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WO2004020641A1 - Procede de manipulation de genie genetique relatif aux plantes, a securite biologique - Google Patents

Procede de manipulation de genie genetique relatif aux plantes, a securite biologique Download PDF

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WO2004020641A1
WO2004020641A1 PCT/CN2002/000625 CN0200625W WO2004020641A1 WO 2004020641 A1 WO2004020641 A1 WO 2004020641A1 CN 0200625 W CN0200625 W CN 0200625W WO 2004020641 A1 WO2004020641 A1 WO 2004020641A1
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gene
sequence
plant
transformation element
genome
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Chinese (zh)
Inventor
Lijia An
Qiao Su
Xiaorong Gao
Liji Jin
Jun Yang
Xiaoying Bi
Xiuying Xia
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DALIAN KEYUAN AGRICULTURAL BIOENGINEERING Co Ltd
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DALIAN KEYUAN AGRICULTURAL BIOENGINEERING Co Ltd
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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)
    • C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
    • C12N15/8213—Targeted insertion of genes into the plant genome by homologous recombination
    • 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)
    • C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
    • 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)
    • C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
    • C12N15/8209—Selection, visualisation of transformants, reporter constructs, e.g. antibiotic resistance markers

Definitions

  • the invention relates to a carrier-free, non-selectable marker gene-generating technical method with biological safety, and belongs to the field of bioengineering.
  • Plant genetic engineering refers to the use of recombinant DNA technology to clone foreign genes and introduce them into plants. Gene expression allows plants to acquire new traits and cultivate new plant varieties. This technology overcomes the limitations of sexual hybridization in plants, and the scope of gene communication is infinitely expanded. It can be introduced into plants from bacteria, viruses, animals, humans, distant plants, and even synthetic genes, so its application prospect is very broad.
  • OECD Organization for Economic Cooperation and Development
  • the area planted with GM soybeans in the United States in 1999 was 15 million hectares, accounting for 50% of the nation ’s soybean planting area; the planting area of genetically modified corn was 10.3 million hectares, accounting for 33% of the country ’s corn planting area.
  • the planting area of genetically modified corn was 10.3 million hectares, accounting for 33% of the country ’s corn planting area.
  • more than 60% of processed foods in the United States contain genetically modified ingredients, and sales of genetically modified foods amount to US $ 10 billion.
  • insect-resistant cotton has the largest planting area. By the end of 2000, the cumulative promotion area of domestic insect-resistant cotton had reached 370,000 hectares, reducing the use of pesticides by 80%, and creating a benefit of 770 million yuan.
  • the root cause of the safety problem lies in: (1) In the current plant gene transformation system, the introduced foreign genes and the used vector elements are usually derived from non-relative species, even artificially synthesized, which may become a new human allergy. (2) The use of antibiotic resistance or herbicide resistance genes as selection marker genes for the selection of transformants may cause potential harm to the ecological environment.
  • the protein encoded by the introduced foreign gene may be allergic in the following cases: (1) the transgene encodes a known allergic protein; (2) the gene source contains an allergic protein; (3) the protein encoded by the imported gene and a known allergic protein The amino acid sequence of the protein has obvious homology in immunology; (4) The protein encoded by the introduced gene is a member of a certain type of protein, and some members of this type of protein family are allergic proteins.
  • the safety problems caused by marker genes mainly depend on: (1) whether the marker genes are directly toxic; (2) horizontal transfer of genes; (3) unexpected gene pleiotropy; (4) marker genes
  • the safety of the encoded protein includes direct toxicity, allergy, and side effects caused by the catalytic function of the protein.
  • the safety problems caused by marker genes mainly include: (1) Will the genes encoding herbicides transform the near-source wild species of transgenic plants into weeds through gene escape channels, destroy the natural ecological environment, and break the dynamic balance of biological populations; (2) whether the transgenic plants will affect the soil; (3) whether the horizontal transfer of antibiotic-encoding genes will lead to resistance to microorganisms and certain pests.
