WO2019154373A1 - 抗虫耐除草剂玉米转化事件 - Google Patents

抗虫耐除草剂玉米转化事件 Download PDF

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WO2019154373A1
WO2019154373A1 PCT/CN2019/074611 CN2019074611W WO2019154373A1 WO 2019154373 A1 WO2019154373 A1 WO 2019154373A1 CN 2019074611 W CN2019074611 W CN 2019074611W WO 2019154373 A1 WO2019154373 A1 WO 2019154373A1
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nucleotides
seq
sequence
corn
plant
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PCT/CN2019/074611
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French (fr)
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刘博林
佘秋明
谭超
许洁婷
王绪霞
田裴秀子
聂东明
韩宇
马崇烈
章旺根
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中国种子集团有限公司
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Priority to US16/968,067 priority Critical patent/US11479790B2/en
Priority to BR112020014816-4A priority patent/BR112020014816A2/pt
Publication of WO2019154373A1 publication Critical patent/WO2019154373A1/zh

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Definitions

  • the present application relates to the field of plant biotechnology, and in particular relates to a method, a detection method and an application method for an insect-resistant herbicide-tolerant corn transformation event.
  • Maize is an important feed and industrial raw material crop. As the largest crop in China, it has long been self-sufficient, but since 2010, imports have increased year by year.
  • Corn borer commonly known as corn borer, is one of the important biological disasters that cause corn to reduce production all year round, seriously affecting the yield and quality of corn, including Asian corn borer (Ostrinia furnacalis) and European corn borer (Ostrinia nubilalis). China is the most frequent and re-emergence area of the Asian corn borer (Ostrinia furnacalis), which occurs on a large scale almost every two years. In general, the corn is reduced by 10%-15% due to the damage of corn borer, and the annual yield reduction can reach more than 30%, even if it is not harvested. Due to the damage of corn borer, the annual loss of corn will reach 6-9 million tons.
  • Corn borer not only directly causes loss of corn yield, but also induces and aggravates the occurrence of corn ear rot, which reduces the quality of corn.
  • the main way to control corn borer is still based on pesticide control.
  • the large-scale use of pesticides not only increases the cost of planting but also destroys the ecological environment.
  • Field weeds and crops compete for water, fertilizer, light energy and growth space, and at the same time are intermediate hosts that are harmful to crop pathogens and pests, and are one of the important biological limiting factors for crop yield increase.
  • the area of crops that are seriously damaged by weeds in China is as high as 1.2 billion mu, including 190 million mu of corn.
  • Herbicides such as glufosinate have the characteristics of high efficiency, low toxicity, easy degradation and no residue, but they are not selective for weeding and cannot be directly used in crop growth period.
  • Plant transgenic breeding technology has the advantages of high purpose, short cycle, high efficiency, and ability to transfer excellent genes between different species. Since the commercialization of the first GM crop in 1996, this technology has brought about tremendous changes in global agriculture.
  • the genes such as Cry1Ac and Cry1F for controlling Lepidoptera corn borers are mainly used.
  • Cry1Ac and Cry1F for controlling Lepidoptera corn borers are mainly used.
  • Cry3 genes to obtain resistance to corn rootworms. Lines such as MON863 are put into commercial production.
  • the application provides a nucleic acid molecule comprising: i) a sequence comprising nucleotides 381-780 of SEQ ID NO: 1 and/or nucleotides 10815-11214, or a fragment thereof, or a variant or a complement thereof; ii) comprising the nucleotides 381 to 780 of SEQ ID NO: 1 and the nucleotides of nucleotides 6239 to 6338, or a fragment thereof or a variant thereof or a complement thereof; a sequence comprising nucleotides 6239-6338 and nucleotides 10815-11214 of SEQ ID NO: 1, or a fragment thereof or variant thereof or a complement thereof; or iv) comprising SEQ ID NO: 1.
  • a nucleic acid molecule provided herein comprises the sequence set forth in SEQ ID NO: 1, or a fragment thereof, or a variant thereof, or a complement thereof.
  • the nucleic acid molecule provided herein comprises an expression cassette: a first expression cassette expressing a glufosinate-resistant gene, as set forth in nucleotides 748-2288 of SEQ ID NO: a second expression cassette for expressing an insect-resistant gene, such as the nucleotides of nucleotides 2620-6959 of SEQ ID NO: 1; and a third expression cassette for expressing a glyphosate-resistant gene, such as SEQ ID NO: 1.
