WO2022094790A1 - 玉米事件2a-7及其鉴定方法 - Google Patents

玉米事件2a-7及其鉴定方法 Download PDF

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WO2022094790A1
WO2022094790A1 PCT/CN2020/126452 CN2020126452W WO2022094790A1 WO 2022094790 A1 WO2022094790 A1 WO 2022094790A1 CN 2020126452 W CN2020126452 W CN 2020126452W WO 2022094790 A1 WO2022094790 A1 WO 2022094790A1
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corn
sequence
seq
nucleic acid
primer
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PCT/CN2020/126452
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English (en)
French (fr)
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赖锦盛
赵海铭
宋伟彬
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中国农业大学
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Priority to PCT/CN2020/126452 priority Critical patent/WO2022094790A1/zh
Priority to US18/020,259 priority patent/US20230304105A1/en
Priority to ARP210103051A priority patent/AR123987A1/es
Publication of WO2022094790A1 publication Critical patent/WO2022094790A1/zh

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    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
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    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
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    • A61K36/88—Liliopsida (monocotyledons)
    • A61K36/899—Poaceae or Gramineae (Grass family), e.g. bamboo, corn or sugar cane
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
    • A61K8/97—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from algae, fungi, lichens or plants; from derivatives thereof
    • A61K8/9783—Angiosperms [Magnoliophyta]
    • A61K8/9794—Liliopsida [monocotyledons]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K8/00—Cosmetics or similar toiletry preparations
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    • A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
    • A61K8/99—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from microorganisms other than algae or fungi, e.g. protozoa or bacteria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
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Definitions

  • the present invention relates to transgenic corn event 2A-7, and to corn plants or parts, seeds, cells or progeny thereof containing nucleic acid molecules that diagnose the corn event.
  • the present invention also provides methods of detecting the presence of nucleic acid molecules unique to the maize event in a sample, as well as probes and primers for detecting the presence of the maize event in a sample.
  • the present invention further relates to methods of producing insect resistant maize plants and seeds.
  • Corn is the most widely planted and most productive cereal crop in the world, ranking first among the three major grains (corn, wheat, and rice).
  • the current corn pests are serious.
  • the stem-boring and leaf-eating Lepidopteran pests, the corn borer is the most widely distributed and the most harmful. It is an important corn pest in the world.
  • the occurrence of hazards has seriously affected the yield and quality of corn, and caused huge economic losses to corn production. Therefore, it is an urgent problem to take effective measures to control its harm to improve corn yield and increase farmers' income.
  • the main method of pest control is to spray chemical pesticides during the growth process.
  • chemical pesticides not only kill pests, but also kill their natural enemies, causing ecological balance damage and environmental pollution. Therefore, there is an urgent need for corn plants that are resistant to corn pests, particularly Lepidopteran pests.
  • Insect-resistant genes can be introduced into maize varieties through transgenic technology, thereby improving the insect resistance of transgenic maize. At the same time, because each plant of transgenic maize has a considerable degree of resistance, its insect-resistance effect is significantly better than that of spraying pesticides. And stable, but also can save manpower and material resources, effectively save social resources.
  • the integration of foreign genes into the host plant genome is random. If the foreign gene happens to be inserted into a very important host gene, and is therefore mutated, the random insertion event can be lethal to the recipient plant. In addition, even if random insertion events do not impair host cell gene function, the expression of inserted foreign genes can be affected by "position effects" caused by surrounding genomic DNA. In some cases, the gene is inserted into sites at which positional effects are strong enough to prevent synthesis of an effective amount of product from the introduced gene. Consequently, large numbers of events must often be screened to identify superior events characterized by optimal expression of the introduced gene of interest. It is necessary to identify one or more unique sequences associated only with the elite event in order to diagnose the presence of the transgenic event.
  • the present invention relates to a maize event, designated 2A-7, comprising nucleic acid molecules stably integrated in its genome encoding Cry1Ab and Cry2Ab proteins that confer insect resistance to 2A-7 maize plants.
  • the seeds of the corn event 2A-7 were deposited in the China General Microorganism Culture Collection and Management Center (CGMCC), and have the deposit number CGMCC NO.17848.
  • CGMCC China General Microorganism Culture Collection and Management Center
  • the transgenic corn plants In the absence of insect stress, the transgenic corn plants have substantially comparable agronomic performance to the non-transgenic isogenic lines. Under insect stress, the transgenic corn plant will have significantly improved resistance to insect attack by corn borer, armyworm, cotton bollworm, peach borer and the like than non-transgenic corn plants.
  • the present invention provides a maize plant or part, seed, cell or progeny thereof having an exogenous nucleic acid molecule incorporated into its genome
  • the exogenous nucleic acid molecule comprises Cry1Ab and Cry2Ab genes, wherein the exogenous nucleic acid molecule is flanked by a 5' flanking region having positions 1-432 or 300-432 of SEQ ID NO:5 and the exogenous nucleic acid molecule is flanked by a 3' flanking region having the nucleotide sequence of positions 8532-9031 or 8532-8800 of SEQ ID NO:5 .
  • the present invention also provides a maize plant or part, seed, cell or progeny thereof having an exogenous nucleic acid molecule incorporated into its genome, the exogenous nucleic acid molecule Comprise Cry1Ab and Cry2Ab gene, wherein, described exogenous nucleic acid molecule is flanked by 5 ' flanking region and 3 ' flanking region, comprises the sequence of the part of 5 ' flanking region and adjacent exogenous nucleic acid molecule such as SEQ ID NO: 1, the sequence comprising the portion of the 3' flanking region and the adjacent exogenous nucleic acid molecule is shown in SEQ ID NO: 2.
  • the corn plants of the present invention or parts, seeds, cells or progeny thereof have resistance to insect attack.
  • the insects are selected from Lepidopteran insects.
  • the insect is selected from one or more of Lepidopteran pests such as armyworm, corn borer (eg, Asian corn borer), cotton bollworm, peach borer, Spodoptera frugiperda.
  • Lepidopteran pests such as armyworm, corn borer (eg, Asian corn borer), cotton bollworm, peach borer, Spodoptera frugiperda.
  • the corn plants of the present invention, or parts, seeds, cells, or progeny thereof also have resistance to herbicides (eg, glyphosate, glufosinate, etc.).
  • the position of the exogenous nucleic acid molecule in the genome corresponds to between Chr3:179141694bp-179141724bp of the B73 reference genome sequence (version V4).
  • the integration of the exogenous nucleic acid molecule results in the deletion of a partial sequence in the original genome, and the deleted sequence is Chr3:179141695bp-179141723bp (B73 reference genome V4 version), a total of 29bp, the deleted region It is the maize endogenous gene Zm00001d042767, the function of which is predicted to be Glucan endo-13-beta-glucosidase 14.
  • the insertion of the target fragment and the deletion of the Chr3:179141695bp-179141723bp sequence will inactivate the gene function.
  • This gene has two copies in maize, and the gene number of another homologous gene is Zm00001d012292.
  • the nucleotide sequences of the Cry1Ab and Cry2Ab genes are codon-optimized for expression in monocotyledonous (e.g., maize) cells.
  • the Cry1Ab gene is shown in SEQ ID NO:12.
  • the Cry2Ab gene is set forth in SEQ ID NO:13.
  • the exogenous nucleic acid molecule further comprises a Bar gene.
  • the Bar gene is shown in SEQ ID NO:14.
  • the exogenous nucleic acid molecule comprises 35S polyA terminator, Bar gene, CAMV 35S promoter, nos polyA terminator, Cry1Ab gene, Gly promoter, CAMV 35S promoter, adh1 enhancer, Cry2Ab gene and nos polyA terminator.
  • the exogenous nucleic acid molecule comprises the sequence of nucleotides 483-8524 of SEQ ID NO:5 or the complement thereof.
  • an unintended integration sequence will be generated, which is either a non-genomic sequence or a target insert (such as a T-border sequence), for example, it is generated by genome repair when the insert is integrated. the sequence of.
  • an exogenous nucleic acid molecule incorporated into its genome contained in a maize plant of the invention, or a part, seed, cell or progeny thereof, may contain this unintended integration sequence.
  • the exogenous nucleic acid molecule comprises the sequence of nucleotides 433-8531 of SEQ ID NO:5 or the complement thereof.
  • the genome of the corn plant or part, seed, cell or progeny thereof comprises the sequence set forth in any one of SEQ ID NOs: 1-5 or the complement thereof. In certain embodiments, the genome of the corn plant or part, seed, cell or progeny thereof comprises the sequence set forth in SEQ ID NO: 5 or the complement thereof.
  • genomic DNA of the maize plant or part, seed, cell or progeny thereof when amplified with SEQ ID NO: 6 and SEQ ID NO: 7 as forward and reverse primers, respectively , resulting in amplicons of about 200-300 bp in length (eg, about 258 bp). In certain embodiments, when the genomic DNA is amplified with SEQ ID NO: 8 and SEQ ID NO: 9 as forward and reverse primers, respectively, the resulting genomic DNA is about 250-350 bp in length (eg, about 310 bp in length). ) amplicons.
  • the corn seeds producing the corn plants or parts, seeds, cells or progeny thereof are deposited in the China General Microorganism Culture Collection and Management Center (CGMCC) and have the deposit number CGMCC NO.17848.
  • CGMCC China General Microorganism Culture Collection and Management Center
  • the maize plant or part, seed, cell or progeny thereof is propagated and/or bred from the maize plant from which the maize seed is grown.
  • the present invention provides a corn seed, which is preserved in the China General Microorganism Culture Collection and Management Center (CGMCC), and has a deposit number of CGMCC NO.17848.
  • CGMCC China General Microorganism Culture Collection and Management Center
  • the present invention also provides a maize plant or part, seed, cell or progeny thereof produced from the maize seed of the second aspect.
  • the maize plant or part, seed, cell or progeny thereof is propagated and/or bred from a maize plant grown from the maize seed of the second aspect.
  • the present invention provides an article of manufacture comprising the maize plant of the first aspect or a part, seed, cell or progeny thereof, or the maize seed of the second aspect, or from the second aspect A corn plant or part, seed, cell or progeny thereof produced from the corn seed.
  • the article of manufacture is a food, agricultural product (eg, feed), cosmetic, pharmaceutical, or industrial product.
  • the article of manufacture comprises the maize plant of the first aspect or a part, seed, cell or progeny thereof, or the maize seed of the second aspect or a maize plant or part, seed thereof , genomic DNA of cells or progeny.
  • a detectable amount of a polynucleotide that is unique to maize event 2A-7 is present in the preparation.
  • the article of manufacture comprises a sequence selected from the group consisting of any one of SEQ ID NOs: 1-5 or the complement thereof.
  • nucleic acids contained in the preparation are amplified with SEQ ID NO: 6 and SEQ ID NO: 7 as forward and reverse primers, respectively, resulting in a length of about 200-300 bp (eg about 258 bp).
  • the resulting length is about 250-350 bp (eg about 310 bp).
  • the article is selected from the group consisting of corn ear ears, husk corn, corn silk, corn pollen, corn grits, corn meal, crushed corn, corn flour, corn oil, corn starch, corn steep liquor, corn Malt, corn sugar, corn syrup, margarine produced from corn oil, unsaturated corn oil, saturated corn oil, corn flakes, popcorn, ethanol and/or liquor produced from corn, produced from corn fermentation Dried distiller's grains (DDGS), animal feed from corn, cosmetics and fillers.
  • DDGS corn fermentation Dried distiller's grains
  • the part of a maize plant of any preceding aspect is selected from the group consisting of kernels, pollen, ovules, flowers, shoots, roots, stalks, silks, tassels, ears and leaves.
  • the present invention also relates to the use of a corn plant according to any of the preceding aspects, or parts, seeds, cells or progeny thereof, for the production of food, agricultural products (eg feed), cosmetics, pharmaceuticals or industrial products.
  • the corn plant or part, seed, cell or progeny thereof is used for the production of food, feed, starch or for winemaking.
  • the part of the corn plant is selected from the group consisting of kernel, pollen, ovule, flower, shoot, root, stem, silk, inflorescence, ear ear and leaf.
  • the corn plant or part, seed, cell or progeny thereof is used to produce an article selected from the group consisting of corn ear ear, husk corn, corn silk, corn pollen, corn grits, corn meal , crushed corn, cornmeal, corn oil, cornstarch, corn steep liquor, corn malt, corn sugar, corn syrup, margarine produced from corn oil, unsaturated corn oil, saturated corn oil, corn flakes, popcorn, made from Corn-produced ethanol and/or liquor, dried distillers grains (DDGS) from corn fermentation, corn-derived animal feed, cosmetics and fillers.
  • DDGS dried distillers grains
  • flanking sequences are specific for a particular transgenic event.
  • specific detection of transgenic events is possible using sequences flanking the insert. For example, hybridization is performed using a probe comprising part of the flanking sequence and part of the exogenous insert sequence, or specific primers containing part of the flank sequence and part of the exogenous insert sequence are designed for PCR amplification, etc.
  • the present invention provides a primer pair comprising a first primer and a second primer, wherein the first primer comprises nucleotides (1-432) of SEQ ID NO:5 ( at least 15 consecutive nucleotides (eg, at least 18; eg, 15-30 consecutive nucleotides, or 18-30 consecutive nucleotides) of the sequence such as nucleotides 200-432) or its complement.
  • the first primer comprises nucleotides (1-432) of SEQ ID NO:5 ( at least 15 consecutive nucleotides (eg, at least 18; eg, 15-30 consecutive nucleotides, or 18-30 consecutive nucleotides) of the sequence such as nucleotides 200-432) or its complement.
  • the second primer comprising a sequence consisting of nucleotides 483-8524 (eg, nucleotides 483-1000) of SEQ ID NO: 5 or
  • a nucleotide sequence consisting of at least 15 consecutive nucleotides (eg, at least 18; eg, 15-30 consecutive nucleotides, or 18-30 consecutive nucleotides) of its complementary sequence or with the nucleoside have at least 80% (eg, 80%-100%, or 90%-100%; or at least 85%, at least 90%, at least 95%, at least 99%, or 100%) sequence identity compared to acid sequences sequence.
  • amplicons of 100-500 bp in length are produced.
  • the amplicon is 200-400 bp in length, such as 200-300 bp, such as about 258 bp.
  • the first primer contains at least 15 contiguous nucleotides (eg, at least 18; eg, 15) of the sequence consisting of nucleotides 300-400 of SEQ ID NO: 5 or the complement thereof. -30 contiguous nucleotides, or 18-30 contiguous nucleotides) consisting of or at least 80% (eg, 80% to 100%, or 90% compared to the nucleotide sequence) % to 100%; or at least 85%, at least 90%, at least 95%, at least 99%, or 100%) of the sequence identity, the second primer contains the sequence from 500-600 of SEQ ID NO:5 Nucleotides consisting of at least 15 consecutive nucleotides (eg, at least 18; eg, 15-30 consecutive nucleotides, or 18-30 consecutive nucleotides) of the sequence of nucleotides at position or their complements The sequence is at least 80% (eg, 80%-100%, or 90%-100%; or at least 85%, at least
  • the first primer contains at least 15 contiguous nucleotides (eg, at least 18; eg, 15) of the sequence consisting of nucleotides 300-350 of SEQ ID NO: 5 or the complement thereof. -30 contiguous nucleotides, or 18-30 contiguous nucleotides) consisting of or at least 80% (eg, 80% to 100%, or 90% compared to the nucleotide sequence) % to 100%; or at least 85%, at least 90%, at least 95%, at least 99%, or 100%) sequence identity.
  • the second primer contains at least 15 consecutive sequences consisting of nucleotides 520-600 (eg, nucleotides 520-570) of SEQ ID NO: 5 or the complement thereof A nucleotide sequence consisting of or at least 80% of the nucleotide sequence (eg, at least 18; eg, 15-30 contiguous nucleotides, or 18-30 contiguous nucleotides) (eg, 80%-100%, or 90%-100%; or at least 85%, at least 90%, at least 95%, at least 99%, or 100%) sequence identity.
  • nucleotides 520-600 eg, nucleotides 520-570
  • a nucleotide sequence consisting of or at least 80% of the nucleotide sequence eg, at least 18; eg, 15-30 contiguous nucleotides, or 18-30 contiguous nucleotides
  • 80%-100%, or 90%-100% eg, 80%-100%, or 90%-100%; or
  • the first primer contains or has at least 80% (eg, 80%-100%, or 90%-100%; or at least 85% compared to the sequence set forth in SEQ ID NO:6) , at least 90%, at least 95%, at least 99%, or 100%) sequence identity
  • the second primer contains or has at least 80% compared to the sequence shown in SEQ ID NO: 7 (eg, 80% to 100%, or 90% to 100%; or at least 85%, at least 90%, at least 95%, at least 99%, or 100%) sequence identity.
  • the present invention provides a primer pair comprising a first primer and a second primer, wherein the first primer contains nucleotides from positions 483 to 8524 of SEQ ID NO: 5 (eg, 8000-8524 nucleotides) or its complement, consisting of at least 15 consecutive nucleotides (eg, at least 18; eg, 15-30 consecutive nucleotides, or 18-30 consecutive nucleotides) The nucleotide sequence of the %, or 100%) sequence identity, the second primer contains a sequence consisting of nucleotides 8532-9031 (eg, nucleotides 8532-8800) of SEQ ID NO:5 or its complement A nucleotide sequence consisting of at least 15 consecutive nucleotides (eg, at least 18; eg, 15-30 consecutive nucleotides, or 18-30 consecutive nucleotides) of the sequence or with said nucleotide sequence compared to sequences having at least 80% (eg, 80%-100%
  • amplicons of 100-500 bp in length are produced.
  • the amplicon is 200-400 bp in length, such as 250-350 bp, such as about 310 bp.
  • the first primer contains at least 15 contiguous nucleotides (eg, at least 18; eg, 15) of the sequence consisting of nucleotides 8400-8500 of SEQ ID NO: 5 or the complement thereof. -30 contiguous nucleotides, or 18-30 contiguous nucleotides) consisting of or at least 80% (eg, 80% to 100%, or 90% compared to the nucleotide sequence) % to 100%; or at least 85%, at least 90%, at least 95%, at least 99%, or 100%) of sequence identity
  • the second primer contains the sequence from 8700-8800 of SEQ ID NO:5 Nucleotides consisting of at least 15 consecutive nucleotides (eg, at least 18; eg, 15-30 consecutive nucleotides, or 18-30 consecutive nucleotides) of the sequence of nucleotides at position or their complements The sequence is at least 80% (eg, 80%-100%, or 90%-100%; or at least 85%, at
  • the first primer comprises nucleotides 8400-8600 of SEQ ID NO:5 (eg, nucleotides 8400-8500, nucleotides 8450-8550, or at least 15 consecutive nucleotides (eg, at least 18; eg, 15-30 consecutive nucleotides, or 18-30 consecutive nucleotides) of the sequence of nucleotides 8450-8500) or its complement.
  • the second primer comprises nucleotides 8500-9000 (eg, nucleotides 8500-8800, nucleotides 8700-8800, or nucleotides 8700-8800) of SEQ ID NO:5 8750-8800 nucleotides) or its complement consisting of at least 15 consecutive nucleotides (eg, at least 18; eg, 15-30 consecutive nucleotides, or 18-30 consecutive nucleotides) The nucleotide sequence of the %, or 100%) sequence identity.
  • the first primer contains or has at least 80% (eg, 80%-100%, or 90%-100%; or at least 85% compared to the sequence set forth in SEQ ID NO: 8) , at least 90%, at least 95%, at least 99%, or 100%) sequence identity
  • the second primer contains or has at least 80% compared to the sequence shown in SEQ ID NO: 9 (e.g., 80% to 100%, or 90% to 100%; or at least 85%, at least 90%, at least 95%, at least 99%, or 100%) sequence identity.
  • the present invention provides a method of detecting nucleic acid molecules unique to corn events 2A-7 in a sample comprising corn nucleic acid, comprising:
  • step (3) detecting the product of step (2) by gel electrophoresis (eg, agarose gel electrophoresis);
  • gel electrophoresis eg, agarose gel electrophoresis
  • maize events 2A-7 are the maize plant or part, seed, cell or progeny thereof as described in any of the above aspects.
  • the maize event 2A-7 comprises exogenous nucleic acid molecules encoding mcry1Ab and mcry2Ab incorporated into its genome, the exogenous nucleic acid molecules flanking 5' flanking regions and 3' flanking regions, comprising The sequence of the part of the 5' flanking region and the adjacent exogenous nucleic acid molecule is shown in SEQ ID NO: 1, and the sequence of the part comprising the 3' flanking region and the adjacent exogenous nucleic acid molecule is shown in SEQ ID NO: :2 shown.
  • the maize event 2A-7 comprises the sequence set forth in any one of SEQ ID NOs: 1-5 or the complement thereof in its genome.
  • step (3) when an expected amplicon is detected, indicating that the sample contains a nucleic acid molecule unique to corn event 2A-7, the expected amplicon refers to when the expected amplicon is The primer pair was used to amplify the nucleic acid from the genomic DNA of maize event 2A-7, resulting in an amplicon.
  • the method includes:
  • step (3) detecting the product of step (2) by gel electrophoresis (eg, agarose gel electrophoresis);
  • gel electrophoresis eg, agarose gel electrophoresis
  • an amplicon with a length of about 250-260 bp eg, about 258 bp
  • the primer pair comprises: contains or has at least 80% (eg, 80%-100%, or 90%-100%; or at least 85% compared to the sequence set forth in SEQ ID NO: 6; %, at least 90%, at least 95%, at least 99%, or 100%) sequence identity to the first primer with the sequence set forth in SEQ ID NO:7 or having at least 80% (e.g., 80% to 100%, or 90% to 100%; or at least 85%, at least 90%, at least 95%, at least 99%, or 100%) sequence identity to the second primer.
  • the method includes:
  • step (3) detecting the product of step (2) by gel electrophoresis (eg, agarose gel electrophoresis);
  • gel electrophoresis eg, agarose gel electrophoresis
  • amplicon when an amplicon with a length of about 305-315 bp (eg, about 310 bp) is detected, it indicates that a nucleic acid molecule unique to maize event 2A-7 is present in the sample.