  • the safety issue caused by the integration of the vector backbone sequence into the plant genome is not clear. Although these sequences have no value in transgenic plants, they may produce proteins with negative effects, or affect the expression of normal genes in plants and promote the rearrangement of transgenes. In addition, these sequences may also escape to the environment and cause potential negative impacts on the ecological environment.
  • Bt toxin
  • the solution to the safety problem of the transgenic plants is to evaluate the safety of the transgenic plants, which mainly includes phenotypic traits (agronomic traits such as morphology and yield), and key nutritional components (fat, protein, carbohydrates or micronutrient components). And anti-nutritional factors, the presence or absence of toxic substances and The presence or absence of allergic proteins, etc., is mainly aimed at the study of the characteristics of proteins encoded by the introduction of foreign genes, and cannot effectively solve the safety problems caused by the gene transformation system, especially by the selection marker genes.
  • phenotypic traits agronomic traits such as morphology and yield
  • key nutritional components fat, protein, carbohydrates or micronutrient components
  • anti-nutritional factors, the presence or absence of toxic substances and The presence or absence of allergic proteins, etc. is mainly aimed at the study of the characteristics of proteins encoded by the introduction of foreign genes, and cannot effectively solve the safety problems caused by the gene transformation system, especially by the selection marker genes.
  • the invention provides a carrier-free, non-selectable marker gene-free plant genetic engineering operation method with biological safety.
  • the method adopted by the present invention can solve the safety problem of the transgenic plant caused by the carrier backbone sequence and the selection marker gene in the conventional plant genetic engineering operation.
  • the main point of the plant genetic engineering operation method provided by the present invention is to construct a gene transformation element composed of a foreign gene and a regulatory sequence, and a boundary sequence on both sides, and then perform a gene transformation operation by means of a pollen tube channel and the like.
  • Expression and border sequences of foreign genes in transformation elements The insertion of the plant genome causes changes in the traits of the transformed plants. Detection is performed at the DNA level, protein level and morphological trait level to identify the transformed plants.
  • the gene transformation element provided by the present invention for transformation is a linear deoxyribonucleotide sequence, which includes a structural gene encoding a functional protein and a plant gene expression control sequence, and a border sequence located on both sides of the expression control sequence.
  • the border sequence in the gene transformation element provided by the present invention may be the border sequence of T-DNA or the entire (or part of) the DNA sequence of the transposon.
  • the transformation rate is 10 -10
  • the present invention may also take all (or part of) the DNA sequence of a coding region in the transformed plant genome as a boundary sequence, or use the regulatory sequence of a coding region as a boundary sequence, and may also use a certain sequence in the transformed plant genome.
  • a DNA sequence of a non-coding region is used as a boundary sequence.
  • the use of a coding region in the plant genome, or a regulatory sequence of a coding region, or a non-coding region DNA sequence as a boundary sequence in a gene transformation element is the basis for homologous recombination of foreign genes and recipient DNA.
  • the main function is to contribute to the gene sequence may be oriented conversion element integrated into the plant genome, constructed by gene conversion rate conversion element 10_ 3-10-4 gene conversion process.
  • the DNA sequence of a coding region in the transformed plant genome as a boundary sequence provided by the present invention may be a structural gene that encodes a certain morphological trait of a plant, and the change may cause the fertility of the plant, whether the leaves have hair, or leaves. Changes in color, dwarfing and other morphological traits.
  • the DNA boundary sequence of a coding region in the transformed plant genome as a boundary sequence provided by the present invention may also encode a structural gene that determines a certain physiological and biochemical trait in a plant, such as a gene that regulates a metabolic link in a plant. Changes in enzyme or protein genes can cause changes in certain amino acids or other metabolites in plants. Select the DNA sequence of a coding region in the transformed plant genome as It is a boundary sequence.
  • the gene transformation element can be inserted into the plant genome in a targeted manner, resulting in the inactivation of the gene encoding a certain trait of the plant.