  • the nucleic acid molecule provided herein is obtained by introducing the following expression cassette into the genome of maize: a first expression cassette expressing a glufosinate-resistant gene, as in paragraph 748 of SEQ ID NO: 1. a sequence represented by nucleotides 2288; a second expression cassette expressing an insect-resistant gene, such as the sequence represented by nucleotides 2620-6959 of SEQ ID NO: 1; and a third expression cassette expressing a glyphosate-resistant gene , as shown in nucleotides 6968-10892 of SEQ ID NO: 1.
  • nucleic acid molecules provided herein are present in a corn plant, seed, plant cell, progeny plant or plant part.
  • the application provides a probe for detecting a maize transformation event comprising the nucleotides 381-780 of SEQ ID NO: 1 or the nucleotides of nucleotides 10815-11214, or a fragment thereof Or a variant thereof or a complement thereof.
  • the present application also provides a primer pair for detecting a maize transformation event, which is capable of specifically amplifying a sequence comprising nucleotides 381 to 780 of SEQ ID NO: 1 or nucleotides 10815 to 11214, Or a fragment thereof or a variant thereof or a complement thereof.
  • the primer pair is: i) a primer pair that specifically recognizes a sequence comprising nucleotides 381 to 780 of SEQ ID NO: 1; ii) a specific recognition comprising SEQ ID NO: Primer pair of the sequence shown in nucleotides 10815-11214; iii) a forward primer that specifically recognizes the sequence of nucleotides 381-780 of SEQ ID NO: 1, and specific recognition comprising SEQ ID NO: a reverse primer of the sequence indicated by nucleotides 681-10915 of SEQ ID NO: 1; and iv) a forward primer which specifically recognizes the sequence of nucleotides 681-10915 of SEQ ID NO: 1, and A reverse primer comprising the sequence shown in nucleotides 10815-11214 of SEQ ID NO: 1 is specifically recognized.
  • the primer pairs provided herein are the nucleotide sequences set forth in SEQ ID No: 8 and SEQ ID No: 9 or the complements thereof; or SEQ ID No: 10 and SEQ ID No: 11 The nucleotide sequence shown or its complement.
  • kits or microarrays for detecting corn transformation events comprising the probes described above and/or the primer pairs described above.
  • the present application provides a method of detecting a corn transformation event, comprising: detecting the presence or absence of the transformation event in a sample to be tested using the probes described above; the primer pairs described above; the probe and primer pairs described above; Or the kit or microarray described above.
  • the present application also provides a method of breeding corn, the method comprising the steps of: 1) obtaining corn comprising the nucleic acid molecule described above; 2) passing the corn obtained in step 1) through pollen culture, unfertilized embryo culture, doubling Culture, cell culture, tissue culture, selfing or hybridization or a combination of the above to obtain progeny plants, seeds, plant cells, progeny plants or plant parts; and, optionally, 3) herbicides of the progeny plants obtained in step 2)
  • the present application also provides corn plants, seeds, plant cells, progeny plants or plant parts and the like obtained by the above method, and articles made from these corn plants, seeds, plant cells, progeny plants or plant parts, and the like. , including food, feed or industrial raw materials.
  • the present application provides a method of controlling a population of lepidopteran pests comprising contacting the lepidopteran pest population with a corn plant, seed, plant cell, progeny plant or plant part obtained by the above method.
  • the present application also provides a method of killing a lepidopteran pest comprising contacting the lepidopteran pest with a pesticidally effective amount of a corn plant, seed, plant cell, progeny plant or plant part obtained by the above method.
  • the present application also provides a method of alleviating damage to a corn by a lepidopteran pest comprising introducing the following expression cassette into the genome of the maize: a first expression cassette expressing a glufosinate-resistant gene, as in paragraph 748 of SEQ ID NO: 1. a sequence represented by nucleotides 2288; a second expression cassette expressing an insect-resistant gene, such as the sequence represented by nucleotides 2620-6959 of SEQ ID NO: 1; and a third expression cassette expressing a glyphosate-resistant gene , as shown in nucleotides 6968-10892 of SEQ ID NO: 1.
