WO2017113573A1 - Soja transgénique résistant au glyphosate et son procédé de préparation et d'application - Google Patents

Soja transgénique résistant au glyphosate et son procédé de préparation et d'application Download PDF

Info

Publication number
WO2017113573A1
WO2017113573A1 PCT/CN2016/083001 CN2016083001W WO2017113573A1 WO 2017113573 A1 WO2017113573 A1 WO 2017113573A1 CN 2016083001 W CN2016083001 W CN 2016083001W WO 2017113573 A1 WO2017113573 A1 WO 2017113573A1
Authority
WO
WIPO (PCT)
Prior art keywords
soybean
transgenic soybean
fragment
dna fragment
transgenic
Prior art date
Application number
PCT/CN2016/083001
Other languages
English (en)
Chinese (zh)
Inventor
邱丽娟
郭兵福
郭勇
张丽娟
洪慧龙
金龙国
王爽
杨慧
Original Assignee
中国农业科学院作物科学研究所
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 中国农业科学院作物科学研究所 filed Critical 中国农业科学院作物科学研究所
Publication of WO2017113573A1 publication Critical patent/WO2017113573A1/fr

Links

Images

Classifications

    • 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)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1085—Transferases (2.) transferring alkyl or aryl groups other than methyl groups (2.5)
    • C12N9/1092—3-Phosphoshikimate 1-carboxyvinyltransferase (2.5.1.19), i.e. 5-enolpyruvylshikimate-3-phosphate synthase
    • 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)
    • 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)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8274—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for herbicide resistance
    • C12N15/8275—Glyphosate
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1025—Acyltransferases (2.3)
    • C12N9/1029—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y205/00—Transferases transferring alkyl or aryl groups, other than methyl groups (2.5)
    • C12Y205/01—Transferases transferring alkyl or aryl groups, other than methyl groups (2.5) transferring alkyl or aryl groups, other than methyl groups (2.5.1)
    • C12Y205/01019—3-Phosphoshikimate 1-carboxyvinyltransferase (2.5.1.19), i.e. 5-enolpyruvylshikimate-3-phosphate synthase