  • the inactivation of the gene usually does not significantly affect the plant. Normal growth.
  • the insertion of the gene transformation element will lead to a change in a certain morphological trait or physiological and biochemical trait.
  • the present invention accordingly establishes a corresponding detection method to effectively identify whether the foreign gene is transformed into the plant.
  • the invention provides the regulatory sequence of a structural gene in the transformed plant genome as a border sequence, which includes a promoter region (and part of the structural gene sequence) and a 3 'end regulatory region (and part of the structural gene sequence).
  • the structural gene can be replaced by the gene transformation element through homologous recombination, so that the foreign gene can be specifically expressed.
  • a certain non-coding DNA sequence provided as a boundary sequence in the transformed plant genome provided by the present invention may be a DNA sequence of a Matrix Attachment Region (MAR) in eukaryotic chromatin that can bind to a nuclear matrix.
  • MAR sequences help to increase the overall expression level of foreign genes and enhance the stability of foreign gene expression. It can also be moderately or highly repetitive sequences in the genome of eukaryotes, such as 18S rRNA sequences.
  • the structural gene encoding a functional protein provided by the present invention includes a phytase gene derived from Aspergillus ficus (A.fic bandit As3.324), and a Suaeda liaotunge is ⁇ d derived from a halophyte ⁇
  • CMO choline monooxygenase
  • BADH betaine aldehyde dehydro-genase
  • the plant gene expression control sequences located on both sides of the structural gene used in the present invention include a promoter, a transcription terminator, an enhancer and the like.
  • the present invention mainly uses a pollen tube channel method to transform a plant, that is, a naked linear exogenous gene
  • the transformation element enters the embryo sac through the pollen tube channel, and directly transforms egg cells, zygotes or early embryonic cells that do not yet have normal cell walls, and does not need to undergo genetic transformation processes such as protoplast culture, cell culture, tissue culture, and regeneration of plants.
  • the invention can also transform plants by seed soaking and embryo sac injection.
  • the constructed gene transformation element is introduced into a plant by using a method such as a pollen tube channel, and a transformed plant seed is obtained. Since the transformed seeds and untransformed seeds are mixed together, the present invention provides three levels of gene-transformed plants (seeds) at the DNA level, protein level, and morphological trait level based on the designed border sequences and the foreign genes used Detection strategy.
  • DNA level detection methods include PCR, DNA sequence determination, SOUTHERN hybridization, NORTHER hybridization, etc .
  • protein level detection methods include physiological and biochemical indicators of expressed proteins (or enzymes), determination of amino acid content changes, WESTERN hybridization, etc .
  • morphological trait levels Detection includes observation and detection of appearance characteristics.
  • the gene transformation element constructed by using the plant gene operation method provided by the present invention is composed of a foreign gene, an expression control sequence and a boundary sequence, so the conventional transformation method is fundamentally solved due to the use of other non-closely related species derived from microorganisms and the like. Vectors, and the safety issues associated with using antibiotic-resistant or herbicide-resistant genes as selectable marker genes.
  • the boundary sequences provided on both sides of the expression control sequence provided by the present invention are the basis for the recombination of genomic DNA of foreign genes and recipient plants.
  • the pollen tube channel method can be used to introduce the gene transformation element into plants. The method is simple and effective, and the transformation speed is fast.
  • transgenic plants can be obtained that year, and can be basically applied to any flowering plant, including any synthetic between any species. Gene transfer is not restricted by plant genotypes, thereby greatly expanding the source of the gene for gene engineering and the range of recipient plants. ⁇ detailed description ⁇
  • Gene (GenBank, AY013315), 11 nucleotides of + 46— + 156 are intron sequences, which contain the characteristic conserved sequences of fungal introns (Doner sequence: GTATGC; Lariat sequence: GCTGAC; Acceptor sequence: CAG) . / ⁇ l encodes a total of 467 amino acids, and the 19 amino acids at the N-terminus are signal peptides. There are 10 potential glycosylation sites in the amino acid sequence encoded by phyl, and amino acids 81-88 are the conserved sequence of the active site of phytase: RHGARYPT.