  • the primer pair comprises: contains or has at least 80% (eg, 80%-100%, or 90%-100%; or at least 85% compared to the sequence set forth in SEQ ID NO: 8; %, at least 90%, at least 95%, at least 99%, or 100%) sequence identity to the first primer with the sequence set forth in SEQ ID NO: 9 or having at least 80% (e.g., 80% to 100%, or 90% to 100%; or at least 85%, at least 90%, at least 95%, at least 99%, or 100%) sequence identity to the second primer.
  • the present invention provides a method of detecting nucleic acid molecules unique to corn events 2A-7 in a sample comprising corn nucleic acid, comprising:
  • maize events 2A-7 are the maize plant or part, seed, cell or progeny thereof as described in any of the above aspects.
  • the maize event 2A-7 comprises exogenous nucleic acid molecules encoding mcry1Ab and mcry2Ab incorporated into its genome, the exogenous nucleic acid molecules flanking 5' flanking regions and 3' flanking regions, comprising The sequence of the part of the 5' flanking region and the adjacent exogenous nucleic acid molecule is shown in SEQ ID NO: 1, and the sequence of the part comprising the 3' flanking region and the adjacent exogenous nucleic acid molecule is shown in SEQ ID NO: :2 shown.
  • the maize event 2A-7 comprises the sequence set forth in any one of SEQ ID NOs: 1-5 or the complement thereof in its genome.
  • the detecting comprises DNA sequencing.
  • step (3) the amplicon is detected using a nucleic acid probe specific for the amplicon.
  • the detection comprises the steps of:
  • step (2) (a) contacting the nucleic acid probe with the amplification product of step (2);
  • hybridization when hybridization is detected, it indicates the presence of nucleic acid molecules unique to maize event 2A-7 in the sample.
  • the present invention provides a method of detecting the presence of a nucleic acid molecule unique to corn events 2A-7 in a sample comprising corn nucleic acid, comprising:
  • maize events 2A-7 are the maize plant or part, seed, cell or progeny thereof as described in any of the above aspects.
  • the presence of a nucleic acid molecule unique to maize event 2A-7 is indicated in the sample.
  • the maize event 2A-7 comprises exogenous nucleic acid molecules encoding mcry1Ab and mcry2Ab incorporated into its genome, the exogenous nucleic acid molecules flanking 5' flanking regions and 3' flanking regions, comprising The sequence of the part of the 5' flanking region and the adjacent exogenous nucleic acid molecule is shown in SEQ ID NO: 1, and the sequence of the part comprising the 3' flanking region and the adjacent exogenous nucleic acid molecule is shown in SEQ ID NO: :2 shown.
  • the maize event 2A-7 comprises the sequence set forth in any one of SEQ ID NOs: 1-5 or the complement thereof in its genome.
  • the nucleic acid probe comprises, or is at least 80% (eg, 80%-100%) the sequence of the 5' flanking region and a portion of the exogenous nucleic acid molecule adjacent thereto. % or 90%-100%, eg, at least 85%, at least 90%, at least 95%, at least 99%, or 100%) sequence identity.
  • the nucleic acid probe has at least 80% (eg, 80%-100% or 90%) of the sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2 or its complement or fragment of any one. %-100%, eg, at least 85%, at least 90%, at least 95%, at least 99%, or 100%) sequence identity.
  • the nucleic acid probe comprises the sequence set forth in SEQ ID NO: 1 or 2 or the complement thereof.
  • the nucleic acid probes are fluorescently labeled, such as Taqman probes.
  • primer is an isolated nucleic acid that is annealed to a complementary target DNA strand by nucleic acid hybridization to form a hybrid between the primer and the target DNA strand, and then passed through a polymerase (e.g. DNA polymerase) is elongated along the target DNA strand.
  • a polymerase e.g. DNA polymerase
  • Pairs or sets of primers can be used for amplification of nucleic acid molecules, eg, by polymerase chain reaction (PCR) or other conventional nucleic acid amplification methods.
  • nucleic acid probe is an isolated nucleic acid linked to a conventional detectable label or reporter molecule, eg, a radioisotope, ligand, chemiluminescent agent, or enzyme. Such a probe complements one strand of the target nucleic acid, in the case of the present invention, hybridizes to one strand of genomic DNA from maize event 2A-7. Probes of the present invention include not only deoxyribonucleic acid or ribonucleic acid, but also polyamide and other probe materials that specifically bind to a target DNA sequence and can be used to detect the presence of the target DNA sequence.
  • PCR primer pairs can be obtained from known sequences, for example, by using computer programs intended for this purpose, such as Primer (version 0.5, 1991, Whitehead Institute for Biomedical Research, Cambridge, MA).
  • nucleic acid probes and primers of the present invention hybridize to target DNA sequences under stringent conditions. Any conventional nucleic acid hybridization or amplification method can be used to identify the presence of DNA for transgenic events in a sample.
  • two nucleic acid molecules are said to be capable of specifically hybridizing to each other if they can form an antiparallel double-stranded nucleic acid structure.
  • a nucleic acid molecule is said to be the "complement (complementary sequence)" of another nucleic acid molecule if it exhibits complete complementarity.
  • molecules are said to exhibit "perfect complementarity" when every nucleotide of one molecule is complementary to a nucleotide of another molecule.
  • Two molecules are said to be “minimally complementary” if they can hybridize to each other with sufficient stability to keep them annealed to each other under at least conventional "low stringency” conditions.
  • molecules are said to be “complementary” if they hybridize to each other with sufficient stability to keep them annealed to each other under conventional "high stringency” conditions.
  • Conventional stringent conditions are described by Sambrook et al., 1989 and Haymes et al., in: Nucleic AcidHybridization, A Practical Approach, IRL Press, Washington, DC (1985). Therefore, deviations from perfect complementarity are allowed, as long as these deviations do not completely preclude the ability of the molecule to form double-stranded structures.
  • a nucleic acid molecule In order for a nucleic acid molecule to be used as a primer or probe, it need only be sufficiently complementary in sequence to be able to form a stable double-stranded structure under the particular solvent and salt concentration used.
  • Suitable stringent conditions to promote DNA hybridization such as 6.0x sodium chloride/sodium citrate (SSC) at about 45°C, followed by a 2.0x SSC wash at 50°C, are known to those skilled in the art, or can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6.
  • the salt concentration in the wash step can be selected from a low stringency of 50°C, about 2.0 ⁇ SSC, to a high stringency of 50°C, about 0.2 ⁇ SSC.
  • the temperature of the wash step can also be increased from low stringency conditions at room temperature of about 22°C to high stringency conditions at about 65°C.
  • Both temperature and salt concentration may be variable, or either temperature or salt concentration may be held constant while the other variable is changed.
  • the probes or primers of the invention specifically hybridize to target sequences under moderately stringent conditions (eg, about 2.0 x SSC and about 65°C). In certain embodiments, the probes or primers of the invention specifically hybridize to target sequences under conditions of high stringency.
  • the probes of the invention are at least 80% (eg, 80%-100%) of the nucleic acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2, or the complement thereof, or a fragment of either or 90%-100%, such as at least 85%, at least 90%, at least 95%, at least 99%, or 100%) sequence identity.
  • Hybridization of probes to target DNA molecules can be detected by any of a number of methods known to those of skill in the art, which may include, but are not limited to, fluorescent labeling, radiolabeling, antibody-based labeling, and chemiluminescent labeling Law.
  • stringent conditions refer to conditions that in a DNA thermal amplification reaction cause the primer pair to interact only with the target nucleic acid sequence (with the corresponding wild A primer of the type sequence (or its complement) should hybridize to the target nucleic acid sequence) and preferably yield a unique amplification product (amplicon).
  • target sequence telomere sequence
  • amplicon refers to a nucleic acid amplification product of a target nucleic acid sequence that is part of a nucleic acid template.
  • primers comprising flanking sequences derived from the plant genome immediately adjacent to the insertion site of the heterologous DNA and derived from A primer pair of a second primer that inserts heterologous DNA
  • the nucleic acid amplification method is performed on DNA extracted from a corn plant tissue sample to generate an amplicon that diagnoses the presence of event DNA.
  • the amplicons are of a certain length and have sequences that also diagnose the event.
  • the length of the amplicon can be plus one nucleotide base pair, preferably about 50 nucleotide base pairs, more preferably about 250 nucleotide base pairs, even more preferably about 450 nucleotide base pairs in the primer pair.
  • primer pairs can be derived from flanking sequences on both sides of the inserted DNA so as to generate an amplicon containing the entire inserted nucleotide sequence.
  • the members of the primer pair from the plant genomic sequence can be positioned at a distance from the inserted DNA molecule, which distance can range from one nucleotide base pair up to about twenty thousand nucleotide base pairs.
  • the use of the term "amplicon" specifically excludes primer dimers that can be formed in DNA thermal amplification reactions.
  • Nucleic acid amplification can be accomplished by any of a variety of nucleic acid amplification methods known in the art, including the polymerase chain reaction (PCR).
  • PCR polymerase chain reaction
  • Various amplification methods are known in the art and are described, inter alia, in U.S. Patent Nos. 4,683,195 and 4,683,202 and PCR Protocols: A Guide to Methods and Applications, eds. Innis et al., Academic Press, San Diego, 1990.
  • PCR amplification methods have been developed to amplify up to 22 kb of genomic DNA and up to 42 kb of phage DNA (Cheng et al., Proc. Natl. Acad. Sci. USA 91:5695-5699, 1994).
  • Heterologous DNA inserts or flanking sequences from maize events 2A-7 can be examined by amplifying these sequences from the events using primers derived from the sequences provided herein, followed by standardization of the PCR amplicons or cloned DNA DNA sequencing.
  • Amplicons produced by these methods can be detected by a variety of techniques.
  • One such method is Genetic Bit Analysis (Nikiforov et al., Nucleic Acid Res. 22:4167-4175, 1994), in which DNA oligos are designed that overlap both adjacent flanking genomic DNA sequences and insert DNA sequences. Nucleotides. The oligonucleotides were immobilized in the wells of a microtiter plate.
  • the single-stranded PCR product can be hybridized to the immobilized oligonucleotide and used as a template for polymerization using DNA Enzyme and single base extension reaction of labeled ddNTPs specific for the next desired base.
  • Readers can be fluorescent or ELISA based. Signals indicate the presence of insert/flanking sequences due to successful amplification, hybridization and single base extension.
  • oligonucleotides are designed that overlap adjacent genomic DNA and intervening DNA junction regions.
  • the oligonucleotides were hybridized to the single-stranded PCR product from the region of interest (one primer on the insert and the other primer on the flanking genomic sequence), and mixed with DNA polymerase, ATP, sulfurylase, luciferase, adenosine triphosphate double Incubation was performed in the presence of phosphatase, adenosine 5' phosphate sulfate and luciferin.
  • the dNTPs were added alone, and the optical signal resulting from the incorporation was measured. The light signal indicates the presence of transgene insert/flanking sequences due to successful amplification, hybridization and single or multiple base extension.
  • Fluorescence polarization is one method that can be used to detect the amplicons of the invention. Use this method to design oligonucleotides that overlap the genomic flanking and intervening DNA junction regions. Oligonucleotides were hybridized to the single-stranded PCR product from the region of interest (one primer on the insert DNA sequence and the other primer on the flanking genomic DNA sequence) and incubated in the presence of DNA polymerase and fluorescently labeled ddNTPs. A single base extension results in the incorporation of ddNTPs. Incorporation can be measured in terms of polarization changes using a fluorometer. Polarization changes indicate the presence of transgene insert/flanking sequences due to successful amplification, hybridization and single base extension.
  • the Taqman probe method (PE Applied Biosystems, Foster City, CA) is described as a method for detecting and quantifying the presence of DNA sequences and is well understood by the manufacturer's instructions. Briefly, FRET oligonucleotide probes were designed that overlap the genomic flanking and intervening DNA junction regions. The FRET probe and PCR primers (one primer on the insert DNA sequence and one primer on the flanking genomic sequence) are cycled in the presence of a thermostable polymerase and dNTPs. FRET probe hybridization results in cleavage of the fluorescent moiety on the FRET probe from the quenching moiety and release of the fluorescent moiety. Fluorescent signals indicate the presence of flanking/transgenic inserts due to successful amplification and hybridization.
  • FRET oligonucleotide probes were designed that overlap the flanking genome and insert DNA junction regions.
  • the unique structure of FRET probes allows them to contain secondary structures that keep the fluorescent and quenching moieties in close proximity.
  • the FRET probe and PCR primers are cycled in the presence of a thermostable polymerase and dNTPs.
  • fluorescent signals indicate the presence of flanking/transgenic inserts due to successful amplification and hybridization.
  • the sample can be any sample containing corn event 2A-7 DNA, so long as the sample contains at least a detectable amount of polynucleotides that diagnose the presence of event 2A-7 in the sample (ie a polynucleotide unique to maize event 2A-7).
  • the sample may be selected from the group consisting of corn ear ears, husk corn, corn silk, corn pollen, corn grits, corn meal, crushed corn, corn flour, corn oil, corn starch, corn steep liquor, Corn malt, corn sugar, corn syrup, margarine produced from corn oil, unsaturated corn oil, saturated corn oil, corn flakes, popcorn, ethanol and/or liquor produced from corn, produced by fermentation of corn Dried distiller's grains (DDGS), animal feed from corn, cosmetics and fillers.
  • DDGS corn Dried distiller's grains
  • kits for identifying corn events 2A-7 comprising the above-described nucleic acid probes and/or primers that specifically bind to any of SEQ ID NOs: 1-5 under high stringency conditions
  • the target sequences shown in item hybridize are shown in item hybridize.
  • the kits also include and other materials necessary to enable nucleic acid hybridization or amplification methods to be performed.
  • the present invention also relates to the use of a primer, primer pair, nucleic acid probe or kit according to any of the above aspects for the detection of nucleic acid molecules unique to maize events 2A-7 in a sample comprising maize nucleic acid, or for the detection of maize The purpose of the existence of Incident 2A-7.
  • the present invention also relates to an isolated nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of the sequence shown in any one of SEQ ID NOs: 1-5 or the complement thereof.
  • the present invention provides a method of producing an insect-resistant corn plant, comprising:
  • progeny plant contains the sequence shown in any one of SEQ ID NOs:1-5 or its complementary sequence in its genome;
  • nucleic acid probe specific for the sequence shown in any one of SEQ ID NOs: 1-5 or its complement for example, a nucleic acid comprising the sequence shown in SEQ ID NO: 1 or 2 or its complement
  • Probes can detect hybridization when the genomic DNA of progeny plants is detected.
  • the first or second parental maize plant comprises exogenous nucleic acid molecules encoding mcry1Ab and mcry2Ab incorporated into its genome, the exogenous nucleic acid molecules flanking 5' flanking regions and 3' flanking regions Region, the sequence that comprises 5 ' flanking region and the part of exogenous nucleic acid molecule adjacent to it is as shown in SEQ ID NO: 1, the sequence that comprises 3 ' flanking region and the part of exogenous nucleic acid molecule adjacent to it is as follows shown in SEQ ID NO:2.
  • the first or second parental maize plant comprises the sequence set forth in any one of SEQ ID NOs: 1-5 or the complement thereof in its genome.
  • the method further comprises the steps of:
  • step (3) (4) selfing the progeny plants obtained in step (3), thereby producing a plurality of second-generation progeny plants;
  • the present invention provides a method of producing hybrid corn seed that can be grown into an insect-resistant corn plant, comprising: crossing a first parental corn plant with a second parental corn plant and harvesting the resulting hybrid seed, wherein the first parental maize plant and/or the second parental maize plant is as defined in the first or second aspect.
  • the first parental maize plant and/or the second parental maize plant comprises exogenous nucleic acid molecules encoding mcry1Ab and mcry2Ab incorporated into its genome, the exogenous nucleic acid molecules flanked by 5' flanks Region and 3 ' flanking region, the sequence of the part comprising 5 ' flanking region and the exogenous nucleic acid molecule adjacent to it is as shown in SEQ ID NO: 1, comprising 3 ' flanking region and the exogenous nucleic acid molecule adjacent to it The part of the sequence is shown in SEQ ID NO:2.
  • the first parental maize plant and/or the second parental maize plant comprises the sequence set forth in any one of SEQ ID NOs: 1-5 or the complement thereof in its genome.
  • the method includes:
  • the first inbred maize line comprises exogenous nucleic acid molecules encoding mcry1Ab and mcry2Ab incorporated into its genome, the exogenous nucleic acid molecules flanking 5' flanking regions and 3' flanking regions, The sequence of the part comprising the 5' flanking region and the adjacent exogenous nucleic acid molecule is shown in SEQ ID NO: 1, and the sequence of the part comprising the 3' flanking region and the adjacent exogenous nucleic acid molecule is shown in SEQ ID NO:2 shown.
  • the first inbred maize line comprises the sequence set forth in any one of SEQ ID NOs: 1-5 or the complement thereof in its genome.
  • the first inbred corn line provides a female parent. In certain embodiments, the first inbred corn line provides a male parent.
  • the insects may be selected from Lepidopteran insects, such as armyworm, corn borer (such as Asian corn borer), cotton bollworm, peach borer, Spodoptera frugiperda and other scales One or more of the order Pterodactyl pests.
  • transgenic genotype of 2A-7 can be introgressed by breeding into other maize lines containing different transgenic genotypes, thereby achieving trait accumulation in the resulting seeds and progeny plants and conferring enlargement to these plants activity spectrum.
  • the transgenic genotypes of the present invention can be introgressed into any maize inbred or hybrid using art-recognized breeding techniques.
  • the goal of plant breeding is to combine different desirable traits in a single variety or hybrid.
  • these characteristics may include resistance to insects and diseases, tolerance to herbicides, tolerance to heat and drought, reduced time for crops to mature, greater yields, and better agronomy quality.
  • uniformity of plant characteristics such as germination and clump establishment, growth rate, maturity, and plant and ear height is important.
  • Field crops are cultivated through techniques that utilize plant pollination methods.
  • a plant is self-pollinating if pollen from a flower is transferred to the same flower or to another flower of the same plant.
  • a plant is cross-pollinated if the pollen comes from flowers of a different plant.
  • Maize can be cultivated by both self-pollination and cross-pollination techniques. Maize has separate male and female flowers on the same plant, on the tassel and ear ears, respectively. Natural pollination of maize occurs when wind blows pollen from the tassels to the filaments that protrude from the top of the ear ears.
  • Reliable methods of controlling male fertility in plants offer opportunities for improved plant breeding. This is especially true for the development of corn hybrids that rely on certain male sterility systems. There are several options available for breeders to control male fertility, for example, manual or mechanical emasculation (or tasselling), cytoplasmic male sterility, genetic male sterility, gametocides, etc. .
  • Hybrid corn seed is typically produced by a male sterility system incorporating manual or mechanical tasseling.
  • Plant breeding techniques known in the art and used in maize plant breeding programs include, but are not limited to, regression selection, backcrossing, pedigree breeding, limited length polymorphism enhanced selection, genetic marker enhanced selection, and transformation.
  • corn refers to maize or maize and includes all plant species that can be cultivated with maize, including wild maize species.
  • Cry2Ab gene has a meaning well known to those skilled in the art, which is an endotoxin gene found in the entomopathogenic bacterium Bacillus thuringiensis (Bt), and can be found in various public databases (eg, GenBank: M23724).
  • the inventors optimized the codons of Cry2Ab for the sequence characteristics of monocotyledonous plants, and obtained a sequence (mCry2Ab) that is particularly suitable for expression in maize cells.
  • the sequence of the mCry2Ab is shown in SEQ ID NO:13, See also Chinese patent application CN201410483143.2.
  • Cry1Ab gene has a meaning well known to those skilled in the art, which is an endotoxin gene found in the entomopathogenic bacterium Bacillus thuringiensis (Bt), and can be found in various public databases (eg, GenBank: M13898).
  • the inventors optimized the codons of Cry1Ab for the sequence characteristics of monocotyledonous plants, and obtained a sequence (mCry1Ab) that is particularly suitable for expression in maize cells.
  • the sequence of the mCry1Ab is shown in SEQ ID NO: 12, See also Chinese patent application CN201710701976.5.
  • Bar gene has the meaning well known to those skilled in the art and is a herbicide resistance gene derived from Streptomyces hygroscopicus. Bar gene is often used as a selectable marker gene, which can produce phosphorylated acetyltransferase, which can acetylate the free amino group of glufosinate herbicides to achieve the purpose of detoxification, thereby facilitating the selection of positive callus in genetic transformation. Bar gene sequences are known in the art, see, eg, Thompson CJ et al., EMBO J. 1987 Sep;6(9):2519-23. In certain exemplary embodiments, the Bar gene has the sequence set forth in SEQ ID NO:14.
  • the terms “corn event,” “event,” “transgenic event,” are used interchangeably and refer to the use of heterologous DNA (eg, including an expression cassette of a related gene) Recombinant plants produced by transformation and regeneration of plant cells or tissues.
  • the term “event” includes the original transformant of heterologous DNA and/or the progeny of the transformant.
  • the term “event” also includes progeny produced by sexual outcrossing between a transformant and another maize line. Even after repeated backcrossing with the recurrent parent, the insert DNA and flanking DNA from the transformed parent are present at the same chromosomal location in the progeny of the hybrid.
  • event also includes DNA from the original transformant, comprising the insert DNA and flanking genomic sequences immediately adjacent to the insert DNA, which is expected to be transferred to progeny as comprising the insert DNA (e.g., the The inserted DNA containing the relevant transgene is received as a result of sexual crosses of the parental line of the original transformant and progeny resulting from selfing) with a parental line that does not contain the inserted DNA.
  • transformation of plant tissue produces multiple events, each event representing the insertion of a DNA construct at a different location in the plant cell's genome. Particular events are selected based on the expression of the transgene or other desired characteristics.
  • “event 2A-7", “2A-7” or “2A-7 event” may be used interchangeably.
  • transformation is the process of introducing a heterologous nucleic acid into a host cell or organism. Specifically, “transformation” refers to the stable integration of a DNA molecule into the genome of a related organism.