Definitions

  • the invention relates to the field of plant genetic engineering technology or breeding, in particular to the transgenic soybean and its application, and more particularly to a glyphosate resistant genetically modified soybean and a preparation method and application thereof.
  • Soybean (Glycine max (L.). Merr) originated in China and is an important oil crop and cash crop in the world. It is also the main source of edible vegetable oil and plant protein. As an important part of the agro-ecosystem, weeds can compete with crops for light, water, nutrients and other resources, thus reducing crop yields. Therefore, effective control of soybean field weeds is one of the key factors for stable yield and high yield of soybean.
  • Traditional manual weeding, including manual weeding and simple agricultural tools, is time-consuming and labor-intensive, and has low efficacy and cannot be controlled in large areas in time.
  • Glyphosate is a systemic, broad-spectrum herbicide with a broad spectrum of herbicides, high efficiency, low toxicity, no residue, especially for humans and animals. It is the most widely used herbicide in the world. one.
  • the mechanism of action of glyphosate is to block the metabolic pathway of shikimic acid in plants and microorganisms by irreversibly binding to EPSPS synthase (5-enolpyruvylshikimate-3-phosphate synthase) in plants. Metabolic processes are disrupted, interfere with protein synthesis, prevent the formation of secondary products, and ultimately cause plant death; another important mechanism of action is inhibition of photosynthetic phosphorylation in plants.
  • glyphosate As a non-selective herbicide, glyphosate also harms soybeans while killing weeds. Therefore, genetically modified glyphosate-tolerant crops are genetically modified to impart glyphosate resistance to crops. Phosphine damage is one of the important ways to improve the efficiency of integrated weed control. The existing research and commercialization history of GM crops prove that soybean tolerance to glyphosate herbicides will be a useful trait for weed control and management.
  • EPSPS genes that increase glyphosate tolerance in plants
  • the EPSPS gene is usually derived from microorganisms, and its structural properties encode glyphosate. It cannot be combined with it and thus retains its catalytic activity in the presence of glyphosate (PCT/CN03/00651).
  • Most of the current globally commercialized glyphosate-resistant GM crops are designed for EPSPS.
  • N-acetyltransferase GAT
  • GAT N-acetyltransferase
  • glyphosate To confer tolerance to glyphosate in plants (ZL 2005 1 0086626.X).
  • the use of N-acetylation to grow transgenic crops can be applied to glyphosate throughout the plant's growth cycle, independent of the growth and development stages.
  • the method provided by the present invention is to insert an exogenous DNA fragment into the 50th, 543th, 770th, and 5th, 543th, 792th positions of chromosome 19 of the target soybean genome, and replace the 24 bp base sequence between the 50th, 543th, and 5th, 543th and 792th positions of the 19th chromosome to obtain the transgenic soybean. ;
  • the transgenic soybean has higher glyphosate resistance than the target soybean
  • the exogenous DNA fragment is a DNA molecule comprising a 5-enolpyruvylshikimate-3-phosphate synthase gene and an N-acetyltransferase gene.
  • the 5-enolpyruvylshikimate-3-phosphate synthase gene is G2-aroA
  • the N-acetyltransferase gene is GAT;
  • the exogenous DNA fragment is SEQ ID NO: 10 or SEQ ID NO: 1 from nucleotides 6217 to 1055 at the 5' end.
  • the exogenous DNA fragment has a length of 0 to 5 Kb extending from the 50th, 543th, and 767th nucleotides of the chromosome 19 of the target soybean genome to the upstream side of the transgenic soybean. Any one of the DNA fragments;
  • the upstream flanking fragment is specifically a nucleotide represented by SEQ ID NO: 8 in the sequence listing;
  • the exogenous DNA fragment is a DNA of a length of 0 to 5 Kb extending from a nucleotide of the 50th, 543,792 nucleotides of the chromosome 19 of the target soybean genome in the downstream flanking fragment of the transgenic soybean. Fragment
  • the downstream flanking fragment is specifically a nucleotide represented by SEQ ID NO: 9 in the Sequence Listing.
  • the upstream flanking fragment is a fragment of the transgenic soybean genome immediately adjacent to the 5' end of the exogenous DNA fragment;
  • the downstream flanking fragment is a fragment of the transgenic soybean genome immediately adjacent to the 3' end of the exogenous DNA fragment;
  • the exogenous DNA fragment is introduced into the soybean of interest by a recombinant vector containing the exogenous DNA fragment;
  • nucleotide sequence of the recombinant vector is specifically Sequence 1 in the Sequence Listing.
  • the target soybean is a conventional soybean variety Jack.
  • transgenic soybean prepared by the above method is also within the scope of protection of the present invention.
  • the transgenic soybean is a homozygous strain.
