WO2016188332A1 - Événement de transformation du maïs et procédé d'identification de spécificité et leur utilisation - Google Patents

Événement de transformation du maïs et procédé d'identification de spécificité et leur utilisation Download PDF

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WO2016188332A1
WO2016188332A1 PCT/CN2016/082025 CN2016082025W WO2016188332A1 WO 2016188332 A1 WO2016188332 A1 WO 2016188332A1 CN 2016082025 W CN2016082025 W CN 2016082025W WO 2016188332 A1 WO2016188332 A1 WO 2016188332A1
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seq
transformation event
gene
nucleotide sequence
resistant
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沈志成
林朝阳
张先文
徐晓丽
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杭州瑞丰生物科技有限公司
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Definitions

  • the invention belongs to the field of plant molecular biology, in particular to the field of breeding of genetically modified crop varieties.
  • the present invention relates to an anti-insect glyphosate resistant maize transformation event "double antibody 12-5" and “double antibody 12-15" and a specific detection method thereof.
  • Insect-resistant transgenic corn can significantly reduce the use of chemical pesticides, thereby reducing production costs and reducing pesticide contamination of the environment and crop products.
  • Herbicide-tolerant corn can significantly reduce the agricultural labor required to control weeds, reduce labor input, and reduce the impact of weeds on corn yield.
  • the use of herbicides to control weeds is also more conducive to the promotion of no-till technology and reduce the loss of soil and fertilizer. Therefore, insect and herbicide resistance properties are of outstanding importance in corn production.
  • the transformation event is a molecular structure composed of a foreign gene at the upstream and downstream flanking regions of the genomic insertion site and a foreign gene.
  • a foreign gene transformed plant can obtain a population of transformants that contain a large number of independent events, each of which is unique.
  • the expression of a foreign gene in a plant is affected by the location of the chromosome into which the foreign gene is inserted. This may be due to the influence of chromatin structure or transcriptional regulatory elements near the integration site.
  • the expression levels of the same genes in different transformation events vary widely, and there may be differences in spatial or temporal patterns of expression.
  • the insertion of foreign genes may also affect the expression of endogenous genes. Therefore, each independent transformation event has a different effect on the recipient plant. Plant transformation events that can effectively express foreign genes without affecting the agronomic traits of the plants themselves have important application value in cultivating new varieties of transgenic crops.
  • transformation event can be introduced into the maize genome of different genetic backgrounds using breeding methods, somatic cell protoplast fusion techniques, thereby conferring maize resistance to glyphosate resistance.
  • the object of the present invention is to provide an excellent maize insect-resistant glyphosate-resistant glucagon conversion event "double antibody 12-5" and “double antibody 12-15", and the design concepts of the foreign genes contained in the two independent transformation events are the same.
  • both of these transformation events can realize the introduction of the foreign gene into the maize line, giving the recipient corn the ability to resist insect glyphosate;
  • the foreign gene of the glyphosate-resistant glyphosate gene can be stably inherited in the recipient maize; the insect-resistant glyphosate-resistant gene Expression does not adversely affect the agronomic traits of the recipient corn.
  • the present invention provides a maize transformation event, wherein the nucleotide sequence shown by SEQ ID NO. 1 is the left wing region of the foreign gene, and the nucleotide sequence shown by SEQ ID NO. 3 is The right wing region of the foreign gene or the nucleotide sequence shown by SEQ ID NO. 14 is the left wing region of the foreign gene, and the nucleotide sequence shown in SEQ ID NO. 15 is the foreign gene. Right wing area.
  • the exogenous gene includes an insect resistance gene and a glyphosate resistance gene, the nucleotide sequence of the insect resistance gene is represented by SEQ ID NO. 4, and the nucleotide sequence of the glyphosate resistance gene is SEQ ID Shown in NO.5.
  • nucleotide sequence of the foreign gene is represented by SEQ ID NO.
  • the maize transformation event is the nucleotide sequence shown in SEQ ID NO. 1 as the left wing region of the foreign gene, and the nucleotide sequence shown in SEQ ID NO. 3 is the right gene of the foreign gene.
  • the flanking region ie, the present invention provides an insect-resistant glyphosate-resistant maize transformation event "double antibody 12-5", the characteristic DNA sequence of which is inserted from the exogenous T-DNA insertion sequence SEQ ID NO.
  • the maize transformation event is the nucleotide sequence shown in SEQ ID NO. 14 as the left wing region of the foreign gene, and the nucleotide sequence shown in SEQ ID NO. 15 is the right side of the foreign gene.
  • Wing region the present invention provides an insect-resistant glyphosate-resistant maize transformation event "double antibody 12-15", the characteristic DNA sequence of which is derived from the exogenous T-DNA insertion sequence SEQ ID NO. 2, the insertion sequence of the left wing region of the maize genome The sequence SEQ ID NO. 14 and the insert sequence right wing region maize genomic sequence SEQ ID NO.