  • the zyl gene sequence is as follows:
  • the PCR reaction conditions are as follows: 94 ⁇ predenaturation for 5min, 94 ° C denaturation for 30sec, 55 ° C annealing for 1min, 72 ° C extension for 1.5min, a total of 30 cycles, and finally 72 ° C extension for 10min.
  • the size of the TCPNT fragment amplified by PCR was about 2.7 kb. After extraction with chloroform / isoamyl alcohol, the supernatant was extracted, precipitated with absolute ethanol, and dried in a 0.1 X SSC solution at a final concentration of 300 ng / ul.
  • a 0.5 kb target gene fragment was obtained. 1019 strains of 137 strains were detected, of which 132 were positive plants; 960 strains of C8605-2 strains were detected, and 94 positive plants were obtained.
  • soybean young leaf genomic DNA as a template for PCR amplification, a 0.5 kb target gene fragment was obtained. 121 early strains of 91025-7 were detected, including 19 positive plants; 259 strains of 16 varieties of Liaodou were detected, 57 of which were positive plants, and 78 strains of Tiefeng 29 were detected, of which 6 were positive plants. ⁇
  • Southern BLOT analysis PCR-detected maize and soybean genomic DNA was digested with EcoRI, agarose electrophoresis, and transmembrane. Using R4 and F4 as primers and pBI121 / phyII plasmid as a template to perform PCR amplification to obtain a 0.5 Kb DNA fragment, using an ECL kit (amersham pharmacia biotech) labeled probe, and performed Southern hybridization. Southern hybridization analysis showed that the transformed phytase gene has been integrated into the genomes of corn and soybean.
  • Enzyme activity analysis Take corn or soybean leaves and control plant leaves that are positive for PCR detection in a mortar, add an appropriate amount of pH 6.5 acetate-sodium acetate buffer solution, grind and centrifuge, and take the supernatant for the determination of phytase activity .
  • Transparent circle screening first prepare calcium phytate solution: 3 g of calcium acetate is dissolved in 100 ml of deionized water, and 1 g of phytic acid is dissolved in 400 ml of deionized water. The calcium acetate solution was slowly added to the phytic acid solution under constant stirring, the mixture was heated to boiling, and continuously stirred, and the mixture was cooled to room temperature at 4 ° C overnight. In this calcium phytate solution, various components were added according to the PDA medium formula, 151bf / in 2 (1.034 X 10 5 Pa) under high pressure steam sterilization for 20min, and the plate was inverted for use.
  • the full-length cDNA of the choline monooxygenase gene (CMO, GenBank, AF354442) of the halophyte Suaeda liaotitchsis kitag was obtained by RT-PCR and RACE.
  • the CMO cDNA is 1820bp in length, 123bp in the 5 ′ non-coding region, and 368bp in the 3 ′ noncoding region.
  • AATAA AATAAA
  • AATTAA open reading frame 1329 nucleotides, encoding 442 amino acids, of which conserved Cys-His pair "CTH” and “CPYH” with Rieske-type (2Fe-S) protein containing mature peptide starting region "AVA”, including a conserved multiiron nuclear binding domain
  • the CMO gene sequence is as follows:
  • the coding region of the CMO gene was obtained by PCR and cloned into the plant expression vector pBI121 to form the pBI121 / CMO recombinant plasmid.
  • the primer sequences are as follows:
  • the MAR sequence (upstream primer 5'-CGATTAAAAATCCCAATTATATTTGG-3 ', downstream primer 5, CCCTTGAAGAAG ACTTTTATCA-3') was obtained from tobacco genomic DNA by PCR, and the length was 1167bp. The two MAR fragments were ligated in the same direction on the pUC19 carrier.
  • the MAR sequence is as follows:
  • the pBI121 / CMO recombinant plasmid was digested with Pstl / EcoRI, and a fragment containing CaMV35S-CMO-os was obtained and inserted between pUC19 MAR fragments to form the gene transformation element MCCNM.