  • the above-mentioned transgenic soybean is a homozygous strain of T2 transgenic soybean GE-J16 (named GE-J16). It was deposited on December 29, 2015 at the General Microbiology Center of China Microbial Culture Collection Management Committee (CGMCC, Address: Beijing No. 3, Beichen West Road, Chaoyang District, Institute of Microbiology, Chinese Academy of Sciences, 100101), the deposit number is CGMCC No.11686, and the classification is named Glycines max.
  • CGMCC General Microbiology Center of China Microbial Culture Collection Management Committee
  • Another object of the present invention is to provide a method for detecting or assisting in detecting whether a plant sample is derived from the transgenic soybean or its progeny prepared by the above method or for detecting or assisting in detecting whether the preparation contains the transgenic soybean or its progeny prepared by the above method.
  • the method provided by the present invention comprises the steps of detecting the genome of the plant sample or its preparation Whether the DNA contains DNA fragment A,
  • the DNA fragment A is as follows 1) or 2):
  • the plant sample is or candidate as the transgenic soybean or its progeny
  • the plant sample is not or the candidate is not the transgenic soybean or its progeny.
  • the method is as follows 1) or 2) or 3):
  • the primer pair 1 is a primer pair capable of amplifying a DNA molecule A consisting of a part or all of the 5' end of the exogenous DNA fragment and the upstream flanking sequence immediately adjacent thereto; the corresponding amplification product of interest is DNA molecule A;
  • the primer pair 2 is a primer pair capable of amplifying a DNA molecule B comprising a part or all of the downstream flanking sequence of the exogenous DNA fragment and the downstream flanking sequence thereof; the corresponding amplification product of interest is DNA molecule B;
  • the primer pair 1 consists of a single-stranded DNA molecule represented by SEQ ID NO: 4 in the Sequence Listing and a single-stranded DNA molecule represented by SEQ ID NO: 11 in the Sequence Listing;
  • the primer-specific corresponding fragment size of the primer pair 1 is 1529 bp, and the nucleotide sequence thereof is specifically the sequence 14;
  • the primer pair 2 is composed of a single-stranded DNA molecule represented by SEQ ID NO: 12 in the Sequence Listing and a single-stranded DNA molecule represented by SEQ ID NO: 13 in the Sequence Listing;
  • the target specific fragment size corresponding to the primer pair 2 is 2203 bp, and the nucleotide sequence thereof is specifically the sequence 15;
  • nucleotide sequence of the probe is sequence 6.
  • the progeny of the transgenic soybean is a transgenic material derived from the transgenic soybean as a parent, and includes progeny derived from the transgenic soybean or derived from hybridization with other soybeans or progeny derived from the mutagenized or hybrid progeny. .
  • a third object of the present invention is to provide a kit for detecting whether a sample is derived from the transgenic soybean or its progeny.
  • the kit provided by the invention comprises 1) the exogenous DNA fragment, 2) the primer pair 1, 3) the primer pair 2 or 4) the probe.
  • the above kit may further include instructions describing the above method.
  • the progeny of the transgenic soybean is a transgenic material derived from the transgenic soybean as a parent, and includes progeny derived from the transgenic soybean or derived from hybridization with other soybeans or progeny derived from the mutagenized or hybrid progeny. .
  • transgenic soybean prepared by the above method in breeding and/or production processing.
  • Figure 1 shows the PCR identification of the T 0 generation transgenic soybean EPSPS (G2-aroA) and GAT genes.
  • T 0 transgenic soybean GE-J16 Analysis of T-DNA copies of the exogenous number 2 is T 0 transgenic soybean GE-J16.
  • Figure 4 shows the resistance of the T 2 generation transgenic soybean GE-J16 homozygous strain under the treatment of 12 L/ha glyphosate isopropylamine salt.
  • FIG 5 is a transgenic soybean GE-J16 T 2 generation of homozygous lines were detected using PCR.
  • Fig. 6 is a schematic diagram showing the exogenous T-DNA insertion site and integration mode of the T 2 generation transgenic soybean GE-J16 homozygous strain.
  • Figure 7 is a schematic diagram showing the location of the genetically modified soybean GE-J16 verification primer.
  • Figure 8 is a qualitative PCR amplification diagram of the transgenic soybean GE-J16 progeny plants.
  • the soybean variety is Jack (Jack, its serial number: WDD01579, which is recorded in the following literature: ⁇ Chinese Soybean Variety Resources Catalogue (Continued 2)>>, Changyi Town, Sun Jianying, Qiu Lijuan, Chen Yiwu, Editor-in-Chief, China Agriculture Publishers, 1996, the public can obtain from the Institute of Crop Sciences of the Chinese Academy of Agricultural Sciences, taking Jack's cotyledonary node as a material for genetic transformation;
  • Agrobacterium tumefaciens strain is Ag10, which is reported in the following literature: Cheng Wei. Preliminary study on ectopic expression of AtMGT4 gene in rice (D). Hunan Normal University, 2012; public available from the Chinese Academy of Agricultural Sciences, Crop Science Institute obtained.