  • transformed/transgenic/recombinant refers to a host organism, such as a bacterium or plant, into which a heterologous nucleic acid molecule has been introduced.
  • the nucleic acid molecule can be stably integrated into the genome of the host or the nucleic acid molecule can also exist as an extrachromosomal molecule. Such extrachromosomal molecules can replicate automatically.
  • Transformed cells, tissues or plants are understood to include not only the final product of the transformation process, but also transgenic progeny thereof.
  • non-transformed refers to a wild-type organism, ie, a bacterium or plant, which does not contain a heterologous nucleic acid molecule.
  • transgenic refers to a plant, plant cell, or a plurality of structured or unstructured plant cells into which a nucleic acid sequence representing a gene of interest has been incorporated by well-known genetic manipulation and gene insertion techniques.
  • Transgenic plants result from manipulation and insertion of such nucleic acid sequences (as opposed to naturally occurring mutations) to produce non-naturally occurring plants or plants with non-naturally occurring genotypes. Transformation techniques for plants and plant cells are well known in the art and may include, for example, electroporation, microinjection, Agrobacterium-mediated transformation, and ballistic transformation.
  • nucleic acids unique to event 2A-7 refers to a characteristic unique to maize event 2A-7.
  • nucleic acids unique to event 2A-7 were not found in other non-2A-7 maize plants.
  • nucleic acids unique to events 2A-7 can be selected from SEQ ID NOs: 1-5.
  • the term "genotype” is the genetic material inherited from a parental maize plant, not all of which is necessarily expressed in the progeny maize plant.
  • the expression “2A-7 genotype” refers to the exogenous genetic material transformed into the genome of the plant in maize event 2A-7 as well as the genetic material that flanks the inserted sequence.
  • identity is used to refer to the match of sequences between two polypeptides or between two nucleic acids.
  • a position in both sequences being compared is occupied by the same base or amino acid monomer subunit (e.g., a position in each of two DNA molecules is occupied by an adenine, or both A position in each of the polypeptides is occupied by a lysine)
  • the molecules are identical at that position.
  • the "percent identity” between two sequences is a function of the number of matched positions shared by the two sequences divided by the number of positions compared x 100. For example, two sequences are 60% identical if 6 out of 10 positions match.
  • the DNA sequences CTGACT and CAGGTT share 50% identity (matching at 3 positions out of a total of 6).
  • comparisons are made when two sequences are aligned for maximum identity.
  • Such alignment can be accomplished using, for example, the method of Needleman et al. (1970) J. Mol. Biol. 48:443-453, which can be conveniently performed by a computer program such as the Align program (DNAstar, Inc.).
  • Align program DNAstar, Inc.
  • Appl Biosci., 4:11-17 (1988)) integrated into the ALIGN program (version 2.0) can also be used, using the PAM120 weight residue table , a gap length penalty of 12, and a gap penalty of 4 to determine the percent identity between two amino acid sequences.
  • the algorithm of Needleman and Wunsch (J MoI Biol. 48:444-453 (1970)) in the GAP program integrated into the GCG software package (available at www.gcg.com), using the Blossum 62 matrix or PAM250 matrix with gap weights of 16, 14, 12, 10, 8, 6, or 4 and length weights of 1, 2, 3, 4, 5, or 6 to determine percent identity between two amino acid sequences .
  • the event 2A-7 provided by the present invention can significantly resist the invasion of Lepidopteran insects and has good herbicide resistance. At the same time, 2A-7 has excellent genetic stability, safety, and agronomic performance comparable to commercially available varieties (eg Zheng 58) without insect stress. Events 2A-7 of the present invention have broad application prospects and achieve positive social and ecological benefits.
  • Figure 1 shows the pCAMBIA3301+mcry1Ab+mcry2Ab vector map.
  • Figure 2 shows the electrophoresis results of the PCR products of the internal reference gene in Example 2.
  • lane 1 industrialized transgenic corn
  • lane 2 industrial transgenic soybean
  • lane 3 industrial transgenic rape
  • lane 4 industrial transgenic cotton
  • lane 5 industrial transgenic rice
  • lane 6 transgenic corn 2A-5
  • Lane 7 Transgenic corn 2A-7
  • Lane 8 Transgenic corn 2A-7
  • Lane 9 Transgenic corn 2A-7
  • Lane 10 Transgenic corn 2A-7
  • Lane 11 Non-transgenic recipient control
  • Lane 12 Blank Control
  • lane 13 positive control T+zSSIIb (Zm00001d052263) plasmid (10 pg)
  • lane M molecular weight marker DL2000plus.
  • Figure 3 shows the electrophoresis results of PCR products of the 5' flanking region-specific reaction system in Example 2.
  • lane 1 industrialized transgenic corn
  • lane 2 industrial transgenic soybean
  • lane 3 industrial transgenic rape
  • lane 4 industrial transgenic cotton
  • lane 5 industrial transgenic rice
  • lane 6 transgenic corn 2A-5
  • Lane 7 Transgenic corn 2A-7
  • Lane 8 Transgenic corn 2A-7
  • Lane 9 Transgenic corn 2A-7
  • Lane 10 Transgenic corn 2A-7
  • Lane 11 Non-transgenic recipient control
  • Lane 12 Blank Control
  • lane 13 previously cloned T+2A-7 5' plasmid (10 pg) containing 5' end flanking sequence and insert
  • Lane M molecular weight marker DL2000plus.
  • Figure 4 shows the electrophoresis results of PCR products of the 3' flanking region-specific reaction system in Example 2.
  • Lane 1 Industrial GMO corn; Lane 2: Industrial GMO soybean; Lane 3: Industrial GMO rape; Lane 4: Industrial GMO cotton; Lane 5: Industrial GMO rice; Lane 6: GMO corn 2A-5; 7: Transgenic maize 2A-7; lane 8: transgenic maize 2A-7; lane 9: transgenic maize 2A-7; lane 10: transgenic maize 2A-7; lane 11: non-transgenic recipient control; lane 12: blank control; Lane 13: T+2A-7 3' plasmid plasmid (10 pg) containing 3' end flanking sequence and insert sequence; Lane M: molecular weight marker DL2000plus.
  • FIG. 5 shows the results of identification of 2A-7 resistance to armyworms in Example 3.
  • FIG. 6A shows the results of identification of the resistance of 2A-7 ears to corn borer in Example 3.
  • FIG. 6B shows the results of the identification of 2A-7 stalks in Example 3 for corn borer resistance. Among them, from left to right are 2A-7, Zheng 58, and Zheng Dan 958.
  • FIG. 7 shows the results of identification of 2A-7 panicle resistance to Helicoverpa armigera in Example 3.
  • FIG. 8 shows the results of the indoor bioassay against Spodoptera frugiperda by leaves of 2A-7 in Example 3.
  • the upper part is the control Zheng 58 leaf
  • the lower part is the 2A-7 leaf.
  • FIG. 9 shows the results of the indoor bioassay of 2A-7 filaments in Example 3 on Spodoptera frugiperda.
  • the upper part is the control Zheng 58 filament
  • the lower part is the 2A-7 filament.
  • Figure 10 shows the field bioassay results of 2A-7 against Spodoptera frugiperda in Example 3. Among them, the left side is 2A-7, and the right side is the control Zheng 58.
  • the present invention relates to the following biological materials that have been preserved in China General Microorganism Culture Collection and Management Center (CGMCC) (No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing):
  • the seeds of corn (Zea mays) 2A-7 which have the deposit number CGMCC NO.17848, and the deposit date is October 28, 2019.
  • the selectable marker gene bar (SEQ ID NO:14), target genes mCry1Ab (SEQ ID NO:12) and mCry2Ab (SEQ ID NO:13) and their supporting regulatory elements were inserted into pCAMBIA3301 (purchased from Hunan Fenghui Biotechnology Co., Ltd. ) in the T-DNA of ), construct a plant expression vector according to conventional molecular biology methods, named pCAMBIA3301+mCry1Ab+mCry2Ab, the supporting regulatory elements of each inserted gene are shown in the following table, the map is shown in Figure 1, wherein the promoter Gly can be See Chinese patent application CN201710702435.4. Positive clones were identified by enzyme digestion and bidirectional sequencing for maize transformation.
  • the above-mentioned vector was transformed into Agrobacterium EHA105 (purchased from Beijing Huayueyang Biotechnology Co., Ltd.), and the young embryos of maize were infected with the Agrobacterium containing the target gene, and the specific transgenic method was as follows:
  • the recipients used in the transgenic process were the F1 generation of the cross of the inbred lines HiIIA and HiIIB (publicly available from the "Maize Genetic Resources Stock Center” (Maize GDB, 2010)).
  • the inbred lines HiIIA and HiIIB are planted in the field, and the inbred lines are bagged when the inbred lines are loose; then ready for pollination, there are two pollination methods: HiIIA as the female parent, HiIIB as the male parent; HiIIA as the male parent, HiIIB as the female parent , 9-11 days after pollination, take the immature embryos on the grains of the pollinated ear, and then carry out Agrobacterium infection indoors.
  • transgenic T0 generation plants After the transgenic T0 generation was obtained, the pollen of the T0 generation transgenic plants was used to cross some female parent for seed production, such as Zheng 58.
  • the inserted sequence was introduced into young embryos of recipient plants by Agrobacterium infection, and transgenic plants were obtained after screening with the herbicide bialaphos. A total of more than 2000 T0 generation transformants were obtained through multiple transformations.
  • mcry1Ab and mcry2Ab protein content the screening of the copy number of inserts, the stability detection of successive generations, and the identification of agronomic traits in different locations, mcry1Ab and mcry2Ab were obtained. High-expressing, single-copy, genetically stable maize transformant 2A-7.
  • the method is as follows: take fresh maize leaves to extract maize genomic DNA. TAIL-PCR was performed, involving a total of 5 degenerate primers: LAD1-1, LAD1-2, LAD1-3, LAD1-4, AC1, 6 specific primers: RB-0a, RB-1a, RB-2a, LB -0a, LB-1a, LB-2a, the sequences of which are shown in the table below.
  • N A/T/C/G
  • B G/T/C
  • V A/G/C
  • D A/G
  • the PCR product was ligated with B vector (Beijing Quanshijin Biotechnology Co., Ltd. CB101-01), and the ligated product was transformed into E. coli competent cells. Single clones were selected for PCR identification, and the amplified products were sent to Beijing Aoke Dingsheng Biotechnology Co., Ltd. for sequencing. The sequencing results were compared with the T-border sequence and the maize genome, and the integration of exogenous fragments was analyzed.
  • B vector Beijing Quanshijin Biotechnology Co., Ltd. CB101-01
  • the 2A-7 transformant is inserted between Chr3: 179141694bp-179141724bp (B73 reference genome V4 version), and the T-border is integrated into the genome sequence As shown in nucleotides 483-8524 of SEQ ID NO:5, the composition of SEQ ID NO:5 is shown in the table below.
  • the exogenous sequence is flanked by a 5' flanking region and a 3' flanking region, the 5' flanking region having nucleotides 1-432 of SEQ ID NO: 5, the 3' flanking region
  • the 'flanking region has nucleotides at positions 8532-9031 of SEQ ID NO:5.
  • the 5' junction sequence covering part of the 5' flanking region and part of the non-genomic sequence is shown in SEQ ID NO: 1 (corresponding to 423-442 of SEQ ID NO: 5), covering part of the non-genomic sequence and part of the 3' flanking region.
  • the 3' junction sequence is shown in SEQ ID NO:2 (corresponding to 8522-8541 of SEQ ID NO:5).
  • the unintended integration sequence is neither the genome sequence nor the T-border sequence, which may be the sequence generated by genome repair when the insert is integrated; (2) The unmarked position in the table is the intergenic sequence in the T-border region .
  • CGMCC General Microorganism Culture Collection and Management Center
  • the 5' and 3' flanking sequences of events 2A-7 are shown in nucleotides 1-432 and 8532-9031 of SEQ ID NO:5, respectively.
  • the forward and reverse primers were respectively involved (Table 5-6), and the PCR reaction was carried out, and the reaction conditions and reaction system were respectively shown in the following Tables 7-8.
  • the maize endogenous gene zSSIIb (Zm00001d052263) was used as the internal reference gene
  • the forward primer zSSIIb-F was shown in SEQ ID NO:10
  • the reverse primer zSSIIb-R was shown in SEQ ID NO:11.
  • PCR amplification products were detected by electrophoresis on an ethidium bromide-stained agarose gel (3%). Appropriate molecular mass standards were added during electrophoresis to determine the size of the amplification products, and the PCR amplification products were visualized using a gel imaging system.
  • genomic DNA extracted from the following samples was used as a template for PCR amplification of the 5' and 3' ends of the maize transformant 2A-7 specific system and the internal standard system to determine the specificity of the method: 4 species Different maize plants containing transformants 2A-7, maize transformants 2A-5, industrialized transgenic corn, industrialized transgenic soybean, industrialized transgenic cotton, industrialized transgenic rice. in:
  • the PCR amplification process can be completed only when the two binding regions are adjacent.
  • the integration of T-DNA is random.
  • the binding regions of other transformants are almost impossible to adjoin, and even if they are contiguous, the product will not match the expected size. Therefore, the above-mentioned 5'-end primer pair and 3'-end primer can be used to detect the maize transformant 2A-7.
  • DNA probes that specifically hybridize thereto can also be used to detect the presence of the 5' junction sequence or the 3' junction sequence to identify the 2A-7 maize event.
  • Transgenic insect-resistant maize 2A-7T5 generation the breeding process is to use Agrobacterium containing the target vector to infect the F1 generation immature embryos of the hybridization of HiIIA and HiIIB, and after obtaining the T0 generation transgenic plants, use Zheng as the recurrent parent to carry out After crossing and backcrossing, after obtaining T3 generation, self-crossing for 2 generations was performed to obtain 2A-7 of homozygous Zheng 58 background;
  • the quality of the above materials meets the requirements of GB4404.1 not lower than the second grade corn seeds.
  • Asian corn borer Ostrinia furnacalis the newly hatched larvae of the corn borer (2-12h incubation time) are the Asian corn borer population that is artificially reared indoors;
  • Cotton bollworm Helicoverpa armigera The newly hatched larvae of cotton bollworm (12-24h incubation time) are artificially reared Helicoverpa armigera populations indoors.
  • Spodoptera frugiperda Spodoptera frugiperda newly hatched larvae (incubation time 12-24h) are indoor artificially reared Spodoptera frugiperda populations.
  • the isolation of the experimental site was carried out by means of isolation for more than 25 days during the flowering period within 300 meters.
  • the field experiment was designed with random block design and repeated three times.
  • the plot area was 30 m 2 (5 m ⁇ 6 m), the row spacing was 60 cm, and the plant spacing was 25 cm.
  • the soil fertility level and tillage management were the same as those in field production. There is an interval of 2m between different pests receiving test plots to avoid the spread of pests between different plots.
  • the field should have a certain humidity before and after the infestation. In case of drought, it should be watered in time.
  • Insect inoculation method The identification of resistance to armyworm was carried out at the heart-leaf stage, and the maize plant developed to the stage of 4-6 leaves, and no less than 40 plants were artificially inoculated in each plot. Each plant receives 30 to 40 newly hatched larvae that are artificially raised, and then inoculates it in the leaves of corn heart. After 3 days of inoculation, the second inoculation is carried out. The number and method of inoculation are the same as the first time.
  • Investigation record 14 days after infestation, the damage re-reading and the number of larvae survived by armyworms on corn leaves were investigated.
  • Result expression According to the damage degree of corn leaves by armyworm, calculate the average value of the damage level (leaf-eating level) of corn leaves by armyworm in each plot. Level of resistance to armyworms.
  • Leaf level Symptom description 1 No damage to the leaves, or only needle-like ( ⁇ 1mm) worm holes on the leaves 2 Only a small number of bullet holes ( ⁇ 5mm) worm holes on individual leaves 3 There are bullet holes ( ⁇ 5mm) on a few leaves 4 Notches on individual blades ( ⁇ 10mm)
  • Inoculation method Identification of resistance to the target pest Asiatic corn borer. Distracted leaf stage generation and ear stage generation, which were carried out at the heart leaf stage (small trumpet stage, the maize plant developed to the 8-leaf to 10-leaf stage) and silking stage, respectively. Artificial inoculation, 2 times in each period, no less than 40 plants in each plot in each period, and 60 to 80 newly hatched larvae of Asian corn borer per plant respectively. Insect picking should be carried out in the evening. If the weather is above moderate rain after picking up the insects, it is necessary to pick up the insects once more.
  • Table 11 Grading standard of damage degree of corn borer to heart leaves
  • a few leaves have 3 to 6 worm holes with a diameter of ⁇ 2mm 6 Some leaves have more than 7 worm holes with a diameter of ⁇ 2mm 7 There are 1 to 2 wormholes with a diameter of >2mm on a few leaves 8 Some leaves have 3 to 6 worm holes > 2mm in diameter 9 Most leaves have more than 7 wormholes > 2mm in diameter
  • Average leaf-eating grade ⁇ (leaf-eating grade ⁇ number of plants at this grade)/total number of plants under investigation
  • Table 12 Evaluation criteria for maize resistance to maize borer
  • Investigation record of silking period Investigate the damage of corn ear, the number of boreholes, the length of borehole tunnel (cm), the age and number of surviving larvae, and evaluate the damage degree of ear and plant damage.
  • Table 13 Grading standard of damage degree of corn borer by Asian corn borer at ear stage
  • Table 14 Evaluation criteria for resistance of maize ears to Asian corn borer
  • Method of inoculation The identification of resistance to cotton bollworm was carried out in the stage of spinning and powdering, and each plant was inoculated with 20 to 30 newly hatched larvae. No less than 40 worms were artificially inoculated in each plot, and they were attached to corn filigree. After 3 days of inoculation, the second inoculation was carried out, and the number of inoculations was the same as the first time. Insect picking should be carried out in the evening. If the weather is above moderate rain after picking up the insects, it is necessary to pick up the insects once more.
  • Table 15 Grading standard of damage degree of cotton bollworm at ear stage of corn
  • Table 16 Evaluation criteria for resistance of maize ears to cotton bollworm
  • Inoculation method identification of resistance to the target pest Spodoptera frugiperda Distracted leaf stage generation and ear stage generation, respectively at the heart leaf stage (small bell mouth stage, maize plants develop to the 8-leaf to 10-leaf stage) and silking stage Artificial inoculation was carried out, 2 times in each period, no less than 40 plants were artificially inoculated in each plot in each period, and 20 to 30 newly hatched larvae of Spodoptera frugiperda were received from each plant. Insect picking should be carried out in the evening. If the weather is above moderate rain after picking up the insects, it is necessary to pick up the insects once more.
  • Table 17 Grading standard of the damage degree of corn leaves by Spodoptera frugiperda
  • Leaf level Symptom description 1 No damage to the leaves, or only needle-like ( ⁇ 1mm) worm holes on the leaves 2 Only a small number of bullet holes ( ⁇ 5mm) worm holes on individual leaves 3 There are bullet holes ( ⁇ 5mm) on a few leaves 4 Notches on individual blades ( ⁇ 10mm) 5 There are nicks on a few leaves ( ⁇ 10mm) 6 Some blades have nicks ( ⁇ 10mm)
  • Table 18 Evaluation criteria for maize resistance to Spodoptera frugiperda
  • Investigation record of silking period Investigate the damage of corn ear, the number of borers, the length of borehole tunnel (cm), the age and number of surviving larvae, and evaluate the damage degree of ear and plant damage.
  • Table 19 Grading standard of the damage degree of Spodoptera frugiperda at ear stage of corn
  • Table 20 Evaluation criteria for resistance of maize ears to Spodoptera frugiperda
  • Table 21 Survey results of the damage level of leaves by armyworms
  • the ear stalk plays an important role in grain development.
  • the ear stalk supports the ear and provides an important guarantee for mechanized harvesting.
  • parameters such as the injury rate of the ear stalk, the number of surviving larvae on the ear stalk, and the length of the ear stalk injury (cm).
  • the number of larvae and the damaged length of ear stalk (cm) were significantly lower than those of the corresponding non-transgenic corn varieties and local common cultivated corn varieties, and reached a significant level of difference (Table 25).
  • the experimental results show that the transgenic maize 2A-7 has a good control effect on the target pest, the corn borer.
  • Table 26 The effect of resistance to cotton bollworm in the spinning stage
  • Figures 8-9 show the results of indoor bioassays for the resistance of 2A-7 leaves or filaments to Spodoptera frugiperda, respectively.
  • Figure 10 shows the results of field bioassays for 2A-7 resistance to Spodoptera frugiperda.
  • Randomized block design with 3-4 repetitions A 1.0m-wide isolation zone is set up between the plots, and the plot area is not less than 24m 2 .
  • the treatments include: no herbicide spraying on GM corn; target herbicide spraying on GM corn; no herbicide spraying on corresponding non-GM corn; no herbicide spraying on corresponding non-GM corn; Corn is sprayed with the target herbicide.
  • the application dose of the herbicides used is divided into: the medium dose (600g active ingredient/ha) of the pesticide registration label, the medium dose 2 times the amount (1200g active ingredient/ha), and the medium dose 4 times the amount (2400g active ingredient/ha).
  • the water volume is 450L/ha.
  • Post-emergence stem and leaf treatment glufosinate-ammonium, general resistance identification should be used in the 3-5 leaf stage of maize.
  • Each treatment needs to be sprayed at one time.
  • the dosage should be calculated according to the actual area of the sprayed area. Rainfall within 12 hours after the drug should be re-tested.
  • the corn seedling rate, plant height and symptoms of phytotoxicity were investigated and recorded at 1 week, 2 weeks and 4 weeks after treatment. 15 corn plants were taken from each plot.
  • the herbicide damage rate was calculated according to the following formula.
  • N the number of affected plants at the same level
  • T total number of plants
  • Symptoms of phytotoxicity are classified according to GB/T 17980.42-2000.
