm for 5-7 days at 26 °C. After the two true leaves were fully expanded, the number of green fluorescent (GFP ⁺ ) and non-green fluorescent (GFF) seedlings was counted under a fluorescence stereo microscope. Then, according to the Mendelian inheritance rule of single gene 3:1 and double gene 15:1, Xc ² analysis was performed on the obtained data.

( Ο represents the number of observations, Ε represents the theoretical number of times): 2 ^ = 3.84. According to the statistical results in Table 3.2, in the 33 affectedfector plants analyzed, except for the copy number of the foreign gene of the L178 transgenic plants, the ratio of the exogenous genes of 16 transgenic plants was 3:1, and the remaining transgenic plants were selected. Both of them met the separation ratio of 15:1, and the separation ratio did not match the phenomenon of 3:1 or 15:1. These results indicate that in the sexual reproduction of pLOCB and pL22720CN transgenic plants, the ρθρ promoter can efficiently maintain the recombination of the recombinase gene and maintain the stable inheritance of the exogenous gene in sexual generation.

 Analysis of Gene Copy Numbers in 3⁄4_2 Transgenic pLOCB and pL22720CN Tobacco

 Vector transgenic assay GFP positive GFP negative separation ratio 2

 A c plant total seed number seed number seed number

 pLOCB L13 310 227 83 3:1 0.11

 L18 395 297 98 3:1 0.00

L19 407 296 111 3:1 0.25

L30 276 196 80 3:1 0.53

L79 176 134 42 3:1 0.02

L91 330 244 86 3:1 0.04

L102 371 271 100 3:1 0.16

L122 157 150 7 15:1 0.04

L131 390 296 94 3:1 0.03

L133 279 211 68 3:1 0.01

L136 196 188 8 15:1 0.08

L155 170 125 45 3:1 0.03

L168 434 407 27 15:1 0.00

L173 179 168 11 15:1 0.00

L174 243 179 64 3: 1 0.04

L175 291 221 70 3:1 0.02

L178 502 502 0 - -

L179 250 241 9 15:1 0.16

L194 220 219 1 15:1 0.73 L198 297 228 69 3:1 0.10

L210 273 212 61 3:1 0.22

L211 320 230 90 3:1 0.38

L213 369 366 3 15:1 1.11

L220 253 195 58 3:1 0.12

2272-1 312 234 78 3:1 0.00

2272-2 328 248 80 3:1 0.01

2272-3 280 216 64 3:1 0.14

2272-4 298 272 26 15:1 0.17 pL22720CN 2272-5 270 255 15 15:1 0.01

 2272-6 300 224 76 3:1 0.00

2272-10 213 203 10 15:1 0.04

2272-11 286 216 70 3:1 0.01

2272-12 375 282 93 3:1 0.00

[Example 7] Screening of single copy transgenic plants

 In order to facilitate the analysis of the efficiency and characteristics of the deletion of the foreign gene in the GAEBS system after hybridization, the single-cury transgenic plants were screened for hybridization experiments by the method of Example 6. The analysis of the copy number of the Effector transgenic plants mainly detected the isolation of GFP green fluorescence in the self-crossing seedlings, and the single-copy plants of the transgenic pLOCB and pL22720CN were initially identified. It can be seen from Table 2 that most of the transgenic plants are single-copy and double-copy plants, of which 16 are single-copy transgenic plants and 6 are pL22720CN.

 For the transgenic pL35SLhG4 tobacco, GUS tissue staining analysis was performed on the seedlings, and the number of blue (GUS+) and white (GUS-) seedlings was counted. Single-copy plants were identified by analyzing the isolation ratio of the Gt/S gene in the self-crossing TV generation. Table 3 shows that the six transformants analyzed were single copy transgenic plants.

Table 3 Analysis of foreign gene copy number in transgenic _P L35SLhG4 tobacco

 Vector transgene detection GFP positive GFP negative separation ratio Xc2

 Plant total seed number seed number seed number

 35S-4 318 234 84 3:1 0.07

35S-5 346 247 99 3:1 0.55

35S-6 344 260 84 3:1 0.01 pL35SLhG4

 35S-7 538 378 160 3:1 1.55

35S-9 484 360 124 3:1 0.02

35S-13 283 210 73 3:1 0.01

The detection of the suitability of the Mendelian separation ratio in the 'To generation seed' in which the GUS stained blue. If: GUS ( + ) : GUS (-) meets 3:1, which is a single copy; if it meets 15:1, it is a double copy.