  • the soybean tissue culture medium was mainly MS and B5 medium, and sterilized at 121 ° C for 15-20 min.
  • Germination medium B5 + 20g / L sucrose + 8g / L agar powder, pH 5.8;
  • Co-culture medium 1/10B5+30g/L sucrose +3.9g/L 2-(N-morpholine)ethanesulfonic acid (MES)+1.67mg/L 6-BA+39mg/L acetosyringone+0.25mg/ L gibberellin (GA 3 ) +1 mmol / L dithiothreitol +1 mmol / L sodium thiosulfate + 1mmol / L cysteine + 5g / L agar powder, pH 5.4;
  • MES 2-(N-morpholine)ethanesulfonic acid
  • GA 3 +1 mmol / L dithiothreitol +1 mmol / L sodium thiosulfate + 1mmol / L cysteine + 5g / L agar powder, pH 5.4;
  • Co-culture solution 1/10B5+30g/L sucrose +3.9g/L 2-(N-morpholine)ethanesulfonic acid (MES)+1.67mg/L 6-BA+39mg/L acetosyringone+0.25mg/ L gibberellin (GA 3 );
  • MES 2-(N-morpholine)ethanesulfonic acid
  • Cluster bud induction medium B5+30g/L sucrose+8g/L agar powder+0.6g/L 2-(N-morpholine)ethanesulfonic acid (MES)+1.67mg/L 6-BA+150mg/L thiophene Sporemycin + 400mg / L carbenicillin + 15mg / L glyphosate, pH 5.7;
  • MES 2-(N-morpholine)ethanesulfonic acid
  • Bud elongation medium MS+B5 organic+30g/L sucrose+8g/L agar powder+0.6g/L 2-(N-morpholine)ethanesulfonic acid (MES)+50mg/L aspartic acid+50mg /L glutamine +0.3mg / L ⁇ -3-acetic acid (IAA) +0.5mg / L gibberellin (GA 3 ) + 150mg / L carbenicillin + 0.1mg / L Zein (Ze) + 5 mg / L glyphosate, pH 5.7;
  • MES 2-(N-morpholine)ethanesulfonic acid
  • IAA 0.5mg / L gibberellin
  • GA 3 150mg / L thiamycin + 400mg / L carbenicillin + 0.1mg / L Zein (Ze) + 5 mg / L glyphosate, pH 5.7;
  • Rooting medium MS+B5 organic+30g/L sucrose+8g/L agar powder+0.6g/L 2-(N-morpholine)ethanesulfonic acid (MES) 50mg/L aspartic acid+50mg/L valley Aminoamide, pH 5.7;
  • MES 2-(N-morpholine)ethanesulfonic acid
  • Agrobacterium cultures were cultured with YEP and LB media.
  • Acetyl syringone, MS and B5 dry powder medium and acetosyringone are products of sigma, 2-(N-morpholine)ethanesulfonic acid (MES), streptomycin, carbenicillin, agar powder, zeatin, day Aspartic acid, glutamine, gibberellin (GA 3 ) and 6-benzylaminoadenine (6-BA) are products of Biodee, and sucrose is a domestic reagent.
  • the gene has a single restriction site XhoI upstream and downstream, and the optimized GAT gene and the intermediate vector vector skeleton obtained in the above (3) are added to the recombinant vector pKT-rGE; Sequence 1.
  • the promoters Rbcs and the glyphosate resistance gene Rbcs-EPSPS (G2-aroA) from the 5' end are the promoter-enhanced 35S and the 5' end of the 8204.
  • nucleotides are the peptide Rbcs and the glyphosate resistance gene Rbcs-EPSPS (G2-aroA), and the 5' end nucleotides 7926-8196 are the glyphosate resistance gene EPSPS (G2-aroA)
  • the NOS at the 5' end of the terminator, the nucleotides 6903-7673 at the 5' end are the glyphosate-degrading gene N-acetyltransferase gene-enhanced 35S, and the nucleotides 6456-6896 at the 5' end are the glyphosate-degrading gene N-
  • the acetyltransferase gene GAT and the nucleotides 6248-6455 at the 5' end are the glyphosate-degrading gene N-acetyltransferase gene terminator CaMV 35S polyA.
  • the recombinant vector pKT-rGE obtained in the above 1 was introduced into Agrobacterium tumefaciens Ag10 to obtain recombinant Agrobacterium Ag10/pKT-rGE.
  • Recombinant Agrobacterium Ag10/pKT-rGE was transformed into cotyledonary explants of in vitro cultured soybean (Glycine max) Jack.
  • Agrobacterium and explants were co-infected for 3 days in a screening environment containing glyphosate.
  • the transgenic plants were induced by the organ regeneration pathway, and the stage of the shoot bud induction was induced by using glyphosate (Sigma) at a concentration of 15 mg/L as a screening agent for 3-6 weeks; the elongation bud induction stage was used at a concentration of 5 mg/L.
  • Phosphine (sigma) is used as a screening agent for 4-8 weeks of induction; in the induction and elongation stages, the medium is changed every 2 weeks to regenerate the transformed cells to inhibit the rapid growth of Agrobacterium tumefaciens in the induction and elongation medium.
  • concentration of 150 mg/L of piperazine and 400 mg/L of carbenicillin were respectively added, when the elongation buds were elongated to 4-6 cm, the rooting medium was transferred to induce rooting to obtain regenerated soybean plants.
  • the regenerated plants were transplanted into pots and cultured in an incubator.
  • the light conditions were 18 h light and 6 h dark.
  • the young leaves were selected and marked with a marker, and 1 ⁇ l of glyphosate isopropylamine was applied.
  • (Roundup) stock solution was subjected to glyphosate tolerance analysis on T 0 regenerated plants.
  • the response of each plant to glyphosate treatment was investigated.
  • One of the transformants GE-J16 showed strong resistance to 1 ⁇ l of glyphosate isopropylamine salt solution.
  • the transformant GE-J16 was T 0 transgenic soybean, and the T 0 generation transgenic soybean was glyphosate resistant. Sex is higher than wild type non-genetically modified soybean Jack.
  • PCR Polymerase chain reaction
