WO2016188332A1 - Corn transformation event and specificity identification method and use thereof - Google Patents

Corn transformation event and specificity identification method and use thereof 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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Chinese (zh)
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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

Disclosed are a corn transformation event and a specificity identification method and use thereof, wherein the transformation event uses the nucleotide sequences shown by SEQ ID NO.1 as the left wing region of the exogenous gene and uses the nucleotide sequences shown by SEQ ID NO.3 as the right wing region of the exogenous gene, or uses the nucleotide sequences shown by SEQ ID NO.14 as the left wing region of the exogenous gene and use the nucleotide sequences shown by SEQ ID NO.15 as the right wing region of the exogenous gene; the corn transformation event can introduce the exogenous gene specifically into a corn strain and endow the acceptor corn with insect-resistant and glyphosate-resistant capabilities; the insect-resistant and glyphosate-resistant gene in the acceptor corn can be stably inherited; and the expression of the insect-resistant and glyphosate-resistant gene would not produce an adverse impact on the agronomic traits of the acceptor corn, and implementing the identification method can provide molecular markers for using the transformation event to breed, thereby increasing the efficiency of the breeding work.

Description

一种玉米转化事件及其特异性鉴定方法和应用Maize transformation event and its specific identification method and application (一)技术领域(1) Technical field
本发明属于植物分子生物学领域，特别是转基因农作物品种培育领域。本发明涉及具有抗虫抗草甘膦玉米转化事件“双抗12-5”和“双抗12-15”及其特异性检测方法。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.
(二)背景技术(2) Background technology
玉米(Zea mays)是一种重要的作物，是世界上很多地区的主要食物来源。随着植物基因工程的发展，通过导入外源基因来进行遗传改造成为遗传育种的主要手段之一。在玉米生产中，抗虫和抗除草剂都是非常需要的农艺性状。抗虫转基因玉米能够大幅度地降低化学杀虫剂的使用量，从而降低生产成本并且减少农药对环境和农作物产品的污染。抗除草剂玉米可以大幅度降低防治杂草所需要的农业劳动力，降低劳动力的投入，减少杂草对玉米产量的影响。此外，利用除草剂防治杂草也更加有利于推广免耕技术，减少土壤和肥料的流失。因此抗虫和抗除草剂性能在玉米生产中具有突出的重要性。Zea mays is an important crop and a major source of food in many parts of the world. With the development of plant genetic engineering, genetic transformation through the introduction of foreign genes has become one of the main means of genetic breeding. In corn production, insect and herbicide resistance are highly desirable agronomic traits. 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. In addition, 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.
转化事件是由外源基因在基因组插入位点的上下游侧翼区和外源基因构成的分子结构。通常，外源基因转化植物可以得到一个转化体群体，这个转化体群体包含大量独立的事件，其中每个事件都是独特的。外源基因在植物中的表达受到外源基因所插入的染色体位置的影响。这可能源自染色质结构或者整合位点附近转录调控元件的影响。相同基因在不同转化事件中的表达水平具有很大的差异，在表达的空间或时间模式上也可能存在差异。而且外源基因的插入也可能会影响内源基因的表达。因此，每个独立转化事件对受体植物的影响都是不同的。获得能有效表达外源基因，同时不影响植株本身农艺性状的植物转化事件在培育转基因作物新品种中具有重要的应用价值。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. Typically, 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. Moreover, 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.
目前并不清楚外源基因在植物中的插入位点整合规律，因此研发过程中需要产生大量转化体并从中筛选理想的转化事件。通常需要产生数百乃至数千的不同转化事件，并在这些转化事件中筛选出具有预期转基因表达水平和模式的单一转化事件。利用育种方法、体细胞原生质融合技术可以将该转化事件导入到不同遗传背景的玉米基因组中，从而赋予玉米抗虫抗草甘膦的能力。It is not clear at this time that the insertion site of the foreign gene in the plant is integrated, so a large number of transformants need to be generated during the development process and the ideal transformation event can be screened therefrom. It is often desirable to generate hundreds or even thousands of different transformation events and to screen for single transformation events with expected transgene expression levels and patterns in these transformation events. The 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.
通过转化事件专利对转基因农作物进行知识产权保护是目前国际上通用方法，能够进入商业化的转化事件是从大量转化事件中筛选鉴定获得，外源基因与受体作物基因组特异位点整合，具有独特的分子特征，同时外源基因表达规律与生产实际需要相吻合，在基因分子特征和表达规律上都与一般转化事件不同，因此具有明显的新颖性、实用性和创造性。目前，国际上商业化的转化事件多获得知识产权保护，部分转化事件在中国申请并获得专利保护。Intellectual property protection of genetically modified crops through conversion event patents is currently a common method in the world. The transformation events that can enter commercialization are screened and identified from a large number of transformation events. The foreign genes are integrated with the specific sites of the recipient crop genome, which is unique. The molecular characteristics, while the expression pattern of exogenous genes are consistent with the actual needs of production, and the molecular characteristics and expression patterns of genes are different from the general transformation events, so they have obvious novelty, practicability and creativity. At present, the international commercialization of conversion events has been protected by intellectual property rights, and some conversion events have been applied for and protected by patents in China.
(三)发明内容(3) Invention content
本发明目的是提供优良的玉米抗虫抗草甘膦转化事件“双抗12-5”和“双抗12-15”，这两个独立转化事件所含外源基因的设计构思相同，是同个植物转化载体侵染玉米后，通过筛选获得的优良转化事件，这两个转化事件都可实现将外源基因引入到玉米品系中，赋予受体玉米具有抗虫抗草甘膦的能力；抗虫抗草甘膦基因外源基因在受体玉米中能够稳定遗传；抗虫抗草甘膦基因的 表达对受体玉米的农艺性状不会产生不良影响。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. After the plant transformation vector infects the maize, through the screening of the excellent transformation events, 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.
本发明提供一种玉米转化事件，所述转化事件以SEQ ID NO.1所示的核苷酸序列为外源基因的左侧翼区，以SEQ ID NO.3所示的核苷酸序列为外源基因的右侧翼区或者以SEQ ID NO.14所示的核苷酸序列为外源基因的左侧翼区，以SEQ ID NO.15所示的核苷酸序列为外源基因的右侧翼区。所述外源基因包括抗虫基因和抗草甘膦基因，所述抗虫基因的核苷酸序列为SEQ ID NO.4所示，所述抗草甘膦基因的核苷酸序列为SEQ ID NO.5所示。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.
进一步，优选所述外源基因的核苷酸序列为SEQ ID NO.2所示。Further, it is preferred that the nucleotide sequence of the foreign gene is represented by SEQ ID NO.
进一步，优选所述玉米转化事件以SEQ ID NO.1所示的核苷酸序列为外源基因的左侧翼区，以SEQ ID NO.3所示的核苷酸序列为外源基因的右侧翼区，即本发明提供抗虫抗草甘膦玉米转化事件“双抗12-5”，其特征DNA序列由外源T-DNA插入序列SEQ ID NO.2、插入序列左侧翼区玉米基因组序列SEQ ID NO.1和插入序列右侧翼区玉米基因组序列SEQ ID NO.3构成。Further, it is preferred that 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 genomic sequence SEQ ID NO. 1 and the insert sequence right wing region maize genomic sequence SEQ ID NO.
进一步，所述玉米转化事件以SEQ ID NO.14所示的核苷酸序列为外源基因的左侧翼区，以SEQ ID NO.15所示的核苷酸序列为外源基因的右侧翼区，即本发明提供抗虫抗草甘膦玉米转化事件“双抗12-15”，其特征DNA序列由外源T-DNA插入序列SEQ ID NO.2、插入序列左侧翼区玉米基因组序列SEQ ID NO.14和插入序列右侧翼区玉米基因组序列SEQ ID NO.15构成。Further, 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.
