WO2013000279A1 - Support binaire d'auto-excision de gène pour contrôler la biosécurité de végétaux transgéniques par reproduction sexuée - Google Patents

Support binaire d'auto-excision de gène pour contrôler la biosécurité de végétaux transgéniques par reproduction sexuée Download PDF

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WO2013000279A1
WO2013000279A1 PCT/CN2012/000892 CN2012000892W WO2013000279A1 WO 2013000279 A1 WO2013000279 A1 WO 2013000279A1 CN 2012000892 W CN2012000892 W CN 2012000892W WO 2013000279 A1 WO2013000279 A1 WO 2013000279A1
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gene
plant
recombinase
promoter
activator
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PCT/CN2012/000892
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Chinese (zh)
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裴炎
邹修平
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西南大学
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
    • C12N15/822—Reducing position variability, e.g. by the use of scaffold attachment region/matrix attachment region (SAR/MAR); Use of SAR/MAR to regulate gene expression

Definitions

  • 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.
  • Another 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.
  • site-specific recombinase system derived from microorganisms is also a good tool for deleting foreign genes.
  • 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) 2 ⁇ plasmid FLP/Fi?r system; raMxif pSRl plasmid R/3 ⁇ 4.
  • 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
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • Keenan et al. proposed a technical idea for producing non-GM foods from genetically modified plants (Keenan and
  • 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.
  • the system consists essentially of a pair of homozygous fusion recognition sites/ ⁇ - ⁇ and a recombinase gene FLP controlled by a tissue (eg pollen or seed) specific promoter. Among them, all foreign genes are placed between the / ⁇ - ⁇ recognition site sequences.
  • tissue eg pollen or seed
  • 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.
  • 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.
  • 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.
  • a trans-transcriptional activator such as the yeast transcription factor GAL4
  • 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 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.
  • 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.
  • the present invention provides an effective control of sexually reproducing plant genetically modified organisms.
  • Safe Gene Automatic Deletion Binary System GENEBS
  • the system uses the green-activated system as a molecular switch to control the expression of the recombinase deletion system.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 / ⁇ , lox2272, tec5 7 ⁇ ⁇ recognition sites and the recombinase gene is selected from FLP, Cre and Cn? '"''Gene.
  • 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.
  • 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).
  • CaMV cauliflower mosaic virus
  • 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.
  • the target promoter is ⁇
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • a transcription activation system transactivation 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.
  • FIG. 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.
  • GFP green fluorescent protein gene
  • Figure 2 is a schematic diagram showing the structure of a cloning vector pUC-35S-bar-nos carrying a 35S-bar-nos element.
  • bar is a phosphinthricin acetyltransferase (PAT) gene.
  • PAT phosphinthricin acetyltransferase
  • 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 ⁇ carrying the loxp-CaMV35S:GFP-MCS-35S:bar-loxp element.
  • ⁇ is a kanamycin resistance gene
  • ⁇ 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.
  • Loxp2272 is a mutant recognition site sequence of ⁇ 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; ⁇ ⁇ 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.
  • 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.
  • 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.
  • 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.
  • Figure 11 is a graph showing the acquisition of Activator transgenic tobacco plants.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • M is a DNA molecular marker
  • G71 15 is a 2272-1 l x35S-5 hybrid genomic DNA as a template
  • ⁇ 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
  • ⁇ 95 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 ⁇ 7115 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 ⁇ 316 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.
  • 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 ⁇ L of TE and stored at -20 °C until use.
  • 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.
  • 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:
  • the molar ratio of the vector DNA fragment to the exogenous ligation product DNA fragment was 1:1.
  • 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 ⁇ 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.
  • 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 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • a pair of primers (shown as SEQ ID N0.24 and SEQ ID NO. 25) were designed based on the ⁇ 2272 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.
  • the three fragments were ligated to obtain the plant expression vector p/o 2272GMN/o 2272.
  • all exogenous gene fragments including the multiple cloning site MCS were located between the ⁇ 22 ⁇ 2 recognition site sequences (as shown in Figure 5). .
  • the pBIN 19 plasmid was used as a template to PCR-amplify the nos-loxp element.
  • the loxp recognition site sequence was added to the 3' end of the nos sequence.
  • 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.
  • 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/ ⁇ 3 ⁇ 4/ ⁇ enzyme to obtain pUC-loxp-35S-GUS: NPTn-nos cloning vector.
  • 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.
  • GUS, ⁇ and The cloning loci MCS are located between a pair of co-directional recognition locus sequences ( Figure 6)).
  • 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.
  • Sacl/Xbal cuts the ⁇ promoter from the pGEM-T-pOp vector, and the ⁇ 3 ⁇ termination sequence is excised from the pUC ⁇ 3 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.
  • 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).
  • 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.
  • 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.
  • a pL35SLhG4 plant expression vector shown in Figure 9) was obtained.
  • 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.
  • Table 1 Agrobacterium tumefaciens-mediated medium for tobacco genetic transformation
  • MSB MSB (MS inorganic salt + B5 organic)
  • 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • the Activator vector contains the Gf/S reporter gene. Therefore, the resistant seedlings were first stained by GUS histochemical staining.
  • 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.
  • 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.
  • transgenic pL35SLhG4 tobacco 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.
  • 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.
  • the GAEBS system can simultaneously remove foreign genes derived from both parents from the hybrid progeny.
  • 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).
  • the efficiency of deleting the foreign gene in the hybrid seed is 23%-100%.
  • the efficiency of systematic deletion of the exogenous gene was between 31% and 100%.
  • the system removed the Effector and Activator exogenous genes in both combinations at an efficiency of 100%.
  • the efficiency of deleting the foreign genes in the hybrid progeny reached 100%.
  • ⁇ , 2 refers to the suitability of GFP-positive seedlings for a 1:1 ratio.
  • ⁇ 2 2 refers to the suitability of GUS-positive seedlings for a ratio of 1:1.
  • Deletion efficiency (%) [(4xG"- 2xT)/T] l00o
  • Deletion efficiency (%) [2xG7T] x l00.
  • G - refers to GFP or GUS negative seedlings
  • T refers The total number of seedlings analyzed.
  • ⁇ , 2 refers to the suitability of GFP-positive seedlings for a 1:1 ratio.
  • ⁇ 2 2 refers to the suitability of GUS-positive seedlings for a ratio of 1:1.
  • the removal efficiency of the system is higher than that of the Activator.
  • 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.
  • 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).
  • the efficiency of deletion of the Activator by the system was significantly improved (p ⁇ 0.05).
  • pLOCBxpL35SLhG4 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.
  • 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.
  • the GFP and GUS activity assays of transgenic progeny of transgenic pLOCB or pL2272 ⁇ 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%.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • Cre/lox-mediated marker gene excision in transgenic maize Zea mays L.