  • the 20 ⁇ l PCR reaction system contained 2 ⁇ l of 10 ⁇ EX-Taq buffer, 2 ⁇ l of 2 mM dNTPs, and 0.5 ⁇ l of 10 ⁇ M gene-specific upstream and downstream primers, EX-Taq. 0.2 ⁇ l of enzyme, hydrated to 20 ⁇ l; PCR reaction procedure was 94 ° C, 4 min (1 cycle); 94 ° C, 30 s (denaturation), 60 ° C, 30 s (annealing), 72 ° C, 45 s (extension) 35 cycles; 72 ° C ( Final extension) 10 min (1 cycle).
  • Amplification of the primer pair (EP-F1/R1) of EPSPS (G2-aroA) can positively amplify the 743 bp product:
  • a primer pair (GAT-F2/R2) that amplifies the GAT gene is capable of amplifying a 257 bp product:
  • M is 200 bp DNA Marker
  • 1 is wild type Jack control
  • 2 is sterile water control
  • 3 is plasmid pKT-rGE positive control
  • 4 is T 0 generation genetically modified soybean GE-J16;
  • the results showed that the PCR results of two exogenous genes of GE-J16 were positive, indicating that the foreign gene EPSPS (G2-aroA) and GAT had been transferred.
  • the transgenic soybeans having glyphosate resistance are propagated by conventional cultivation and breeding methods, and the transgenic soybean seeds are harvested.
  • the phenotype of non-resistant plants is leaf chlorosis, leaf curling, shrinkage, apical meristem necrosis, until the whole plant dies; resistant plants phenotype is strong growth trend, leaves are not chlorotic, no curl, no shrinkage .
  • T 1 generation transgenic soybean GE-J16 The results of T 1 generation transgenic soybean GE-J16 are shown in Figure 3.
  • (1, 2, 5: T 1 generation of genetically modified soybean GE-J16 anti- Single plant of glyphosate; 3, 4: T 1 generation transgenic soybean GE-J16 is not resistant to glyphosate alone) indicates that T 1 generation transgenic soybean GE-J16 is in the first to third compound leaf stage to 3L/ ha (200ml / acre) in the presence of glyphosate isopropylamine salt resistance (of Roundup) was
  • T 2 generation transgenic soybean GE-J16 strains were sprayed with 3L/ha, 6L/ha and 9L/ha glyphosate isopropylamine solution (Roundup) solution, and the T 2 generation with no resistance was selected.
  • the T 2 generation transgenic soybean GE-J16 homozygous strain and the wild type control Jack were sprayed with 12 liters/ha of glyphosate isopropylamine solution (Roundup) solution, and the transgenic homozygous strain GE-J16 was investigated 2 weeks later. Its negative control Jack responded to the phytotoxicity of glyphosate.
  • T 2 generation transgenic soybean GE-J16 homozygous strain has important breeding prospects and utilization value.
  • Glyphosate-tolerant transgenic soybean GE-J16 obtained a homozygous strain, and its resistance was stably inherited in the offspring.
  • the probe for Southern blotting was to select a 338 bp vector DNA sequence design probe from the T-DNA region of the vector (sequence 6), and refer to the instruction manual of the PCR method DIG labeling kit produced by Beijing Meilaibo Medical Technology Co., Ltd. High-six-labeled probe.
  • the recombinant plasmid pKT-rGE was used as a positive control, and the genomic DNA of the non-transgenic wild type genetic transformation receptor Jack was a negative control.
  • the genomic DNA of the T 0 transgenic soybean GE-J16 obtained in the above Example 2 was extracted, and 50-70 ⁇ g of genomic DNA was digested with 5 units of restriction enzyme in a total volume of 200 ⁇ l at 37 ° C for 5-10 h to digest The DNA was precipitated and redissolved in 25 ⁇ l of sterile water, and each sample was spiked with 6 ⁇ l of 6 ⁇ Loading Buffer, digested DNA, positive control, negative control, and standard molecular size marker III (Digoxigenin-labeled (Roche)). And ⁇ Hind III marker), DNA was separated on a 0.8-1.0% agarose gel electrophoresis at 45V.
  • the DNA was observed with ethidium bromide and included on a fluorescent scale for imaging recording.
  • the DNA was then transferred to a Hybond nylon membrane by referring to the instructions of the What Down Schleicher & Schuell Corporation's Rapid Downward transfer system, which hybridized with the probe and displayed by hybridization (Beijing Meilaibo Medical Technology Co., Ltd.).
  • Copy number determination can be performed by analyzing genomic DNA adjacent to the left and right border regions, and digesting genomic DNA with restriction enzymes DraI, HindIII, and XbaI.
  • the genomic DNA of the different plants of the T 2 generation transgenic soybean GE-J16 homozygous strain obtained in Example 2 was extracted, and about 50 ng of genomic DNA was used as template DNA in a 20 ul PCR reaction system, and the examples were used.
  • the amplified EPSPS (G2-aroA) primer pair (EP-F1/R1) and the amplified GAT gene primer pair (GAT-F2/R2) were subjected to PCR amplification, respectively.
  • the genome-wide resequencing analysis of the above-mentioned T 2 generation transgenic soybean GE-J16 homozygous strain was carried out.
  • the binding region of the genomic DNA of the foreign T-DNA and the insertion site was PCR-amplified to verify the insertion position of the foreign T-DNA, and the result confirmed the correctness of the T-DNA insertion site.
  • the T 2 generation transgenic soybean GE-J16 The results of T-DNA insertion of homozygous strains are shown in Figure 6.
  • the 5'-end flanking sequence of the exogenous T-DNA inserted in the T 2 generation transgenic soybean GE-J16 homozygous strain is sequence 8
  • the 3'-end downstream flanking sequence of DNA is sequence 9.