  • the Lepidopteran pest described in the above method is Ostrinia furnacalis, Ostrinia nubilalis or Mythimna separate (Walker).
  • Figure 1 is a schematic view of the structure of the carrier pZHZH35006, wherein:
  • Kozak sequence A sequence of eukaryotic mRNAs used for translation initiation
  • CaMV 35S promoter Cauliflower Mosaic Virus 35S Promoter
  • ⁇ sequence1 is derived from tobacco etch virus gene expression enhancing element
  • Figure 2 is a photograph of a plant after 4-5 days of spraying 250 ml/mu of glufosinate herbicide "guaranteed test", wherein:
  • A is ZZM030, the plant grows normally, without any symptoms of injury;
  • B is a non-transgenic negative control wild type Xiang 249, leaves dry, chlorotic, plaque, growth stagnation, showing obvious symptoms of phytotoxicity.
  • Figure 3 is a photograph of a plant after a week of spraying 200 ml/mu of glyphosate herbicide "Nongda", where:
  • A is ZZM030, the plant grows normally, without any symptoms of injury;
  • B is a non-transgenic negative control wild type Zhaoxiang 249, leaves dry, chlorotic, growth stagnation, showing obvious symptoms of phytotoxicity.
  • Figure 4 is a field experiment photograph of the identification of plant aphid resistance.
  • the white spots in the figure are wormholes, among which:
  • A is a ZZM030 leaf, a pinhole-like wormhole, and the wormhole is sparse and scattered;
  • B is a non-transgenic negative control wild type Xiang 249 leaves, mung bean size wormholes, individual short-spotted mosaics.
  • Fig. 5 is a field experiment photograph of the identification of plant armyworm resistance.
  • the leaf edge and the missing part on the leaf are the wormholes after being eaten by the armyworm, among which:
  • A is a ZZM030 blade
  • B is a non-transgenic negative control wild type Xiang 249 leaf.
  • Figure 6 shows the results of ZZM030 Southern hybrid copy number detection, in which:
  • Figure 6A shows the results of insertion copy number detection of the cry1Ab/cry1AcZM gene, wherein: lane 1, DNA molecular marker; lane 2, blank; lane 3, HindIII-digested ZZM030 genomic DNA hybridized with cry1Ab/cry1AcZM-specific probe; lane 4, HindIII-cleaved wild-type 249 genomic DNA was hybridized with cry1Ab/cry1AcZM-specific probe as a negative control; Lane 5, KpnI-digested ZZM030 genomic DNA was hybridized with cry1Ab/cry1AcZM-specific probe; Lane 6, KpnI digestion Wild type 249 genomic DNA was hybridized with cry1Ab/cry1AcZM-specific probe as a negative control; Lane 7, EcoRI-digested ZZM030 genomic DNA was hybridized with cry1Ab/cry1AcZM-specific probe as a positive control; Lane 8, EcoRI enzyme The wild type 249 genomic DNA was crossed with the cry1Ab/c
  • Figure 6B shows the results of bar gene insertion copy number detection, wherein: lane 1, DNA molecular marker; lane 2, blank; lane 3, HindIII digested ZZZ030 genomic DNA hybridized with bar-specific probe; lane 4, HindIII digested Wild type 249 genomic DNA was hybridized with bar-specific probe as a negative control; Lane 5, EcoRI-digested ZZM030 genomic DNA was hybridized with bar-specific probe; Lane 6, EcoRI digested wild-type 249 genomic DNA and Bar-specific probe hybridization as a negative control; Lane 7, KpnI-cleaved ZZM030 genomic DNA hybridized with bar-specific probe as a positive control; Lane 8, KpnI digested wild-type 249 genomic DNA and bar specificity Hybridization of the probe as a negative control; Lane 9, KpnI digested plasmid and wild type 249 genomic DNA were hybridized with bar specific probe as a positive control;
  • Figure 6C shows the results of cp4 epspsZM gene insertion copy number detection, wherein: lane 1, DNA molecular marker; lane 2, blank; lane 3, HindIII digested ZZM030 genomic DNA hybridized with cp4 epspsZM-specific probe; lane 4, HindIII enzyme
  • the wild type 249 genomic DNA was crossed with the cp4 epspsZM specific probe as a negative control; Lane 5, KpnI digested ZZZ030 genomic DNA was hybridized with cp4 epspsZM specific probe; Lane 6, KpnI digested wild type 249 genomic DNA was hybridized with cp4 epspsZM-specific probe as a negative control; Lane 7, EcoRI-digested ZZM030 genomic DNA was hybridized with cp4epspsZM-specific probe as a positive control; Lane 8, EcoRI digested wild-type 249 genome DNA was hybridized with cp4
  • Figure 7 shows the results of ZZM030 event-specific PCR assays, in which:
  • Figure 7A is a left boundary detection result
  • Figure 7B is a result of the right border detection
  • Lanes 1-4 are: sterile water, 249 genomic DNA, ZZM030 genomic DNA, non-ZZM030 event genomic DNA obtained after transformation of the same vector.