  • Level 2 Slight phytotoxicity of corn, less than 10% phytotoxicity
  • Level 3 moderate phytotoxicity of corn, which can be recovered later without affecting yield
  • Level 4 The damage to the corn is heavy, it is difficult to recover, and the yield is reduced
  • Level 5 The corn has been seriously damaged by phytotoxicity and cannot be recovered, resulting in a significant reduction or failure of production.
  • the injury rate of transformants was investigated at 1 week, 2 weeks and 4 weeks after spraying, and the results are shown in the table below.
  • the GM corn 2A-7 grows normally without any harmful symptoms after spraying the medium dose of glufosinate-ammonium, and the resistance level is 1. After spraying 2 times the medium dose of glufosinate, the corn has slight phytotoxicity, and the phytotoxicity is less than 10%, the resistance grade is grade 2, after spraying 4 times the medium dose of glufosinate-ammonium, the corn has moderate phytotoxicity, the plant height can be recovered, and the yield is lower than that of the micro-spraying treatment, and the resistance grade is grade 4.
  • the above results show that 2A-7 has excellent resistance to herbicides such as glufosinate-ammonium.

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Abstract

提供了转基因玉米事件2A-7，以及含有诊断该玉米事件的核酸分子的玉米植物或其部分、种子、细胞或后代。该事件表现出对鳞翅目昆虫侵袭的抗性。还提供检测样品中对所述玉米事件独特的核酸分子的存在情况的方法，以及用于检测样品中的所述玉米事件的存在情况的探针和引物。还提供了产生含有该玉米事件的核酸分子的玉米植株及种子的方法。

Description

玉米事件2A-7及其鉴定方法 技术领域
本发明涉及转基因玉米事件(transgenic corn event)2A-7，以及含有诊断该玉米事件的核酸分子的玉米植物或其部分、种子、细胞或后代。本发明还提供检测样品中对所述玉米事件独特的核酸分子的存在情况的方法，以及用于检测样品中的所述玉米事件的存在情况的探针和引物。本发明进一步涉及产生昆虫抗性玉米植株及种子的方法。
背景技术
玉米是全球种植范围最广、产量最大的谷类作物，居三大粮食(玉米、小麦、水稻)之首。然而当前玉米虫害严重，世界范围内玉米害虫约350种，其中以蛀茎性和食叶性的鳞翅目害虫──玉米螟分布最广、危害最重，为世界性的重要玉米害虫，它的发生危害严重地影响了玉米的产量和品质，对玉米生产造成巨大的经济损失。因此采取有效措施控制其危害对提高玉米产量、增加农民收入是亟待解决的问题。
长期以来由于没有合适的抗虫品种，虫害防治的主要方法是在生长过程中喷施化学杀虫剂。但是化学杀虫剂不但杀死害虫，还杀死了害虫的天敌，造成生态平衡破坏和环境污染。因此，存在对针对玉米害虫特别是鳞翅目害虫具有抗性的玉米植物的迫切需求。
通过转基因技术可以将抗虫基因导入玉米品种中，进而提高转基因玉米的抗虫性，同时由于转基因玉米的每株植株都具有相当程度的抗性，因而其抗虫效果比喷洒农药防治效果显著要好且稳定，还能够节省人力和物力的投入，有效的节约社会资源。
然而，外来基因在宿主植物基因组中的整合是随机的。如果该外来基因碰巧插入一个非常重要的宿主基因，并且因此突变，则该随机插入事件对该受体植物可以是致命的。另外，即使随机插入事件不损害宿主细胞基因的功能，插入的外源基因的表达也可以受到由周围基因组DNA引起的“位置效应”的影响。在一些情况下，该基因被插入至一些位点中，在这些位点的位置效应足够强以致阻止从该引入基因合成有效量的产物。因此，经常必须筛选大量事件，以鉴别以最佳表达所导入的目标基因为特征的优良事件。鉴定仅与该优良事件相关的一种或多种独有序列从而诊断该转基因事件的存在是必要的。
发明内容
本发明涉及称为2A-7的玉米事件，其包含稳定整合在其基因组中的编码Cry1Ab和 Cry2Ab蛋白的核酸分子，所述蛋白给予2A-7玉米植物以昆虫抵抗性。所述玉米事件2A-7的种子保藏于中国普通微生物菌种保藏管理中心(CGMCC)，并具有保藏号CGMCC NO.17848。在无昆虫压力下，该转基因玉米植物具有与非转基因的同基因系基本上相当的农学性能。在有昆虫压力下，该转基因玉米植物将具有比非转基因玉米植物显著提高的抵抗玉米螟、粘虫、棉铃虫、桃蛀螟等昆虫侵袭的性能。
玉米转化体2A-7及其制品
因此，在第一方面，本发明提供了一种玉米植物或其部分、种子、细胞或后代，所述玉米植物或其部分、种子、细胞或后代具有掺入其基因组中的外源核酸分子，所述外源核酸分子包含Cry1Ab和Cry2Ab基因，其中，所述外源核酸分子侧接5’侧翼区，所述5’侧翼区具有SEQ ID NO:5的第1-432位或第300-432位的核苷酸序列，并且所述外源核酸分子侧接3’侧翼区，所述3’侧翼区具有SEQ ID NO:5的第8532-9031位或第8532-8800位的核苷酸序列。
本发明还提供了一种玉米植物或其部分、种子、细胞或后代，所述玉米植物或其部分、种子、细胞或后代具有掺入其基因组中的外源核酸分子，所述外源核酸分子包含Cry1Ab和Cry2Ab基因，其中，所述外源核酸分子侧接5’侧翼区和3’侧翼区，包含5’侧翼区和与之相邻的外源核酸分子的部分的序列如SEQ ID NO:1所示，包含3’侧翼区和与之相邻的外源核酸分子的部分的序列如SEQ ID NO:2所示。
本发明的玉米植物或其部分、种子、细胞或后代具有对昆虫侵袭的抗性。在某些实施方案中，所述昆虫选自鳞翅目昆虫。在某些实施方案中，所述昆虫选自黏虫、玉米螟(如亚洲玉米螟)、棉铃虫、桃蛀螟、草地贪夜蛾等鳞翅目害虫中的一种或多种。在一些实施方案中，本发明的玉米植物或其部分、种子、细胞或后代还具有对除草剂(例如草甘膦、草铵膦等)的抗性。
在某些实施方案中，所述外源核酸分子在基因组中的位置对应于B73参考基因组序列(V4版本)的Chr3:179141694bp-179141724bp之间。在某些实施方案中，所述外源核酸分子的整合导致原基因组中的部分序列被删除，所述被删除的序列是Chr3:179141695bp-179141723bp(B73参考基因组V4版本)，共计29bp，删除区域为玉米内源基因Zm00001d042767，该基因的功能预测为Glucan endo-13-beta-glucosidase 14，目标片段的插入和Chr3:179141695bp-179141723bp序列的删除将使该基因功能失活。该基因在玉米中为双拷贝，另外一个同源基因的基因号为Zm00001d012292。
在某些实施方案中，所述Cry1Ab和Cry2Ab基因的核苷酸序列经密码子优化用于在 单子叶植物(例如玉米)细胞中进行表达。在某些实施方案中，所述Cry1Ab基因如SEQ ID NO:12所示。在某些实施方案中，所述Cry2Ab基因如SEQ ID NO:13所示。
在某些实施方案中，所述外源核酸分子进一步包含Bar基因。在某些实施方案中，所述Bar基因如SEQ ID NO:14所示。
在某些实施方案中，所述外源核酸分子包含35S polyA终止子、Bar基因、CAMV 35S启动子、nos polyA终止子、Cry1Ab基因、Gly启动子、CAMV 35S启动子、adh1增强子、Cry2Ab基因和nos polyA终止子。
在某些实施方案中，所述外源核酸分子包含SEQ ID NO:5的第483-8524位核苷酸的序列或其互补序列。
易于理解的是，在外源核酸整合入基因组的过程中会产生非预期整合序列，该序列即非基因组序列也目的插入片段(例如T-border序列)，例如是插入片段整合时由基因组修复所产生的序列。因此，本发明的玉米植物或其部分、种子、细胞或后代中所包含的掺入其基因组中的外源核酸分子可以包含该非预期整合序列。因此，在某些示例性实施方案中，所述外源核酸分子包含SEQ ID NO:5的第433-8531位核苷酸的序列或其互补序列。
在某些实施方案中，所述玉米植物或其部分、种子、细胞或后代的基因组包含SEQ ID NOs:1-5任一项所示的序列或其互补序列。在某些实施方案中，所述玉米植物或其部分、种子、细胞或后代的基因组包含SEQ ID NO:5所示的序列或其互补序列。
在某些实施方案中，当以SEQ ID NO:6及SEQ ID NO:7分别作为正向引物和反向引物对所述玉米植物或其部分、种子、细胞或后代的基因组DNA进行扩增时，产生长度约为200-300bp(例如约258bp)的扩增子。在某些实施方案中，当以SEQ ID NO:8及SEQ ID NO:9分别作为正向引物和反向引物对所述基因组DNA进行扩增时，产生长度约为250-350bp(例如约310bp)的扩增子。
在某些实施方案中，产生所述玉米植物或其部分、种子、细胞或后代的玉米种子保藏于中国普通微生物菌种保藏管理中心(CGMCC)，并具有保藏号CGMCC NO.17848。在某些实施方案中，所述玉米植物或其部分、种子、细胞或后代由所述玉米种子长成的玉米植物繁衍和/或育种得到。
在第二方面，本发明提供了一种玉米种子，其保藏于中国普通微生物菌种保藏管理中心(CGMCC)，并具有保藏号CGMCC NO.17848。本发明还提供了由第二方面所述的玉米种子产生的玉米植物或其部分、种子、细胞或后代。在某些实施方案中，所述玉米植物或其部分、种子、细胞或后代由第二方面所述的玉米种子长成的玉米植物繁衍和/或育种得到。
在第三方面，本发明提供了一种制品，其包含第一方面所述的玉米植物或其部分、种子、细胞或后代，或第二方面所述的玉米种子，或由第二方面所述的玉米种子产生的玉米植物或其部分、种子、细胞或后代。
在某些实施方案中，所述制品是食品、农产品(例如饲料)、化妆品、药品或工业产品。
在某些实施方案中，所述制品包含第一方面所述的玉米植物或其部分、种子、细胞或后代、或第二方面所述的玉米种子或由其产生的玉米植物或其部分、种子、细胞或后代的基因组DNA。
在某些实施方案中，所述制品中存在可检测量的多核苷酸，所述多核苷酸对玉米事件2A-7是独特的。
在某些实施方案中，所述制品包含选自SEQ ID NOs:1-5任一项所示的序列或其互补序列。
在某些实施方案中，当以SEQ ID NO:6及SEQ ID NO:7分别作为正向引物和反向引物对所述制品中所含有的核酸进行扩增时，产生长度约为200-300bp(例如约258bp)的扩增子。在某些实施方案中，当以SEQ ID NO:8及SEQ ID NO:9分别作为正向引物和反向引物对所述制品中所含有的核酸进行扩增时，产生长度约为250-350bp(例如约310bp)的扩增子。
在某些实施方案中，所述制品选自玉米耳穗、去苞叶玉米、玉米穗丝、玉米花粉、玉米糁、玉米粉、压碎玉米、玉米面、玉米油、玉米淀粉、玉米浆、玉米麦芽、玉米糖、玉米糖浆、由玉米油生产的人造黄油、不饱和玉米油、饱和玉米油、玉米片、爆玉米花、由玉米生产的乙醇和/或汁(liquor)、由玉米发酵产生的干酒糟(DDGS)、来自玉米的动物饲料、化妆品和填充剂。
在某些实施方案中，前述任一方面所述的玉米植物的部分选自籽粒、花粉、胚珠、花、枝条(shoots)、根、茎(stalks)、穗丝、花序(tassels)、耳穗和叶。
本发明还涉及前述任一方面所述的玉米植物或其部分、种子、细胞或后代，用于生产食品、农产品(例如饲料)、化妆品、药品或工业产品的用途。
在某些实施方案中，所述玉米植物或其部分、种子、细胞或后代用于生产食品、饲料、淀粉或用于酿酒。
在某些实施方案中，所述玉米植物的部分选自选自籽粒、花粉、胚珠、花、枝条、根、茎、穗丝、花序、耳穗和叶。
在某些实施方案中，所述玉米植物或其部分、种子、细胞或后代用于生产选自下列的制品：玉米耳穗、去苞叶玉米、玉米穗丝、玉米花粉、玉米糁、玉米粉、压碎玉米、玉米面、玉米油、玉米淀粉、玉米浆、玉米麦芽、玉米糖、玉米糖浆、由玉米油生产的人造黄油、不饱和玉米油、饱和玉米油、玉米片、爆玉米花、由玉米生产的乙醇和/或汁(liquor)、由玉米发酵产生的干酒糟(DDGS)、来自玉米的动物饲料、化妆品和填充剂。
鉴定事件2A-7的方法
鉴于转基因事件中外源片段在植物基因组中的整合具有随机性的特点，不同转基因事件的外源片段在基因组中的插入位点不同。对于特定的转基因事件来说，其侧翼序列是特异的。因此，利用插入片段侧翼序列可以对转基因事件进行特异性检测。如利用包含部分侧翼序列和部分外源插入片段序列的探针进行杂交，或是设计包含部分侧翼序列和部分外源插入片段序列的特异性引物进行PCR扩增等。
因此，在第四方面，本发明提供了一种引物对，其包含第一引物和第二引物，其中，所述第一引物含有由SEQ ID NO:5的第1-432位核苷酸(例如第200-432位核苷酸)的序列或其互补序列的至少15个连续核苷酸(例如至少18个；例如15-30个连续核苷酸，或18-30个连续核苷酸)所组成的核苷酸序列或与所述核苷酸序列相比具有至少80％(例如，80％～100％，或90％～100％；或至少85％，至少90％，至少95％，至少99％，或100％)的序列同一性的序列，所述第二引物含有由SEQ ID NO:5的第483-8524位核苷酸(例如第483-1000位核苷酸)的序列或其互补序列的至少15个连续 核苷酸(例如至少18个；例如15-30个连续核苷酸，或18-30个连续核苷酸)所组成的核苷酸序列或与所述核苷酸序列相比具有至少80％(例如，80％～100％，或90％～100％；或至少85％，至少90％，至少95％，至少99％，或100％)的序列同一性的序列。
在某些实施方案中，当使用所述第一引物和第二引物扩增SEQ ID NO:5所示的序列时，产生长度为100-500bp的扩增子。在某些实施方案中，所述扩增子的长度为200-400bp，例如200-300bp，例如约258bp。
在某些实施方案中，所述第一引物含有由SEQ ID NO:5的第300-400位核苷酸的序列或其互补序列的至少15个连续核苷酸(例如至少18个；例如15-30个连续核苷酸，或18-30个连续核苷酸)所组成的核苷酸序列或与所述核苷酸序列相比具有至少80％(例如，80％～100％，或90％～100％；或至少85％，至少90％，至少95％，至少99％，或100％)的序列同一性的序列，所述第二引物含有由SEQ ID NO:5的第500-600位核苷酸的序列或其互补序列的至少15个连续核苷酸(例如至少18个；例如15-30个连续核苷酸，或18-30个连续核苷酸)所组成的核苷酸序列或与所述核苷酸序列相比具有至少80％(例如，80％～100％，或90％～100％；或至少85％，至少90％，至少95％，至少99％，或100％)的序列同一性的序列。
在某些实施方案中，所述第一引物含有由SEQ ID NO:5的第300-350位核苷酸的序列或其互补序列的至少15个连续核苷酸(例如至少18个；例如15-30个连续核苷酸，或18-30个连续核苷酸)所组成的核苷酸序列或与所述核苷酸序列相比具有至少80％(例如，80％～100％，或90％～100％；或至少85％，至少90％，至少95％，至少99％，或100％)的序列同一性的序列。
在某些实施方案中，所述第二引物含有由SEQ ID NO:5的第520-600位核苷酸(例如第520-570位核苷酸)的序列或其互补序列的至少15个连续核苷酸(例如至少18个；例如15-30个连续核苷酸，或18-30个连续核苷酸)所组成的核苷酸序列或与所述核苷酸序列相比具有至少80％(例如，80％～100％，或90％～100％；或至少85％，至少90％，至少95％，至少99％，或100％)的序列同一性的序列。
在某些实施方案中，所述第一引物含有SEQ ID NO:6所示的序列或与其相比具有至少80％(例如，80％～100％，或90％～100％；或至少85％，至少90％，至少95％，至少99％，或100％)的序列同一性的序列，所述第二引物含有SEQ ID NO:7所示的序列或与其相比具有至少80％(例如，80％～100％，或90％～100％；或至少85％，至 少90％，至少95％，至少99％，或100％)的序列同一性的序列。
在第五方面，本发明提供了一种引物对，其包含第一引物和第二引物，其中，所述第一引物含有由SEQ ID NO:5的第483-8524位核苷酸(例如第8000-8524位核苷酸)的序列或其互补序列的至少15个连续核苷酸(例如至少18个；例如15-30个连续核苷酸，或18-30个连续核苷酸)所组成的核苷酸序列或与所述核苷酸序列相比具有至少80％(例如，80％～100％，或90％～100％；或至少85％，至少90％，至少95％，至少99％，或100％)的序列同一性的序列，所述第二引物含有由SEQ ID NO:5的第8532-9031位核苷酸(例如第8532-8800位核苷酸)的序列或其互补序列的至少15个连续核苷酸(例如至少18个；例如15-30个连续核苷酸，或18-30个连续核苷酸)所组成的核苷酸序列或与所述核苷酸序列相比具有至少80％(例如，80％～100％，或90％～100％；或至少85％，至少90％，至少95％，至少99％，或100％)的序列同一性的序列。
在某些实施方案中，当使用所述第一引物和第二引物扩增SEQ ID NO:5所示的序列时，产生长度为100-500bp的扩增子。在某些实施方案中，所述扩增子的长度为200-400bp，例如250-350bp，例如约310bp。
在某些实施方案中，所述第一引物含有由SEQ ID NO:5的第8400-8500位核苷酸的序列或其互补序列的至少15个连续核苷酸(例如至少18个；例如15-30个连续核苷酸，或18-30个连续核苷酸)所组成的核苷酸序列或与所述核苷酸序列相比具有至少80％(例如，80％～100％，或90％～100％；或至少85％，至少90％，至少95％，至少99％，或100％)的序列同一性的序列，所述第二引物含有由SEQ ID NO:5的第8700-8800位核苷酸的序列或其互补序列的至少15个连续核苷酸(例如至少18个；例如15-30个连续核苷酸，或18-30个连续核苷酸)所组成的核苷酸序列或与所述核苷酸序列相比具有至少80％(例如，80％～100％，或90％～100％；或至少85％，至少90％，至少95％，至少99％，或100％)的序列同一性的序列。
在某些实施方案中，所述第一引物含有由SEQ ID NO:5的第8400-8600位核苷酸(例如，第8400-8500位核苷酸，第8450-8550位核苷酸，或第8450-8500位核苷酸)的序列或其互补序列的至少15个连续核苷酸(例如至少18个；例如15-30个连续核苷酸，或18-30个连续核苷酸)所组成的核苷酸序列或与所述核苷酸序列相比具有至少80％(例如，80％～100％，或90％～100％；或至少85％，至少90％，至少95％，至 少99％，或100％)的序列同一性的序列。
在某些实施方案中，所述第二引物含有由SEQ ID NO:5的第8500-9000位核苷酸(例如第8500-8800位核苷酸，第8700-8800位核苷酸，或第8750-8800位核苷酸)的序列或其互补序列的至少15个连续核苷酸(例如至少18个；例如15-30个连续核苷酸，或18-30个连续核苷酸)所组成的核苷酸序列或与所述核苷酸序列相比具有至少80％(例如，80％～100％，或90％～100％；或至少85％，至少90％，至少95％，至少99％，或100％)的序列同一性的序列。