  • the size of the MCC bandage obtained by enzymatic digestion was about 4.9 kb, which was extracted with chloroform / isoamyl alcohol, the supernatant was extracted, and the ethanol was precipitated. After drying, it was dissolved in a 0.1 X SSC solution with a final concentration of 300 ng / ul. Used for conversion.
  • the rice varieties Liaojing 294, Liaojing 454 and Tiejing 4 were selected. Within 1-3 hours after flowering of the rice, cut off the flowers that have been opened and the flowers that cannot be opened on the day. Select the flowers that are open on the day, cut out one-third of the leaves, and inject the MCCNM solution with a micro syringe. , 10ul per flower, bagging. Control plants transformed only 0.1 X SSC solution (without MCCNM). Harvest seeds after maturity.
  • the amplified rice seedling genomic DNA was used as a template for PCR amplification to obtain a 1.4 kb target gene fragment.
  • Standard curve of betaine Prepare QACs precipitation solution. Add 0.5ml of each concentration of standard solution and add 0.2ml of QACs precipitation solution. (Incubate at TC for 90min, shake intermittently. Add 2ml of pre-cooled water, quickly add 20ml of pre-cooled dichloroethane at 10 ° C, and shake vigorously at 4 ° C. Let it stand at 5 ° C and 4 ° C until the two phases are completely separated. Return to room temperature, remove the phase and measure OD 365 .
  • Choline standard curve The steps are the same as above, but the reaction reagent is a choline precipitation solution.
  • Salt Tolerance Test At the beginning of the three-leaf stage of transformed rice, it was irrigated with 1% -1.5% Nacl solution, once every 2-3 days, and screened for a total of 4-6 weeks. Surviving plants were transplanted after slow seedlings.
  • the progeny of rice plant ⁇ was analyzed using the above-mentioned detection methods.
  • Experimental data show that the foreign CM0 gene is stably inherited in the rice genome.
  • BADH betaine aldehyde dehydrogenase
  • RT-PCR and RACE techniques were used to obtain the entire cDNA sequence of betaine aldehyde dehydrogenase gene (BADH, GenBank, AF359282) of Da liaotungensis kitag).
  • the BADH cDNA is 1901 bp in length, 66 bp in the 5 'non-coding region, and 329 bp in the 3' non-coding region. It contains 2 possible potyA signals: AATAA, open reading frame 1506 bp, encoding 502 amino acids, including aldehydes conserveed sequences of dehydrogenases VTLELGGKSP and cysteine residue C.
  • the BADH sequence is as follows:
  • the structure of the gene transformation element MCBNM is:
  • the coding region of the BADH gene was obtained by PCR and cloned into the plant expression vector PBI121 to form the pBI121 / BADH recombinant plasmid.
  • the primer sequences are as follows:
  • Recombinant plasmid pBI121 / BADH was digested with SpW / EcoRI to obtain CaMV35S- CMO-
  • the MCB Li fragment was about 5. lkb in size after digestion with enzyme, and was extracted with chloroform / isoamyl alcohol. The supernatant was extracted and precipitated with absolute ethanol. After being dried, it was dissolved in a 0.1 X SSC solution with a final concentration of 300ng / ul. Used for conversion.
  • the wheat varieties Liaochun 9, Liaochun 10 and Liaochun 13 were selected. Within 1-3 hours after flowering of wheat, cut off the glory flowers that have been opened and the glory flowers that cannot be opened on that day. Select the glory flowers that are open that day and cut the glumes one by one. Drop 1/3, inject the MCCNM solution with a micro-syringe, .10ml per flower, bagging. Control plants transformed only 0.1 X SSC solution (without MCBNM). Harvest seeds after maturity.
  • PCR detection Using BF and BR primers, PCR amplification was performed using wheat seedling genomic DNA as a template to obtain a 1.5 kb target gene fragment.