  • corn is any corn plant and includes all plant species that can be bred with corn, including whole plants, plant cells, plant organs, plant protoplasts, plant cell tissue cultures from which plants can be regenerated, plant healing Infected tissue, and intact plant cells in a plant or plant part, such as embryos, pollen, ovules, seeds, leaves, flowers, branches, fruits, stems, roots, root tips, anthers, and the like.
  • Excellent transformation events can be transferred to germplasm of other genetic backgrounds by conventional breeding methods, ie, sexual hybridization, and the progeny maintain the transgene expression characteristics of the original transformants.
  • the present application relates to the excellent transformation event ZZM030 by screening from a number of transformation events.
  • transformation event ZZM030 refers to Agrobacterium-mediated genetic transformation using a maize inbred line 249 as a receptor to obtain insertion of a foreign gene insert (T-DNA insert) at a specific genomic site.
  • a maize plant in which the exogenous gene insert comprises the following three genes: an insect resistance gene, an anti- glufosinate gene, and a glyphosate resistant gene.
  • the transformation event ZZM030 obtained in the present application the inserted foreign gene is located in the non-functional site of the maize genome, does not affect the expression of other genes of the recipient plant itself, and enables the transgenic corn plant to obtain insect-resistant and herbicide-tolerant properties. Maintained its good agronomic traits.
  • the T-DNA insert obtained after transgene has the sequence shown in nucleotides 681-10915 of SEQ ID NO: 1.
  • the transformation event ZZM030 can refer to this transgenic process, and can also refer to the T-DNA insert in the genome obtained by this process, or the combination of the T-DNA insert and the flanking sequence, or can be derived from this transgenic process. Corn plants.
  • the transformation event ZZM030 may also refer to a progeny plant obtained by vegetative propagation, sexual reproduction, doubling or doubling of the above plants or a combination thereof.
  • the event is also applicable to the transformation of a T-DNA insert by transforming the same foreign gene (the sequence shown in nucleotides 681-10915 of SEQ ID NO: 1) into another plant recipient variety.
  • T- which is the flanking sequence of nucleotides 1-480 of SEQ ID NO: 1 and the nucleotides 10916-11375 of SEQ ID NO: 1 are obtained as the right flanking sequence.
  • DNA insert (nucleotides 681-10915).
  • the flanking sequences are not limited to nucleotides 1-480 and 10916-11375 of SEQ ID NO: 1, since the listed flanking sequences are only used to indicate the location of the T-DNA insert in the genome. That is, the insertion point on the left side of the T-DNA insert is located on chromosome 4, 40636901 bp; the insertion point on the right side of the T-DNA insert is located on chromosome 4, 40, 636, 883 bp.
  • flanking sequences of the present application may extend to both sides according to the genomic sequence, that is, the left flanking sequence may extend downstream of chromosome 4, 40636901 bp, and the right flanking sequence may extend upstream of chromosome 4, 40, 636, 883 bp.
  • any sequence comprising a T-DNA insert of the transformation event ZZM030 and a junction site of a flanking sequence can be used to detect the transformation event ZZM030 of the present application, including but not limited to, including an upstream insertion site (left side)
  • One or more of: i) comprises the sequence of nucleotides 381-780 of SEQ ID NO: 1; ii) comprises the sequence of nucleotides 1-898 of SEQ ID NO: 1; iii) Included is the sequence of nucleotides
  • the sequence that can be used to detect the transformation event ZZM030 of the present application is a sequence comprising an upstream insertion site or a fragment thereof or a variant thereof or a complement thereof, such as a nucleus from position 381 to 780 comprising SEQ ID NO:
  • the sequence shown by the nucleotide sequence either comprises the sequence shown in nucleotides 1078-11373 of SEQ ID NO: 1, or a combination of the sequence comprising the upstream insertion site and the sequence comprising the downstream insertion site.