在某些实施方案中，所述第一引物含有SEQ ID NO:8所示的序列或与其相比具有至少80％(例如，80％～100％，或90％～100％；或至少85％，至少90％，至少95％，至少99％，或100％)的序列同一性的序列，所述第二引物含有SEQ ID NO:9所示的序列或与其相比具有至少80％(例如，80％～100％，或90％～100％；或至少85％，至少90％，至少95％，至少99％，或100％)的序列同一性的序列。
在第六方面，本发明提供了在包含玉米核酸的样品中检测对玉米事件2A-7独特的核酸分子的方法，其包括：
(1)将第四或第五方面所述的引物对与该样品接触；
(2)进行核酸扩增反应；和
(3)通过凝胶电泳(例如琼脂糖凝胶电泳)检测步骤(2)的产物；
其中，所述玉米事件2A-7是如上任一方面中所述的玉米植物或其部分、种子、细胞或后代。
在某些实施方案中，所述玉米事件2A-7包含掺入其基因组中的编码mcry1Ab和mcry2Ab的外源核酸分子，所述外源核酸分子侧接5’侧翼区和3’侧翼区，包含5’侧翼区和与之相邻的外源核酸分子的部分的序列如SEQ ID NO:1所示，包含3’侧翼区和与之相邻的外源核酸分子的部分的序列如SEQ ID NO:2所示。
在某些实施方案中，所述玉米事件2A-7在其基因组中包含SEQ ID NOs:1-5任一项所示的序列或其互补序列。
在某些实施方案中，在步骤(3)中当检测到预期扩增子时，表明所述样品包含对玉米事件2A-7独特的核酸分子，所述预期扩增子是指，当所述引物对用于扩增玉米事件2A-7的基因组DNA的核酸时，所产生的扩增子。
在某些实施方案中，所述方法包括：
(1)将第四方面所述的引物对与该样品接触；
(2)进行核酸扩增反应；和
(3)通过凝胶电泳(例如琼脂糖凝胶电泳)检测步骤(2)的产物；
其中，当检测到长度为约250-260bp(例如约258bp)的扩增子时，表明在所述样品中存在对玉米事件2A-7独特的核酸分子。
在某些实施方案中，所述引物对包含：含有SEQ ID NO:6所示的序列或与其相比具有至少80％(例如，80％～100％，或90％～100％；或至少85％，至少90％，至少95％，至少99％，或100％)的序列同一性的序列的第一引物和含有SEQ ID NO:7所示的序列或与其相比具有至少80％(例如，80％～100％，或90％～100％；或至少85％，至少90％，至少95％，至少99％，或100％)的序列同一性的序列的第二引物。
在某些实施方案中，所述方法包括：
(1)将第五方面所述引物对与该样品接触；
(2)进行核酸扩增反应；和
(3)通过凝胶电泳(例如琼脂糖凝胶电泳)检测步骤(2)的产物；
其中，当检测到长度为约305-315bp(例如约310bp)的扩增子时，表明在所述样品中存在对玉米事件2A-7独特的核酸分子。
在某些实施方案中，所述引物对包含：含有SEQ ID NO:8所示的序列或与其相比具有至少80％(例如，80％～100％，或90％～100％；或至少85％，至少90％，至少95％，至少99％，或100％)的序列同一性的序列的第一引物和含有SEQ ID NO:9所示的序列或与其相比具有至少80％(例如，80％～100％，或90％～100％；或至少85％，至少90％，至少95％，至少99％，或100％)的序列同一性的序列的第二引物。
在第七方面，本发明提供了在包含玉米核酸的样品中检测对玉米事件2A-7独特的核酸分子的方法，其包括：
(1)将引物对与该样品接触；其中，当该引物对被用于扩增玉米事件2A-7的基因组DNA的核酸时产生含有选自下列的核苷酸序列的扩增子：SEQ ID NOs:1-5任一项所示的序列或其互补序列；
(2)进行核酸扩增反应，由此产生所述扩增子；和
(3)检测该扩增子；
其中，所述玉米事件2A-7是如上任一方面中所述的玉米植物或其部分、种子、细胞或后代。
在某些实施方案中，所述玉米事件2A-7包含掺入其基因组中的编码mcry1Ab和mcry2Ab的外源核酸分子，所述外源核酸分子侧接5’侧翼区和3’侧翼区，包含5’侧翼区和与之相邻的外源核酸分子的部分的序列如SEQ ID NO:1所示，包含3’侧翼区和与之相邻的外源核酸分子的部分的序列如SEQ ID NO:2所示。
在某些实施方案中，所述玉米事件2A-7在其基因组中包含SEQ ID NOs:1-5任一项所示的序列或其互补序列。
在某些实施方案中，在步骤(3)中，所述检测包括DNA测序。
在某些实施方案中，在步骤(3)中，使用特异于该扩增子的核酸探针对所述扩增子进行检测。在某些实施方案中，所述检测包括以下步骤：
(a)将所述核酸探针与步骤(2)的扩增产物接触；
(b)对所述扩增产物和核酸探针实施严格杂交条件；和
(c)检测所述核酸探针与扩增产物的杂交；
其中，当检测到杂交时，说明在所述样品中存在对玉米事件2A-7独特的核酸分子。
在第八方面，本发明提供了在包含玉米核酸的样品中检测对玉米事件2A-7独特的核酸分子的存在的方法，其包括：
(1)将特异于靶序列的核酸探针与所述样品接触，所述靶序列含有选自下列的核苷酸序列：SEQ ID NOs:1-5任一项所示的序列或其互补序列；
(2)对所述样品和核酸探针实施严格杂交条件；和
(3)检测所述核酸探针与样品的杂交；
其中，所述玉米事件2A-7是如上任一方面中所述的玉米植物或其部分、种子、细胞或后代。
在某些实施方案中，当检测到杂交时，说明在所述样品中存在对玉米事件2A-7独特的核酸分子。
在某些实施方案中，所述玉米事件2A-7包含掺入其基因组中的编码mcry1Ab和mcry2Ab的外源核酸分子，所述外源核酸分子侧接5’侧翼区和3’侧翼区，包含5’侧翼区 和与之相邻的外源核酸分子的部分的序列如SEQ ID NO:1所示，包含3’侧翼区和与之相邻的外源核酸分子的部分的序列如SEQ ID NO:2所示。
在某些实施方案中，所述玉米事件2A-7在其基因组中包含SEQ ID NOs:1-5任一项所示的序列或其互补序列。
在某些实施方案中，所述核酸探针包含：5’侧翼区和与之相邻的外源核酸分子的部分的序列，或与所述序列相比具有至少80％(例如80％-100％或90％-100％，例如至少85％，至少90％，至少95％，至少99％，或100％)的序列同一性的序列。
在某些实施方案中，所述核酸探针与SEQ ID NO：1或SEQ ID NO：2所示的序列或其互补序列或任一个的片段具有至少80％(例如80％-100％或90％-100％，例如至少85％，至少90％，至少95％，至少99％，或100％)的序列同一性。
在某些实施方案中，所述核酸探针包含SEQ ID NO:1或2所示的序列或其互补序列。
在某些实施方案中，所述核酸探针带有荧光标记，例如Taqman探针。
在本文中，术语“引物”是分离的核酸，它们通过核酸杂交被退火为补合的(complimentary)靶DNA链，以在该引物与该靶DNA链之间形成杂交，并且然后通过聚合酶(例如DNA聚合酶)沿着该靶DNA链延长。多对或多组引物可以用于核酸分子的扩增，例如通过聚合酶链式反应(PCR)或者其他常规核酸扩增方法。
术语“核酸探针”是连接常规可检测的标记或报告分子的分离核酸，所述标记或报告分子例如为放射性同位素、配体、化学发光剂或酶。这样的探针与靶核酸的一条链补合，在本发明的情况下，与来自玉米事件2A-7的基因组DNA的一条链杂交。本发明的探针不仅包括脱氧核糖核酸或核糖核酸，而且包括特异性地结合靶DNA序列并可用于检测该靶DNA序列的存在情况的聚酰胺和其它探针材料。
制备与使用探针和引物的方法描述于例如MolecularCloning：A Laboratory Manual，第2版，1-3卷，Sambrook等编辑，ColdSpring Harbor Laboratory Press，Cold Spring Ha r bor，NY，1 989(在下文，“Sam brook等，1 989”)；Current Protocols in Molecular Biology，Ausubel等编辑，Greene Publishing and Wiley-Interscience，New York，1992(定期更新)(在下文，“Ausubel等，1992”)；和Innis等，PCR Protocols：A Guide to Methods and Applications，Academic Press：San Diego，1990。PCR引物对可从已知序列得到，例如，通过使用拟用于该用途的计算机程序，如Primer(0.5 版，1991，Whitehead Institute for Biomedical Research，Cambridge，MA)。
本发明的核酸探针和引物在严格条件下同靶DNA序列杂交。任何常规的核酸杂交或扩增方法均可用于鉴定样品中转基因事件的DNA的存在情况。在本文中，如果两个核酸分子能形成反向平行的双链核酸结构，则这两个分子被称为能够彼此特异性杂交。如果核酸分子显示出完全互补，则一个核酸分子被称为另一个核酸分子的“互补物(互补序列)”。在本文中，当一个分子的每个核苷酸均与另一个分子的核苷酸互补时，这些分子被称为表现出“完全互补”。如果两个分子可相互杂交，具有的稳定性足以使它们在至少常规的“低严格”条件下保持互相退火，则它们被称为“最低限度互补”。类似的，如果分子可相互杂交，具有的稳定性足以使它们在常规的“高严格”条件下保持互相退火，则这些分子被称为“互补”。常规严格条件由Sambrook等，1989和Haymes等，描述于：Nucleic AcidHybridization，A Practical Approach，IRL Press，Washington，DC(1985)。因此，允许相对于完全互补有偏离，只要这些偏离没有完全排除分子形成双链结构的能力。为了使核酸分子用作引物或探针，仅仅需要在序列上足够互补，以能够在所用的特定溶剂和盐浓度下形成稳定的双链结构。
促进DNA杂交的适宜严格条件，如在约45℃下的6.0×氯化钠/柠檬酸钠(SSC)，随后在50℃以2.0×SSC洗涤，是本领域技术人员已知的，或者可见于Current Protocols in Molecular Biology，John Wiley&Sons，N.Y.(1989)，6.3.1-6.3.6。例如，洗涤步骤中的盐浓度可选自低严格的50℃、约2.0×SSC到高严格的50℃、约0.2×SSC。此外，洗涤步骤的温度也可以从约22℃室温的低严格条件升高到约65℃的高严格条件。温度和盐浓度都是可变的，或者温度或盐浓度中的任一个可保持恒定，而改变另一个变量。在某些实施方案中，在中度严格条件(如约2.0×SSC和约65℃)下，本发明的探针或引物与靶序列特异性杂交。在某些实施方案中，在高严格条件下，本发明的探针或引物与靶序列特异性杂交。
在某些实施方案中，本发明的探针与SEQ ID NO：1或SEQ ID NO：2中所示的核酸序列或其互补序列或任一个的片段具有至少80％(例如80％-100％或90％-100％，例如至少85％，至少90％，至少95％，至少99％，或100％)的序列同一性。探针与靶DNA分子的杂交可通过本领域技术人员已知的众多方法中的任一种检测，这些方法可包括但不限于荧光标签法、放射性标签法、基于抗体的标签法和化学发光标签法。
关于利用特定扩增引物对进行靶核苷酸序列扩增(例如通过PCR)，“严格条件”是指这样的条件：在DNA热扩增反应中使引物对仅与靶核酸序列(具有相应野生型序列 (或其互补物)的引物应与该靶核酸序列结合)杂交并优选产生独特的扩增产物(扩增子)。
术语“特异于(靶序列)”是指在严格杂交条件下探针或引物仅同含有靶序列的样品中的该靶序列杂交。
术语“扩增子”是指为核酸模板一部分的靶核酸序列的核酸扩增产物。例如，为了确定玉米植物是否由含有本发明玉米植物的转基因事件基因组DNA的有性杂交产生，可使用包含来源于紧邻插入异源DNA的插入位点的植物基因组中的侧翼序列的引物和来源于插入异源DNA的第二个引物的引物对，对从玉米植物组织样品提取的DNA实施核酸扩增方法，以产生诊断事件DNA的存在情况的扩增子。所述扩增子具有一定长度，并具有也诊断所述事件的序列。扩增子的长度可在引物对加一个核苷酸碱基对、优选加大约50个核苷酸碱基对、更优选加大约250个核苷酸碱基对、甚至更优选加大约450个核苷酸碱基对的组合长度范围内。或者，引物对可来源于插入DNA两边的侧翼序列，以便产生包含全部插入核苷酸序列的扩增子。来自植物基因组序列的引物对的成员可以与插入DNA分子相距一定距离定位，该距离可以在一个核苷酸碱基对至最高大约两万个核苷酸碱基对的范围内。术语“扩增子”的使用特别排除了可以在DNA热扩增反应中形成的引物二聚体。
核酸扩增可以通过本领域已知的多种核酸扩增方法中的任一种来实现，包括聚合酶链式反应(PCR)。多种扩增方法是本领域已知的，尤其是描述于美国专利号4,683,195和4,683,202以及PCR Protocols：A Guide to Methods and Applications，Innis等编辑，Academic Press，San Diego，1990。PCR扩增方法已发展成扩增最多22kb的基因组DNA和最多42kb的噬菌体DNA(Cheng等，Proc.Natl.Acad.Sci.USA91：5695-5699，1994)。这些方法以及DNA扩增领域已知的其它方法可用于实施本发明。来自玉米事件2A-7的异源DNA插入序列或侧翼序列可如下检验：使用源于本文提供的序列的引物扩增来自所述事件的这些序列，随后对PCR扩增子或克隆的DNA进行标准DNA测序。
可以通过多种技术来检测通过这些方法产生的扩增子。一个这样的方法是遗传比特分析(Genetic Bit Analysis)(Nikiforov等，Nucleic Acid Res.22：4167-4175，1994)，其中设计与相邻侧翼基因组DNA序列和插入DNA序列这二者重叠的DNA寡核苷酸。将寡核苷酸固定在微量滴定板的孔中。在对目的区域进行PCR后(使用位于插入序列的一个引物和位于相邻侧翼基因组序列的一个引物)，可以将单链PCR产物同固定的寡核苷酸杂交，并作为模板用于利用DNA聚合酶和特异于下一个期望碱基的标记ddNTP的单碱基延伸反应。 读数器可以是荧光的或基于ELISA的。信号指示归因于成功的扩增、杂交和单碱基延伸的插入序列/侧翼序列的存在情况。
另一个方法是Winge(Innov.Pharma.Tech.00：18-24，2000)描述的焦磷酸测序技术(pyrosequencing technique)。在该方法中，设计与相邻基因组DNA和插入DNA接合区重叠的寡核苷酸。使寡核苷酸与来自目的区域的单链PCR产物杂交(一引物位于插入序列，另一引物位于侧翼基因组序列)，并在DNA聚合酶、ATP、硫酸化酶、萤光素酶、三磷酸腺苷双磷酸酶、腺苷5’磷酸硫酸酯以及萤光素存在下温育。单独加入dNTP，测量掺入产生的光信号。光信号指示归因于成功的扩增、杂交和单碱基或多碱基延伸的转基因插入序列/侧翼序列的存在情况。
如Chen等描述的荧光偏振(Genome Res.9：492-498，1999)是一种可用于检测本发明的扩增子的方法。使用此方法设计与基因组侧翼和插入DNA接合区重叠的寡核苷酸。将寡核苷酸与来自目的区域的单链PCR产物杂交(一引物位于插入DNA序列，另一引物位于侧翼基因组DNA序列)，并在DNA聚合酶和荧光标记的ddNTP存在下温育。单碱基延伸导致掺入ddNTP。可以使用荧光计根据偏振变化测量掺入。偏振变化指示归因于成功的扩增、杂交和单碱基延伸的转基因插入序列/侧翼序列的存在情况。
Taqman探针法(PE Applied Biosystems，Foster City，CA)被描述为检测并定量DNA序列的存在情况的方法，并通过生产商所提供的说明书得以充分了解。简而言之，设计与基因组侧翼和插入DNA接合区重叠的FRET寡核苷酸探针。使FRET探针和PCR引物(一引物位于插入DNA序列，一引物位于侧翼基因组序列)在热稳定聚合酶和dNTP存在下循环。FRET探针杂交导致将FRET探针上的荧光部分与猝灭部分裂开并释放出荧光部分。荧光信号指示归因于成功的扩增和杂交的侧翼序列/转基因插入序列的存在情况。
如Tyangi等(Nature Biotech.14：303-308，1996)所述，业已描述了分子信标可用于序列检测。简而言之，设计与侧翼基因组和插入DNA接合区重叠的FRET寡核苷酸探针。FRET探针的独特结构使其含有保持荧光部分和淬灭部分紧邻的二级结构。将FRET探针和PCR引物(一引物位于插入DNA序列，一引物位于侧翼基因组序列)在热稳定聚合酶和dNTP存在下循环。在成功PCR扩增之后，FRET探针与靶序列杂交导致除去探针二级结构以及荧光和猝灭部分空间分离，这导致产生荧光信号。荧光信号指示归因于成功的扩增和杂交的侧翼序列/转基因插入序列的存在情况。
在上述任一方面所述的方法中，所述样品可以为任何含有玉米事件2A-7DNA的 样品，只要该样品含有至少可检测量的、诊断样品中事件2A-7的存在情况的多核苷酸(即对玉米事件2A-7独特的多核苷酸)。在某些实施方案中，所述样品可以选自玉米耳穗、去苞叶玉米、玉米穗丝、玉米花粉、玉米糁、玉米粉、压碎玉米、玉米面、玉米油、玉米淀粉、玉米浆、玉米麦芽、玉米糖、玉米糖浆、由玉米油生产的人造黄油、不饱和玉米油、饱和玉米油、玉米片、爆玉米花、由玉米生产的乙醇和/或汁(liquor)、由玉米发酵产生的干酒糟(DDGS)、来自玉米的动物饲料、化妆品和填充剂。
本发明还提供了用于鉴定玉米事件2A-7的试剂盒，其包括以上所述的核酸探针和/或引物，它们在高严格条件下特异性地与SEQ ID NOs:1-5任一项所示的靶序列杂交。在某些实施方案，所述试剂盒还包含以及使核酸杂交或者扩增方法能够完成所必要的其他材料。
本发明还涉及如上任一方面所述的引物、引物对、核酸探针或试剂盒用于在包含玉米核酸的样品中检测对玉米事件2A-7独特的核酸分子的用途，或者用于检测玉米事件2A-7的存在的用途。
本发明还涉及一种分离的核酸分子，其含有选自下列的核苷酸序列：SEQ ID NOs:1-5任一项所示的序列或其互补序列。
产生抗虫玉米植物的方法
在第九方面，本发明提供了一种生产昆虫抗性玉米植物的方法，其包括：
(1)将第一亲代玉米植物与第二亲代玉米植物杂交；其中，所述第一或第二亲代玉米植物如第一方面或第二方面所定义；
(2)由(1)的杂交获得第一代子代植物；和
(3)从这些第一代子代植物中选择对昆虫具有抗性的子代植物，其中，当所述子代植物满足下述(3a)-(3c)中的至少一种时，表明其为昆虫抗性玉米植物：
(3a)子代植物在其基因组中含有SEQ ID NOs:1-5任一项所示的序列或其互补序列；
(3b)当以SEQ ID NO:6及SEQ ID NO:7分别作为正向引物和反向引物对子代植物的基因组DNA进行扩增时，产生长度约为200-300(例如约258bp)bp的扩增子；和/或，
(3c)当以SEQ ID NO:8及SEQ ID NO:9分别作为正向引物和反向引物对子代植物的基因组DNA进行扩增时，产生长度约为250-350bp(例如约310bp)的扩增子；或，
(3d)当使用特异于SEQ ID NOs:1-5任一项所示的序列或其互补序列的核酸探针(例如，包含SEQ ID NO:1或2所示的序列或其互补序列的核酸探针)对子代植物的基因组DNA进行检测时，能够检测到杂交。
在某些实施方案中，所述第一或第二亲代玉米植物包含掺入其基因组中的编码mcry1Ab和mcry2Ab的外源核酸分子，所述外源核酸分子侧接5’侧翼区和3’侧翼区，包含5’侧翼区和与之相邻的外源核酸分子的部分的序列如SEQ ID NO:1所示，包含3’侧翼区和与之相邻的外源核酸分子的部分的序列如SEQ ID NO:2所示。
在某些实施方案中，所述第一或第二亲代玉米植物在其基因组中包含SEQ ID NOs:1-5任一项所示的序列或其互补序列。
在某些实施方案中，所述方法还包括以下步骤：
(4)将步骤(3)获得的子代植物自交，由此产生多个第二代子代植物；
(5)从这些第二代子代植物中选择对昆虫具有抗性的植物，其中，当所述植物满足所述(3a)-(3c)中的至少一种时，表明其为昆虫抗性玉米植物。
在第十方面，本发明提供了一种产生可生长为昆虫抗性玉米植物的杂种玉米种子的方法，其包括：将第一亲代玉米植物与第二亲代玉米植物杂交和收获产生的杂种种子，其中所述第一亲代玉米植物和/或第二亲代玉米植物如第一方面或第二方面所定义。
在某些实施方案中，所述第一亲代玉米植物和/或第二亲代玉米植物包含掺入其基因组中的编码mcry1Ab和mcry2Ab的外源核酸分子，所述外源核酸分子侧接5’侧翼区和3’侧翼区，包含5’侧翼区和与之相邻的外源核酸分子的部分的序列如SEQ ID NO:1所示，包含3’侧翼区和与之相邻的外源核酸分子的部分的序列如SEQ ID NO:2所示。
在某些实施方案中，所述第一亲代玉米植物和/或第二亲代玉米植物在其基因组中包含SEQ ID NOs:1-5任一项所示的序列或其互补序列。
在某些实施方案中，所述方法包括：
(1)种植第一近交玉米系的种子，该近交玉米系是第一方面或第二方面中所定义的玉米植物；以及种植具有不同基因型的第二近交系的种子；
(2)培育产生自所述种植的玉米植物直到开花的时期；
(3)对其中一个玉米近交系的植物的花去雄；
(4)将该两种不同近交系彼此有性杂交；和