  • the maize transformation events "double antibody 12-5" and “double antibody 12-15" of the present invention are introduced into corn by agrobacterium infection method using an exogenous T-DNA sequence containing an insect resistance glyphosate gene expression cassette.
  • the maize transgenic population is obtained by regenerating corn cells containing the foreign gene, and molecular transformation and bioassay methods are used to screen for corn transformation events that can meet production needs.
  • the exogenous T-DNA provided by the present invention contains an insect resistance gene expression cassette and a glyphosate resistant gene expression cassette.
  • the insect resistance gene expression cassette provided by the present invention comprises a maize polyubiquitin-1 gene promoter (pZmUbi-1), an insect resistance fusion gene cry1Ab-cry2Aj and a maize PEP carboxylase gene (pepc) terminator.
  • the maize polyubiquitin-1 gene promoter (pZmUbi-1) is 2.1 kb in size and has a nucleotide sequence of SEQ ID NO. 7, which is a constitutive promoter that drives expression of the target gene in all tissues of maize.
  • the PEP carboxylase gene (pepc) terminator, derived from maize, is 0.2 kb in size and the nucleotide sequence is SEQ ID NO.
  • the insect resistance gene is a fusion gene of cry1Ab and cry2Aj, and the nucleotide sequence is SEQ ID NO. 4, wherein 1-1947 bp is the nucleotide sequence of cry1Ab; and the 1948-1965 bp nucleotide sequence is CCCGGGAAGGGTGGAGGA, encoding A linker peptide between Cry1Ab and Cry2Aj, the linker peptide sequence is PGKGGG; 1966-3861bp is the nucleotide sequence of the cry2Aj modified gene.
  • the fusion gene has a full length of 3861 bp and the encoded amino acid sequence is SEQ ID NO.
  • the encoded protein consists of 1287 amino acid residues with a protein molecular weight of 142.8 kDa.
  • Cry1 and Cry2 are two widely used insect-resistant genes. Among them, Cry1Ab, Cry1Ac, Cry1F and Cry2Ab have been widely used in corn and cotton.
  • Cry1Ab is a Bt crystal insecticidal protein with strong insecticidal ability. In particular, its insecticidal activity against corn borer is particularly high.
  • Cry2Aj has a relatively high insecticidal ability against the main crops of Lepidoptera pests. It is similar to the insecticidal spectrum of Cry2Ab, which is currently widely produced, and the amino acid sequences are similar. The safety and insect resistance of these genes have been well documented.
  • the invention uses The fusion gene of cry1Ab and cry2Aj is characterized by the simultaneous use of two different insect-resistant Bt genes, and they are expressed in the same amount in maize. Current research suggests that simultaneous expression of two different types of insect-resistant genes may slow the development of pest resistance (Zhao et al., 2003, Nat. Biotechnol. 21: 1493-1497), which is beneficial to the long-term effectiveness of insect-resistant transgenic maize. use.
  • the glyphosate-resistant expression cassette provided by the present invention is a composite promoter consisting of the 35S promoter of Cauliflower Mosaic Virus (CaMV) and the maize polyubiquitin-1 gene promoter (p35S-pZmUbi-1, nucleoside)
  • the acid sequence is SEQ ID NO. 9
  • the 5' end is ligated with a G10evo (EPSPS) encoding the AHAS gene chloroplast signal peptide (the nucleotide sequence is SEQ ID NO. 5, and the encoded amino acid sequence is SEQ ID NO. 10). It is composed of the 35S gene terminator (SEQ ID NO. 11) of CaMV.
  • the glyphosate resistant gene is a 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene.
  • EPSPS 5-enolpyruvylshikimate-3-phosphate synthase
  • Glyphosate is a broad-spectrum herbicide that kills plants by inhibiting the activity of 5-enolpyruvylshikimate-3-phosphate synthase in plants, causing plants to fail to synthesize aromatic amino acids.
  • the crop can obtain glyphosate-tolerant ability by transferring the EPSPS which is resistant to glyphosate, and selectively weed the crop while growing.
  • the invention also relates to a recombinant vector containing a maize transformation event comprising a T-DNA insertion sequence of the invention.
  • the vector map is as shown in Figure 1, and the vector sequence is SEQ ID NO.
  • the present invention provides a recombinant cell containing a maize transformation event, the recombinant cell comprising the recombinant vector of the present invention.
  • the recombinant cell is a recombinant Agrobacterium cell comprising the recombinant vector of the invention.
  • the invention provides a primer pair for detecting a transformation event "double antibody 12-5", the primer pair is identifiable by a first primer that specifically recognizes a T-DNA insertion sequence and any specific recognition of SEQ ID NO. 1 or SEQ ID NO
  • the first primer sequence is: SP1 or R1
  • the second primer sequence is: RB-Test or LB-test.