[Example 8] Hybridization test of transgenic tobacco For the hybridization method, see Liu Renxiang et al. (2000). During the flowering period of tobacco, the anthers to be split on the second day of the afternoon are stored in the laboratory. After the morning of the second day, the anthers have been split, and the field is picked up with a brush. The pollen is applied to the stigma (to the stamen) to be pollinated, and the pollination process is completed.

 [Example 9] Analysis of the efficiency of deleting foreign genes by hybridization pathway of GAEBS system

 1. The GAEBS system can simultaneously remove foreign genes derived from both parents from the hybrid progeny.

According to the Mendelian inheritance law, only one quarter of the progeny of the To-generation pLOCB (parent) and _P L35SLhG4 (parental) transgenic hybrids contain both the pLOCB and pL35SLhG4 transgenes. Therefore, deletions only occur in these zygotes and Produced in the plant. According to the expression activity of GFP and GUS in the hybrid progeny, if the foreign gene is completely deleted in the hybrid progeny seed, three phenotypes will be produced: GFP+/GUS-, GFP-/GUS+ and -/- (the foreign gene is completely The deleted plant); if the deletion is not complete, it is possible to produce a fourth phenotype: GFP+/GUS+. Therefore, the GFP and GUS activities of the hybrid progeny seedlings were first tested to determine the phenotype of the hybrid progeny seedlings, and the number of seedlings of different phenotypes was counted. Then, according to the Mendelian inheritance law, the efficiency of the gene deletion system to delete the foreign gene through the hybridization pathway is calculated. In the present study, the efficiency of deletion of the Effector and Activator elements was evaluated using the efficiency of deletion of GFT and Gf/reporter genes, respectively. The statistical results are shown in Tables 4 and 5.

The 35S-4 and 35S-13 transformants were randomly selected to hybridize with different pLOCB independent transformants, respectively, and the statistical system removed the average efficiency of the foreign genes in these hybrid combinations (the system removed the average of the Efficiency and Activator efficiencies). As shown in Figure 12, in the hybrid combination of 35S-4 with L13, L19, L79, L91, L102, L13 K L198 and L213 transgenic plants, the efficiency of deleting the foreign gene in the hybrid seed is 23%-100%. Similarly, in the hybrid progeny of 35S-13 and the above 10 pLOCB transgenic plants, the efficiency of systematic deletion of the exogenous gene was between 31% and 100%. The same strain of pLOCB was crossed with different pL35SLhG4 transgenic plants, and the efficiency of deleting the exogenous gene in these hybrid combinations was compared. The results showed that the hybrid combinations of L91, L79, L131 and L213 with 5 _P L35SLhG4 independent transgenic plants, respectively. Among them, the system removal efficiency is: 23%-49%, 83%-100%, 91%-100% and 36%-50%. Among them, L198 hybridized with 5 transgenic plants of pL35SLhG4, the deletion efficiency of the system was 100% (as shown in Figure 13). From the above results, it can be seen that the efficiency of deleting exogenous genes between different pLOCB transgenic plants is more obvious; while the same pLOCB transgenic plants hybridize with different pL35SLhG4, the efficiency of deleting exogenous genes is relatively stable. Therefore, relative to the Activator, the insertion position of the Effector in the genome is a key factor affecting the efficiency of system deletion.

Using a similar method, the efficiency of deletion of the foreign gene in the hybrid progeny of pL22720CN and pL35SLhG4 was analyzed. The results are shown in Fig. 14. Nine strains of pL22720CN were crossed as maternal counterparts with the 35S-4 male parent, and the statistical system removed the average efficiency of the foreign gene in these hybrid combinations. As can be seen from Figure 11, the system's removal efficiency is between 20% and 100%. Among them, the removal efficiency of the two combinations of 2272-4x35S-4 and 2272-ll x35S-4 reached 100%. However, when 2272-4 and 2272-11 plants were crossed with other pL35SLhG4 transgenic plants (such as 35S-5, 35S-6 and 35S-7), the average efficiency of deleting exogenous genes in the seeds did not reach 100%. (table 5). As can be seen from Table 5, in the 9 pL22720CN Effectors analyzed, no transformants capable of hybridizing with different Activators and systematically deleting the foreign genes were 100%. In comparison, 2272-1, 2272-3, 2272-4, 2273-6, 2272-11 Both 2272-12 and the 2272-12 are relatively efficient responders with a deletion efficiency of over 88%.