  • the sequence useful for detecting the transformation event ZZM030 of the present application is a sequence comprising an upstream insertion site, or a fragment thereof, or a variant thereof, or a complement thereof, and a sequence comprising the T-DNA insert or a fragment thereof or A combination of a variant or a complement thereof, for example, comprising the sequence shown in nucleotides 381-780 of SEQ ID NO: 1 or the sequence comprising nucleotides 1-898 of SEQ ID NO: 1, A sequence comprising the nucleotides of nucleotides 6239-6338 of SEQ ID NO: 1 or a sequence comprising the nucleotides of nucleotides 681-10915 of SEQ ID NO: 1.
  • the sequence useful for detecting the transformation event ZZM030 of the present application is a sequence comprising a downstream insertion site, or a fragment thereof, or a variant thereof, or a complement thereof, and a sequence comprising the T-DNA insert or a fragment thereof or A combination of a variant or a complement thereof, for example, comprising the sequence set forth in nucleotides 10815-11214 of SEQ ID NO: 1 or comprising the sequence of nucleotides 1078-11373 of SEQ ID NO: 1, A sequence comprising the nucleotides of nucleotides 6239-6338 of SEQ ID NO: 1 or a sequence comprising the nucleotides of nucleotides 681-10915 of SEQ ID NO: 1.
  • sequence useful for detecting the transformation event ZZM030 of the present application is a sequence comprising the nucleotides 381-11241 of SEQ ID NO: 1 or a fragment thereof, or a variant thereof, or a complement thereof, or The sequence of SEQ ID NO: 1 or a fragment thereof or a variant thereof or a complement thereof is included.
  • primer pairs, probes, and combinations of primer pairs and probes capable of specifically detecting the T-DNA insert of the transformation event ZZM030 with the flanking sequence can be used to detect the transformation event ZZM030 of the present application.
  • nucleotide sequence includes a deoxyribonucleotide or ribonucleotide polymer involving a single-stranded or double-stranded form, and unless otherwise limited, the nucleotide sequence is in the 5' to 3' direction. Write from left to right.
  • the application also relates to a fragment of a nucleic acid sequence, which refers to a portion of an incomplete smaller fragment in the entire portion.
  • a fragment of SEQ ID NO: 1 comprises at least about 10 nucleotides, at least about 20 nucleotides, at least about 30 nucleotides, at least about 40 nucleotides of the entire sequence of SEQ ID NO: 1. Or a sequence of at least about 50 nucleotides or more.
  • the nucleic acid sequences of the present application can be altered to make conservative amino acid substitutions.
  • the nucleotide sequence of the present application can be subjected to substitution without altering the amino acid sequence in accordance with monocot codon preference, for example, a codon encoding the same amino acid sequence can be replaced with a monocot preferred codon.
  • the amino acid sequence encoded by the nucleotide sequence is not altered.
  • the application also relates to variants of a nucleic acid sequence. Generally, a variant of a particular nucleic acid fragment will have at least about 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91% of the particular nucleotide sequence.
  • nucleotide sequence variants of the embodiments may differ from the sequences of the present application by as little as 1-15 nucleotides, as few as 1-10 (e.g., 6-10), as few as 5, as little as 4, 3, 2 or even 1 nucleotide.
  • a "probe” is an isolated polynucleotide to which a conventional detectable label or reporter molecule such as a radioisotope, ligand, chemiluminescent agent or enzyme is added, which is complementary to the strand of the target polynucleotide.
  • the DNA probe provided by the present application for detecting the transformation event ZZM030 comprises a sequence comprising a contiguous nucleotide of sufficient length of SEQ ID NO: 1 or a fully complementary sequence thereof, the DNA probe being A nucleotide sequence comprising an upstream insertion site or a downstream insertion site is hybridized under stringent hybridization conditions and does not hybridize under stringent hybridization conditions to a nucleotide sequence that does not contain an upstream insertion site or a downstream insertion site.
  • the probe provided herein comprises the nucleotides 381-780 of SEQ ID NO: 1 or the nucleotides of nucleotides 10815-11214, or a fragment thereof or variant thereof or complement thereof sequence.