  • the present invention provides a method for identifying a maize transformation event "double antibody 12-5" comprising:
  • the present invention also provides a method for PCR identification of a maize transformation event "double antibody 12-5" specific for the maize transformation event "double antibody 12-5" specific PCR identification primers (SP1 and RB-Test are To detect whether the right side of the foreign gene is linked to the maize genome-specific site, and R1 and LB-test are to detect whether the left side of the foreign gene is linked to the maize genome-specific site):
  • SP1 5'-TTTCTCCATAATAATGTGTGAGTAGTTCCC-3' (SEQ ID NO. 17);
  • RB-Test 5'-CTCGTCATCGACCAAGTCATGAAG-3' (SEQ ID NO. 18).
  • R1 5'-CGTCGTTTTACAACGTCGTGACTGG-3' (SEQ ID NO. 19);
  • the PCR reaction system of the present invention is:
  • the PCR reaction conditions were: 32 cycles, each cycle being 95 ° C, 45 seconds; 65 ° C, 50 seconds; 72 ° C, 30 seconds.
  • the invention provides a primer pair for detecting a transformation event "double antibody 12-15", the primer pair is identifiable by a first primer that specifically recognizes a T-DNA insertion sequence and any specific recognition of SEQ ID NO. 14 or SEQ ID NO
  • the second primer of .15 is composed.
  • the first primer sequence is: LB-15T or RB-15T
  • the second primer sequence is: LB-15G or RB-15G.
  • the present invention provides a method for identifying a maize transformation event "double antibody 12-15" comprising:
  • the present invention also provides a method for PCR identification of a maize transformation event "double antibody 12-15" specific for the maize transformation event "double antibody 12-15” specific PCR identification primers (RB-15T and RB- 15G is to detect whether the right side of the foreign gene is linked to the maize genome-specific site, and LB-15T and LB-15G are to detect whether the left side of the foreign gene is linked to the maize genome-specific site):
  • LB-15T 5'-CTAAAACCAAAATCCAGTACTAAAATCC-3' (SEQ ID NO. 21);
  • LB-15G 5'-GCCGTACGTTTCCCAGCC-3' (SEQ ID NO. 22).
  • RB-15T 5'-AGCTTGAGCTTGGATCAGATTGTCGT-3' (SEQ ID NO. 23);
  • RB-15G 5'-CGTACAGGGAGCTTAGGGGG-3' (SEQ ID NO. 24);
  • the PCR reaction system of the present invention is:
  • the PCR reaction conditions were: 32 cycles, each cycle being 95 ° C, 45 seconds; 65 ° C, 50 seconds; 72 ° C, 30 seconds.
  • the present invention provides an application of the corn transformation event in preparing an insect-resistant glyphosate-resistant maize cell, and further hybridizing the corn material and the corn breeding material containing the corn transformation event, and further performing the backcrossing Insect resistant glyphosate resistant jade Rice cells.
  • the present invention provides a method for cultivating insect-resistant glyphosate-resistant corn using the insect-resistant glyphosate-resistant transformation event "double antibody 12-15", comprising: using corn material containing the transformation event "double antibody 12-15” and other corn breeding After the material is hybridized, backcrossing is further carried out to obtain a new material containing the conversion event "double antibody 12-15" of the present invention.
  • the plant cell containing the "double antibody 12-5" transformation event (ie SEQ ID NO. 12) of the present invention namely Zea mays L. double-resistant 12-5 seed, is deposited in the China Center for Type Culture Collection. Deposit No. CCTCC NO: P201506, date of deposit: April 27, 2015, deposit address: Wuhan University, Wuhan, China, 430072.
  • the plant cell containing the "double antibody 12-15" transformation event (ie SEQ ID NO. 16) of the present invention namely Zea mays L. double-resistant 12-15 seed, is deposited in the China Center for Type Culture Collection. Deposit No. CCTCC NO: P201607, date of deposit: April 11, 2016, deposit address: Wuhan University, Wuhan, China, 430072.
  • the beneficial effects of the present invention are mainly embodied in that the present invention provides excellent transformation events "double antibody 12-5" and "double antibody 12-15", and the transformation event can realize the foreign gene Specific introduction into maize lines, confers resistance to glyphosate resistance to recipient corn; insect-resistant glyphosate-resistant gene can be stably inherited in recipient maize; expression of insect-resistant glyphosate-resistant gene to receptor The agronomic traits of corn do not adversely affect.
  • Figure 2 Fluorescence development of Southern blot after double-resistance 12-5 glyphosate resistance gene; BamHI is a single digestion of BamHI genome; XbaI is a single digestion of XbaI genome; G10evo full-length DNA labeled with digoxigenin Fluorescence development after hybridization as a probe onto a nylon matrix membrane; the right side of the figure is the DNA length standard (bp) marker, and the arrow refers to the specific band produced by the hybridization.
  • BamHI is a single digestion of BamHI genome
  • XbaI is a single digestion of XbaI genome
  • the right side of the figure is the DNA length standard (bp) marker, and the arrow refers to the specific band produced by the hybridization.