 In summary, in the 35 cross combinations, the system removed the Effector and Activator exogenous genes in both combinations at an efficiency of 100%. Among them, after the L198 plants were crossed with 5 different pL35SLhG4, the efficiency of deleting the foreign genes in the hybrid progeny reached 100%. These results indicate that the GAEBS system can efficiently delete all foreign genes from different chromosomes of the parents from the zygote, and the deletion efficiency is as high as 100%.

Table 4 Statistical analysis of system deletion efficiency in To-transgenic pLOCB and _P L35SLhG4 hybrids

Hybrid analysis of GFP+ seedlings / GUS + number of seedlings / ^a G+G + number of seedlings 'delete

 Total number of buds GFP-number of seedlings GUS - number of seedlings GFP and GUS

 Gene efficiency (%)

35S-4xL13 384 144/240 169/215 69 1 1.75/2.64 50/0

35S-4xL19 299 82/217 148/151 9 30.03/0.01 90/0

35S-4xL79 278 73/205 45/233 0 30.87/62.89 95/100

35S-4xL91 281 72/209 159/122 2 32.91/2.31 98/0

35S-4xL102 377 87/290 129/248 3 54.12/18.47 100/63

35S-4xL131 448 109/339 1 14/334 0 58.53/53.53 100/98

35S-4xL174 398 160/238 156/242 3 7.45/9.08 39/43

35S-4xL198 160 38/122 16/144 0 21.53/50.4 100/100

35S-4xL213 195 61/134 84/1 1 1 0 13.29/1.73 75/0

35S-4xL220 189 73/1 16 86/103 50 4.67/0.68 46/0

L13x35S-13 106 33/73 50/56 17 7.17/0.12 75/0

L19x35S-13 234 55/179 1 19/1 15 0 32.33/0.02 100/0

L79x35S-13 194 48/146 47/147 0 24.25/25.26 100/100

L91 x35S-13 246 76/170 108/138 4 17.58/1.71 76/0

L102x35S-13 291 75/216 120/171 0 33.68/4.3 97/35

L131 x35S-13 352 86/266 89/263 0 45.51/42.51 100/99

L174x35S-13 298 123/175 1 19/179 40 4.36/5.84 35/40

L198x35S-13 328 77/251 77/251 0 45.62/45.62 100/100

L213x35S-13 78 25/53 42/36 6 4.67/0.16 72/0

L220x35S-13 260 72/188 124/136 60 25.43/0.23 89/0

L131 x35S-6 280 66/214 51/229 0 38.59/55.94 100/100

L131 x35S-7 375 97/278 107/268 2 43.20/34.13 97/86

35S-5xL131 423 1 12/31 1 1 15/308 0 46.34/43.57 94/91

35S-6xL198 284 71/213 51/233 0 35.00/57.68 100/100

L198x35S-5 447 102/345 78/369 0 65.51/94.07 100/100

L198x35S-7 570 129/441 132/438 0 84.84/81.6 100/100

L79x35S-7 256 76/180 74/178 4 20.72/22.36 81/84 L79x35S-6 221 56/165 67/154 10 26.39/16.73 99/79

L79x35S-5 208 48/160 45/163 0 29.62/39.91 100/100

L213x35S-7 377 137/240 139/238 4 13.8/12.7 55/53

35S-6xL213 274 67/207 124/150 1 35.26/1.14 100/0

35S-5xL213 249 58/191 118/131 2 34.99/0.29 100/0

35S-5xL91 227 87/140 118/109 32 5.96/0.14 47/0

L91 x35S-7 208 58/150 96/112 16 19.91/0.54 88/0

35S-6xL91 78 25/53 42/36 6 4.67/0.16 72/0

L131 pLGN 293 133/160 129/164 50 1.15/1.97 0/0

L198xpLGN 230 100/130 100/130 40 1.83/1.83 0/0 pLGBx35S-5 220 106/1 14 102/118 49 0.14/0.14 0/0 pLGBx35S-7 294 130/164 124/170 50 1.85/3.44 0/0