  • the complete exogenous T-DNA sequence inserted into the T2 generation transgenic soybean GE-J16 is obtained as sequence 10, and the exogenously inserted exogenous DNA molecule is integrated into 4639 bp, and the transgenic soybean exogenous DNA molecule integration does not contain a vector. Skeleton sequence.
  • the T2 generation transgenic soybean GE-J16 homozygous strain was a glyphosate-resistant plant, which was analyzed by flanking sequences and insertion sites, which was sequence 10 or sequence 1 from the 5' end of the sequence table. 10855 nucleotides were inserted between the 50th, 543th, 767th, and 5th, 543th and 792th positions of chromosome 19 of the non-transgenic wild-type soybean Jack genome, and the 24 bp base sequence between the 50th, 543th, and 543th, 543th and 792th positions of the 17th chromosome was replaced, and the transgenic soybean was obtained.
  • nucleotide sequence of the upstream flanking fragment of nucleotides 50, 543, 767 upstream and immediately adjacent to nucleotides 50, 543, 767 is sequence 8, downstream from the 50th, 543, 792 and immediately downstream of the 50th, 543th, 792th nucleotide
  • the nucleotide sequence of the fragment is SEQ ID NO: 9;
  • SEQ ID NO: 10 is a DNA molecule containing a 5-enolpyruvylshikimate-3-phosphate synthase gene and an N-acetyltransferase gene.
  • the insertion position of the foreign DNA molecule and the flanking sequences on both sides can be used to identify whether or not the target transgenic soybean (T2 generation transgenic soybean GE-J16 homozygous strain).
  • the T2 generation genetically modified soybean GE-J16 homozygous strain (named GE-J16) was deposited on December 29, 2015 in the General Microbiology Center of China Microbial Culture Collection Management Committee (CGMCC, Address: Beichen West, Chaoyang District, Beijing) No. 3, No. 1 Hospital, Institute of Microbiology, Chinese Academy of Sciences, 100101), the accession number is CGMCC No.11686, and the classification is named Glycines max.
  • Example 4 According to the position of exogenous DNA molecule insertion in Example 4 and its upstream and downstream flanking gene sequences, specific primers were developed, and a qualitative PCR identification method for T2 generation transgenic soybean GE-J16 homozygous strain and self-crossing or hybrid progeny was established. .
  • Primer GAT-F2 5'GCGATTTACTTCGTGGTGCAT 3' (SEQ ID NO: 4).
  • Primer G2EP-R2 5'ACCACCATCAATCTCGAAACG 3' (sequence 12)
  • the amplification results showed that the genomic DNA amplification of roots, stems, leaves, flowers and seeds of T 2 generation transgenic soybean GE-J16 homozygous strains obtained a specific 1529 bp target fragment, and the specific nucleotide sequence of the target fragment was as follows. Sequence 14 is shown.
  • the amplification results showed that the genomic DNA amplification of the roots, stems, leaves, flowers and seeds of the T 2 generation transgenic soybean GE-J16 homozygous strains gave a specific 2203 bp target fragment, and the specific nucleotide sequence of the target fragment was obtained. As shown in sequence 15.
  • the genomic DNA of the sample to be tested is extracted as a template, and PCR amplification is performed using primer pair 1 and primer pair 2 to detect PCR amplification products:
  • the transgenic soybean to be detected is the T2 generation transgenic soybean ZH10-6 homozygous strain obtained in Example 4;
  • the transgenic soybean to be tested is not the T2 generation transgenic soybean ZH10-6 homozygous strain obtained in Example 4.
  • the experiment of the present invention proves that the present invention adopts the insertion of an exogenous DNA fragment between 50, 543, 767 and 50, 543, 792 of soybean chromosome 19 to obtain transgenic soybean containing the foreign DNA fragment; the exogenous DNA fragment includes the glyphosate resistance gene G2-aroA. And glyphosate degrading gene N-acetyltransferase gene GAT gene; transgenic soybean has higher resistance to glyphosate than wild-type soybean without transgenic.
  • the transgenic soybean is tolerant to high doses of glyphosate, which can be further modified by hybridization with elite soybean lines to optimize other agronomic traits such as yield, quality, pest resistance and the like.
  • the exogenous DNA fragment soybean co-expressed the glyphosate resistance gene EPSPS (G2-aroA) and the glyphosate degradation gene N-acetyltransferase gene (GAT) at the seedling stage (the cotyledon was unearthed and the true leaves were not unfolded)
  • the plants were tolerant to 1-1.5 ⁇ l of the glyphosate isopropylamine salt solution, and the field was tolerant to about 3 to 12 liters of glyphosate isopropylamine per hectare.
  • the total standard measure of good weed control varies between 3-6 liters per hectare depending on weed pressure.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Microbiology (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Plant Pathology (AREA)
  • Cell Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Analytical Chemistry (AREA)
  • Immunology (AREA)
  • Medicinal Chemistry (AREA)
  • Mycology (AREA)
  • Botany (AREA)
  • Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