  • Figure 3 Fluorescence development of Southern blotting of the double-resistant 12-5 insect-resistant gene; genomic DNA of KpnI as double antibody 12-5 was digested with KpnI; genomic DNA of SmaI was "double antibody 12-5" was digested with SmaI The digoxigen-labeled Cry1Ab full-length DNA was used as a probe, and hybridized to a nylon matrix membrane for fluorescence development.
  • the right side of the figure is the DNA length standard (bp).
  • Figure 4 Fluorescence development of Southern blotting of the 12-15 anti-insect gene; SacI is the single-digestion of the SacI gene, and the AclI double-antibody 12-15 genome is digested with AclI, and M is the DNA length standard (bp). ).
  • Figure 6 PCR validation electrophoresis map of the insertion site; 1 is the transgenic maize double-antibody 12-5 left wing PCR; 2 is the non-transgenic control left wing PCR; 3 is the transgenic maize right wing PCR; 4 is the non-transgenic control Right wing PCR; M is the DNA size standard (PCR product between 200-500 bp).
  • Figure 7 PCR validation electrophoresis map of the insertion site; maize "double antibody 12-15" left wing PCR; 1 and 2 are non-transgenic controls; lanes 3-6 are transgenic maize “double antibody 12-15”; DNA size standard (PCR product is around 1170 bp).
  • Figure 8 PCR validation electrophoresis map of the insertion site; maize “double antibody 12-15" right-wing PCR; 1 and 2 are non-transgenic controls; lanes 3-6 are transgenic maize “double antibody 12-15”; DNA size standard (PCR product is around 600 bp).
  • FIG. 9 Electrophoretic map of DNA genetic stability in the recipient maize; M is a DNA size standard sample (100 bp gradient); - is a negative control (non-transgenic corn); + is a positive control (added Non-transgenic corn samples of PCR-positive products verified by sequence determination; 1, 2, 3, 4, 5, 6, and 7 are receptor corn samples of 1, 2, 3, 4, 5, 6, and 7 generations, respectively.
  • the expected size of the PCR product is 145 bp, which is substantially consistent with the size of the electrophoresis analysis of the PCR product.
  • FIG. 10 Electrophoretic map of DNA genetic stability in "Recombinant 12-15" in recipient maize; M is a DNA size standard sample (100 bp gradient); - is a negative control (non-transgenic corn); + is a positive control ( Non-transgenic corn samples with PCR-positive products verified by sequence determination); 1, 2, 3, 4, 5, 6, and 7 are receptors for 1, 2, 3, 4, 5, 6, and 7 generations, respectively
  • the corn sample; the expected size of the PCR product is 1000 bp, which is basically consistent with the size of the electrophoresis analysis of the PCR product.
  • the vector map of the present invention for maize transformation is shown in Figure 1.
  • the nucleotide sequence of the transforming plasmid vector is SEQ ID NO. 13, and the names and positions of the specific vector constituent elements are shown in Table 1.
  • the nucleotide sequence of the T-DNA gene is shown in SEQ ID NO: 2.
  • SEQ ID NO: 2 comprises the complete anti-glyphosate expression cassette and the insect resistance expression cassette, specifically consisting of the following part, the glyphosate resistant expression cassette: the complex of the 35S promoter derived from CaMV and the maize Polyubiqutin-1 promoter Promoter (nucleotide sequence shown in SEQ ID NO.
  • EPSPS 5'-end glyphosate-resistant gene G10evo
  • the terminator is the 35S gene terminator of CaMV (0.2 kb in length and nucleotide sequence SEQ ID NO. 11).
  • Insect resistance expression cassette: Cry1Ab-PGKGGG-Cry2Aj fusion gene, the promoter driving the Cry1Ab-Cry2Aj fusion gene is derived from the maize polyubiquitin-1 gene promoter (pZmUbi-1) (obtained from the maize genome by PCR, the size is 2.1 kb, The nucleotide sequence is SEQ ID NO.
  • pZmUbi-1 can drive the expression of the target gene in all plant tissues
  • the terminator is a PEP carboxylase gene (pepc) terminator derived from maize (0.2 kb in size, nucleotides).
  • the sequence is SEQ ID NO. 8).
  • the obtained plant transformation plasmid was introduced into Agrobacterium LBA4404 by an electric shock method (2500 V) to obtain Agrobacterium containing a plant transformation vector.
  • Table 1 Name, location and function of the components of the corn transformation vector
  • the method used to obtain the maize transformation event in this study was Agrobacterium-mediated method, which was transformed according to the method reported by Frame et al. and the medium formulation (Plant Physiol, 2002, 129: 13-22), using glyphosate as a screening reagent. Specific steps are as follows:
  • step (2) Transfer the immature embryos cultured in step (1) to a callus induction medium containing a final concentration of 200 mg/L longtime antibiotic (GlaxoSmithKline, USA), dark culture at 28 ° C for 10-14 Killing Agrobacterium.
  • a callus induction medium containing a final concentration of 200 mg/L longtime antibiotic (GlaxoSmithKline, USA), dark culture at 28 ° C for 10-14 Killing Agrobacterium.