本发明所述玉米转化事件“双抗12-5”和“双抗12-15”是利用农杆菌侵染法，将含有抗虫抗草甘膦基因表达框的外源T-DNA序列导入玉米细胞中，通过再生含有外源基因的玉米细胞获得玉米转基因群体，并利用分子生物学和生物测定的方法筛选得到能够满足生产需要的玉米转化事件。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. In the cells, 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.
本发明提供的外源T-DNA含有抗虫基因表达框和抗草甘膦基因表达框。The exogenous T-DNA provided by the present invention contains an insect resistance gene expression cassette and a glyphosate resistant gene expression cassette.
本发明提供的抗虫基因表达框由玉米polyubiquitin-1基因启动子(pZmUbi-1)、抗虫融合基因cry1Ab-cry2Aj和玉米PEP carboxylase基因(pepc)终止子构成。其中玉米polyubiquitin-1基因启动子(pZmUbi-1)大小为2.1kb，核苷酸序列为SEQ ID NO.7，是组成型启动子，可驱动目的基因在玉米所有组织中表达。PEP carboxylase基因(pepc)终止子，来源于玉米，大小为0.2kb，核苷酸序列为SEQ ID NO.8。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.
进一步，所述抗虫基因是cry1Ab和cry2Aj的融合基因，核苷酸序列为SEQ ID NO.4，其中，1-1947bp为cry1Ab的核苷酸序列；1948-1965bp核苷酸序列为CCCGGGAAGGGTGGAGGA，编码Cry1Ab和Cry2Aj之间的连接肽，连接肽序列为PGKGGG；1966-3861bp为cry2Aj改良基因的核苷酸序列。融合基因全长为3861bp，编码的氨基酸序列为SEQ ID NO.6。所编码的蛋白质由1287个氨基酸残基组成，蛋白分子量大小为142.8kDa。Cry1和Cry2是两类被广泛应用的抗虫基因，其中Cry1Ab，Cry1Ac，Cry1F和Cry2Ab等在玉米、棉花中已经大规模推广应用，Cry1Ab是一种杀虫能力比较强的Bt晶体杀虫蛋白质，特别是其对玉米螟的杀虫活性尤为高。Cry2Aj对主要农作物鳞翅目害虫具有比较高的杀虫能力，与目前生产大量推广的Cry2Ab的杀虫谱比较接近，氨基酸序列也比较相似。这些基因的安全性和抗虫能力已经得到充分证明。本发明使用了 cry1Ab和cry2Aj的融合基因，其特点是同时利用两种不同的抗虫Bt基因，并且它们在玉米中表达量相同。目前的研究认为，二个不同类型抗虫基因同时表达有可能减缓害虫抗性的发生(Zhao et al.,2003,Nat.Biotechnol.21:1493-1497)，有利于抗虫转基因玉米的长期有效利用。Further, 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.
本发明提供的抗草甘膦表达框是由花椰菜花斑病病毒(Cauliflower Mosaic Virus,CaMV)的35S启动子和玉米polyubiquitin-1基因启动子组成的复合启动子(p35S-pZmUbi-1，核苷酸序列为SEQ ID NO.9)，5’端连有一段编码AHAS基因叶绿体信号肽的G10evo(EPSPS)(核苷酸序列为SEQ ID NO.5，编码的氨基酸序列为SEQ ID NO.10)和CaMV的35S基因终止子(SEQ ID NO.11)构成。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), and 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.
进一步，所述抗草甘膦基因是5-烯醇丙酮莽草酸-3-磷酸合成酶(EPSPS)基因。草甘膦是一种广谱灭生性除草剂，它通过抑制植物中5-烯醇丙酮莽草酸-3-磷酸合成酶的活性，导致植物不能合成芳香族氨基酸而死亡。为了方便杂草控制，通过在作物转入对草甘膦具有抗性的EPSPS可以使作物获得抗草甘膦的能力，实现在农作物生长的同时选择性地除草。Further, the glyphosate resistant gene is a 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene. 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. In order to facilitate weed control, the crop can obtain glyphosate-tolerant ability by transferring the EPSPS which is resistant to glyphosate, and selectively weed the crop while growing.
本发明还涉及一种含玉米转化事件的重组载体，其含有本发明所述T-DNA插入序列。在一个实施方案中，所述载体图谱如附图1所示，载体序列为SEQ ID NO.13。The invention also relates to a recombinant vector containing a maize transformation event comprising a T-DNA insertion sequence of the invention. In one embodiment, the vector map is as shown in Figure 1, and the vector sequence is SEQ ID NO.
本发明提供一种含玉米转化事件的重组细胞，重组细胞中含有本发明所述的重组载体。在一个实施方案中，所述重组细胞为含有本发明所述重组载体的重组农杆菌细胞。The present invention provides a recombinant cell containing a maize transformation event, the recombinant cell comprising the recombinant vector of the present invention. In one embodiment, the recombinant cell is a recombinant Agrobacterium cell comprising the recombinant vector of the invention.
本发明提供用于检测转化事件“双抗12-5”的引物对，所述引物对由特异性识别T-DNA插入序列的第一引物和任意特异性识别SEQ ID NO.1或SEQ ID NO.3的第二引物组成。在一些实施方案中，所述第一引物序列为：SP1或R1，所述第二引物序列为：RB-Test或LB-test。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 second primer composition of .3. In some embodiments, the first primer sequence is: SP1 or R1, and the second primer sequence is: RB-Test or LB-test.
本发明提供一种鉴定玉米转化事件“双抗12-5”的方法，其包括：The present invention provides a method for identifying a maize transformation event "double antibody 12-5" comprising:
1)从待鉴定的玉米样品提取基因组DNA；1) extracting genomic DNA from the corn sample to be identified;
2)以提取的DNA样品为模板，使用本发明提供的引物对进行PCR扩增；2) using the extracted DNA sample as a template, using the primer pair provided by the present invention for PCR amplification;
3)检测PCR扩增产物，如果扩增产物长度与转化事件上所述PCR引物对的序列之间的理论长度一致，则说明所述样品中含有“双抗12-5”。3) Detecting the PCR amplification product, if the length of the amplification product is consistent with the theoretical length between the sequences of the PCR primer pair on the transformation event, it indicates that the sample contains "double antibody 12-5".
本发明还提供一种玉米转化事件“双抗12-5”特异性的PCR鉴定方法，所述用于玉米转化事件“双抗12-5”特异性PCR鉴定的引物(SP1和RB-Test是检测外源基因右侧是否与玉米基因组特异性位点连接，R1和LB-test是检测外源基因左侧是否与玉米基因组特异性位点连接)为：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)；SP1: 5'-TTTCTCCATAATAATGTGTGAGTAGTTCCC-3' (SEQ ID NO. 17);
RB-Test：5’-CTCGTCATCGACCAAGTCATGAAG-3’(SEQ ID NO.18)。RB-Test: 5'-CTCGTCATCGACCAAGTCATGAAG-3' (SEQ ID NO. 18).
R1：5’-CGTCGTTTTACAACGTCGTGACTGG-3’(SEQ ID NO.19)；R1: 5'-CGTCGTTTTACAACGTCGTGACTGG-3' (SEQ ID NO. 19);
LB-test：5’-AAGACGTCCGGGGGAACCGTTGTTC-3’(SEQ ID NO.20)；LB-test: 5'-AAGACGTCCGGGGGAACCGTTGTTC-3' (SEQ ID NO. 20);
本发明所述PCR反应体系为：The PCR reaction system of the present invention is:
Figure PCTCN2016082025-appb-000001
Figure PCTCN2016082025-appb-000001
Figure PCTCN2016082025-appb-000002
Figure PCTCN2016082025-appb-000002
PCR反应条件为：32个循环，每个循环是95℃，45秒；65℃，50秒；72℃，30秒。The PCR reaction conditions were: 32 cycles, each cycle being 95 ° C, 45 seconds; 65 ° C, 50 seconds; 72 ° C, 30 seconds.