  • a "primer” is an isolated polynucleotide that anneals to a complementary target DNA strand by nucleic acid hybridization to form a hybrid between the primer and the target DNA strand, and then stretches along the target DNA strand by, for example, a DNA polymerase. Primer pairs are involved in their target polynucleotide amplification applications, such as by polymerase chain reaction (PCR) or other conventional nucleic acid amplification methods.
  • PCR polymerase chain reaction
  • the primer pair provided by the present application for detecting the transformation event ZZM030 comprises a first DNA molecule and a second DNA molecule different from the first DNA molecule, wherein the first DNA molecule and the second DNA molecule a nucleotide sequence comprising a contiguous nucleotide of sufficient length of SEQ ID NO: 1 or a fully complementary sequence thereof, and wherein the first DNA molecule is present in the T-DNA insert of SEQ ID NO: 1
  • the second DNA molecule is present in the flanking sequence of SEQ ID NO: 1, and when used in conjunction with DNA from the transformation event ZZZ030 in an amplification reaction, the DNA primer produces an amplicon for detecting the transformation event ZZZ030 DNA in the sample.
  • the amplicon comprises the nucleotides 381-780 of SEQ ID NO: 1 or the nucleotides of nucleotides 10815-11214, or a fragment thereof or a variant thereof or a complement thereof.
  • the primer pair provided herein is a primer pair that specifically recognizes a sequence comprising nucleotides 381 to 780 or nucleotides 1-898 of SEQ ID NO: 1.
  • the primer pair provided herein is a primer pair that specifically recognizes a sequence comprising nucleotides 10815-11214 or 10578-11373 of SEQ ID NO: 1.
  • the primer pairs provided herein are: i) a primer pair that specifically recognizes a sequence comprising nucleotides 381 to 780 or nucleotides 1-898 of SEQ ID NO: 1; a primer pair specifically comprising a sequence represented by nucleotides 10815-11214 or 10578-11373 of SEQ ID NO: 1; or the primer pair comprises: specifically recognizing comprising SEQ ID NO: 1 a forward primer of the sequence indicated by nucleotides 381-780 or 1-898, and a sequence specifically recognizing a nucleotide comprising nucleotides 10815-11214 or 10578-11373 of SEQ ID NO: 1.
  • Reverse primer a primer pair that specifically recognizes a sequence comprising nucleotides 381 to 780 or nucleotides 1-898 of SEQ ID NO: 1
  • a primer pair specifically comprising a sequence represented by nucleotides 10815-11214 or 10578-11373 of SEQ ID NO: 1
  • the primer pairs provided herein are: i) a primer pair that specifically recognizes a sequence comprising nucleotides 381-780 or nucleotides 1-898 of SEQ ID NO: 1; And ii) specifically identifying a primer pair comprising the sequence of nucleotides 6239-6338 or 681-10915 of SEQ ID NO: 1; or, the primer pair comprises: specific recognition comprising SEQ ID NO: a forward primer of the sequence of nucleotides 381 to 780 or nucleotides 1-898 of 1 and specifically recognizes nucleotides 6239-6338 or 681-10915 of SEQ ID NO: 1.
  • the reverse primer of the sequence is shown.
  • the primer pairs provided herein are: i) a primer pair that specifically recognizes a sequence comprising nucleotides 6239-6338 or 681-10915 of SEQ ID NO: 1; And ii) specifically identifying a primer pair comprising a sequence represented by nucleotides 10815-11214 or 10578-11373 of SEQ ID NO: 1; or, the primer pair comprises: specifically recognizing comprising SEQ ID NO: a forward primer of the sequence indicated by nucleotides 6239-6338 or 681-10915, and a nucleotide which specifically recognizes nucleotides 10815-11214 or 10578-11373 of SEQ ID NO: 1.
  • the reverse primer of the sequence is shown.
  • the primer pairs provided herein are: i) a primer pair that specifically recognizes a sequence comprising nucleotides 381 to 780 or nucleotides 1-898 of SEQ ID NO: 1, Ii) specifically identifying a primer pair comprising the sequence of nucleotides 6239-6338 or 681-10915 of SEQ ID NO: 1, and iii) specifically identifying 10815-11214 comprising SEQ ID NO: 1.