  • step (4) transfer the step (4) to the living embryonic tissue to the regeneration medium, and darkly culture at 28 ° C for 10-14 days, one strain per dish.
  • 240 insect-resistant glyphosate-resistant maize independent transformants were obtained by Agrobacterium-mediated (Example 1).
  • the primers were designed according to the vector sequence (SEQ ID NO. 13 and the glucan-containing gene G10eve (the nucleotide sequence is shown in SEQ ID NO. 5) and the insect-resistant gene cry1Ab-cry2Aj (the nucleotide sequence are SEQ ID NO.
  • T3 and T5 transgenic maize containing transformants containing the transformation event "double antibody 12-1"-transformation event "double antibody 12-15” were selected for glyphosate resistance comparison.
  • Transgenic maize and parental non-transgenic control seeds were germinated 20-30 days, growing to 4-5 leaf stage, spraying a final concentration of 0.4 wt% glyphosate (Nongda, Monsanto, USA), using 25 L/ Mu, 7 days later recorded corn growth and mortality and mortality.
  • the glyphosate spray test results are shown in Table 2.
  • the transgenic lines have obvious glyphosate resistance.
  • T1 and T5 transgenic maize containing transformants containing the transformation events "double antibody 12-1" to "double antibody 12-15" were selected for insect resistance analysis.
  • the final concentration of 0.4% by weight of glyphosate was determined after 20 days of germination.
  • 10 strains were taken, 10 Asian corn borers per plant, and Asian corn borer from China.
  • Corn Pest Group Institute of Plant Protection, Academy of Agricultural Sciences.
  • Eggs were incubated at 28 ⁇ 1°C, RH 70 ⁇ 5%, 16h:8h (L:D), and larvae for 12 hours incubation were selected for bioassay experiments.
  • the corn borer was investigated for damage and the insect resistance was graded.
  • the insect-resistant grading adopts the 9-level standard (Marcon et al., 1999): Grades 1-3: wormhole needle-like (Grade 1: rare, dispersed; Grade 2: medium amount; Grade 3: large amount). Grades 4-6: Size of wormhole matches (Level 4: rare, scattered; Level 5: medium quantity; Level 6: Large amount). Grades 7-9: The wormhole is larger than the match head (7: less scattered; 8: medium; 9: large). Classification of resistance levels: 1 to 2 (high resistance), 3 to 4 (insect resistance), 5 to 6 (infestation), 7 to 9 (high sense). The results are shown in Table 3. "Double antibody 12-1", “double antibody 12-5", “double antibody 12-9”, “double antibody 12-10", “double antibody 12-11", “double antibody” 12-13", “double antibody 12-14” and “double antibody 12-15” have high insect resistance.
  • T5 and T5 transgenic maize containing transformants containing the transformation events "double antibody 12-1" to "double antibody 12-15” were selected, respectively, in the middle of the transgenic maize and control corn (6-8 leaves fully developed)
  • Corn borer resistance assays were performed in the laboratory by selecting leaves that were not fully deployed. Two days after picking up corn mash, the area of feeding and the death of corn pupa were investigated. The results are shown in Table 4. The results show that most of the transgenic corn is well tolerated. Their leaves are eaten very little, especially the double-anti- 12-5, double-anti 12-9, double-anti 12-11, double-anti 12-14 and double-anti 12-15 insects.
  • Table 4 Insect resistance of Asian corn borer in the heart leaf stage of transgenic corn
  • the maize genomic DNA is extracted and digested with restriction endonucleases BamHI and XbaI (the restriction enzymes have a single recognition site in the foreign gene), and the digested fragments are separated by electrophoresis on agarose gel, and then The DNA was transferred to a nylon matrix membrane and hybridized to the nylon substrate membrane using the digoxigenin-labeled G10eve (nucleotide sequence SEQ ID NO. 5) full-length DNA as a probe, followed by fluorescence development.
  • the results showed that the transformation event "double antibody 12-5" obtained a signal band of about 14 kb when BamHI was digested, and a signal band of about 5.0 kb when digested with XbaI (Fig.
  • the glyphosate resistant gene is a single copy insert.
  • the transformation event "double antibody 12-15” obtained a signal band of about 2.5 kb when BamHI was digested, and a signal band of about 8.7 kb when digested with XbaI (Fig. 5), which proved "double antibody 12-15".
  • the glyphosate resistant gene is a single copy insert.
  • Southern blot analysis was performed on the copy number of the "double-antibody 12-5" insect-resistant gene using the Cry1Ab of the right anti-insect fusion gene in the T-DNA as a probe, and the restriction enzymes KpnI and SmaI were used to bind the double antibody 12-
  • the 5" genomic DNA was subjected to single digestion, and after separation on agarose gel, it was transferred to a nylon matrix membrane, and then the digoxigen-labeled Cry1Ab (1-1947 bp in nucleotide sequence SEQ ID NO. 4) was used as a probe.