  • SOUTHERN BLOT analysis The wheat genomic DNA of the positive plants detected by PCR was digested with EcoRI, agarose electrophoresis, and transferred to a membrane. Using BF and BR1 (5'-TAGGCTGCCTGAGAACAT QAC-3 ') as primers and pBI121 / BADH plasmid as template for PCR amplification to obtain about 0.45Kb DNA fragment, using ECL kit (Amersham Pharmacia Biotech) labeled probe, SOUTHERN hybrid. Southern hybridization analysis showed that the transformed BADH gene had been integrated into the wheat genome.
  • the progeny wheat plants were analyzed using the above detection methods. Experimental data show that the exogenous BADH gene is stably inherited in the wheat genome.
  • the structure of the gene transformation element LCPNL is:
  • -LKRSDH5 'end partial sequence -CaMV35S-p / z_yII-Nos-LKRSDH3, end partial sequence-
  • the specific strategy is: Design and synthesize primers based on the LKRSDH gene sequence, and obtain the 5 and 3 'end partial sequences from the genomic DNA of maize, respectively, and ligate to pUC19.
  • the DNA Extraction Kit for GMO Detection kit was used to extract the DNA of maize, and then the primers L1 (5, -TGGCTACTACTGAGAGTGATCGTTG-3,) and L2 (5'-GTCATC ATACTTACGCTGTCCGAGAC-3 ') were used to obtain the 5, terminal portion of the LKRSDH gene. , The length is 1350bp.
  • the sequence of the 5 'end of the LKRSDH gene is as follows:
  • the ⁇ 121 / ⁇ ⁇ ⁇ recombinant plasmid in Example 1 was digested with enzyme to obtain a fragment containing CaMV35Si I-Nos and inserted between the pUC19 / LKRSDH fragments to form the gene transformation element LCPNL.
  • the size of the LCPNL fragment obtained by enzymatic digestion was about 4.55 kb.
  • the supernatant was extracted, precipitated with absolute ethanol, and then dried and dissolved in xssc solution at a final concentration of 300 ng / ul.
  • Lysine content detection is expressed as grams of lysine per 100 grams of corn or grams of lysine per 100 grams of total corn protein. After the corn seeds were pulverized, the total protein content was determined by the Nitrogen determination method. Enzymatic hydrolysis (6mol / L HC1 at 110 ° C, hydrolysis in vacuum or nitrogen-filled ampoules for 10-24 hours) to obtain a sample solution, and HPLC (derived from ninhydrin column) with amino acid analyzer to detect amino acid composition And calculate the amino acid content.
  • Example 5 Construction of the MZm3-4 Gene Boundary Sequence and Phytase Gene Transformation Element Containing Specific Expression of the Corn Tapetum and Transformation of Corn with Pollen Tube Pathway Method
  • the base sequence is as follows:
  • the primers MZ3 (5, -TGCACCTGCAAGTGAGGA-3,) and MZ4 (5'-CCATGTG GATTAGGCGTTATTGAGTCG-3 ') were used for PCR amplification to obtain the 3' end partial sequence of the MZm3-4 gene with a length of about 0.41 Kb.
  • the base sequence is as follows:
  • the pBI121 / II recombinant plasmid constructed in Example 1 was digested to obtain a fragment containing CaMV35S— / z ⁇ II—Nos and inserted between the pUC19 / MZm3- 4 fragments to form the gene transformation element MCPNMo.
  • Example 6 Construction of a rice gliadin (seed storage protein) gene ssp boundary sequence and phytase gene transformation element and transformation of rice by a pollen tube channel method
  • the gene transformation element SPS structure is-
  • -ssp5 'regulatory sequence (including the promoter region) a / ⁇ II- ssp3' regulatory sequence -3
  • the specific strategy is: design primers based on the rice ssp gene sequence, and obtain ssp gene 5 from the rice genomic DNA, the regulatory sequence (Including the promoter region) and the 3 'end regulatory sequence, linked to pUC19.