Seedlings that tested positive for both GFP and GUS. ^b r -∑ ^U (O stands for the number of observations, E stands for the theoretical number of times): ^. ₅ 3.84. If you do not delete the hair

 E '

GUS (+): GUS (-) =1 : 1 and GFP ( + ): GFP ( -) =1 : 1 in the hybrid seedlings. χ, ² refers to the suitability of GFP-positive seedlings for a 1:1 ratio. Χ2 ² refers to the suitability of GUS-positive seedlings for a ratio of 1:1.

e Only when χ ^{2 is} greater than 3.84, the efficiency of deletion of ^ and (7) foreign genes is estimated separately. When both parents are single copies: The deletion efficiency is calculated according to the following formula: Deletion efficiency (%) = [(4xG"- 2xT)/T] l00o For a parental transgenic pLOCB double copy, the efficiency of GFP exogenous gene deletion is calculated as follows: Deletion efficiency (%) = [2xG7T] x l00. G - refers to GFP or GUS negative seedlings, T refers The total number of seedlings analyzed.

Table 5 Statistical analysis of exogenous gene deletion efficiency in To-transgenic transgenic pL22720CN and pL35SLhG4 hybrids GFP+ seedling number/GUS+ seedling number of hybridization analysis/ ^a G+G ⁺ V/Z2 ^{2 e} deletion of GFP and total number of buds GFF seedlings GUS-miao The efficiency of the number of seedlings GUS gene (%)

2272-1 X35S-4 274 85/189 69/205 10 19.36/33.26 76 /99

2272-2x35S-4 238 70/168 63/175 0 19.77/25.88 82 /94

2272-3x35S-4 226 58/168 50/176 1 26.29/34.57 97/100

2272-4x35S-4 260 40/220 40/220 0 61.62/61.62 100/100

2272-5x35S-4 217 87/130 102/115 42 4.06/0.33 40 /0

2272-6x35S-4 276 72/204 62/214 1 31.09/41.31 96/100

2272-10x35S-4 235 91/144 47/188 0 5.75 /41.70 45 /100

2272-11 X35S-4 264 63/201 57/207 0 35.55/42.05 100/100

2272-12x35S-4 310 70/240 85/225 5 46.07/31.16 100/90

2272-3x35S-6 263 63/200 75/188 0 35.16/23.85 100/86 2272-3x35S-7 232 50/182 104/128 0 36.98 /1.14 100/0

2272-4x35S-6 208 60/148 40/168 1 18.19/38.77 85/100

2272-4x35S-5 479 157/422 106/373 0 28.08/73.86 69/100

2272-4x35S-7 332 114/218 121/211 1 15.98/11.93 63/54

2272-6x35S-6 179 42/137 47/132 0 24.68/19.71 100/95

35S-5x2272-6 153 45/108 46/107 3 12.56/1 1.76 82/80

2272-1 l x35S-6 236 56/180 72/164 0 32.0/17.54 100/78

2272-1 l x35S-5 328 85/243 79/249 0 37.57/43.54 96/100

2272-1 l x35S-7 222 66/156 71/151 0 17.84/14.06 81/72

35S-7x2272-12 338 77/261 105/233 7 49.54/23.86 100/76

2272- 12x35S-5 242 60/182 77/165 0 30.25/15.64 100/73

2272-12x35S-6 181 44/1 37 60/121 1 23.38/9.94 100/67

2272- 12x35S- 13 109 3 1/78 27/82 0 9.71/13.38 86/100

2272-5x35S-6 244 98/146 138/106 67 4.53/1.97 39/0

2272-10x35S-6 151 56/95 25/126 1 4.78/33.11 52/100

35S-5x2272-10 370 140/230 68/302 3 10.70/73.36 49/100

35S-7x2272-10 252 95/157 1 15/137 1 7.38/0.88 49/0

Seedlings that tested positive for both GFP and GUS. ^b ^ ₍ ² =∑ ^UXJ (O stands for observations, E stands for theoretical number of times): ^. ₅ , =3.84. If not deleted

 E '

Health, hybrid! ^GUS (+): GUS ( - ) =1 : 1 and GFP ( + ): GFP ( - ) =1 : 1 in the seedlings. χ, ² refers to the suitability of GFP-positive seedlings for a 1:1 ratio. Χ2 ² refers to the suitability of GUS-positive seedlings for a ratio of 1:1.