本发明提供用于检测转化事件“双抗12-15”的引物对，所述引物对由特异性识别T-DNA插入序列的第一引物和任意特异性识别SEQ ID NO.14或SEQ ID NO.15的第二引物组成。在一些实施方案中，所述第一引物序列为：LB-15T或RB-15T，所述第二引物序列为：LB-15G或RB-15G。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. In some embodiments, the first primer sequence is: LB-15T or RB-15T, and the second primer sequence is: LB-15G or RB-15G.
本发明提供一种鉴定玉米转化事件“双抗12-15”的方法，其包括：The present invention provides a method for identifying a maize transformation event "double antibody 12-15" comprising:
4)从待鉴定的玉米样品提取基因组DNA；4) extracting genomic DNA from the corn sample to be identified;
5)以提取的DNA样品为模板，使用本发明提供的引物对进行PCR扩增；5) using the extracted DNA sample as a template, using the primer pair provided by the present invention for PCR amplification;
6)检测PCR扩增产物，如果扩增产物长度与转化事件上所述PCR引物对的序列之间的理论长度一致，则说明所述样品中含有“双抗12-15”。6) Detecting the PCR amplification product, if the length of the amplification product is consistent with the theoretical length between the sequences of the PCR primer pair on the transformation event, the sample contains "double antibody 12-15".
本发明还提供一种玉米转化事件“双抗12-15”特异性的PCR鉴定方法，所述用于玉米转化事件“双抗12-15”特异性PCR鉴定的引物(RB-15T和RB-15G是检测外源基因右侧是否与玉米基因组特异性位点连接，LB-15T和LB-15G是检测外源基因左侧是否与玉米基因组特异性位点连接)为：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-15T: 5'-CTAAAACCAAAATCCAGTACTAAAATCC-3' (SEQ ID NO. 21);
LB-15G：5’-GCCGTACGTTTCCCAGCC-3’(SEQ ID NO.22)。LB-15G: 5'-GCCGTACGTTTCCCAGCC-3' (SEQ ID NO. 22).
RB-15T：5’-AGCTTGAGCTTGGATCAGATTGTCGT-3’(SEQ ID NO.23)；RB-15T: 5'-AGCTTGAGCTTGGATCAGATTGTCGT-3' (SEQ ID NO. 23);
RB-15G：5’-CGTACAGGGAGCTTAGGGGG-3’(SEQ ID NO.24)；RB-15G: 5'-CGTACAGGGAGCTTAGGGGG-3' (SEQ ID NO. 24);
本发明所述PCR反应体系为：The PCR reaction system of the present invention is:
Figure PCTCN2016082025-appb-000003
Figure PCTCN2016082025-appb-000003
PCR反应条件为：32个循环，每个循环是95℃，45秒；65℃，50秒；72℃，30秒。The PCR reaction conditions were: 32 cycles, each cycle being 95 ° C, 45 seconds; 65 ° C, 50 seconds; 72 ° C, 30 seconds.
本发明提供一种所述玉米转化事件在制备抗虫抗草甘膦玉米细胞中的应用，利用含有所述玉米转化事件的玉米材料与玉米育种材料进行杂交后，进一步进行回交，获得所述抗虫抗草甘膦玉 米细胞。具体利用抗虫抗草甘膦转化事件“双抗12-5”培育抗虫抗草甘膦玉米的方法，包括：利用含有转化事件“双抗12-5”的玉米材料与其它玉米育种材料进行杂交后，进一步进行回交，获得含有本发明所述转化事件“双抗12-5”的新材料。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. A method for cultivating insect-resistant glyphosate-resistant corn by using the insect-resistant glyphosate-resistant transformation event "double-antibiotic 12-5", comprising: using corn material containing the transformation event "double antibody 12-5" and other corn breeding materials After hybridization, backcrossing is further carried out to obtain a new material containing the transformation event "double antibody 12-5" of the present invention.
本发明提供利用抗虫抗草甘膦转化事件“双抗12-15”培育抗虫抗草甘膦玉米的方法，包括：利用含有转化事件“双抗12-15”的玉米材料与其它玉米育种材料进行杂交后，进一步进行回交，获得含有本发明所述转化事件“双抗12-15”的新材料。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.
本发明所述含“双抗12-5”转化事件(即SEQ ID NO.12)的植物细胞，即玉蜀黍属玉米Zea mays L.双抗12-5种子，保藏于中国典型培养物保藏中心，保藏号CCTCC NO：P201506，保藏日期：2015年4月27日，保藏地址：中国武汉武汉大学，邮编430072。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.
本发明所述含“双抗12-15”转化事件(即SEQ ID NO.16)的植物细胞，即玉蜀黍属玉米Zea mays L.双抗12-15种子，保藏于中国典型培养物保藏中心，保藏号CCTCC NO:P201607，保藏日期：2016年4月11日，保藏地址：中国武汉武汉大学，邮编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.
与现有技术相比，本发明的有益效果主要体现在：本发明提供了优良的转化事件“双抗12-5”和“双抗12-15”，所述转化事件可实现将外源基因特异性引入到玉米品系中，赋予受体玉米具有抗虫抗草甘膦的能力；抗虫抗草甘膦基因在受体玉米中能够稳定遗传；抗虫抗草甘膦基因的表达对受体玉米的农艺性状不会产生不良影响。Compared with the prior art, 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.
(四)附图说明(4) Description of the drawings
图1、实施例1含目的基因的质粒结构图，LB和RB：T-DNA的边界序列；pZmUbi-1：玉米polyubiqutin-1启动子；p35S：35S启动子(CaMV病毒)；G10evo：抗草甘膦EPSPS基因；Cry1Ab-Cry2Aj：抗虫Bt融合基因cry1Ab/cry2Aj。Figure 1. Example 1 plasmid structure map containing the gene of interest, LB and RB: border sequence of T-DNA; pZmUbi-1: maize polyubiqutin-1 promoter; p35S: 35S promoter (CaMV virus); G10evo: anti-grass Glyphosate EPSPS gene; Cry1Ab-Cry2Aj: insect-resistant Bt fusion gene cry1Ab/cry2Aj.
图2、双抗12-5抗草甘膦基因的Southern杂交后的荧光显影图；BamHI为基因组经BamHI单酶切；XbaI为基因组经XbaI单酶切；以地高辛标记的G10evo全长DNA作为探针杂交到尼龙基质膜上以后荧光显影；图右侧为DNA长度标准(bp)标记，箭头指杂交产生的特异性条带。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.
图3、双抗12-5抗虫基因的Southern杂交后荧光显影图；KpnI为双抗12-5的基因组DNA经过KpnI酶切；SmaI为“双抗12-5”的基因组DNA经过SmaI酶切，地高辛标记的Cry1Ab全长DNA作为探针，杂交到尼龙基质膜上以后荧光显影，图右侧为DNA长度标准(bp)。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).
图4、双抗12-15抗虫基因的Southern杂交后的荧光显影图；SacI为基因组经SacI单酶切,AclI位双抗12-15基因组经AclI单酶切，M为DNA长度标准(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). ).