(5)收获由此生产的杂种种子。
在某些实施方案中，所述第一近交玉米系包含掺入其基因组中的编码mcry1Ab和mcry2Ab的外源核酸分子，所述外源核酸分子侧接5’侧翼区和3’侧翼区，包含5’侧翼区和与之相邻的外源核酸分子的部分的序列如SEQ ID NO:1所示，包含3’侧翼区和与之相邻的外源核酸分子的部分的序列如SEQ ID NO:2所示。
在某些实施方案中，所述第一近交玉米系在其基因组中包含SEQ ID NOs:1-5任一项所示的序列或其互补序列。
在某些实施方案中，该第一近交玉米系提供了母本。在某些实施方案中，该第一近交玉米系提供了父本。
在第九或第十方面所述的方法中，所述昆虫可以选自鳞翅目昆虫，例如黏虫、玉米螟(如亚洲玉米螟)、棉铃虫、桃蛀螟、草地贪夜蛾等鳞翅目害虫中的一种或多种。
本领域的普通技术人员将认识到2A-7的转基因基因型能通过育种渗入至包含不同转基因基因型的其他玉米系中，从而在得到的种子和子代植物中实现特征堆积并给予这些植物增大的活性谱。
本发明的转基因基因型能够采用本领域公认的育种技术渗入至任何玉米近交或杂种中。植物育种的目标是将不同的希望的特征结合在单一品种或杂交种中。对于大田作物而言，这些特征可能包括对昆虫和疾病的抵抗性、对除草剂的耐受、对热和旱的耐受、减少农作物成熟的时间、更大的产量，以及更好的农艺学质量。随着许多农作物的机械收获，植物特征(例如发芽和植物丛建立，生长速率、成熟，以及植物和耳穗的高度)的均一性是很重要的。
大田作物是通过利用了植物授粉方法的技术来培育。如果来自一朵花的花粉被转移至同一植物的同一朵花或另一朵花，则该植物是自我授粉的。如果花粉来自不同植物的花，则该植物是异花授粉的。
玉米(玉蜀黍)能通过自花受粉和异花授粉两种技术培育。玉米在同一个植物上具有单独的雄花和雌花，分别位于雄花穗和耳穗上。当风将花粉从雄花穗吹到从耳穗顶突出的花丝时，发生玉米的自然授粉。
在植物中控制雄性能育性的可靠方法为改善的植物育种提供了机会。这对依赖于某些雄性不育系统的玉米杂种的发展尤其是成立的。对培育者而言有几个可用的控制雄性能育性的选择，例如，手工或机械去雄(或去雄花穗)、细胞质的雄性不育性、遗传雄性不育性、杀配子药等等。
杂种玉米种子通常通过结合手工或机械去雄花穗的雄性不育系统而典型地产生。
两个玉米近交在交错的条中种植在一块地上，并且从一个近交(雌性)中去除带花粉的雄花穗。假如能足够隔离外来玉米花粉源，则该去雄花穗近交系的耳穗将只从另一个近交系(雄性)受精，并且因此得到的种子是杂种，并将形成杂交植物。
使用雄性不育近交只是玉米杂种生产中的一个因素。本领域已知的且在玉米植物育种计划中使用的植物育种技术包括但不限于，回归选择、回交、系谱育种、限制长度多态性增强的选择、遗传标记物增强的选择和转化。
术语定义
在本发明中，除非另有说明，否则本文中使用的科学和技术名词具有本领域技术人员所通常理解的含义。同时，为了更好地理解本发明，下面提供相关术语的定义和解释。
如本文中所使用的，术语“玉米”是指玉蜀黍或玉米并且包括可以用玉米培育的所有植物品种，包含野生玉蜀黍种类。
如本文中所使用的，术语“Cry2Ab基因”具有本领域技术人员公知的含义，其是从昆虫病原菌苏云金芽胞杆菌(Bacillus thuringiensis,简称Bt)中发现的内毒素基因，并可参见各种公共数据库(例如，GenBank:M23724)。本发明人对Cry2Ab的密码子针对单子叶植物的序列特征进行了优化改造，获得了特别适于在玉米细胞中表达的序列(mCry2Ab)，所述mCry2Ab的序列如SEQ ID NO:13所示，并可参见中国专利申请CN201410483143.2。
如本文中所使用的，术语“Cry1Ab基因”具有本领域技术人员公知的含义，其是从昆虫病原菌苏云金芽胞杆菌(Bacillus thuringiensis,简称Bt)中发现的内毒素基因，并可参见各种公共数据库(例如，GenBank:M13898)。本发明人对Cry1Ab的密码子针对单子叶植物的序列特征进行了优化改造，获得了特别适于在玉米细胞中表达的序列(mCry1Ab)，所述mCry1Ab的序列如SEQ ID NO:12所示，并可参见中国专利申请CN201710701976.5。
如本文中所使用的，术语“Bar基因”具有本领域技术人员公知的含义，其是来源于吸水链霉菌的抗除草剂基因。Bar基因常作为选择标记基因，其可以产生磷酸化乙酰转移酶，可以使草胺膦类除草剂的自由氨基乙酰化，达到解毒目的，从而方便与遗传转化中阳性愈伤的筛选。Bar基因序列是本领域已知的，例如参见Thompson CJ et al.,EMBO J.1987 Sep；6(9):2519-23.。在某些实例性实施方案中，Bar基因具有如SEQ ID NO:14所示的序列。
如本文中所使用的，术语“玉米事件(corn event)”、“事件(event)”“转基因事件”可互换使用，其是指通过用异源DNA(例如，包括相关基因的表达盒)转化和再生植物细胞或组织而产生的重组植物。术语“事件”包含异源DNA的原始转化体和/或该转化体的子代。术语“事件”也包含在转化体与另一玉米系之间通过有性异型杂交而产生的子代。即使在重复与回归亲本回交后，来自该转化的亲本的插入DNA和侧翼DNA存在于在该杂交子代的同样的染色体位置。术语“事件”也包括来自原始转化体的DNA，包含该插入DNA以及直接邻近该插入DNA的侧翼基因组序列，其预期将被转移至子代中，该子代作为包含该插入DNA(例如，该原始转化体及由自交产生的子代)的亲本品系与不含该插入DNA的亲本品系的有性杂交的结果而接收了包含相关转基因的插入DNA。通常，植物组织的转化产生多个事件，每个事件代表DNA构建体插入至植物细胞的基因组的不同位置上。基于该转基因的表达或其他希望的特征，选择特殊的事件。因此，“事件2A-7”、“2A-7”或者“2A-7事件”可以互换地使用。
如本文中所使用的，术语“转化”是将异源核酸引入至宿主细胞或生物体中的过程。具体地说，“转化”是指将DNA分子稳定整合进相关生物体的基因组中。
如本文中所使用的，术语“转化的/转基因的/重组”是指已经引入了异源核酸分子的宿主生物体，例如细菌或植物。该核酸分子能够被稳定地整合进入该宿主的基因组中或者该核酸分子也能作为染色体外的分子而存在。这样的染色体外的分子能够自动复制。转化的细胞、组织或者植物应理解为不仅包含转化过程的最后产物，还包含其转基因的子代。“非转化的”、“非转基因的”，或“非重组”宿主是指野生型生物体，即，细菌或植物，它不包含异源核酸分子。如此处所用，“转基因的”是指植物，植物细胞，或多个结构化或没有结构化的植物细胞，这些细胞通过熟知的基因操作和基因插入技术已将代表相关基因的核酸序列整合进该植物基因组中，并典型地整合进细胞核的染色体、线粒体或其他包含染色体的细胞器中，这是在与天然植物或植物细胞中正常存在的位点不同的位点，或在多于在天然植物或植物细胞中正常存在的拷贝数中的位点 发生的。转基因植物来自这种核酸序列的操作和插入(与自然发生的突变相反)，以产生非自然发生的植物或具有非自然发生的基因型的植物。植物和植物细胞的转化技术在本领域是熟知的，并可能包含例如电穿孔法、显微注射法、土壤杆菌属介导的转化法，以及生物射弹转化(ballistict ransformation)。
如本文中所使用的，表述“对玉米事件2A-7独特”是指玉米事件2A-7特有的特征。因此，对事件2A-7独特的核酸在其他非2A-7的玉米植物中没有发现。在某些实施方案中，对事件2A-7独特的核酸可以选自SEQ ID NOs:1-5。
如本文中所使用的，术语“基因型”是由亲本玉米植物遗传的遗传物质，并不是所有遗传物质都必然地表达在该后代玉米植物中。因此，表述“2A-7基因型”是指在玉米事件2A-7中转化进植物的基因组的外源遗传物质以及位于该插入的序列侧翼的遗传物质。
如本文中所使用的，术语“同一性”用于指两个多肽之间或两个核酸之间序列的匹配情况。当两个进行比较的序列中的某个位置都被相同的碱基或氨基酸单体亚单元占据时(例如，两个DNA分子的每一个中的某个位置都被腺嘌呤占据，或两个多肽的每一个中的某个位置都被赖氨酸占据)，那么各分子在该位置上是同一的。两个序列之间的“百分数同一性”是由这两个序列共有的匹配位置数目除以进行比较的位置数目×100的函数。例如，如果两个序列的10个位置中有6个匹配，那么这两个序列具有60％的同一性。例如，DNA序列CTGACT和CAGGTT共有50％的同一性(总共6个位置中有3个位置匹配)。通常，在将两个序列比对以产生最大同一性时进行比较。这样的比对可通过使用，例如，可通过计算机程序例如Align程序(DNAstar,Inc.)方便地进行的Needleman等人(1970)J.Mol.Biol.48：443-453的方法来实现。还可使用已整合入ALIGN程序(版本2.0)的E.Meyers和W.Miller(Comput.Appl Biosci.，4:11-17(1988))的算法，使用PAM120权重残基表(weight residue table)、12的缺口长度罚分和4的缺口罚分来测定两个氨基酸序列之间的百分数同一性。此外，可使用已整合入GCG软件包(可在www.gcg.com上获得)的GAP程序中的Needleman和Wunsch(J MoI Biol.48:444-453(1970))算法，使用Blossum 62矩阵或PAM250矩阵以及16、14、12、10、8、6或4的缺口权重(gap weight)和1、2、3、4、5或6的长度权重来测定两个氨基酸序列之间的百分数同一性。
发明的有益效果
本发明所提供的事件2A-7能够显著抵抗鳞翅目昆虫的侵袭并具备良好的除草剂抗性。同时2A-7具备优良的遗传稳定性、安全性，与商业上可获得的品种(例如郑58)在无昆虫压力下的农学性能相当。本发明的事件2A-7具备广阔的应用前景，实现积极的社会和生态效益。
附图说明
图1显示了pCAMBIA3301+mcry1Ab+mcry2Ab载体图谱。
图2显示了实施例2中的内参基因PCR产物的电泳结果。其中，泳道1：产业化转基因玉米；泳道2：产业化转基因大豆；泳道3：产业化转基因油菜；泳道4：产业化转基因棉花；泳道5：产业化转基因水稻；泳道6：转基因玉米2A-5；泳道7：转基因玉米2A-7；泳道8：转基因玉米2A-7；泳道9：转基因玉米2A-7；泳道10：转基因玉米2A-7；泳道11：非转基因受体对照；泳道12：空白对照；泳道13：阳性对照T+zSSIIb(Zm00001d052263)质粒(10pg)；泳道M：分子量标志物DL2000plus。
图3显示了实施例2中的5’侧翼区特异性反应体系的PCR产物电泳结果。其中，泳道1：产业化转基因玉米；泳道2：产业化转基因大豆；泳道3：产业化转基因油菜；泳道4：产业化转基因棉花；泳道5：产业化转基因水稻；泳道6：转基因玉米2A-5；泳道7：转基因玉米2A-7；泳道8：转基因玉米2A-7；泳道9：转基因玉米2A-7；泳道10：转基因玉米2A-7；泳道11：非转基因受体对照；泳道12：空白对照；泳道13：前期克隆的含有5’端侧翼序列和插入序列的T+2A-7 5’质粒(10pg)；泳道M：分子量标志物DL2000plus。
图4显示了实施例2中的3’侧翼区特异性反应体系的PCR产物电泳结果。泳道1：产业化转基因玉米；泳道2：产业化转基因大豆；泳道3：产业化转基因油菜；泳道4：产业化转基因棉花；泳道5：产业化转基因水稻；泳道6：转基因玉米2A-5；泳道7：转基因玉米2A-7；泳道8：转基因玉米2A-7；泳道9：转基因玉米2A-7；泳道10：转基因玉米2A-7；泳道11：非转基因受体对照；泳道12：空白对照；泳道13：含有3’端侧翼序列和插入序列的T+2A-7 3’质粒质粒(10pg)；泳道M：分子量标志物DL2000plus。
图5显示了实施例3中2A-7对黏虫抗性的鉴定结果。
图6A显示了实施例3中2A-7穗部对玉米螟抗性的鉴定结果。其中，从上至下依次为2A-7、郑58、郑单958。
图6B显示了实施例3中2A-7茎秆对玉米螟抗性的鉴定结果。其中，从左至右依次为2A-7、郑58、郑单958。
图7显示了实施例3中2A-7穗部对棉铃虫抗性的鉴定结果。其中，从上至下依次为2A-7、郑58、郑单958。
图8显示了实施例3中2A-7叶片对草地贪夜蛾室内生测结果。其中，上部为对照郑58叶片，下部为2A-7叶片。
图9显示了实施例3中2A-7花丝对草地贪夜蛾室内生测结果。其中，上部为对照郑58花丝，下部为2A-7花丝。
图10显示了实施例3中2A-7对草地贪夜蛾田间生测结果。其中，左侧为2A-7，右侧为对照郑58。
序列信息
本发明涉及的部分序列的信息提供于下面的表1中。
表1：序列的描述
Figure PCTCN2020126452-appb-000001
关于生物材料保藏的说明
本发明涉及下列已在中国普通微生物菌种保藏管理中心(CGMCC)(北京市朝阳区北辰西路1号院3号)进行保藏的生物材料：
玉米(Zea mays)2A-7的种子，其具有保藏号CGMCC NO.17848，且保藏日期为2019年10月28日。
具体实施方式
现参照下列意在举例说明本发明(而非限定本发明)的实施例来描述本发明。
除非特别指明，否则基本上按照本领域内熟知的以及在各种参考文献中描述的常规方法进行实施例中描述的实验和方法。另外，实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。本领域技术人员知晓，实施例以举例方式描述本发明，且不意欲限制本发明所要求保护的范围。本文中提及的全部公开案和其他参考资料以其全文通过引用合并入本文。
实施例1.玉米事件2A-7的转化和选择
将选择标记基因bar(SEQ ID NO:14)、目的基因mCry1Ab(SEQ ID NO:12)和mCry2Ab(SEQ ID NO:13)及其配套调控元件插入到pCAMBIA3301(购自湖南丰晖生物科技有限公司)的T-DNA中，按照常规分子生物学方法构建植物表达载体，命名为pCAMBIA3301+mCry1Ab+mCry2Ab，各插入基因的配套调控元件如下表所示，图谱如图1所示，其中启动子Gly可参见中国专利申请CN201710702435.4。用酶切和双向测序的方法鉴定阳性克隆，用于玉米转化。
表2：转化载体中的调控元件
调控元件 功能 来源
Gly 目的基因mcry1Ab的启动子 玉米
nos 目的基因mcry1Ab的终止子 根癌农杆菌
CaMV35s 目的基因mcry2Ab的启动子 花椰菜花叶病毒
adh1 增强mCry2Ab的表达 玉米
nos 目的基因mcry2Ab的终止子 根癌农杆菌
CaMV35s 选择标记基因bar的启动子 花椰菜花叶病毒
35S ployA 选择标记基因bar的终止子 花椰菜花叶病毒
将上述载体转化农杆菌EHA105(购自北京华越洋生物科技有限公司)，再用含有目的基因的农杆菌侵染玉米幼胚，具体的转基因方法如下：
在转基因过程中所用受体为自交系HiIIA和HiIIB(公众可从“玉米遗传资源原种中心”获得(Maize GDB，2010))的杂交F1代。首先在田间种植自交系HiIIA和HiIIB，到自交系散粉时分别套袋；然后准备授粉，有两种授粉方式：HiIIA作母本，HiIIB作父本；HiIIA作父本，HiIIB作母本，授粉后9-11天，取授粉果穗籽粒上的未成熟幼胚，然后在室内进行农杆菌侵染，将被农杆菌侵袭的幼胚放在选择培养基上进行多次筛选，获得抗性愈伤，将抗性愈伤再生成苗，得到转基因T0代植株。获得转基因T0代以后，用T0代转基因植株的花粉对一些制种母本，如郑58等进行杂交。采用农杆菌侵染法将插入序列导入受体植株的幼胚，经除草剂双丙胺磷筛选后获得转基因植株。通过多次转化共获得2000余份T0代转化体，通过对mcry1Ab、mcry2Ab蛋白含量检测、插入片段拷贝数筛选、连续多代的稳定性检测以及不同地点农艺性状鉴定，由此获得mcry1Ab、mcry2Ab均高表达、单拷贝、遗传稳定的玉米转化体2A-7。
进一步，鉴定玉米转化体2A-7基因组中外源DNA的整合情况，方法如下：取新鲜玉米叶片提取玉米基因组DNA。进行TAIL-PCR，共涉及5种简并引物：LAD1-1、LAD1-2、LAD1-3、LAD1-4、AC1，6种特异性引物：RB-0a、RB-1a、RB-2a、LB-0a、LB-1a、LB-2a，其序列如下表所示。
表3：TAIL-PCR中所使用的引物
Figure PCTCN2020126452-appb-000002
Figure PCTCN2020126452-appb-000003
注：N＝A/T/C/G，B＝G/T/C，V＝A/G/C，D＝A/G
将PCR产物与B载体(北京全式金生物技术有限公司CB101-01)连接，并将连接产物转化入大肠杆菌感受态细胞。挑选单克隆进行PCR鉴定，并将扩增产物送北京奥科鼎盛生物科技有限公司测序。将测序结果与T-border序列和玉米基因组进行比对，对外源片段整合情况进行分析。
经确定，外源DNA的整合导致受体基因组中的部分序列被删除，所述被删除的序列是Chr3:179141695bp-179141723bp(B73参考基因组V4版本)，共计29bp，删除区域为玉米内源基因Zm00001d042767，该基因的功能预测为Glucan endo-13-beta-glucosidase 14，目标片段的插入和Chr3:179141695bp-179141723bp序列的删除将使该基因功能失活。该基因在玉米中为双拷贝，另外一个同源基因的基因号为Zm00001d012292。2A-7转化体插入在Chr3:179141694bp-179141724bp(B73参考基因组V4版本)之间，T-border整合入基因组的序列如SEQ ID NO:5的第483-8524位核苷酸所示，SEQ ID NO:5的组成如下表所示。在2A-7基因组中，所述外源序列侧接5’侧翼区和3’侧翼区，所述5’侧翼区具有SEQ ID NO:5的第1-432位的核苷酸，所述3’侧翼区具有SEQ ID NO:5的第8532-9031位的核苷酸。涵盖部分5’侧翼区及部分非基因组序列的5’接合序列如SEQ ID NO:1所示(对应于SEQ ID NO:5的423-442)，涵盖部分非基因组序列和部分3’侧翼区的3’接合序列如SEQ ID NO:2所示(对应于SEQ ID NO:5的8522-8541)。
表4：玉米转化体2A-7整合入基因组序列信息
Figure PCTCN2020126452-appb-000004
Figure PCTCN2020126452-appb-000005
注：(1)非预期整合序列即非基因组序列也非T-border序列，可能是插入片段整合时由基因组修复产生的序列；(2)表中未标注的位置为T-border区基因间序列。
2A-7种子的制备
通过对3301+mcry1Ab+mcry2Ab载体进行遗传转化，获得2A-7T0代植株，经过连续2代自交，获得纯合的2A-7种子，并在中国普通微生物菌种保藏管理中心(CGMCC)进行保藏，保藏号为CGMCC NO.17848，保藏日期为2019年10月28日。
实施例2.玉米事件2A-7的鉴定方法
1.材料和方法
1.1、玉米基因组DNA提取方法
(1)取CTAB溶液，预先65℃水浴；
(2)取约0.1g新鲜玉米叶片，剪成碎块，放在预冷的研钵当中，在液氮中迅速研磨成粉末状并立即转入预冷的2mL EP管中(一般不超过1/2管体积)；
(3)迅速向EP管中加入0.8mL 65℃温浴的CTAB缓冲液，轻轻摇荡均匀，65℃水浴30min，并不时轻摇；
(4)放置于通风橱约15min，冷却至室温；
(5)加入等体积的氯仿：异戊醇(24：1)，混匀，轻微振荡15min；
(6)室温下，12000rpm离心8min；
(7)吸取上清液至新的1.5mL EP管；
(8)加入等体积预冷的异丙醇(4℃预冷)；
(9)室温下，12000rpm离心8min；
(10)弃上清，加入75％乙醇1mL，混匀，弃上清(乙醇沉淀)；
(11)在通风橱放置直至乙醇完全挥发(1～2h)；
(12)用300μL的TE Buffer溶解DNA，4℃过夜备用。
1.2、转化事件特异性PCR方法
事件2A-7的5’及3’侧翼序列分别如SEQ ID NO:5的第1-432位核苷酸以及第8532-9031位核苷酸所示。针对玉米转化体2A-7的5'端及3’端插入位点序列分别涉及正反向引物(表5-6)，进行PCR反应，反应条件及反应体系分别如下表7-8所示。
表5：5'端引物信息
Figure PCTCN2020126452-appb-000006
表6：3'端引物信息
Figure PCTCN2020126452-appb-000007
表7：PCR反应条件
Figure PCTCN2020126452-appb-000008
Figure PCTCN2020126452-appb-000009
表8：PCR反应条件
反应体系的成分 终浓度 每一反应的体积(μL)
1.无核酸酶的水   12.6
2.反应缓冲液 1× 2
3.dNTP’s 0.25mM 2
4.正向引物 0.25 0.5
5.反向引物 0.25 0.5