Only when _χι ² when e and Χ2 ² greater than 3.84, respectively, was estimated efficiency exogenous gene deleted. When both parents are single copy: The deletion efficiency is calculated as follows: Deletion efficiency (%) = [(4xG"-2xT)/T] xl 00 _o For a parental transgene pL22720CB double copy, the efficiency of GFP exogenous gene deletion is The following formula is used to calculate the Deletion efficiency (%) = [2xG7T] x 100. G is the GFP or GUS negative seedling, and the T refers to the total number of seedlings analyzed.

 2. Effect of different recognition site sequences in Effector on system deletion efficiency

To further compare the effect of mutant lox2272 and wild-type loxp recognition site sequences in the subtractor on the deletion efficiency of the automatic deletion system, the average deletion efficiency of the GFP and GUS genes in the systems in Tables 4 and 5 were calculated. The results are shown in Figure 15. Show. The average deletion efficiency of the Effectsor system based on GFP was 85% for pLOCBxpL35SLhG4 and 81% for _P L22720CNxpL35SLhG4. There is no significant difference between the two. The average deletion efficiency of Activator was based on GUS: pL22720CNxpL35SLhG4 was 73% higher, while pLOCBxpL35SLhG4 was 48%. It can be seen from the above that the removal efficiency of the system is higher than that of the Activator. In the _P L22720CNxpL35SLhG4 system, the deletion efficiency of the effector containing the mutant recognition site sequence (lox2272) was higher than that of the Activator containing the wild type recognition site (loxp), but there was no significant difference. In the pLOCBxpL35SLhG4 system, which contains wild-type recognition sites, the efficiency of deletion of the system is reduced. The removal efficiency of Activator was 37% higher, and the difference between the two reached a very significant level (p<0.01). In addition, when the loxp recognition site in the Effector element was replaced with the mutant 1οχ2272, the efficiency of deletion of the Activator by the system (from 48% to 73%) was significantly improved (p<0.05).

 Although in the pLOCBxpL35SLhG4 hybrid combination, the average deletion efficiency of the Activator was significantly lower than that of the Effector, a pLOCB transgenic plant with a deletion efficiency of 100%, such as L198, was obtained. No transgenic plants with 100% deletion efficiency were obtained in the 9 pL22720CN transgenic plants analyzed. However, in the pL22720CNxpL35SLhG4 hybrid combination, the system can efficiently delete the Effector and Activator, and the average deletion efficiency is above 73%. Therefore, pLOCBxpL35SLhG4 and pL22720CNxpL35SLhG4 are efficient combinations of gene automatic deletion binary systems.

 [Example 10] Detection of exogenous DNA fragments by GAEBS system

 PCR analysis of deletion of foreign genes in hybrid generation

 The GFP and GUS activity assays of transgenic progeny of transgenic pLOCB or pL2272〇CN and pL35SLhG4 tobacco have demonstrated that the GAEBS system can efficiently delete foreign genes derived from both parents, and the deletion efficiency reaches 100%. In order to further analyze the system to delete the foreign gene in the hybrid generation, the genomic DNA of the hybrid progeny and the hybrid parent were extracted and PCR analysis was performed. A pair of primers (designated as SEQ ID N0.38 and SEQ ID N0.39) were designed based on the DNA sequence between the T-DNA both-end boundary sequence of the plant expression vector backbone (pBIN 19) and the recombinase recognition site sequence. Conventional PCR screens for plants with exogenous gene deletion. If deletion of the foreign gene occurs, only about 800 bp of non-functional residual fragments will be detected (as shown in Figure 16A).