图5、双抗12-15抗草甘膦基因的Southern杂交后的荧光显影图；BamHI为双抗12-15基因组经BamHI单酶切；XbaI为双抗12-15基因组经XbaI单酶切；以地高辛标记的G10evo全长DNA作为探针杂交到尼龙基质膜上以后荧光显影；M为DNA长度标准(bp)标记。Figure 5. Fluorescence development of Southern blot after double-anti 12-15 glyphosate resistance gene; BamHI is double-antibody 12-15 genome by BamHI single digestion; XbaI is double antibody 12-15 genome by XbaI single digestion; The digoxigenin-labeled G10evo full-length DNA was hybridized to a nylon matrix membrane as a probe for fluorescence development; M was a DNA length standard (bp) marker.
图6、插入位点的PCR验证电泳图；1为转基因玉米双抗12-5左侧翼PCR；2为非转基因对照左侧翼PCR；3为转基因玉米右侧翼PCR；4为非转基因对照右侧翼PCR；M为DNA大小标准(PCR产物在200-500bp之间)。 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).
图7、插入位点的PCR验证电泳图；玉米“双抗12-15”左侧翼PCR；1和2为非转基因对照；泳道3-6为转基因玉米“双抗12-15”；M为DNA大小标准(PCR产物在1170bp左右)。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).
图8、插入位点的PCR验证电泳图；玉米“双抗12-15”右侧翼PCR；1和2为非转基因对照；泳道3-6为转基因玉米“双抗12-15”；M为DNA大小标准(PCR产物在600bp左右)。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).
图9、双抗12-5在受体玉米中的DNA遗传稳定性检测电泳图；M为DNA大小标准样品(100bp梯度)；-为阴性对照(非转基因玉米)；+为阳性对照(加入了经过序列测定验证的PCR阳性产物的非转基因玉米样品)；1，2，3，4，5，6，7分别是第1，2，3，4，5，6，7代的受体玉米样品；PCR产物的预期大小是145bp，与PCR产物电泳分析的大小基本一致。Figure 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.
图10、“双抗12-15”在受体玉米中的DNA遗传稳定性检测电泳图；M为DNA大小标准样品(100bp梯度)；-为阴性对照(非转基因玉米)；+为阳性对照(加入了经过序列测定验证的PCR阳性产物的非转基因玉米样品)；1，2，3，4，5，6，7分别是第1，2，3，4，5，6，7代的受体玉米样品；PCR产物的预期大小是1000bp，与PCR产物电泳分析的大小基本一致。Figure 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.
(五)具体实施方式(5) Specific implementation methods
下面结合具体实施例对本发明进行进一步描述，但本发明的保护范围并不仅限于此：The present invention will be further described below in conjunction with specific embodiments, but the scope of protection of the present invention is not limited thereto:
实施例1：转化事件的获得Example 1: Acquisition of a conversion event
(1)含有外源基因的质粒载体的获得(1) Acquisition of a plasmid vector containing a foreign gene
本发明用于玉米转化的载体图谱如图1所示，转化质粒载体核苷酸序列为SEQ ID NO.13，具体载体组成元件的名称、位置如表1所示。其中T-DNA基因的核苷酸序列为SEQ ID NO:2所示。SEQ ID NO:2包含完整的抗草甘膦表达框和抗虫表达框，具体由如下部分组成，抗草甘膦表达框：来源于CaMV的35S启动子与玉米Polyubiqutin-1启动子形成的复合启动子(核苷酸序列为SEQ ID NO.9所示)，该复合启动子驱动5’端连有一段编码AHAS基因叶绿体信号肽的抗草甘膦基因G10evo(EPSPS)(核苷酸序列为SEQ ID NO.5所示)，终止子是CaMV的35S基因终止子(长度为0.2kb，其核苷酸序列为SEQ ID NO.11)。抗虫表达框：Cry1Ab-PGKGGG-Cry2Aj融合基因，驱动Cry1Ab-Cry2Aj融合基因的启动子为来源于玉米polyubiquitin-1基因启动子(pZmUbi-1)(通过PCR从玉米基因组获得，大小为2.1kb，核苷酸序列为SEQ ID NO.7，pZmUbi-1可驱动目的基因在所有植物组织中表达)，终止子为来源于玉米的PEP carboxylase基因(pepc)终止子(大小为0.2kb，核苷酸序列为SEQ ID NO.8)。利用电击法(2500V)将获得的植物转化质粒导入农杆菌LBA4404中，获得含有植物转化载体的农杆菌。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. 9), which drives a 5'-end glyphosate-resistant gene G10evo (EPSPS) encoding the AHAS chloroplast signal peptide (the nucleotide sequence is SEQ ID NO. 5), 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. 7, pZmUbi-1 can drive the expression of the target gene in all plant tissues, and 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.
表1玉米转化载体组成元件的名称、位置和功能Table 1 Name, location and function of the components of the corn transformation vector
Figure PCTCN2016082025-appb-000004
Figure PCTCN2016082025-appb-000004
Figure PCTCN2016082025-appb-000005
Figure PCTCN2016082025-appb-000005
(2)玉米遗传转化(2) Genetic transformation of maize
本研究中获得玉米转化事件所用的方法是农杆菌介导方法，根据Frame等报道的方法和培养基配方(Plant Physiol,2002,129:13-22)进行转化，使用草甘膦为筛选试剂，具体步骤如下：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:
(1)取授粉后8～10天的亲本玉米穗，收集大小为1.0-1.5mm未成熟胚。将含有转化载体的农杆菌与未成熟胚在22℃共培养2-3天。(1) Parent corn ears 8 to 10 days after pollination, and 1.0-1.5 mm immature embryos were collected. Agrobacterium containing the transformation vector was co-cultured with immature embryos at 22 ° C for 2-3 days.
(2)将步骤(1)培养后的未成熟胚转移到含有终浓度200mg/L特美汀抗生素(葛兰素史克，美国)的愈伤组织诱导培养基上，28℃暗培养10-14天杀灭农杆菌。(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.
(3)将步骤(2)诱导培养后的所有愈伤组织转到含有终浓度2mM草甘膦的筛选培养基上，28℃暗培养2-3周。(3) All the calli obtained after the induction of the step (2) were transferred to a screening medium containing a final concentration of 2 mM glyphosate, and cultured at 28 ° C for 2-3 weeks.
(4)转移步骤(3)所有的愈伤组织到新鲜的含有(2mM)草甘膦的筛选培养基上，28℃暗培养2-3周。(4) Transfer step (3) All calli tissues were cultured on fresh screening medium containing (2 mM) glyphosate at 28 ° C for 2-3 weeks.
(5)然后，转移步骤(4)成活的胚性组织到再生培养基上，28℃暗培养10-14天，每皿一个株系。(5) Then, 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.
(6)转移步骤(5)胚性组织到新鲜的再生培养基上，26℃光照培养10-14天。(6) Transfer step (5) Embryogenic tissue onto fresh regeneration medium, and cultured at 26 ° C for 10-14 days.
(7)转移步骤(6)所有发育完全的植株到生根培养基上，26℃光照培养直到根发育完全，将生根后的再生苗移植到温室中生长繁种，用于筛选分析。(7) Transfer step (6) All fully-developed plants were cultured on rooting medium, and cultured at 26 ° C until the roots were fully developed. The rooted regenerated shoots were transplanted into the greenhouse for growth and breeding for screening analysis.