  • the needle was hybridized to a nylon matrix membrane and then developed by fluorescence. The result shows that with KpnI A signal band of approximately 7 kb was obtained upon digestion, and a signal band of approximately 6.5 kb was obtained when SmaI was digested (Fig. 3).
  • the genomic DNA of the transformation event "double antibody 12-15” was digested with restriction endonucleases SacI and AclI, separated on agarose gel, transferred to a nylon matrix membrane, and then labeled with digoxin.
  • Cry1Ab (1-1947 bp in nucleotide sequence SEQ ID NO. 4) was fluorescently developed as a probe after hybridization onto a nylon substrate membrane. The results showed that a signal band of about 7.8 kb was obtained when SacI was digested, and a signal band of about 5.0 kb was obtained when digested with AclI (Fig. 4).
  • Table 5 Grain number per panicle, 100 grain weight and growth period of transgenic corn
  • the DNA fragment of the left wing region of the transformation event "double antibody 12-5" T-DNA was sequenced and aligned, and the obtained sequence was SEQ ID NO. 1, wherein the sequence between nucleotides 1-576 bp corresponds to corn.
  • Genomic DNA a sequence between 577 and 826 bp of nucleotides corresponds to exogenous DNA.
  • the DNA fragment of the right wing region of T-DNA was sequenced and aligned, and the obtained sequence was SEQ ID NO. 3, wherein the sequence of nucleotides 1-102 bp corresponds to exogenous DNA, and the nucleotides were 211-1007 bp.
  • the sequence corresponds to maize genomic DNA.
  • sequenced alignment and verified insertion site upstream and downstream flanking sequences and exogenous T-DNA sequences are integrated to form a specific nucleotide sequence of the transformation event "double antibody 12-5" according to the present invention, the sequence The number is SEQ ID NO. 12 (ie, spliced from SEQ ID NO. 1, SEQ ID NO. 2, and SEQ ID NO. 3).
  • the DNA fragment of the left wing region of the transformation event "double antibody 12-15" T-DNA was sequenced and aligned, and the obtained sequence was SEQ ID NO. 14, wherein the sequence between nucleotides 1-164 bp corresponds to corn.
  • Genomic DNA between nucleotides 1065-1172bp The sequence corresponds to the foreign DNA.
  • the DNA fragment of the right wing region of T-DNA was sequenced and aligned, and the obtained sequence was SEQ ID NO. 15, wherein the nucleotide 1-54 bp sequence corresponded to the foreign DNA, and the nucleotide was 55-604 bp.
  • the sequence corresponds to maize genomic DNA.
  • sequence number Is SEQ ID NO. 16 ie, spliced from SEQ ID NO. 14, SEQ ID NO. 2, and SEQ ID NO. 15.
  • a PCR primer (see Table 6) that can be used to specifically detect "double antibody 12-5" was designed based on the nucleotide sequence of the transformation event (SEQ ID NO. 12).
  • the PCR reaction system is: 10 ⁇ amplification buffer 5 ⁇ L, dNTP mixture 200 ⁇ mol/L, forward primer 10 pmol, reverse primer 10 pmol, genomic DNA 0.1-2 ⁇ g, Taq DNA polymerase 2.5 ⁇ L, MgCl 2 1.5 mmol/ L, add double distilled water to 50 ⁇ L.
  • the PCR conditions were: 32 cycles, each cycle being 95 ° C, 45 seconds; 65 ° C, 50 seconds; 72 ° C, 30 seconds.
  • the conditions of the PCR can be adjusted depending on the enzyme used and the reaction system.
  • the obtained PCR product was subjected to agarose electrophoresis analysis.
  • the specific PCR products at the left and right borders were 304 bp and 350 bp, respectively (Fig. 6).
  • the PCR product can also be further verified by sequencing the sequence if necessary.
  • a PCR primer (see Table 7) that can be used to specifically detect the double antibody 12-15 was designed based on the nucleotide sequence of the transformation event (SEQ ID NO. 16).
  • the PCR reaction system is: 10 ⁇ amplification buffer 5 ⁇ L, dNTP mixture 200 ⁇ mol/L, forward primer 10 pmol, reverse primer 10 pmol, genomic DNA 0.1-2 ⁇ g, Taq DNA polymerase 2.5 ⁇ L, MgCl 2 1.5 mmol/ L, add double distilled water to 50 ⁇ L.
  • the PCR conditions were: 32 cycles, each cycle being 95 ° C, 45 seconds; 65 ° C, 50 seconds; 72 ° C, 30 seconds.
  • the conditions of the PCR can be adjusted depending on the enzyme used and the reaction system.
  • the obtained PCR product was subjected to agarose electrophoresis analysis.
  • the specific PCR products at the left and right borders were 1171 bp (Fig. 7) and 604 bp, respectively (Fig. 8).
  • the PCR product can also be further verified by sequence determination if necessary.
  • primers were designed for insertion site-specific PCR detection, and 1-7 generations of receptor maize extraction genomes were taken and identified by PCR. T-DNA integration.