To have been confirmed plants exogenous gene deletion occurs, into a ho using PCR detected from the parents of the exogenous gene _Cre ^'nt and LhG4 ^ATO like deletion situation, wherein the detection cre ^mt gene primers used as _{SEQ ID NO.} 40 and SEQ ID N0.41; primers used to detect the LhG4 ^ATO gene are shown as 8 £0 10 «).42 and 8 £0 10 > 0.43. The results showed that in the hybrid progeny f27115, f795, Π316 and f!987 of 2272-1 l x35S-5, L79x35S-5, L131 and L198x35S-7, the creint gene from the female parent and the LhG4ATO gene from the male parent were indeed It has been successfully removed from the genome of these hybrid progeny (as shown in Figure 17).

On this basis, the amplification conditions were changed, the fragment extension time was increased, and the molecular characteristics of deletion of the foreign gene were further analyzed by PCR. The PCR reaction conditions used were: 94 ° C for 5 min; 98 ° C lOsec; 68 ° C for 8 min, 35 cycles. The DNA polymerase is LA Taq (TaKaRa) polymerase. If deletion occurs, a non-functional T-DNA residual fragment of about 800 bp will be detected, otherwise a large T-DNA fragment of about 7.0 kb or / and 9.0 kb will be detected (as shown in A of Figure 16). As can be seen from Fig. 16B, a specific large fragment of about 7.0 kb size was amplified using the parental 35S-13 genome as a template, and the fragment includes a Loxp recognition site, 35S-GUS: NPTII-nos, and 35S-LhG4ATO-T3A. Fragment. Similarly, a large 9.0 kb fragment was amplified using the parental L13 genome as a template, including the loxp recognition site, 35S-GFP-nos, pOp-creint-T3A and 35S-bar-nos fragments. Only the remaining fragments of about 0.8 kb in the G7115, f795, fl316 and fl987 plants which have been confirmed to have completely deleted the foreign gene were detected, indicating that all the foreign genes derived from the male and female in these plants were Completely removed (including Effector and Activator). In addition, in the fl 7413 plant, in addition to the detection of a specific band of 0.8 kb, a large fragment of about 7.0 kb was detected, which was large. The small agreement with the parental 35S-13 indicates that the foreign gene of Activator has not been deleted; also in the f413 plant, a large fragment of about 9.0 kb long, which is consistent with the size of the parent L13, was detected, indicating that the foreign gene of the Effector was not been deleted. These results demonstrate at the molecular level that the GAEBS system can simultaneously completely remove all exogenous functional genes derived from both parents from the hybrid progeny genome.

 2. Sequence analysis of residual fragments after deletion of foreign genes in the F1 generation

 According to the principle of Creint/loxP system recombination (Lee and Saito, 1998; Ennifar et al., 2003; Ghosh et al., 2005), the gene-automatic deletion of the binary system removes the foreign gene and leaves only about the plant genome. A non-functional T-DNA fragment of 800 bp in length containing a loxp recognition site sequence (shown in Figure 14). In order to understand the exact sequence information of the residual fragment after deletion of the foreign gene by the automatic deletion binary system, about 800 bp fragments amplified by the G7115 and fl 987 lanes in Fig. 14B were separately recovered, cloned and sequenced. The specific process and sequencing results are shown in Figure 18.

From the sequencing results, it was found that in the hybridization of L2272-11 and 35S-LhG4 transgenic plants, there was only one ίοχΡ and 10x2272 recognition site sequence and the non-functional region sequence at both ends of the T-DNA, and another xP and 1οχ2272 recognition sites. The sequence as well as the 35S-GUS NPTII-nos, 35S-GFP-nos p0p-cre ^int -3TA and nosP-NTPII-nos sequences have been completely deleted. In the flage plants of the L198 and 35S-LhG4 transgenic plants, the sequencing results were the same as those of Ω7115, except that the recognition site sequence from the Effector parent was hxP. These results were further confirmed at the molecular level, in the transgenic pLOCB or pL22720CN and the transgenic pL35SLhG4 parent hybrid generation seed cells, after systemic combination, all foreign genes can be completely removed.