实施例2：转化事件的筛选Example 2: Screening of transformation events
通过农杆菌介导获得了240个抗虫抗草甘膦玉米独立转化体(实施例1)。根据载体序列(SEQ ID NO.13设计引物，用PCR方法筛选含有抗草甘膦基因G10eve(核苷酸序列为SEQ ID NO.5所示)和抗虫基因cry1Ab-cry2Aj(核苷酸序列为SEQ ID NO.4所示)，同时不含载体骨架序列的玉米转化体，并在温室中进行抗虫抗草甘膦性能的初步测试：通过喷施0.4wt％的草甘膦，去除草甘膦抗性差的转化体，在剩余的转化体上接玉米螟，筛选没有玉米螟为害的转化体，共计获得15 个含转化事件编号为“双抗12-1”～“双抗12-15”的具有较强抗虫抗草甘膦能力的玉米转化体。分别将含双抗独立转化事件的转化体与玉米自交系B73杂交留种，进行随后的筛选分析。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. 4), a maize transformant containing no vector backbone sequence, and preliminary testing of insect-resistant glyphosate resistance in a greenhouse: removal of glyphosate by spraying 0.4 wt% of glyphosate A transformant with poor phosphine resistance, the corn borer was attached to the remaining transformants, and the transformants without the corn borer were screened for a total of 15 A maize transformant with a strong resistance to insect glyphosate resistant to transformation events numbered "double antibody 12-1" to "double antibody 12-15". The transformants containing the double-independent independent transformation event were separately crossed with the maize inbred line B73 for subsequent screening analysis.
(1)草甘膦抗性筛选(1) Glyphosate resistance screening
选择15个含转化事件“双抗12-1”-转化事件“双抗12-15”的转化体的T3和T5代转基因玉米进行草甘膦抗性对比。转基因玉米和亲本非转基因对照种子发芽后20-30天，长至4-5叶期，喷施终浓度为0.4wt％的草甘膦(农达，孟山都公司，美国)，使用量为25L/亩，7天后记录玉米生长发育情况和死亡率。草甘膦喷施试验结果如表2所示，转基因品系都具有明显的抗草甘膦能力。其中含有转化事件“双抗12-1”、“双抗12-3”、“双抗12-4”、“双抗12-5”、“双抗12-7”、“双抗12-9”、“双抗12-10”、“双抗12-12”、“双抗12-13”、“双抗12-14”和“双抗12-15”的草甘膦抗性水平比较高。Thirteen 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. It contains the transformation events "double antibody 12-1", "double antibody 12-3", "double antibody 12-4", "double antibody 12-5", "double antibody 12-7", "double antibody 12-9" "Glyphosate 12-10", "double antibody 12-12", "double antibody 12-13", "double antibody 12-14" and "double antibody 12-15" have higher levels of glyphosate resistance .
表2.双抗玉米草甘膦抗性试验Table 2. Double-resistance maize glyphosate resistance test
Figure PCTCN2016082025-appb-000006
Figure PCTCN2016082025-appb-000006
(2)抗虫能力测试(2) Insect resistance test
1)田间抗虫能力测试 1) Field insect resistance test
分别选择15个含转化事件“双抗12-1”～“双抗12-15”的转化体的T3和T5代转基因玉米进行抗虫性能分析。转基因玉米和亲本非转基因对照温室中发芽后20天喷洒终浓度为0.4wt％的草甘膦确定是转基因后，各取10株，每株接1龄亚洲玉米螟10个，亚洲玉米螟来自中国农业科学院植物保护研究所玉米害虫组。将卵块至于28±1℃，RH 70±5％，16h:8h(L:D)条件下孵化，选取孵化12小时内的幼虫供生测实验用。接虫6天后调查玉米螟为害情况，进行抗虫分级。抗虫分级采用9级标准(Marcon et al.,1999)：1～3级：虫孔针刺状(1级：稀少、分散；2级：中等数量；3级：大量)。4～6级：虫孔火柴头大小(4级：稀少、分散；5级：中等数量；6级：大量)。7～9级：虫孔大于火柴头(7级：稀少分散；8级：中等数量；9级：大量)。抗性级别分类：1～2级(高抗)，3～4级(抗虫)，5～6级(感虫)，7～9级(高感)。结果如表3所示，“双抗12-1”、“双抗12-5”、“双抗12-9”、“双抗12-10”、“双抗12-11”、“双抗12-13”、“双抗12-14”和“双抗12-15”具有高抗虫性能。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. In the transgenic corn and parental non-transgenic control greenhouses, the final concentration of 0.4% by weight of glyphosate was determined after 20 days of germination. After transgenic, 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. Six days after the inoculation, 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.
表3：不同转化事件对玉米螟的抗性等级鉴定Table 3: Identification of resistance levels of corn borer by different transformation events
Figure PCTCN2016082025-appb-000007
Figure PCTCN2016082025-appb-000007
2)实验室抗虫性能测试2) Laboratory insect resistance test
分别选择15个含转化事件“双抗12-1”～“双抗12-15”的转化体的T3和T5代转基因玉米，在转基因玉米和对照玉米心叶中期(6-8叶完全展开)选择未完全展开的心叶叶片在实验室进行玉米螟抗性测定。接玉米螟2天后调查取食面积和玉米螟死亡情况。结果见表4所示，结果显示 大部分转基因玉米抗性良好。它们的叶片被取食非常少，特别是双抗12-5、双抗12-9、双抗12-11、双抗12-14和双抗12-15接虫2天后玉米螟取食面积不到10mm2。接虫2天以后玉米螟死亡率在80％以上，4天以后大部分转基因玉米螟死亡率到达100％。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. To 10mm 2 . After 2 days of inoculation, the mortality rate of corn borer was above 80%. After 4 days, the mortality rate of most genetically modified corn meal reached 100%.
表4：转基因玉米心叶期对亚洲玉米螟的抗虫性Table 4: Insect resistance of Asian corn borer in the heart leaf stage of transgenic corn
Figure PCTCN2016082025-appb-000008
Figure PCTCN2016082025-appb-000008
注：表4中a、b表示差异显著Note: a and b in Table 4 indicate significant differences
(3)外源基因插入拷贝数鉴定(3) Identification of foreign gene insertion copy number
综合抗虫和抗草甘膦测定结果，选择抗虫和耐草甘膦能力都比较强的双抗12-1、双抗12-5、双抗12-9、双抗12-10、双抗12-14和双抗12-15进行外源基因插入拷贝数鉴定。使用罗氏公司(DIG High Prime DNA Labeling and Detection Starter Kit II，Roche)进行Southern分析，遵照试剂盒提供的操作步骤进行杂交探针制作和DNA杂交检测。即提取玉米基因组DNA，分别经过限制性内切酶BamHI和XbaI酶切(这些限制性内切酶在外源基因中具有单个的识别位点)，在琼脂糖胶上电泳分离酶切片段，然后将DNA转移到尼龙基质膜上，利用地高辛标记的G10eve(核苷酸序列SEQ ID NO.5)全长DNA作为探针杂交到尼龙基质膜上以后荧光显影。结果显示转化事件“双抗12-5”在BamHI酶切时获得一条大约14kb的信号带，利用XbaI酶切时获得大约5.0kb的信号带(图2)，证明“双抗12-5”中抗草甘膦基因是单拷贝插入。转化事件“双抗12-15”在BamHI酶切时获得一条大约2.5kb的信号带，而在XbaI酶切时获得大约8.7kb的信号带(图5)，证明“双抗12-15”中抗草甘膦基因是单拷贝插入。Comprehensive anti-insect and glyphosate-resistant results, selection of anti-insect and glyphosate-tolerant double-antibody 12-1, double-anti 12-5, double-anti 12-9, double-anti 12-10, double-antibody Exogenous gene insertion copy number identification was performed on 12-14 and double antibody 12-15. Southern analysis was performed using DIG High Prime DNA Labeling and Detection Starter Kit II (Roche), and hybridization probe preparation and DNA hybridization detection were performed following the procedures provided in the kit. That is, 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. 2), which proved "double antibody 12-5". 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.