La présente invention concerne un soja transgénique résistant au glyphosate ainsi qu'un procédé de préparation et une application de ce dernier. La présente invention décrit également un procédé d'amélioration génétique d'un soja transgénique, comprenant l'insertion d'un fragment d'ADN exogène entre la position 50,543,767 et la position 50,543,792 du chromosome no 19 d'un génome de soja cible pour obtenir un soja transgénique tolérant au glyphosate sous hautes doses. Sont en outre prévus des procédés de détection de cellules, tissus, organes, produits, etc., du soja transgénique et de sa descendance.
PCT/CN2016/083001 2015-12-30 2016-05-23 Soja transgénique résistant au glyphosate et son procédé de préparation et d'application WO2017113573A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201511022465.8A CN105505981B (zh) 2015-12-30 2015-12-30 一种抗草甘膦转基因大豆及其制备方法与应用
CN201511022465.8 2015-12-30

Publications (1)

Publication Number Publication Date
WO2017113573A1 true WO2017113573A1 (fr) 2017-07-06

Family

ID=55714274

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2016/083001 WO2017113573A1 (fr) 2015-12-30 2016-05-23 Soja transgénique résistant au glyphosate et son procédé de préparation et d'application

Country Status (3)

Country Link
CN (1) CN105505981B (fr)
AR (1) AR107260A1 (fr)
WO (1) WO2017113573A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112359123A (zh) * 2020-10-10 2021-02-12 浙江省农业科学院 转g10evo-epsps基因大豆实时荧光定量检测方法及其试剂盒

Families Citing this family (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105505981B (zh) * 2015-12-30 2018-12-25 中国农业科学院作物科学研究所 一种抗草甘膦转基因大豆及其制备方法与应用
CN106350532A (zh) * 2016-08-28 2017-01-25 浙江大学 一种抗草甘膦融合基因、编码蛋白及其应用
CN108179147B (zh) * 2018-02-03 2022-02-11 吉林省农业科学院 高油酸转基因大豆事件e2d9050外源插入片段侧翼序列及其应用
CN108239639B (zh) * 2018-02-03 2022-02-11 吉林省农业科学院 耐逆转基因大豆事件wb1外源插入片段侧翼序列及其应用
CN108179146B (zh) * 2018-02-03 2022-02-11 吉林省农业科学院 抗病转基因大豆事件b5c9120-3外源插入片段侧翼序列及其应用
CN112322631B (zh) * 2020-11-06 2022-08-16 武汉天问生物科技有限公司 一种抗草甘膦转基因大豆的培育方法
CN114807221B (zh) * 2022-04-28 2023-06-06 宁波大学科学技术学院 水稻根系伸长调控基因OsDRP1C的突变体、蛋白质、表达载体及其应用

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101278053A (zh) * 2005-07-29 2008-10-01 孟山都技术有限公司 利用来自转基因杂交体的分离子来开发新的种质
WO2012082542A2 (fr) * 2010-12-15 2012-06-21 Syngenta Participations Ag Soja comprenant le mécanisme de transformation syht04r, et compositions et procédés de détection de ce mécanisme
CN104450755A (zh) * 2014-11-14 2015-03-25 重庆大学 一种东亚飞蝗ATP合酶α亚基基因及其dsRNA在害虫防治中的应用
CN105505981A (zh) * 2015-12-30 2016-04-20 中国农业科学院作物科学研究所 一种抗草甘膦转基因大豆及其制备方法与应用

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN100365133C (zh) * 2005-10-21 2008-01-30 天津师范大学 一种转基因大豆的检测方法及所用引物
CN101100676B (zh) * 2007-06-15 2010-12-22 中国农业科学院生物技术研究所 培育抗草甘膦植物的双价表达载体