6.DNA聚合酶(5U/μL) 0.1U/μL 0.4
DNA样品(50ng，25ng/μL) 2.5ng/μL 2
总体积   20
同时以玉米内源基因zSSIIb(Zm00001d052263)作为内参基因，其正向引物zSSIIb-F如SEQ ID NO:10所示，反向引物zSSIIb-R如SEQ ID NO:11所示。
用溴化乙锭染色的琼脂糖凝胶(3％)电泳检测PCR扩增产物。在电泳时加入合适的分子质量标准，以确定扩增产物的大小，利用凝胶成像系统使PCR扩增产物可见。
2.方法验证数据
以下样品中提取的1000ng左右的基因组DNA为模板，用于5’端和3’端的玉米转化体2A-7特异性体系和内标体系的PCR扩增，以确定该方法的特异性：4种含有转化体2A-7的不同的玉米单株、玉米转化体2A-5、产业化转基因玉米、产业化转基因大豆、产业化转基因棉花、产业化转基因水稻。其中：
1)产业化转基因玉米(Bt-11、Bt-176、MON863、MON810、GA21、NK603、T25、TC1507、MON89034、MON88017、59122、MIR604、3272、MON87460，混合制成1个样品，含量各1％)
2)产业化转基因大豆(MON87769、356043、305423、CV127、MON89788、A5547-127、A2704-12，混合制成1个样品，含量各1％)
3)产业化转基因油菜(MS1、MS8、RF1、RF2、RF3、T45、Oxy235、Topas19/2混合制成1个样品，含量各1％)
4)产业化转基因棉花(MON1445、MON531、MON15985、LLCOTTON25、MON88913，混合制成1个样品，含量各1％)
5)产业化转基因水稻(KF-6、KMD-1、M12、KF-2、KF-8混合制成1个样品， 含量各1％)。
内参基因的检测结果如图2所示，结果显示，所有玉米样品显示出预期扩增产物，所有非玉米样品未显示出扩增产物。
使用表5所示的5’端引物对进行扩增的检测结果如图3所示，结果显示，对于所有含转化体2A-7的样品，都观察到清晰的、与预期大小片段一致的单一条带，无非特异性扩增；对于所有其他玉米和非玉米样品，未观察到预期扩增产物。
使用表6所示的3’端引物对进行扩增的检测结果如图4所示，结果显示，对于所有含转化体2A-7的样品，都观察到清晰的、与预期大小片段一致的单一条带，无非特异性扩增；对于所有其他玉米和非玉米样品，未观察到预期扩增产物。
由于特异性PCR的两条引物分别与T-DNA及受体基因组的特定区域结合，因此仅当两段结合区域邻接时，才能完成PCR扩增过程。而转基因过程中，T-DNA的整合具有随机性，除2A-7转化体外，其它转化体的结合区域几乎不可能邻接，而即使邻接，其产物也将与预期大小不符。因此，上述5’端引物对及3’端引物可用于检测玉米转化体2A-7。
此外，鉴于5’接合序列和3’接合序列的独特性还可以利用与其特异性杂交的DNA探针来检测所述5’接合序列或3’接合序列的存在从而鉴定2A-7玉米事件。
实施例3.玉米事件2A-7的田间抗虫性状鉴定
1.试验依据
本试验参照的依据是：《农业部953号公告-10.1-2007》。
2.试验材料
2.1供试玉米
(1)转基因抗虫玉米2A-7T5代，其选育过程为利用含有目标载体的农杆菌，侵染HiIIA和HiIIB杂交的F1代幼胚，获得T0代转基因植株后，以郑为轮回亲本进行杂交和回交，获得T3代后，连续自交2代，获得纯合的郑58背景的2A-7；
(2)受体对照郑58；和
(3)当地生产应用的常规玉米品种郑单958。
上述材料的质量达到GB4404.1中不低于二级玉米种子的要求。
2.2供试昆虫
黏虫Mythimna separata：黏虫初孵幼虫(孵化时间12-24h)室内人工饲料或玉米幼 苗饲养的黏虫种群；
亚洲玉米螟Ostrinia furnacalis：玉米螟初孵幼虫(孵化时间2-12h)为室内人工饲养的亚洲玉米螟种群；
棉铃虫Helicoverpa armigera：棉铃虫初孵幼虫(孵化时间12-24h)为室内人工饲养的棉铃虫种群。
草地贪夜蛾Spodoptera frugiperda：草地贪夜蛾初孵幼虫(孵化时间12-24h)为室内人工饲养的草地贪夜蛾种群。
3.隔离措施
试验地隔离采用300米内花期隔离25天以上的方式进行。
4.实验方法
抗虫性人工接虫鉴定：按照《农业部953号公告-10.1-2007》的标准。
田间试验设计采用随机区组设计，三次重复，小区面积为30m 2(5m×6m)，行距60cm，株距25cm，土壤肥力水平和耕作管理与大田生产相同，全生育期不喷施杀虫剂。不同害虫接虫试验小区之间有2m的间隔，避免害虫在不同小区之间的扩散。接虫前和接虫后应保证田间具有一定湿度，如遇干旱，应及时浇水。
4.1黏虫
接虫方法：对黏虫的抗性鉴定在心叶期进行，玉米植株发育至展4叶-6叶期进行，每小区人工接虫不少于40株。每株接人工饲养的初孵幼虫30～40头，接于玉米心叶中，接虫3天后，进行第二次接虫，接虫数量和方法同第一次，接虫选择在傍晚进行。
调查记录：接虫14天后，调查玉米叶片受黏虫的为害重读和幼虫存活数。
结果表述：根据玉米叶片受黏虫的为害程度，计算各小区黏虫对玉米叶片为害级别(食叶级别)的平均值，其判断标准见表9，然后按照表10的规定判定转基因抗虫玉米对黏虫的抗性水平。
表9：玉米叶片受黏虫为害程度的分级标准
食叶级别 症状描述
1 叶片无被害，或仅叶片上有针刺状(≤1mm)虫孔
2 仅个别叶片上有少量弹孔大小(≤5mm)虫孔
3 少数叶片上有弹孔大小(≤5mm)虫孔
4 个别叶片上缺刻(≤10mm)
5 少数叶片上有缺刻(≤10mm)
6 部分叶片上有缺刻(≤10mm)
7 个别叶片部分被取食，少数叶片上有大片缺刻(≤10mm)
8 少数叶片被取食，部分叶片上有大片缺刻(≤10mm)
9 大部分叶片被取食
表10：玉米对黏虫的抗性评价标准
心叶期食叶级别平均值 抗性类型
1.0～2.0 高抗HR
2.1～4.0 抗R
4.1～6.0 中抗MR
6.1～8.0 感S
8.1～9.0 高感HS
4.2亚洲玉米螟
接虫方法：对靶标害虫亚洲玉米螟的抗性鉴定分心叶期世代和穗期世代，分别在心叶期(小喇叭口期，玉米植株发育至8叶～10叶期)和吐丝期进行人工接虫，每个时期各接虫2次，每个时期每小区人工接虫不少于40株，每株分别接60～80头亚洲玉米螟初孵幼虫。接虫选择在傍晚进行，若接虫后遇中雨以上的天气，需再接虫1次。
心叶期调查记录：心叶期接虫2～3周后逐株调查中上部叶片被玉米螟取食的状况。每份鉴定材料随机选取15～20株/行，逐株按表11中的描述记载玉米螟食叶级别(根据玉米螟幼虫取食心叶后所形成的叶片虫孔直径大小和数量划分食叶级别)。
表11：玉米螟对心叶为害程度的分级标准
食叶级别 症状描述
1 仅个别叶片上有1个～2个孔径≤1mm虫孔
2 仅个别叶片上有3个～6个孔径≤1mm虫孔
3 少数叶片上有7个以上孔径≤1mm虫孔
4 个别叶片上有1个～2个孔径≤2mm虫孔
5 少数叶片上有3个～6个孔径≤2mm虫孔
6 部分叶片上有7个以上孔径≤2mm虫孔
7 少数叶片上有1个～2个孔径＞2mm的虫孔
8 部分叶片上有3个～6个孔径＞2mm虫孔
9 大部分叶片上有7个以上孔径＞2mm虫孔
计算玉米螟对鉴定材料群体叶片为害程度(食叶级别)的平均值。计算方法如下：
平均食叶级别＝∑(食叶级别×该级别植株数)/调查总株数
心叶期结果表述：根据食叶级别的平均值，划分各鉴定材料的虫害级别，见表12。
表12：玉米对玉米螟的抗性评价标准
虫害级别 心叶期食叶级别平均值 抗性
1 1.0～2.9 高抗HR
3 3.0～4.9 抗R
5 5.0～6.9 中抗MR
7 7.0～8.9 感S
9 9.0 高感HS
吐丝期调查记录：调查玉米雌穗被害情况、蛀孔数量、蛀孔隧道长度(cm)以及存活幼虫龄期和存活数量，评价雌穗被害程度及植株被害情况。
吐丝期结果表述：玉米穗期抗虫性评价根据雌穗被害情况、蛀孔数量、蛀孔隧道长度(cm)以及存活幼虫龄期和存活数量，计算各小区穗期玉米螟对雌穗的抗性被害级别平均值，判断标准见表13，根据表14的规定辨别玉米穗期对玉米螟的抗性水平。
表13：玉米穗期受亚洲玉米螟为害程度的分级标准
雌穗被害级别 症状描述
1 雌穗没有受害
2 花丝被害＜50％
3 大部分花丝被害≥50％；有幼虫存活，龄期≤2龄
4 穗尖被害≤1cm，有幼虫存活，龄期≤3龄
5 穗尖被害≤2cm；或有幼虫存活，龄期≤4龄；隧道长度≤2cm
6 穗尖被害≤3cm；或有幼虫存活，龄期≥4龄；隧道长度≤4cm
7 穗尖被害≤4cm，隧道长度≤6cm
8 穗尖被害≤5cm，隧道长度≤8cm
9 穗尖被害＞5cm，隧道长度＞8cm
表14：玉米雌穗对亚洲玉米螟的抗性评价标准
雌穗被害叶级别平均值 抗性类型
1.0～2.0 高抗HR
2.1～3.0 抗R
3.1～5.0 中抗MR
5.1～7.0 感S
≥7.1 高感HS
4.3棉铃虫
接虫方法：对棉铃虫的抗性鉴定在抽丝散粉期进行，每株分别接20～30头初孵幼虫。每小区人工接虫不少于40株，接于玉米花丝上，接虫3天后，第二次接虫，接虫数量同第一次。接虫选择在傍晚进行，若接虫后遇中雨以上的天气，需再接虫1次。
危害程度调查：在人工接虫第14～21天进行，逐株调查雌穗被害率，每个雌穗存活幼虫数、雌穗被害长度。
结果表述：玉米穗期抗虫性评价根据雌穗被害率、存活幼虫数、雌穗被害长度(cm)，计算各小区玉米穗期棉铃虫对雌穗的为害级别平均值，判断标准见表15，根据表16的规定判别玉米穗期对棉铃虫的抗性水平。
表15：玉米穗期受棉铃虫为害程度的分级标准
雌穗被害级别 症状描述
0 雌穗没有被害
1 仅花丝被害
2 穗顶被害1cm
3+ 穗顶下被害每增加1cm，相应的被害级别增加1级
…N  
表16：玉米雌穗对棉铃虫的抗性评价标准
雌穗被害叶级别平均值 抗性类型
0～1.0 高抗HR
1.1～3.0 抗R
3.1～5.0 中抗MR
5.1～7.0 感S
≥7.1 高感HS
4.4草地贪夜蛾
接虫方法：对靶标害虫草地贪夜蛾的抗性鉴定分心叶期世代和穗期世代，分别在心叶期(小喇叭口期，玉米植株发育至8叶～10叶期)和吐丝期进行人工接虫，每个时期各接虫2次，每个时期每小区人工接虫不少于40株，每株分别接20～30头草地贪夜蛾初孵幼虫。接虫选择在傍晚进行，若接虫后遇中雨以上的天气，需再接虫1次。
调查记录：接虫14天后，调查玉米叶片受草地贪夜蛾的为害程度。
结果表述：根据玉米叶片受草地贪夜蛾的为害程度，计算各小区草地贪夜蛾对玉米叶片为害级别(食叶级别)的平均值，其判断标准见下表，然后按照表17、表18的规定判定转基因抗虫玉米对草地贪夜蛾的抗性水平。
表17：玉米叶片受草地贪夜蛾为害程度的分级标准
食叶级别 症状描述
1 叶片无被害，或仅叶片上有针刺状(≤1mm)虫孔
2 仅个别叶片上有少量弹孔大小(≤5mm)虫孔
3 少数叶片上有弹孔大小(≤5mm)虫孔
4 个别叶片上缺刻(≤10mm)
5 少数叶片上有缺刻(≤10mm)
6 部分叶片上有缺刻(≤10mm)
7 个别叶片部分被取食，少数叶片上有大片缺刻(≤10mm)
8 少数叶片被取食，部分叶片上有大片缺刻(≤10mm)
9 大部分叶片被取食
表18：玉米对草地贪夜蛾的抗性评价标准
心叶期食叶级别平均值 抗性类型
1.0～2.0 高抗HR
2.1～4.0 抗R
4.1～6.0 中抗MR
6.1～8.0 感S
8.1～9.0 高感HS
吐丝期调查记录：调查玉米雌穗被害情况、蛀孔数量、蛀孔隧道长度(cm)以及存活幼虫龄期和存活数量，评价雌穗被害程度及植株被害情况。
吐丝期结果表述：玉米穗期抗虫性评价根据雌穗被害情况、蛀孔数量、蛀孔隧道长度(cm)以及存活幼虫龄期和存活数量，计算各小区穗期草地贪夜蛾对雌穗的抗性被害级别平均值，判断标准见表19，根据表20的规定辨别玉米穗期对草地贪夜蛾的抗性水平。
表19：玉米穗期受草地贪夜蛾为害程度的分级标准
雌穗被害级别 症状描述
1 雌穗没有受害
2 花丝被害＜50％
3 大部分花丝被害≥50％；有幼虫存活，龄期≤2龄
4 穗尖被害≤1cm，有幼虫存活，龄期≤3龄
5 穗尖被害≤2cm；或有幼虫存活，龄期≤4龄；隧道长度≤2cm
6 穗尖被害≤3cm；或有幼虫存活，龄期≥4龄；隧道长度≤4cm
7 穗尖被害≤4cm，隧道长度≤6cm
8 穗尖被害≤5cm，隧道长度≤8cm
9 穗尖被害＞5cm，隧道长度＞8cm
表20：玉米雌穗对草地贪夜蛾的抗性评价标准
雌穗被害叶级别平均值 抗性类型
1.0～2.0 高抗HR
2.1～3.0 抗R
3.1～5.0 中抗MR
5.1～7.0 感S
≥7.1 高感HS
5.结果分析
5.1黏虫抗性鉴定结果分析
接虫后2-3周调查接虫鉴定结果。结果如图5及表21所示，通过对叶片危害率、虫孔或缺刻大小等参数的分析表明，5％显著水平范围内，转基因玉米2A-7的叶片危害率、虫孔或缺刻大小都显著低于其对应的非转基因玉米品种和当地普通栽培玉米品种，且达到显著差异水平。实验结果表明，转基因玉米2A-7对靶标害虫黏虫具有较好的控制作用。
表21：叶片受黏虫危害级别调查结果
Figure PCTCN2020126452-appb-000010
5.2玉米螟抗性鉴定结果分析
5.2.1叶片危害级别调查
心叶期(V6-V8)接虫，接虫后14天逐株调查食叶级别。叶片危害级别调查结果显示，转基因2A-7玉米对靶标害虫玉米螟有很好的控制作用，5％显著水平范围内，2A-7叶片危害级别显著低于其对应的非转基因玉米品种和当地普通栽培玉米品种，且达到显著差异水平(表22)。结果表明，转基因玉米2A-7亚洲玉米螟抗性优于其对应的非转基因玉米品种和当地普通栽培玉米品种。
表22：心叶期叶片受玉米螟危害级别调查结果
Figure PCTCN2020126452-appb-000011
Figure PCTCN2020126452-appb-000012
5.2.2雌穗危害级别调查
吐丝期接虫，于收获前调查接虫鉴定结果。结果如图6A-6B及表23-24所示，通过对雌穗危害率、单株蛀孔数、单株隧道长度和单株活虫数及存活幼虫龄期等五个参数的分析表明，5％显著水平范围内，转基因玉米2A-7的雌穗危害率、单株蛀孔数、单株隧道长度和单株活虫数及存活幼虫龄期都显著低于其对应的非转基因玉米品种和当地普通栽培玉米品种，且达到显著差异水平。实验结果表明，转基因玉米2A-7对靶标害虫亚洲玉米螟具有较好的控制作用。
表23：吐丝期抗玉米螟效果
Figure PCTCN2020126452-appb-000013
表24：雌穗受玉米螟危害级别调查结果
Figure PCTCN2020126452-appb-000014
由以上结果可知，2A-7、对照及主栽品种郑单958的雌穗平均被害级别分别为1.00、7.57和7.40。综合以上各项数据，2A-7抗虫玉米和两种对照玉米对玉米螟的抗性效果存在 显著差异，表现为高抗水平。
5.2.3穗柄危害级别调查
穗柄作为玉米灌浆中养分运输的唯一通道，对籽粒发育起着重要作用，同时，在收获期，穗柄对果穗起到支撑作用，为机械化收获提供的重要保障。为此我们调查了穗柄被害率、穗柄存活幼虫数、穗柄被害长度(cm)等参数，分析表明，5％显著水平范围内，转基因玉米2A-7的穗柄被害率、穗柄存活幼虫数、穗柄被害长度(cm)都显著低于其对应的非转基因玉米品种和当地普通栽培玉米品种，且达到显著差异水平(表25)。实验结果表明，转基因玉米2A-7对靶标害虫玉米螟具有较好的控制作用。
表25：穗柄对玉米螟抗虫效果
Figure PCTCN2020126452-appb-000015
5.3棉铃虫抗性鉴定结果分析
吐丝期接虫，于接虫后2-3周调查接虫鉴定结果。结果如图7及表26所示，通过对雌穗被害率、存活幼虫数、雌穗被害长度(cm)参数的分析表明，5％显著水平范围内，转基因玉米2A-7的雌穗被害率、存活幼虫数、雌穗被害长度(cm)都显著低于其对应的非转基因玉米品种和当地普通栽培玉米品种，且达到显著差异水平。实验结果表明，转基因玉米2A-7对靶标害虫棉铃虫具有较好的控制作用。
表26：吐丝期抗棉铃虫效果
Figure PCTCN2020126452-appb-000016
以上结果充分说明2A-7对玉米螟、粘虫、棉铃虫等鳞翅目害虫的侵袭具有显著的抗性。
5.4草地贪夜蛾抗性鉴定结果
5.4.1叶片危害级别调查
心叶期(小喇叭口期，玉米植株发育至8叶～10叶期)接虫，接虫后2-3周调查接虫鉴定结果。结果如表27所示，通过对叶片危害率、虫孔或缺刻大小等参数的分析表明，5％显著水平范围内，2A-7转化体的叶片危害率、虫孔或缺刻大小都显著低于其对应的非转基因玉米对照品种，且达到显著差异水平。实验结果表明，转化体叶片对靶标害虫草地贪夜蛾具有良好的控制作用，达到高抗级别。
表27：心叶期叶片受草地贪夜蛾危害级别调查结果
Figure PCTCN2020126452-appb-000017
5.4.2雌穗危害级别调查
吐丝期接虫，于收获前调查接虫鉴定结果。结果表28-29所示，通过对雌穗危害率、单株蛀孔数、单株隧道长度和单株活虫数及存活幼虫龄期等五个参数的分析表明，5％显著水平范围内，转基因玉米2A-7的雌穗危害率、单株蛀孔数、单株隧道长度和单株活虫数及存活幼虫龄期都显著低于其对应的非转基因玉米品种和当地普通栽培玉米品种，且达到显著差异水平。实验结果表明，转基因玉米2A-7对靶标害虫草地贪夜蛾具有较好的控制作用。
表28：吐丝期抗草地贪夜蛾效果
Figure PCTCN2020126452-appb-000018
Figure PCTCN2020126452-appb-000019
表29：雌穗受草地贪夜蛾危害级别调查结果
Figure PCTCN2020126452-appb-000020
此外，图8-图9分别显示了2A-7叶片或花丝对草地贪夜蛾抗性的室内生测结果。图10显示了2A-7对草地贪夜蛾抗性的田间生测结果。
以上结果表明，转基因玉米2A-7对草地贪夜蛾具有良好的抗性。
实施例4.玉米事件2A-7的草铵膦抗性鉴定
1.试验方案
1.1试验材料
保试达Basta(18％草铵膦可溶液剂)，拜耳公司生产。
2A-7：转化体T5代材料，同实施例3。
1.2试验设计
(1)试验设计
随机区组设计，3-4次重复。小区间设1.0m宽隔离带，小区面积不小于24m 2，处理包括：转基因玉米不喷施除草剂；转基因玉米喷施目标除草剂；对应的非转基因玉米不喷施除草剂；对应的非转基因玉米喷施目标除草剂。
(2)草铵膦施用剂量
所用除草剂的施用剂量分为：农药登记标签的中剂量(600g有效成分/公顷)、中剂量2倍量(1200g有效成分/公顷)、中剂量4倍量(2400g有效成分/公顷)。对水量450L/公顷。
1.3施药时期
按耐草铵膦玉米推荐时间施用。
苗后茎叶处理草铵膦，一般抗性鉴定应在玉米3-5叶期使用。
1.4喷雾器械要求
(1)喷雾器的选择
要求选择压力均恒、喷幅较宽、流速稳定的人工背负式喷雾器，或CO 2压缩喷雾器。喷雾均匀。
(2)喷头
选择扇形喷头。
(3)喷施方法
每种处理需一次性喷施完成。用药量应按照喷施小区的实际面积计算。药后12小时内降雨应重新进行试验。
1.5抗性鉴定调查
分别在用药后1周、2周和4周调查和记录玉米成苗率、植株高度、药害症状。每小区取15株玉米。
玉米收获后取每小区中间2行玉米测产。
1.6结果分析与表述
除草剂受害率按下式计算。
Figure PCTCN2020126452-appb-000021
式中：
X—受害率，单位为百分率(％)；
N—同级受害株数；
S—级别数；
T—总株数；
M—最高级别。
药害症状分级按GB/T 17980.42-2000。
1级：玉米生长正常、无任何危害症状；
2级：玉米轻微药害，药害少于10％；
3级：玉米中等药害，以后能恢复，不影响产量；
4级：玉米药害较重，难以恢复，造成减产；
5级：玉米药害严重，不能恢复，造成明显减产或绝产。
2.实验结果
2.1受害率
分别在喷施1周、2周和四周对转化体的受害率进行调查，结果见下表。
表30：草铵膦处理受害率
Figure PCTCN2020126452-appb-000022
从上表可以看出在喷施2倍中剂量草铵膦后有轻微药害，药害少于10％，喷施4倍中剂量浓度草铵膦后，转基因玉米2A-7中等药害，药害大于10％。
2.2 2A-7株高调查
分别在喷施1周、2周和四周对转化体的株高进行调查，结果见下表。
表31：草铵膦处理对株高的影响
Figure PCTCN2020126452-appb-000023
从上表可以看出，转基因玉米2A-7在喷施不同浓度草铵膦后与未喷施对照株高有少量降低，但无显著性差异。
2.3 2A-7产量调查
在收获时对单穗籽粒产量和含水量进行调查，并计算亩产(含14％水分)结果见下表。
表32：草铵膦处理对产量的影响
Figure PCTCN2020126452-appb-000024
Figure PCTCN2020126452-appb-000025
从上表可以看出，转基因玉米2A-7在喷施不同浓度草铵膦后较与未喷施对照产量有所降低，但无显著性差异。