利用T-DNA中右边的抗虫融合基因的Cry1Ab作为探针对“双抗12-5”抗虫基因拷贝数进行Southern杂交分析，分别用限制性内切酶KpnI和SmaI对“双抗12-5”的基因组DNA进行单酶切，在琼脂糖胶上分离以后，转移到尼龙基质膜上，然后利用地高辛标记的Cry1Ab(核苷酸序列SEQ ID NO.4中1-1947bp)作为探针杂交到尼龙基质膜上以后荧光显影。结果显示，用KpnI 酶切时获得一条大约7kb的信号带，而在SmaI酶切时获得大约6.5kb的信号带(图3)。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).
分别用限制性内切酶SacI和AclI对转化事件“双抗12-15”的基因组DNA进行单酶切，在琼脂糖胶上分离以后，转移到尼龙基质膜上，然后利用地高辛标记的Cry1Ab(核苷酸序列SEQ ID NO.4中1-1947bp)作为探针杂交到尼龙基质膜上以后荧光显影。结果显示，SacI酶切时获得一条大约7.8kb的信号带，而用AclI酶切时获得大约5.0kb的信号带(图4)。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).
因此上述实验证明转化事件“双抗12-5”和“双抗12-15”是一个抗虫基因和抗草甘膦基因都是单拷贝插入的转化事件。Thus, the above experiments demonstrate that the transformation events "double antibody 12-5" and "double antibody 12-15" are transformation events in which both the insect resistance gene and the glyphosate resistant gene are single copy insertions.
(4)转基因玉米的农艺性状(4) Agronomic traits of genetically modified corn
根据抗虫性能、抗草甘膦能力和外源基因插入拷贝数，筛选获得具有高抗虫抗草甘膦能力，外源基因单拷贝插入的“双抗12-5”和“双抗12-15”进行农艺性状分析。结果显示转化事件“双抗12-5”和“双抗12-15”生长期、花期与非转基因亲本没有差异，在转基因玉米上使用草甘膦除草对“双抗12-5”和“双抗12-15”的产量没有影响(表5)。According to insect resistance, glyphosate resistance and foreign gene insertion copy number, "double antibody 12-5" and "double antibody 12-" with high resistance to glyphosate and single copy insertion of foreign genes were screened. 15" for agronomic trait analysis. The results showed that the transformation events "double antibody 12-5" and "double antibody 12-15" growth period, flowering period and non-transgenic parents did not differ, using glyphosate herbicide on GM corn for "double antibody 12-5" and "double The yield of anti-12-15" had no effect (Table 5).
表5：转基因玉米的每穗粒数、100粒重量和生育期Table 5: Grain number per panicle, 100 grain weight and growth period of transgenic corn
Figure PCTCN2016082025-appb-000009
Figure PCTCN2016082025-appb-000009
注：*表示由于严重的虫害造成粒重和粒数降低。Note: * indicates that the grain weight and the number of grains are reduced due to severe pests.
实施例3：外源基因插入位点侧翼序列的鉴定Example 3: Identification of flanking sequences of foreign gene insertion sites
使用Liu等报道的TAIL-PCR(Thermal asymmetric interlaced PCR)方法(Liu，Plant Journal1995，8(3):457-463)对优良转化事件“双抗12-5”和“双抗12-15”外源转基因DNA插入点两侧的区域序列进行测定。该方法通过3个嵌套的特异性引物分别和简并引物组合进行连续的PCR扩增，利用不同退火温度选择性地扩增目标片段。对扩增获得的DNA片段进行克隆、测序并与网上玉米基因组数据库(http://www.maizegdb.org)进行比对分析。Using the TAIL-PCR (Thermal asymmetric interlaced PCR) method reported by Liu et al. (Liu, Plant Journal 1995, 8(3): 457-463), the excellent transformation events were "double antibody 12-5" and "double antibody 12-15". The sequence of the region flanking the insertion point of the source transgenic DNA was determined. The method performs continuous PCR amplification by combining three nested specific primers with degenerate primers, and selectively amplifies the target fragments by different annealing temperatures. The amplified DNA fragments were cloned, sequenced and aligned with the online maize genome database (http://www.maizegdb.org).
对转化事件“双抗12-5”T-DNA左侧翼区的DNA片段进行测序和比对，获得的序列为SEQ ID NO.1，其中核苷酸1-576bp之间的序列对应于玉米基因组DNA，核苷酸577-826bp之间的序列对应于外源DNA。对T-DNA右侧翼区的DNA片段进行测序和比对，获得的序列为SEQ ID NO.3，其中，核苷酸1-210bp的序列对应于外源DNA，核苷酸211-1007bp的序列对应于玉米基因组DNA。将上述经测序比对和验证过的插入位点上下游旁侧序列和外源T-DNA序列整合形成本发明所述的转化事件“双抗12-5”的特异性核苷酸序列，序列编号为SEQ ID NO.12(即SEQ ID NO.1、SEQ ID NO.2和SEQ ID NO.3的拼接而成)。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. The above-described 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).
对转化事件“双抗12-15”T-DNA左侧翼区的DNA片段进行测序和比对，获得的序列为SEQ ID NO.14，其中核苷酸1-1064bp之间的序列对应于玉米基因组DNA，核苷酸1065-1172bp之间 的序列对应于外源DNA。对T-DNA右侧翼区的DNA片段进行测序和比对，获得的序列为SEQ ID NO.15，其中，核苷酸1-54bp的序列对应于外源DNA，核苷酸55-604bp的序列对应于玉米基因组DNA。将上述经测序比对和验证过的插入位点上下游旁侧序列外源T-DNA序列整合形成本发明所述的转化事件“双抗12-15”的特异性核苷酸序列，序列编号为SEQ ID NO.16(即SEQ ID NO.14、SEQ ID NO.2和SEQ ID NO.15的拼接而成)。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. Integrating the above sequenced and verified insertion site upstream and downstream flanking sequence exogenous T-DNA sequence to form a specific nucleotide sequence of the transformation event "double antibody 12-15" of the present invention, sequence number Is SEQ ID NO. 16 (ie, spliced from SEQ ID NO. 14, SEQ ID NO. 2, and SEQ ID NO. 15).
(2)外源基因特异性检测(2) Exogenous gene-specific detection
根据转化事件的核苷酸序列(SEQ ID NO.12)设计可以用于特异性检测“双抗12-5”的PCR引物(如表6所示)。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).
表6.“双抗12-5”特异性检测引物Table 6. "Double antibody 12-5" specific detection primers
Figure PCTCN2016082025-appb-000010
Figure PCTCN2016082025-appb-000010
所述PCR反应体系为：10×扩增缓冲液5μL，dNTP混合物200μmol/L，正向引物10pmol，反向引物10pmol，基因组DNA0.1～2μg，Taq DNA聚合酶2.5μL，MgCl21.5mmol/L，加双蒸水至50μL。The PCR reaction system is: 10× amplification buffer 5 μL, dNTP mixture 200 μmol/L, forward primer 10 pmol, reverse primer 10 pmol, genomic DNA 0.1-2 μg, Taq DNA polymerase 2.5 μL, MgCl 2 1.5 mmol/ L, add double distilled water to 50 μL.
PCR条件是：32个循环，每个循环是95℃，45秒；65℃，50秒；72℃，30秒。PCR的条件可以根据使用的酶和反应体系进行调整。对获得PCR产物进行琼脂糖电泳分析。左右边界的特异PCR产物分别是304bp和350bp(图6)。必要的时候也可以对PCR产物进行序列测定进一步验证。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.
根据转化事件的核苷酸序列(SEQ ID NO.16)设计可以用于特异性检测双抗12-15的PCR引物(如表7所示)。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).