 The above examples show that the GAEBS system of the present invention for controlling the safety hazards of foreign genetic organisms can not only effectively maintain the stable inheritance of foreign genes in the sexual reproduction of the parents, but also efficiently extract all foreign genes from the hybrid zygote. Completely deleted, the removal efficiency reaches 100%. The method of the invention is simple and convenient, and can delete 100% of all foreign genes when the foreign gene needs to be deleted, and the effect is remarkable, and has a good application prospect.

The above description of the present invention is not intended to limit the invention, and various modifications and changes can be made by those skilled in the art without departing from the spirit of the invention. .

references

 Arumugam N, Gupta V, Jagannath A, Mukhopadhyay A, Pradhan A, Burma P, Pental D

(2007) A passage through in vitro culture leads to efficient production of marker-free transgenic plants in Brassicajuncea using the Cre/loxP system. Transgenic Res. 16: 703-712

 Coppoolse E, de Vroomen M, Roelofs D, Smit J, van Gennip F, Hersmus B, Nijkamp H, van Haaren M (2003) Cre recombinase expression can result in phenotypic aberrations in plants. Plant mol. biol 51: 263-279

 Corneille S, Lutz K, Svab Z, Maliga P (2001) Efficient elimination of selectable marker genes from the plastid genome by the CRE-lox site-specific recombination system. Plant J. 27: 171 -178

 Craft J, Samalova M, Baroux C, Townley H, Martinez A, Jepson I, Tsiantis M, Moore I

(2005) New pOp/LhG4 vectors for stringent glucocorticoid-dependent transgene expression in Arabidopsis. Plant J. 41: 899-918

 Ennifar E, Meyer J, Buchholz F, Stewart A, Suck D (2003) Crystal structure of a wild-type

Cre recombinase-loxP synapse reveals a novel spacer conformation suggesting an alternative mechanism for DNA cleavage activation. Nucleic Acids Res. 31: 5449-5460

 Ghosh K, Lau C, Gupta K, Van Duyne G (2005) Preferential synapsis of loxP sites drives ordered strand exchange in Cre-loxP site-specific recombination. Nat. Chem. Biol 1: 275-282

 Gidoni D, Srivastava V, Carmi N (2008) Site-specific excisional recombination strategies for elimination of unwanted transgenes from crop plants. In Vitro Cellular & Developmental Biology-Plant 44: 457-467

 Haseloff J (1999) GFP variants for multispectral imaging of living cells. Methods Cell Biol. 58: 139-151

 Jefferson RA, Kavanagh TA, Bevan MW (1987) GUS fusions: beta-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J. 6: 3901 -3907

 Johnson AA, Hibberd JM, Gay C, Essah PA, Haseloff J, Tester M, Guiderdoni E (2005) Spatial control of transgene expression in rice (Oryza sativa L.) using the GAL4 enhancer trapping system. Plant J. 41: 779- 789

 Keenan R, Stemmer W (2002) Nontransgenic crops from transgenic plants. Nature biotechnology 20: 215-216

 Lee Q Saito I (1998) Role of nucleotide sequences of loxP spacer region in Cre-mediated recombination. Gene 216: 55-65

Luo K, Duan H, Zhao D, Zheng X, Deng W, Chen Y, Stewart CN, Jr., McAvoy R, Jiang X, Wu Y, He A, Pei Y, Li Y (2007) 'GM-gene-deletor ': fused loxP-FRT recognition sequences The improvement of the efficiency of FLP or CRE recombinase on transgene excision from pollen and seed of tobacco plants. Plant Biotechnol. J. 5: 263-274

 Marjanac Q De Paepe A, Peck I, Jacobs A, De Buck S, Depicker A (2008) Evaluation of CRE-mediated excision approaches in Arabidopsis thaliana. Transgenic Res. 17: 239-250

 Moore I, Samalova M, Kurup S (2006) Transactivated and chemically inducible gene expression in plants. Plant J. 45: 651-683

 Russell S, Hoopes J, Odell J (1992) Directed excision of a transgene from the plant genome. Mol Gen. Genet. 234: 49-59

 Samalova M, Brzobohaty B, Moore I (2005) ρΟρό/LhGR: a stringently regulated and highly responsive dexamethasone-inducible gene expression system for tobacco. Plant J. 41 : 919-935

Sambrook J, Lasel D (2002) Guide to Molecular Cloning. Vol 26. Beijing: Science Press, ρ 1723