  • the primers were: BR-1 (5'GGCGAATGCTAGAGCAGCTTGAGCT-3') (SEQ ID NO. 25) and GN-1 (5' CCTACTGCGATGACGTTCGGTGCC-3') (SEQ ID NO. 26),
  • Reaction system 10 ⁇ L of amplification buffer 5 ⁇ L, dNTP mixture 200 ⁇ mol/L, BR-110 pmol, 10 pmol GN-1, genomic DNA 0.1-2 ⁇ g, Taq DNA polymerase 2.5 ⁇ L, MgCl 2 1.5 mmol/L, double distilled water Up to 50 ⁇ L.
  • the corn borer bioassay (Marcon et al., 1999) showed that the first, second, third, fourth, fifth, sixth, and seventh generations of transgenic corn had 100% insecticidal effects on the first-instar corn borer, and the insect resistance was stable. .
  • Spraying glyphosate tests showed that the first, second, third, fourth, fifth, sixth, and seventh generations of transgenic corn were resistant to 100 g of active glyphosate-containing glyphosate pesticide per acre.
  • the ability to resist glyphosate is stable and inherited.
  • the results demonstrate that the resistance to insects and glyphosate is stable and inherited.
  • primers were designed for insertion site-specific PCR detection, and 1-7 generations of receptor maize extraction genomes were taken and identified by PCR. T-DNA integration.
  • the primers were: LB-SP4: 5' CTAAAACCAAAATCCAGTACTAAAATCC (SEQ ID NO. 27) and LB-M: CTGTTCTGATGGTGGCAGGCAGG (SEQ ID NO. 28),
  • Reaction system 10 ⁇ L of amplification buffer 5 ⁇ L, dNTP mixture 200 ⁇ mol/L, BR-110 pmol, 10 pmol GN-1, genomic DNA 0.1-2 ⁇ g, Taq DNA polymerase 2.5 ⁇ L, MgCl 2 1.5 mmol/L, double distilled water Up to 50 ⁇ L.
  • the corn borer bioassay showed that the first, second, third, fourth, and fifth generations of transgenic corn had 100% insecticidal effects on the first-instar corn borer, and the insect resistance was stable and inherited.
  • Spraying glyphosate tests showed that the first, second, third, fourth, and fifth generations of transgenic corn were resistant to 100 g of active glyphosate-containing glyphosate pesticide per acre. Glyphosate resistance is stable and inherited. The results demonstrate that the resistance to insects and glyphosate is stable and inherited.
  • the maize containing the "double antibody 12-5" transformation event was used as the donor parent, and the maize inbred line B73 was used as the recipient parent for one hybridization, four backcrosses and three selfings. Glyphosate was used during the backcrossing to remove isolates that did not contain the "double antibody 12-5" transgenic complex.
  • a stable inbred line B735 containing the composite transgene structure of the present invention was obtained in the BC4F3 generation (selfed for 3 generations after 4 generations of backcrossing).
  • the DNA was extracted from the leaf tissue of the strain, and the exogenous insertion gene and its flanking DNA fragment were amplified.
  • the sequencing analysis confirmed that the sequence derived from the composite transgene structure of the donor parent was consistent, indicating the "double antibody 12-5" transformation event.
  • the maize containing the "double antibody 12-15" transformation event was used as the donor parent, and the maize inbred line MR-1 was used as the recipient parent for one cross, four backcrosses and three times of selfing. Glyphosate was used during the backcrossing to remove isolates that did not contain the "double-antibody 12-15" transgenic complex.
  • a stable inbred line M45 containing the composite transgene structure of the present invention was obtained in the BC4F3 generation (selfed for 3 generations after 4 generations of backcrossing). The DNA was extracted from the leaf tissue of the strain, and the exogenous insert gene and its flanking DNA fragment were amplified.
  • the sequence analysis confirmed that the sequence derived from the composite transgene structure of the donor parent was consistent, indicating the "double antibody 12-15" transformation event. Stable transfer to new receptor materials. M45 was used as the female parent, and RF1 was used as the male parent to obtain the hybrid M7R. PCR analysis and sequencing of the exogenous insertion gene and its flanking DNA of M7R showed that the M7R contained the transformation event "double antibody 12-15". Field resistance tests showed that M7R has good glyphosate resistance and insect resistance.

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Abstract

La présente invention concerne un évènement de transformation du maïs et un procédé d'identification de spécificité et leur utilisation, où l'évènement de transformation utilise les séquences nucléotidiques présentées dans SEQ ID no : 1 comme région côté gauche du gène exogène et les utilisations des séquences nucléotidiques présentées par SEQ ID no : 3 comme région côté droit du gène exogène, où les utilisations des séquences nucléotidiques présentées par SEQ ID no : 14 comme région côté gauche du gène exogène et l'utilisation des séquences nucléotidiques présentées par SEQ ID no : 15 comme région côté droit du gène exogène; l'événement de transformation du maïs peut introduire le gène exogène spécifiquement dans une souche de maïs et doter le maïs accepteur de capacités de résistance aux insectes et de résistance au glyphosate; le gène de résistance aux insectes et de résistance au glyphosate dans le maïs accepteur peut être hérité de manière stable; et l'expression du gène de résistance aux insectes et de résistance au glyphosate ne produirait pas d'impact défavorable sur les traits agronomiques du maïs accepteur, et la mise en œuvre du procédé d'identification peut fournir des marqueurs moléculaires pour l'utilisation de l'événement de transformation pour la sélection, accroissant de là l'efficacité du travail de sélection.