表7.双抗12-15特异性检测引物Table 7. Dual-antibody 12-15 specific detection primers
Figure PCTCN2016082025-appb-000011
Figure PCTCN2016082025-appb-000011
所述PCR反应体系为：10×扩增缓冲液5μL，dNTP混合物200μmol/L，正向引物10pmol，反向引物10pmol，基因组DNA0.1～2μg，Taq DNA聚合酶2.5μL，MgCl21.5mmol/L，加双蒸水至50μL。 The PCR reaction system is: 10× amplification buffer 5 μL, dNTP mixture 200 μmol/L, forward primer 10 pmol, reverse primer 10 pmol, genomic DNA 0.1-2 μg, Taq DNA polymerase 2.5 μL, MgCl 2 1.5 mmol/ L, add double distilled water to 50 μL.
PCR条件是：32个循环，每个循环是95℃，45秒；65℃，50秒；72℃，30秒。PCR的条件可以根据使用的酶和反应体系进行调整。对获得PCR产物进行琼脂糖电泳分析。左右边界的特异PCR产物分别是1171bp(图7)和604bp(图8)。必要的时候也可以将PCR产物进行序列测定进一步验证。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.
实施例4：遗传稳定性检测Example 4: Genetic stability testing
1)转化事件“双抗12-5”在受体玉米中的遗传稳定性检测1) Genetic stability test of transformation event "double antibody 12-5" in recipient corn
根据转化事件“双抗12-5”的重组DNA分子(SEQ ID NO.12)设计引物进行插入位点特异性的PCR检测，分别取1-7代次的受体玉米提取基因组，利用PCR鉴定T-DNA整合情况。引物为：BR-1(5’GGCGAATGCTAGAGCAGCTTGAGCT-3’)(SEQ ID NO.25)和GN-1(5’CCTACTGCGATGACGTTCGGTGCC-3’)(SEQ ID NO.26)，According to the transformation event "double antibody 12-5" recombinant DNA molecule (SEQ ID NO. 12), 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),
反应体系：10×扩增缓冲液5μL，dNTP混合物200μmol/L，BR-110pmol，10pmolGN-1，基因组DNA0.1～2μg，Taq DNA聚合酶2.5μL，MgCl21.5mmol/L，加双蒸水至50μL。Reaction system: 10 μL of amplification buffer 5 μL, dNTP mixture 200 μmol/L, BR-110 pmol, 10 pmol GN-1, genomic DNA 0.1-2 μg, Taq DNA polymerase 2.5 μL, MgCl 2 1.5 mmol/L, double distilled water Up to 50 μL.
反应条件：95℃1分钟，60℃40秒，72℃30秒，30个循环。TAQ酶从上海生工获得。结果显示，所有代次的转基因玉米PCR结果都为阳性。琼脂糖电泳分析显示，PCR产物大小与预期大小一致是145bp(图9)。Reaction conditions: 95 ° C for 1 minute, 60 ° C for 40 seconds, 72 ° C for 30 seconds, 30 cycles. The TAQ enzyme was obtained from Shanghai Biotech. The results showed that the PCR results of all the transgenic maize were positive. Agarose electrophoresis analysis showed that the PCR product size was 145 bp in agreement with the expected size (Figure 9).
“双抗12-5”抗虫抗草甘膦生物测定"Double-resistant 12-5" insect-resistant glyphosate bioassay
玉米螟生物测定(Marcon et al.,1999)表明第1,2,3,4,5,6,7代的转基因玉米对一龄玉米螟的杀虫效果都是100％，抗虫能力稳定遗传。喷施草甘膦试验表明，第1,2,3,4,5,6,7代的转基因玉米都能抗每亩100克活性草甘膦含量的草甘膦农药。抗草甘膦能力稳定遗传。结果证明抗虫和抗草甘膦能力稳定遗传。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.
2)转化事件“双抗12-15”在受体玉米中的遗传稳定性检测2) Genetic stability test of transformation event "double antibody 12-15" in recipient corn
根据转化事件“双抗12-15”的重组DNA分子(SEQ ID NO.16)设计引物进行插入位点特异性的PCR检测，分别取1-7代次的受体玉米提取基因组，利用PCR鉴定T-DNA整合情况。引物为：LB-SP4：5’CTAAAACCAAAATCCAGTACTAAAATCC(SEQ ID NO.27)和LB-M:CTGTTCTGATGGTGGCAGGCAGG(SEQ ID NO.28)，According to the transformation event "double antibody 12-15" recombinant DNA molecule (SEQ ID NO. 16), 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),
反应体系：10×扩增缓冲液5μL，dNTP混合物200μmol/L，BR-110pmol，10pmolGN-1，基因组DNA0.1～2μg，Taq DNA聚合酶2.5μL，MgCl21.5mmol/L，加双蒸水至50μL。Reaction system: 10 μL of amplification buffer 5 μL, dNTP mixture 200 μmol/L, BR-110 pmol, 10 pmol GN-1, genomic DNA 0.1-2 μg, Taq DNA polymerase 2.5 μL, MgCl 2 1.5 mmol/L, double distilled water Up to 50 μL.
反应条件：95℃1分钟，60℃40秒，72℃30秒，30个循环。TAQ酶从上海生工获得。结果显示，所有代次的转基因玉米PCR结果都为阳性。琼脂糖电泳分析显示，PCR产物大小与预期大小一致是1000bp(图10)。Reaction conditions: 95 ° C for 1 minute, 60 ° C for 40 seconds, 72 ° C for 30 seconds, 30 cycles. The TAQ enzyme was obtained from Shanghai Biotech. The results showed that the PCR results of all the transgenic maize were positive. Agarose electrophoresis analysis showed that the PCR product size was 1000 bp consistent with the expected size (Figure 10).
“双抗12-15”抗虫抗草甘膦生物测定"Double-resistant 12-15" insect-resistant glyphosate bioassay
玉米螟生物测定表明第1,2,3,4,5代的转基因玉米对一龄玉米螟的杀虫效果都是100％，抗虫性能稳定遗传。喷施草甘膦试验表明，第1,2,3,4,5代的转基因玉米都能抗每亩100克活性草甘膦含量的草甘膦农药。抗草甘膦性能稳定遗传。结果证明抗虫和抗草甘膦能力稳定遗传。 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.
实施例5：抗虫抗草甘膦玉米品种培育Example 5: Breeding of insect-resistant glyphosate-resistant maize varieties
1)“双抗12-5”杂交转育1) "Double-resistant 12-5" hybridization
以含有“双抗12-5”转化事件的玉米为供体亲本，以玉米自交系B73为受体亲本进行一次杂交，4次回交和3次自交。在回交过程中使用草甘膦来清除不含“双抗12-5”转基因复合结构的分离株系。于BC4F3代(回交4代后自交3代)获得了含有本发明所述的复合转基因结构的稳定自交系B735。从该株系叶片组织中提取DNA，扩增出外源插入基因及其侧翼的DNA片断，经测序分析证实，来自与供体亲本的复合转基因结构序列一致，说明“双抗12-5”转化事件稳定转育到新的受体材料中。以B735为母本，Mo17为父本组配获得杂交种B7M，对B7M进行外源插入基因及其侧翼DNA的PCR分析和测序，结果显示B7M含有转化事件“双抗12-5”。田间抗性试验显示，B7M具有良好的抗草甘膦能力和抗虫性能。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. Stable transfer to new receptor materials. Using B735 as the female parent and Mo17 as the male parent, the hybrid B7M was obtained, and the B7M was subjected to PCR analysis and sequencing of the exogenous insertion gene and its flanking DNA. The results showed that B7M contained the transformation event "double antibody 12-5". Field resistance tests showed that B7M has good glyphosate resistance and insect resistance.