  • the primer 1 (5, -CTTGCATGGTGiCAGTAGTGCCTG-3,) and primer 2 (5'-GGG CAAAGATCTTGCTGGTGTATG-3 ') were used for PCR amplification to obtain the 5' end regulatory sequence (including the promoter region sequence) of the ssp gene, and the length was about 0.8Kb.
  • the base sequence is as follows:
  • the PBI121 / phyll recombinant plasmid constructed in ⁇ in Example 1 was digested with enzyme to obtain a fragment containing phyll and inserted between the pUC19 / ssp fragments to form a gene transformation element SPS.
  • the size of SPS obtained by enzyme digestion was about 2.2kb. After extraction with chloroform / isoamyl alcohol, the supernatant was extracted, and the precipitate was precipitated with anhydrous ethanol. After being dried, it was dissolved in a 0.1 X SSC solution with a final concentration of 300ng / ul.

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Abstract

L'invention concerne un procédé de manipulation de génie génétique relatif aux plantes, à sécurité biologique, caractérisé par une absence de vecteur et de gène marqueur de sélection, qui permet de résoudre le problème de sécurité lié aux plantes transgéniques, sous un double aspect environnemental et alimentaire, résultant de la séquence principale de vecteur et du gène marqueur de sélection dans la technique actuelle de manipulation de génie génétique relative aux plantes. Les caractéristiques techniques de l'invention sont les suivantes: construction de l'élément de transformation génique comprenant le gène étranger et la séquence de régulation, avec la séquence de délimitation des deux côtés, conduite de la manipulation de transformation génique par canal de tube pollinique, etc., détection selon l'expression du gène étranger dans l'élément de transformation génique et la modification du caractère de plante modifié résultant de l'insertion de la séquence de délimitation dans le génome de plante. On résout ainsi le problème de sécurité lié à la technique de transformation existante, ce problème venant du fait que la technique existante fait appel à un vecteur dérivé d'autres espèces de micro-organismes non apparentées, etc., et à un gène marqueur de sélection qui est un gène résistant à des médicaments de type antibiotique ou à des produits de type herbicide.
PCT/CN2002/000625 2002-09-02 2002-09-06 Procede de manipulation de genie genetique relatif aux plantes, a securite biologique Ceased WO2004020641A1 (fr)

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS61135588A (ja) * 1984-12-04 1986-06-23 Norin Suisanshiyou Nogyo Seibutsu Shigen Kenkyusho 植物細胞の選択マ−カ−の遺伝子，シヤトルベクタ−，アグロバクテリア，植物細胞及び植物個体
DE4309203C1 (de) * 1993-03-22 1994-04-21 Holt Claus Von Prof Dr Verfahren zur Produktion von transgenischen Pflanzen
JPH06261769A (ja) * 1993-03-17 1994-09-20 Res Dev Corp Of Japan プラスミド
WO1995005471A2 (fr) * 1993-08-13 1995-02-23 Ciba-Geigy Ag Procede de transformation stable de plantes
WO1999001563A1 (fr) * 1997-06-30 1999-01-14 Mogen International N.V. Plasmides servant a transformer des plantes et leur procede d'utilisation

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS61135588A (ja) * 1984-12-04 1986-06-23 Norin Suisanshiyou Nogyo Seibutsu Shigen Kenkyusho 植物細胞の選択マ−カ−の遺伝子，シヤトルベクタ−，アグロバクテリア，植物細胞及び植物個体
JPH06261769A (ja) * 1993-03-17 1994-09-20 Res Dev Corp Of Japan プラスミド
DE4309203C1 (de) * 1993-03-22 1994-04-21 Holt Claus Von Prof Dr Verfahren zur Produktion von transgenischen Pflanzen
WO1995005471A2 (fr) * 1993-08-13 1995-02-23 Ciba-Geigy Ag Procede de transformation stable de plantes
WO1999001563A1 (fr) * 1997-06-30 1999-01-14 Mogen International N.V. Plasmides servant a transformer des plantes et leur procede d'utilisation

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