Schmidt E, Taylor D, Prigge J, Barnett S, Capecchi M (2000) Illegitimate Cre-dependent chromosome rearrangements in transgenic mouse spermatids. Proceedings of the National Academy of Sciences of the United States of America 97: 13702

 Zhang W, Subbarao S, Addae P, Shen A, Armstrong C, Peschke V, Gilbertson L (2003)

Cre/lox-mediated marker gene excision in transgenic maize (Zea mays L.) plants. Theor. Appl. Genet 107: 1 157-1 168

 Liu Renxiang, Ding Wei, Yang Jun, Feng Yonggang (2000) Seed Production Techniques of Male Sterile Hybrids in Flue-cured Tobacco[J]. Tobacco Science of China 21: 7

Claims

Claim

 1. A gene automatic deletion binary system for the safe control of exogenous genetic organisms for sexually reproducing plants, including

 1) an effector element for constructing a first plant expression vector, the effector element comprising

 Two homologous recombinase specific recognition sites; inserting the following genes or nucleotides between the two recombinase specific recognition sites:

 a promoter that controls expression of a foreign gene;

 a multiple cloning site for introduction of a foreign gene;

 Recombinase gene;

 a target promoter that maintains recombinase gene silencing;

 2) an activator element for constructing a second plant expression vector, the activator element comprising:

 Two homologous recombinase specific recognition sites; inserting the following genes or nucleotides between the two recombinase specific recognition sites:

 a promoter that controls expression of a foreign gene;

 a multiple cloning site for introduction of a foreign gene;

 a promoter that controls the transcriptional activator gene;

 a transcriptional activator gene that cooperates with the target promoter;

 3) A plant expression vector comprising a first plant vector and a second plant vector for introducing the effector element and the activator element into the plant genome, respectively.

The gene automatic deletion binary system according to claim 1, wherein the recombinase specific recognition site is selected from the group consisting of ΙοχΡ, 1οχ2272, 1οχ5171 and an FRT recognition site, and the recombinase gene is selected from the group consisting of FLP, Cre and Cre. ^Int recombinase gene.

 The gene automatic deletion binary system according to claim 1, wherein the promoter for controlling the transcription activator gene is a CaMV 35S promoter.

 The gene automatic deletion binary system according to claim 1, wherein the target promoter and the transcription activator gene coordinated thereto are selected from the group consisting of pOp/LhG4, tTA/TOP10/pTAX, Gal4: VP16/UAS or mGal4: VP16/UAS transcriptional activation system.

The gene automatic deletion binary system according to claim 1, wherein the recombinase specific recognition site is ΙοχΡ or 1οχ2272, the recombinase gene is Cre ^int , and the target promoter and the transcription thereof are coordinated. The activator gene is pOp/LhG4, and the promoter for controlling expression of the foreign gene and the promoter for controlling the transcription activator gene are the CaMV35S promoter.

 6. A first plant expression vector comprising an effector element, comprising: two homologous recombinase specific recognition sites; inserting the following genes or nucleotides between the two recombinase specific recognition sites:

 a promoter that controls expression of a foreign gene;

 a multiple cloning site for introduction of a foreign gene;

 Recombinase gene;

A target promoter that maintains recombinase gene silencing.

7. A second plant expression vector comprising an activator element comprising two homologous recombinase specific recognition sites; inserting the following gene or nucleotide between the two recombinase specific recognition sites - control a promoter for expression of a foreign gene;

 a multiple cloning site for introduction of a foreign gene;

 a promoter that controls the transcriptional activator gene;

 a transcriptional activator gene that cooperates with the target promoter;

 8. The use of the automatic gene deletion binary system of claim 1 for the preparation of a transgenic plant.

 9. A method of preparing a safe transgenic plant, comprising the steps of: - 1) constructing the first plant expression vector of claim 6;

 2) constructing the second plant expression vector of claim 7;

 3) introducing the first plant expression vector described in the above step 1) into the plant genome to prepare the first transgenic plant;

4) introducing the second plant expression vector described in the step 2) into the plant genome to prepare a second transgenic plant;

5) The first transgenic plant and the second transgenic plant are crossed as a parent to obtain a safe transgenic plant from which the foreign gene is deleted.