1)“双抗12-15”有性杂交转育1) Sexual hybridization of "double antibody 12-15"
以含有“双抗12-15”转化事件的玉米为供体亲本，以玉米自交系MR-1为受体亲本进行一次杂交，4次回交和3次自交。在回交过程中使用草甘膦来清除不含“双抗12-15”转基因复合结构的分离株系。于BC4F3代(回交4代后自交3代)获得了含有本发明所述的复合转基因结构的稳定自交系M45。从该株系叶片组织中提取DNA，扩增出外源插入基因及其侧翼的DNA片断，经测序分析证实，来自与供体亲本的复合转基因结构序列一致，说明“双抗12-15”转化事件稳定转育到新的受体材料中。以M45为母本，RF1为父本组配获得杂交种M7R，对M7R进行外源插入基因及其侧翼DNA的PCR分析和测序，结果显示M7R含有转化事件“双抗12-15”。田间抗性试验显示，M7R具有良好的抗草甘膦能力和抗虫性能。 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.

Claims (10)

  1. 一种玉米转化事件，其特征在于所述转化事件以SEQ ID NO.1所示的核苷酸序列为外源基因的左侧翼区，以SEQ ID NO.3所示的核苷酸序列为外源基因的右侧翼区或者以SEQ ID NO.14所示的核苷酸序列为外源基因的左侧翼区，以SEQ ID NO.15所示的核苷酸序列为外源基因的右侧翼区。A maize transformation event characterized in that the 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 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.
  2. 如权利要求1所述的玉米转化事件，其特征在于所述外源基因包括抗虫基因和抗草甘膦基因。The maize transformation event of claim 1 wherein said exogenous gene comprises an insect resistance gene and a glyphosate resistant gene.
  3. 如权利要求2所述的玉米转化事件，其特征在于所述抗虫基因的核苷酸序列为SEQ ID NO.4所示。The maize transformation event according to claim 2, wherein the nucleotide sequence of the insect-resistant gene is represented by SEQ ID NO.
  4. 如权利要求2所述的玉米转化事件，其特征在于所述抗草甘膦基因的核苷酸序列为SEQ ID NO.5所示。The maize transformation event according to claim 2, wherein the nucleotide sequence of the glyphosate resistant gene is represented by SEQ ID NO.
  5. 如权利要求1所述的玉米转化事件，其特征在于所述外源基因的核苷酸序列为SEQ ID NO.2所示。The maize transformation event of claim 1 wherein the nucleotide sequence of said foreign gene is set forth in SEQ ID NO.
  6. 一种权利要求1所述玉米转化事件的特异性PCR鉴定方法，其特征在于以SEQ ID NO.1所示的核苷酸序列为外源基因的左侧翼区，以SEQ ID NO.3所示的核苷酸序列为外源基因的右侧翼区，用于特异性PCR鉴定的引物：A method for specific PCR identification of a maize transformation event according to claim 1, characterized in that the nucleotide sequence shown in SEQ ID NO. 1 is the left wing region of the foreign gene, as shown in SEQ ID NO. The nucleotide sequence shown is the right wing region of the foreign gene, and the primers for specific PCR identification:
    SP1：5’-TTTCTCCATAATAATGTGTGAGTAGTTCCC-3’；SP1: 5'-TTTCTCCATAATAATGTGTGAGTAGTTCCC-3';
    RB-Test：5’-CTCGTCATCGACCAAGTCATGAAG-3’。RB-Test: 5'-CTCGTCATCGACCAAGTCATGAAG-3'.
    R1：5’-CGTCGTTTTACAACGTCGTGACTGG-3’；R1: 5'-CGTCGTTTTACAACGTCGTGACTGG-3';
    LB-test：5’-AAGACGTCCGGGGGAACCGTTGTTC-3’。LB-test: 5'-AAGACGTCCGGGGGAACCGTTGTTC-3'.
  7. 一种权利要求1所述玉米转化事件的特异性PCR鉴定方法，其特征在于以SEQ ID NO.14所示的核苷酸序列为外源基因的左侧翼区，以SEQ ID NO.15所示的核苷酸序列为外源基因的右侧翼区，用于特异性PCR鉴定的引物：A method for specific PCR identification of a maize transformation event according to claim 1, wherein the nucleotide sequence shown by SEQ ID NO. 14 is the left wing region of the foreign gene, as set forth in SEQ ID NO. The nucleotide sequence shown is the right wing region of the foreign gene, and the primers for specific PCR identification:
    LB-15T：5’-CTAAAACCAAAATCCAGTACTAAAATCC-3’；LB-15T: 5'-CTAAAACCAAAATCCAGTACTAAAATCC-3';
    LB-15G：5’-GCCGTACGTTTCCCAGCC-3’。LB-15G: 5'-GCCGTACGTTTCCCAGCC-3'.
    RB-15T：5’-AGCTTGAGCTTGGATCAGATTGTCGT-3’；RB-15T: 5'-AGCTTGAGCTTGGATCAGATTGTCGT-3';
    RB-15G：5’-CGTACAGGGAGCTTAGGGGG-3’。RB-15G: 5'-CGTACAGGGAGCTTAGGGGG-3'.
  8. 一种含权利要求1所述玉米转化事件的植物细胞，其特征在于所述植物细胞含以SEQ ID NO.1所示的核苷酸序列为外源基因的左侧翼区，以SEQ ID NO.3所示的核苷酸序列为外源基因的右侧翼区的转化事件，保藏于中国典型培养物保藏中心，保藏号CCTCC NO:P201506，保藏日期：2015年4月27日，保藏地址：中国武汉武汉大学，邮编430072。A plant cell comprising the maize transformation event of claim 1, wherein the plant cell comprises a nucleotide sequence represented by SEQ ID NO. 1 as a left wing region of a foreign gene, SEQ ID NO The nucleotide sequence shown in .3 is the transformation event of the right wing region of the foreign gene, deposited in the China Center for Type Culture Collection, deposit number CCTCC NO: P201506, deposit date: April 27, 2015, deposit address : Wuhan University, Wuhan, China, 430072.
  9. 一种含权利要求1所述玉米转化事件的植物细胞，其特征在于所述植物细胞含以SEQ ID NO.14所示的核苷酸序列为外源基因的左侧翼区，以SEQ ID NO.15所示的核苷酸序列为外源基因的右侧翼区的转化事件，保藏于中国典型培养物保藏中心，保藏号CCTCC NO:P201607，保藏日期：2016年4月11日，保藏地址：中国武汉武汉大学，邮编430072。A plant cell comprising the maize transformation event of claim 1, wherein the plant cell comprises a nucleotide sequence represented by SEQ ID NO. 14 as a left wing region of a foreign gene, SEQ ID NO The nucleotide sequence shown in .15 is the transformation event of the right wing region of the foreign gene, deposited in the China Center for Type Culture Collection, deposit number CCTCC NO: P201607, deposit date: April 11, 2016, deposit address : Wuhan University, Wuhan, China, 430072.
  10. 一种权利要求1所述玉米转化事件在制备抗虫抗草甘膦玉米细胞中的应用，其特征在于所述方法为：利用含有所述玉米转化事件的玉米材料与玉米育种材料进行杂交后，进一步进行回交，获得所述抗虫抗草甘膦玉米细胞。 Use of a corn transformation event according to claim 1 for the preparation of insect-resistant glyphosate-resistant maize cells, characterized in that the method comprises: using a corn material containing the corn transformation event to hybridize with a corn breeding material, Further backcrossing is performed to obtain the insect resistant glyphosate resistant maize cells.
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