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111394500A (zh) * 2020-04-22 2020-07-10 中国农业科学院作物科学研究所 一种用于鉴定待测植物样品是否来源于SbSNAC1-382事件或其后代的方法
US11479790B2 (en) 2018-02-11 2022-10-25 China National Seed Group Corporation, Ltd. Insect-resistant herbicide-tolerant corn transformation event

Families Citing this family (15)

* Cited by examiner, † Cited by third party
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CN116103332A (zh) * 2022-09-07 2023-05-12 杭州瑞丰生物科技有限公司 转基因大豆事件cal16及其检测方法

Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102031266A (zh) * 2010-03-25 2011-04-27 浙江大学 抗虫融合基因、融合蛋白质及其应用
CN102146371A (zh) * 2011-01-17 2011-08-10 杭州瑞丰生物科技有限公司 高抗草甘膦特变基因及其改良方法和应用
CN102321640A (zh) * 2011-08-18 2012-01-18 杭州瑞丰生物科技有限公司 抗除草剂基因及其在转基因农作物中的应用
WO2013014241A1 (fr) * 2011-07-28 2013-01-31 Genective Événement de maïs tolérant au glyphosate vco-ø1981-5 et kit et procédé pour détecter celui-ci
CN103266132A (zh) * 2013-05-31 2013-08-28 中国农业科学院生物技术研究所 苏云金芽胞杆菌cry1Ah/cry1Ie双价基因表达载体及其应用
CN104946631A (zh) * 2015-05-22 2015-09-30 杭州瑞丰生物科技有限公司 玉米转化事件“双抗12-5”及其特异性鉴定方法
CN105002180A (zh) * 2015-07-16 2015-10-28 杭州瑞丰生物科技有限公司 一种增强外源基因表达的方法
CN105200067A (zh) * 2015-09-25 2015-12-30 浙江大学 水稻转化事件g6h1及其特异性pcr鉴定方法

Patent Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102031266A (zh) * 2010-03-25 2011-04-27 浙江大学 抗虫融合基因、融合蛋白质及其应用
CN102146371A (zh) * 2011-01-17 2011-08-10 杭州瑞丰生物科技有限公司 高抗草甘膦特变基因及其改良方法和应用
WO2013014241A1 (fr) * 2011-07-28 2013-01-31 Genective Événement de maïs tolérant au glyphosate vco-ø1981-5 et kit et procédé pour détecter celui-ci
CN102321640A (zh) * 2011-08-18 2012-01-18 杭州瑞丰生物科技有限公司 抗除草剂基因及其在转基因农作物中的应用
CN103266132A (zh) * 2013-05-31 2013-08-28 中国农业科学院生物技术研究所 苏云金芽胞杆菌cry1Ah/cry1Ie双价基因表达载体及其应用
CN104946631A (zh) * 2015-05-22 2015-09-30 杭州瑞丰生物科技有限公司 玉米转化事件“双抗12-5”及其特异性鉴定方法
CN105002180A (zh) * 2015-07-16 2015-10-28 杭州瑞丰生物科技有限公司 一种增强外源基因表达的方法
CN105200067A (zh) * 2015-09-25 2015-12-30 浙江大学 水稻转化事件g6h1及其特异性pcr鉴定方法

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
CHANG, XUE ET AL.: "Evaluation of Transgenic crylAb/cry2LAMBDA maize for its resistance to Ostrinia Furnacalis", JOURNAL OF PLANT PROTECTION, vol. 40, no. 4, 31 August 2013 (2013-08-31), pages 339 - 344 *
JIN, DAN: "Selection and Molecular Detection for the High Expression Strains from Transgenic Maize Events Carrying Fusion Bt Cry Toxin Gene and Cp4-EPSPS Glyphosate Resistance Gene", CHINA MASTER'S THESES FULL-TEXT DATABASE, vol. S1, 15 December 2013 (2013-12-15), pages D047 - 9 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11479790B2 (en) 2018-02-11 2022-10-25 China National Seed Group Corporation, Ltd. Insect-resistant herbicide-tolerant corn transformation event
CN111394500A (zh) * 2020-04-22 2020-07-10 中国农业科学院作物科学研究所 一种用于鉴定待测植物样品是否来源于SbSNAC1-382事件或其后代的方法
CN111394500B (zh) * 2020-04-22 2024-05-07 中国农业科学院作物科学研究所 一种用于鉴定待测植物样品是否来源于SbSNAC1-382事件或其后代的方法

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