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101278053A (zh) * 2005-07-29 2008-10-01 孟山都技术有限公司 利用来自转基因杂交体的分离子来开发新的种质
WO2012082542A2 (fr) * 2010-12-15 2012-06-21 Syngenta Participations Ag Soja comprenant le mécanisme de transformation syht04r, et compositions et procédés de détection de ce mécanisme
CN104450755A (zh) * 2014-11-14 2015-03-25 重庆大学 一种东亚飞蝗ATP合酶α亚基基因及其dsRNA在害虫防治中的应用
CN105505981A (zh) * 2015-12-30 2016-04-20 中国农业科学院作物科学研究所 一种抗草甘膦转基因大豆及其制备方法与应用

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
GUO B, GUO Y, HONG H, JIN L, ZHANG L, CHANG RZ, LU W, LIN M, QIU LJ: "Co-expression of G2-EPSPS and glyphosate acetyltransferase GAT genes conferring high tolerance to glyphosate in soybean.", FRONTIERS IN PLANT SCIENCE, vol. 6, 15 October 2015 (2015-10-15), pages 847, XP055653307, ISSN: 1664-462X, DOI: 10.3389/fpls.2015.00847 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112359123A (zh) * 2020-10-10 2021-02-12 浙江省农业科学院 转g10evo-epsps基因大豆实时荧光定量检测方法及其试剂盒

Also Published As

Publication number Publication date
AR107260A1 (es) 2018-04-11
CN105505981B (zh) 2018-12-25
CN105505981A (zh) 2016-04-20

Similar Documents

Publication Publication Date Title
WO2017113573A1 (fr) Soja transgénique résistant au glyphosate et son procédé de préparation et d'application
WO2016086884A1 (fr) Soja transgénique résistant au glyphosate ainsi qu'un procédé de préparation et une utilisation associée
RU2636021C2 (ru) Линии трансгенной сои, генетическое событие 8264.42.32.1, устойчивое к гербицидам с пакетированными генами на его основе, и их детектирование
CN107129993B (zh) 一种修饰的抗草甘膦基因及抗草甘膦水稻的培育方法
CN100429310C (zh) 控制水稻分蘖的基因及用途
CN110144363B (zh) 抗虫耐除草剂玉米转化事件
WO2017215327A1 (fr) Séquence d'acide nucléique utilisée pour détecter la plant de soja tolérant aux herbicides dbn9008, et procédé de détection associé
WO2014036946A1 (fr) Gène bph9 de résistance au fulgore du riz brun et marqueurs moléculaires, et leurs utilisations
WO2016165243A1 (fr) Gène bph6 de riz résistant à la delphacide brune du riz et marqueur moléculaire étroitement lié correspondant
JP7039493B2 (ja) 生物学的試料中の遺伝子導入ダイズイベントｄｂｎ９００４の有無を検出するための核酸配列、それを含有するキット、およびその検出方法
CN111206031A (zh) 一种用于检测玉米植物naz-4的核酸序列及其检测方法
US11597944B2 (en) Gene conferring resistance to Cercospora beticola in beets
CN111247255A (zh) 用于检测大豆植物dbn8007的核酸序列及其检测方法
CN111406117A (zh) 用于检测大豆植物dbn8002的核酸序列及其检测方法
US20220154206A1 (en) Gene for resistance to plant disease
CN110881367A (zh) 一种玉米事件t抗-4及其使用方法
AU2021328620A1 (en) Plant resistance gene and means for its identification
CN116287384B (zh) 抗虫耐除草剂玉米转化事件ld05的核酸分子、检测方法及其应用
IL293400A (en) cca gene for resistance to viruses
WO2024051077A1 (fr) Événement de soja transgénique cal16 et son procédé de détection
US20230227841A1 (en) Gene conferring resistance to cercospora beticola in beets
WO2022213520A1 (fr) Vecteur d'expression des gènes gr79 et gat de résistance au glyphosate, maïs à haute résistance au glyphosate, et procédé de détection associé
CN105906697B (zh) 水稻OsMTOPVIB蛋白及其编码基因在调控花粉育性中的应用
CN117512162A (zh) 转基因大豆事件xp-2及其检测方法
BR112017011898B1 (pt) Método para melhoramento genético de uma soja transgênica, método e kit para detectar se uma amostra de planta e/ou seus produtos é derivada da soja trangênica preparada pelo dito método e uso da planta trangênica ou progênies da mesma preparada pelo dito método

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 16880381

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 16880381

Country of ref document: EP

Kind code of ref document: A1