基于上述结果，根据GB/T 17980.42-2000的药害症状分级结果如下表所示。转基因玉米2A-7在喷施中剂量草铵膦后玉米生长正常、无任何危害症状，抗性等级为1级，在喷施2倍中剂量草铵膦后玉米轻微药害，药害少于10％，抗性等级为2级，在喷施4倍中剂量草铵膦后玉米中等药害，株高能够恢复，产量较微喷处理有所降低，抗性等级为4级。以上结果表明2A-7对草铵膦等除草剂具备优良的抗性。
表33：草铵膦抗性等级划分
Figure PCTCN2020126452-appb-000026
尽管本发明的具体实施方式已经得到详细的描述，本领域技术人员将会理解。根据已经公开的所有教导，可以对那些细节进行各种修改和替换，这些改变均在本发明的保护范围之内。本发明的全部范围由所附权利要求及其任何等同物给出。

Claims (35)

  1. 一种玉米植物或其部分、种子、细胞或后代，所述玉米植物或其部分、种子、细胞或后代具有掺入其基因组中的外源核酸分子，所述外源核酸分子包含Cry1Ab和Cry2Ab基因，其中，所述外源核酸分子侧接5’侧翼区，所述5’侧翼区具有SEQ ID NO:5的第1-432位或第300-432位的核苷酸序列，并且所述外源核酸分子侧接3’侧翼区，所述3’侧翼区具有SEQ ID NO:5的第8532-9031位或第8532-8800位的核苷酸序列。
  2. 一种玉米植物或其部分、种子、细胞或后代，所述玉米植物或其部分、种子、细胞或后代具有掺入其基因组中的外源核酸分子，所述外源核酸分子包含Cry1Ab和Cry2Ab基因，其中，所述外源核酸分子侧接5’侧翼区和3’侧翼区，包含5’侧翼区和与之相邻的外源核酸分子的部分的序列如SEQ ID NO:1所示，包含3’侧翼区和与之相邻的外源核酸分子的部分的序列如SEQ ID NO:2所示。
  3. 权利要求1或2所述的玉米植物或其部分、种子、细胞或后代，其中，所述外源核酸分子在基因组中的位置对应于B73 V4版本参考基因组序列的Chr3:179141694bp-179141724bp之间。
  4. 权利要求1-3任一项所述的玉米植物或其部分、种子、细胞或后代，其中，所述Cry1Ab和Cry2Ab基因的核苷酸序列经密码子优化用于在单子叶植物(例如玉米)细胞中进行表达；
    优选地，所述Cry1Ab基因如SEQ ID NO:12所示；
    优选地，所述Cry2Ab基因如SEQ ID NO:13所示。
  5. 权利要求1-4任一项所述的玉米植物或其部分、种子、细胞或后代，其中，所述外源核酸分子进一步包含Bar基因；
    优选地，所述外源核酸分子包含35S polyA终止子、Bar基因、CAMV 35S启动子、nos polyA终止子、Cry1Ab基因、Gly启动子、CAMV 35S启动子、adh1增强子、Cry2Ab基因和nos polyA终止子；
    优选地，所述外源核酸分子包含SEQ ID NO:5的第483-8524位核苷酸的序列或 其互补序列。
  6. 权利要求1-5任一项所述的玉米植物或其部分、种子、细胞或后代，其中所述玉米植物或其部分、种子、细胞或后代的基因组包含SEQ ID NOs:1-5任一项所示的序列或其互补序列。
  7. 权利要求1-6任一项所述的玉米植物或其部分、种子、细胞或后代，其中，当以SEQ ID NO:6及SEQ ID NO:7分别作为正向引物和反向引物对所述玉米植物或其部分、种子、细胞或后代的基因组DNA进行扩增时，产生长度约为200-300bp(例如约258bp)的扩增子；和/或，当以SEQ ID NO:8及SEQ ID NO:9分别作为正向引物和反向引物对所述基因组DNA进行扩增时，产生长度约为250-350bp(例如约310bp)的扩增子。
  8. 权利要求1-7任一项所述的玉米植物或其部分、种子、细胞或后代，其中，产生所述玉米植物或其部分、种子、细胞或后代的玉米种子保藏于中国普通微生物菌种保藏管理中心(CGMCC)，并具有保藏号CGMCC NO.17848。
  9. 权利要求1-8任一项所述的玉米植物或其部分、种子、细胞或后代，其具有对昆虫侵袭的抗性；
    优选地，所述昆虫选自鳞翅目昆虫；
    优选地，所述昆虫选自黏虫、玉米螟(如亚洲玉米螟)、棉铃虫、桃蛀螟、草地贪夜蛾中的一种或多种。
  10. 玉米种子，其保藏于中国普通微生物菌种保藏管理中心(CGMCC)，并具有保藏号CGMCC NO.17848。
  11. 由权利要求10所述的玉米种子产生的玉米植物或其部分、种子、细胞或后代；
    优选地，所述玉米植物或其部分、种子、细胞或后代具有对昆虫侵袭的抗性；
    优选地，所述昆虫选自鳞翅目昆虫；
    优选地，所述昆虫选自黏虫、玉米螟(如亚洲玉米螟)、棉铃虫、桃蛀螟、草地贪 夜蛾中的一种或多种。
  12. 包含权利要求1-9、11任一项所述的玉米植物或其部分、种子、细胞或后代、或权利要求9所述的玉米种子的制品；
    优选地，所述制品包含所述玉米植物或其部分、种子、细胞或后代、或玉米种子的基因组DNA。
  13. 权利要求12所述的制品，其中，所述制品包含选自SEQ ID NOs:1-5任一项所示的序列或其互补序列。
  14. 权利要求12所述的制品，其中，当以SEQ ID NO:6及SEQ ID NO:7分别作为正向引物和反向引物对所述制品中所含有的核酸进行扩增时，产生长度约为200-300bp(例如约258bp)的扩增子；和/或，当以SEQ ID NO:8及SEQ ID NO:9分别作为正向引物和反向引物对所述制品中所含有的核酸进行扩增时，产生长度约为250-350bp(例如约310bp)的扩增子。
  15. 权利要求12-14任一项所述的制品，其中，所述制品选自玉米耳穗、去苞叶玉米、玉米穗丝、玉米花粉、玉米糁、玉米粉、压碎玉米、玉米面、玉米油、玉米淀粉、玉米浆、玉米麦芽、玉米糖、玉米糖浆、由玉米油生产的人造黄油、不饱和玉米油、饱和玉米油、玉米片、爆玉米花、由玉米生产的乙醇和/或汁(liquor)、由玉米发酵产生的干酒糟(DDGS)、来自玉米的动物饲料、化妆品和填充剂。
  16. 权利要求1-9、11任一项所述的玉米植物或其部分、种子、细胞或后代，或权利要求11-14所述的制品，其中，所述玉米植物的部分选自籽粒、花粉、胚珠、花、枝条、根、茎、穗丝、花序、耳穗和叶。
  17. 一种引物对，其包含第一引物和第二引物，其中，所述第一引物含有由SEQ ID NO:5的第1-432位核苷酸(例如第200-432位核苷酸)的序列或其互补序列的至少15个连续核苷酸(例如15-30个连续核苷酸)所组成的核苷酸序列或与所述核苷酸序列相比具有至少80％同一性的序列，所述第二引物含有由SEQ ID NO:5的第483- 8524位核苷酸(例如第483-1000位核苷酸)的序列或其互补序列的至少15个连续核苷酸(例如15-30个连续核苷酸)所组成的核苷酸序列或与所述核苷酸序列相比具有至少80％同一性的序列。
  18. 权利要求17所述的引物对，其中，当使用所述第一引物和第二引物扩增SEQ ID NO:5所示的序列时，产生长度为100-500bp的扩增子；
    优选地，所述扩增子的长度为200-400bp，例如200-300bp，例如约258bp。
  19. 权利要求17或18所述的引物对，其中，所述第一引物含有由SEQ ID NO:5的第300-400位核苷酸的序列或其互补序列的至少15个连续核苷酸(例如15-30个连续核苷酸)所组成的核苷酸序列或与所述核苷酸序列相比具有至少80％同一性的序列，所述第二引物含有由SEQ ID NO:5的第500-600位核苷酸的序列或其互补序列的至少15个连续核苷酸(例如15-30个连续核苷酸)所组成的核苷酸序列或与所述核苷酸序列相比具有至少80％同一性的序列；
    优选地，所述第一引物含有由SEQ ID NO:5的第300-350位核苷酸的序列或其互补序列的至少15个连续核苷酸所组成的核苷酸序列或与所述核苷酸序列相比具有至少80％同一性的序列；
    优选地，所述第二引物含有由SEQ ID NO:5的第520-570位核苷酸的序列或其互补序列的至少15个连续核苷酸所组成的核苷酸序列或与所述核苷酸序列相比具有至少80％同一性的序列；
    优选地，所述第一引物含有SEQ ID NO:6所示的序列或与其相比具有至少80％同一性的序列，所述第二引物含有SEQ ID NO:7所示的序列或与其相比具有至少80％同一性的序列。
  20. 一种引物对，其包含第一引物和第二引物，其中，所述第一引物含有由SEQ ID NO:5的第483-8524位核苷酸(例如第8000-8524位核苷酸，例如第8300-8524位核苷酸)的序列或其互补序列的至少15个连续核苷酸(例如15-30个连续核苷酸)所组成的核苷酸序列或与所述核苷酸序列相比具有至少80％同一性的序列，所述第二引物含有由SEQ ID NO:5的第8532-9031位核苷酸(例如第8532-8800位核苷酸)的序列或其互补序列的至少15个连续核苷酸(例如15-30个连续核苷酸)所组成的核苷酸 序列或与所述核苷酸序列相比具有至少80％同一性的序列。
  21. 权利要求20所述的引物对，其中，当使用所述第一引物和第二引物扩增SEQ ID NO:5所示的序列时，产生长度为100-500bp的扩增子；
    优选地，所述扩增子的长度为200-400bp，例如250-350bp，例如约310bp。
  22. 权利要求20或21所述的引物对，其中，所述第一引物含有由SEQ ID NO:5的第8400-8500位核苷酸的序列或其互补序列的至少15个连续核苷酸(例如15-30个连续核苷酸)所组成的核苷酸序列或与所述核苷酸序列相比具有至少80％同一性的序列，所述第二引物含有由SEQ ID NO:5的第8700-8800位核苷酸的序列或其互补序列的至少15个连续核苷酸(例如15-30个连续核苷酸)所组成的核苷酸序列或与所述核苷酸序列相比具有至少80％同一性的序列；
    优选地，所述第一引物含有由SEQ ID NO:5的第8450-8500位核苷酸的序列或其互补序列的至少15个连续核苷酸所组成的核苷酸序列或与所述核苷酸序列相比具有至少80％同一性的序列；
    优选地，所述第二引物含有由SEQ ID NO:5的第8750-8800位核苷酸的序列或其互补序列的至少15个连续核苷酸所组成的核苷酸序列或与所述核苷酸序列相比具有至少80％同一性的序列；
    优选地，所述第一引物含有SEQ ID NO:8所示的序列或与其相比具有至少80％同一性的序列，所述第二引物含有SEQ ID NO:9所示的序列或与其相比具有至少80％同一性的序列。
  23. 在包含玉米核酸的样品中检测对玉米事件2A-7独特的核酸分子的方法，其包括：
    (1)将权利要求17-22任一项所述引物对与该样品接触；
    (2)进行核酸扩增反应；和
    (3)通过凝胶电泳检测步骤(2)的产物；
    其中，所述玉米事件2A-7是权利要求1-9任一项所述的玉米植物或其部分、种子、细胞或后代；
    优选地，所述玉米事件2A-7包含掺入其基因组中的编码mcry1Ab和mcry2Ab的 外源核酸分子，所述外源核酸分子侧接5’侧翼区和3’侧翼区，包含5’侧翼区和与之相邻的外源核酸分子的部分的序列如SEQ ID NO:1所示，包含3’侧翼区和与之相邻的外源核酸分子的部分的序列如SEQ ID NO:2所示；
    优选地，所述玉米事件2A-7在其基因组中包含SEQ ID NOs:1-5任一项所示的序列或其互补序列；
    优选地，在步骤(3)中当检测到预期扩增子时，表明所述样品包含对玉米事件2A-7独特的核酸分子。
  24. 权利要求23所述的方法，其包括：
    (1)将权利要求17-19任一项所述引物对与该样品接触；
    (2)进行核酸扩增反应；和
    (3)通过凝胶电泳检测步骤(2)的产物；
    其中，当检测到长度为约250-260bp(例如约258bp)的扩增子时，表明在所述样品中存在对玉米事件2A-7独特的核酸分子；
    优选地，所述引物对包含：含有SEQ ID NO:6所示的序列或与其相比具有至少80％同一性的序列的第一引物和含有SEQ ID NO:7所示的序列或与其相比具有至少80％同一性的序列的第二引物。
  25. 权利要求23所述的方法，其包括：
    (1)将权利要求20-22任一项所述引物对与该样品接触；
    (2)进行核酸扩增反应；和
    (3)通过凝胶电泳检测步骤(2)的产物；
    其中，当检测到长度为约305-315bp(例如约310bp)的扩增子时，表明在所述样品中存在对玉米事件2A-7独特的核酸分子；
    优选地，所述引物对包含：含有SEQ ID NO:8所示的序列或与其相比具有至少80％同一性的序列的第一引物和含有SEQ ID NO:9所示的序列或与其相比具有至少80％同一性的序列的第二引物。
  26. 在包含玉米核酸的样品中检测对玉米事件2A-7独特的核酸分子的方法，其包括：
    (1)将引物对与该样品接触；其中，当该引物对被用于扩增玉米事件2A-7的基因组DNA的核酸时产生含有选自下列的核苷酸序列的扩增子：SEQ ID NOs:1-5任一项所示的序列或其互补序列；
    (2)进行核酸扩增反应，由此产生所述扩增子；和
    (3)检测该扩增子；
    其中，所述玉米事件2A-7是权利要求1-9任一项所述的玉米植物或其部分、种子、细胞或后代；
    优选地，所述玉米事件2A-7包含掺入其基因组中的编码mcry1Ab和mcry2Ab的外源核酸分子，所述外源核酸分子侧接5’侧翼区和3’侧翼区，包含5’侧翼区和与之相邻的外源核酸分子的部分的序列如SEQ ID NO:1所示，包含3’侧翼区和与之相邻的外源核酸分子的部分的序列如SEQ ID NO:2所示；
    优选地，所述玉米事件2A-7在其基因组中包含SEQ ID NOs:1-5任一项所示的序列或其互补序列。
  27. 在包含玉米核酸的样品中检测对玉米事件2A-7独特的核酸分子的存在的方法，其包括：
    (1)将特异于靶序列的核酸探针与所述样品接触，所述靶序列含有选自下列的核苷酸序列：SEQ ID NOs:1-5任一项所示的序列或其互补序列；
    (2)对所述样品和核酸探针实施严格杂交条件；和
    (3)检测所述核酸探针与样品的杂交；
    其中，所述玉米事件2A-7是权利要求1-9任一项所述的玉米植物或其部分、种子、细胞或后代；
    优选地，所述玉米事件2A-7包含掺入其基因组中的编码mcry1Ab和mcry2Ab的外源核酸分子，所述外源核酸分子侧接5’侧翼区和3’侧翼区，包含5’侧翼区和与之相邻的外源核酸分子的部分的序列如SEQ ID NO:1所示，包含3’侧翼区和与之相邻的外源核酸分子的部分的序列如SEQ ID NO:2所示；
    优选地，所述玉米事件2A-7在其基因组中包含SEQ ID NOs:1-5任一项所示的序列或其互补序列；
    优选地，当检测到杂交时，说明在所述样品中存在对玉米事件2A-7独特的核酸分子；
    优选地，所述核酸探针含有：包含所述5’侧翼区和与之相邻的外源核酸分子的部分的序列，或与所述序列相比具有至少80％序列同一性的序列；
    优选地，所述核酸探针与SEQ ID NO：1或SEQ IDNO：2所示的序列或其互补序列或任一个的片段具有至少80％的序列同一性；
    优选地，所述核酸探针包含SEQ ID NO:1或2所示的序列或其互补序列；
    优选地，所述核酸探针带有荧光标记。
  28. 权利要求23-27任一项所述的方法，其中，所述样品选自玉米耳穗、去苞叶玉米、玉米穗丝、玉米花粉、玉米糁、玉米粉、压碎玉米、玉米面、玉米油、玉米淀粉、玉米浆、玉米麦芽、玉米糖、玉米糖浆、由玉米油生产的人造黄油、不饱和玉米油、饱和玉米油、玉米片、爆玉米花、由玉米生产的乙醇和/或汁(liquor)、由玉米发酵产生的干酒糟(DDGS)、来自玉米的动物饲料、化妆品和填充剂。
  29. 一种试剂盒，其包含：(i)权利要求17-22任一项所述的引物对，和/或(ii)核酸探针，所述核酸探针特异于SEQ ID NOs:1-5任一项所示的序列或其互补序列；
    优选地，所述核酸探针与SEQ ID NO：1或SEQ IDNO：2所示的序列或其互补序列或任一个的片段具有至少80％的序列同一性；
    优选地，所述核酸探针包含SEQ ID NO:1或2所示的序列或其互补序列；
    优选地，所述核酸探针带有荧光标记。
  30. 分离的核酸分子，其含有选自下列的核苷酸序列：SEQ ID NOs:1-5任一项所示的序列或其互补序列。
  31. 一种生产昆虫抗性玉米植物的方法，其包括：
    (1)将第一亲代玉米植物与第二亲代玉米植物杂交；其中，所述第一或第二亲代玉米植物如权利要求1-9、11任一项中所定义；
    (2)由(1)的杂交获得第一代子代植物；和
    (3)从这些第一代子代植物中选择对昆虫具有抗性的子代植物，其中，当所述子代植物满足下述(3a)-(3c)中的至少一种时，表明其为昆虫抗性玉米植物：
    (3a)子代植物在其基因组中含有SEQ ID NOs:1-5任一项所示的序列或其互补序 列；
    (3b)当以SEQ ID NO:6及SEQ ID NO:7分别作为正向引物和反向引物对子代植物的基因组DNA进行扩增时，产生长度约为200-300bp(例如约258bp)的扩增子；和/或，
    (3c)当以SEQ ID NO:8及SEQ ID NO:9分别作为正向引物和反向引物对子代植物的基因组DNA进行扩增时，产生长度约为250-350bp(例如约310bp)的扩增子；或，
    (3d)当使用特异于SEQ ID NOs:1-5任一项所示的序列或其互补序列的核酸探针(例如，包含SEQ ID NO:1或2所示的序列或其互补序列的核酸探针)对子代植物的基因组DNA进行检测时，能够检测到杂交；
    优选地，所述第一或第二亲代玉米植物包含掺入其基因组中的编码mcry1Ab和mcry2Ab的外源核酸分子，所述外源核酸分子侧接5’侧翼区和3’侧翼区，包含5’侧翼区和与之相邻的外源核酸分子的部分的序列如SEQ ID NO:1所示，包含3’侧翼区和与之相邻的外源核酸分子的部分的序列如SEQ ID NO:2所示；
    优选地，所述第一或第二亲代玉米植物在其基因组中包含SEQ ID NOs:1-5任一项所示的序列或其互补序列；
    优选地，所述昆虫选自鳞翅目昆虫，例如黏虫、玉米螟(如亚洲玉米螟)、棉铃虫、桃蛀螟、草地贪夜蛾等鳞翅目害虫中的一种或多种。
  32. 权利要求31所述的方法，其中，所述方法还包括以下步骤：
    (4)将步骤(3)获得的子代植物自交，由此产生多个第二代子代植物；
    (5)从这些第二代子代植物中选择对昆虫具有抗性的植物，其中，当所述植物满足所述(3a)-(3c)中的至少一种时，表明其为昆虫抗性玉米植物。
  33. 一种产生可生长为昆虫抗性玉米植物的杂种玉米种子的方法，其包括：将第一亲代玉米植物与第二亲代玉米植物杂交和收获产生的杂种种子，其中所述第一亲代玉米植物和/或第二亲代玉米植物如权利要求1-9、11任一项中所定义；
    优选地，所述第一亲代玉米植物和/或第二亲代玉米植物包含掺入其基因组中的编码mcry1Ab和mcry2Ab的外源核酸分子，所述外源核酸分子侧接5’侧翼区和3’侧翼区，包含5’侧翼区和与之相邻的外源核酸分子的部分的序列如SEQ ID NO:1所示， 包含3’侧翼区和与之相邻的外源核酸分子的部分的序列如SEQ ID NO:2所示；
    优选地，所述第一亲代玉米植物和/或第二亲代玉米植物在其基因组中包含SEQ ID NOs:1-5任一项所示的序列或其互补序列；
    优选地，所述昆虫选自鳞翅目昆虫，例如黏虫、玉米螟(如亚洲玉米螟)、棉铃虫、桃蛀螟、草地贪夜蛾等鳞翅目害虫中的一种或多种。
  34. 权利要求33所述的方法，其包括：
    (1)种植第一近交玉米系的种子，该近交玉米系是权利要求1-9、11任一项中所定义的玉米植物；以及种植具有不同基因型的第二近交系的种子；
    (2)培育产生自所述种植的玉米植物直到开花的时期；
    (3)对其中一个玉米近交系的植物的花去雄；
    (4)将该两种不同近交系彼此有性杂交；和
    (5)收获由此生产的杂种种子；
    优选地，所述第一近交玉米系包含掺入其基因组中的编码mcry1Ab和mcry2Ab的外源核酸分子，所述外源核酸分子侧接5’侧翼区和3’侧翼区，包含5’侧翼区和与之相邻的外源核酸分子的部分的序列如SEQ ID NO:1所示，包含3’侧翼区和与之相邻的外源核酸分子的部分的序列如SEQ ID NO:2所示；
    优选地，所述第一近交玉米系在其基因组中包含SEQ ID NOs:1-5任一项所示的序列或其互补序列。
  35. 权利要求1-9、11任一项所述的玉米植物或其部分、种子、细胞或后代、或权利要求10所述的玉米种子，用于生产食品、饲料、化妆品、药品或工业产品的用途。
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