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Abstract

L'invention concerne la construction d'un nouveau système binaire d'autoexcision de gène (GAEBS) pour contrôler le risque de biosécurité d'un gène exogène végétal dans la reproduction sexuée. Principalement à l'aide du système d'activation de la transcription en tant que commutateur moléculaire pour contrôler un système d'excision de la recombinase, d'un côté, le gène de recombinase localisé entre les sites de reconnaissance de la recombinase est maintenu silencieux dans la génération sexuée d'un végétal et la stabilité héréditaire du gène exogène introduit par la technologie de « délétion du gène GM » dans la reproduction sexuée du végétal parent est réalisée ; et d'un autre côté, en effectuant l'hybridation des végétaux parents portant un élément effecteur et un élément activateur respectivement, l'élément effecteur et l'élément activateur peuvent être liés à l'activité du facteur d'activation de la transcription présente. En même temps, en utilisant un promoteur spécifique du tissu végétal pour promouvoir l'expression de la recombinase, l'excision spécifique du tissu du gène exogène est réalisée et un végétal transgénique sûr est obtenu.
PCT/CN2012/000892 2011-06-29 2012-06-29 Support binaire d'auto-excision de gène pour contrôler la biosécurité de végétaux transgéniques par reproduction sexuée WO2013000279A1 (fr)

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