  • the primer pair provided herein is a primer pair that specifically recognizes the sequence comprising SEQ ID NO: 1.
  • the primer pair is the nucleotide sequence represented by SEQ ID No: 8 and SEQ ID No: 9 or a complement thereof; or the nucleoside represented by SEQ ID No: 10 and SEQ ID No: The acid sequence or its complement.
  • kit refers to a reagent set or chip for the identification and/or detection purposes of the maize transformation event ZMM030 in a biological sample.
  • a kit or a chip may be used for the purpose of quality control (eg purity of a seed lot), in a plant material or in a plant material or a material derived from a plant material such as, but not limited to, the detection of event ZMM030 in a food or feed product, and The composition thereof can be specifically adjusted.
  • the kit or probe provided herein includes any of the probes or any of the primer pairs provided herein. In another specific embodiment, the kit or probe provided herein includes any of the probes provided herein or a combination of any of the primer pairs.
  • the present application also provides transgenic corn plants, progeny, seeds, plant cells or plant parts and articles thereof, including but not limited to food, feed or industrial materials.
  • Nucleic acid molecular sequences of detectable T-DNA inserts and flanking sequences of the flanking sequences provided herein are included in these plants, progeny, seeds, plant cells, plant parts, and articles thereof.
  • the present application also provides a method for breeding corn, comprising the steps of: 1) obtaining a nucleic acid molecule sequence comprising a T-DNA insert and a flanking sequence of a flanking sequence provided by the present application; 2) Step 1) The obtained corn is obtained from pollen culture, unfertilized embryo culture, double culture, cell culture, tissue culture, selfing or hybridization or a combination of the above to obtain progeny plants, seeds, plant cells, progeny plants or plant parts; Step 3), the corn plants obtained in the step 2) are subjected to the herbicide-resistant glufosinate and glyphosate and the insect resistance identification, and the probes, primer pairs, kits or arrays provided by the application are used for detection. Whether there is a conversion event ZZM030.
  • the present application provides methods for controlling weeds in the field, as well as methods for controlling or killing lepidopteran pests.
  • the method of controlling weeds in the field comprises planting a corn plant comprising a transformation event ZMM030 in the field, and applying an effective dose in the field without damaging the inclusion event ZZZ030 Weed control of glyphosate and glufosinate herbicides in the case of transgenic maize plants.
  • the method for controlling or killing lepidopteran pests comprises contacting the lepidopteran pest with an effective amount of a corn plant of transformation event ZZZ030, or feeding the lepidopteran pest An effective amount of a corn plant of the transformation event ZZM030, or a corn plant that causes the lepidopteran pest to feed an effective amount of the transformation event ZZM030.
  • the lepidopteran pests include, but are not limited to, Ostrinia furnacalis, Ostrinia nubilalis, Mythimna separate (Walker), and the like.
  • ⁇ ективное amount or “insecticidal effective amount” refers to the amount of a pesticidal substance or organism present in a pest environment.
  • corn variety materials involved in the following examples are all provided by China Seed Group Co., Ltd., in which the maize inbred line Xiang 249 is the female parent of the corn variety Great Wall 799, which is based on the foreign introduced corn germplasm resources. Separation and strict selection, after 10 generations, was selected in 1996.
  • the insect-resistant gene cry1Ab/cry1AcZM which is expressed in plants and produces an insect-resistant effect disclosed in the PCT International Application WO2017012577A1 by the Applicant is used.
  • the gene is based on the 608 amino acid sequences of the fusion and engineered Cry1Ab and Cry1Ac N-terminal, using the plant-preferred codons to replace the coding sequence, and by replacing the codons, the enrichment of the plant transcriptional instability caused by the DNA sequence
  • the AT sequence and the commonly used restriction sites are corrected for elimination.
  • a 67-nucleotide ⁇ sequence and a 3-nucleotide (ACC) Kozak sequence was added at the 5' end to enhance the translation efficiency of the eukaryotic gene.
  • a 135 bp polyA sequence was added to the 3' end.
  • the protein encoded by this gene contains three functional segments, wherein the two functional regions at the N-terminus are highly homologous to the corresponding portion of the Cry1Ab, and the functional region at the C-terminus is highly homologous to Cry1Ac.
  • the insect resistance gene cry1Ab/cry1AcZM as shown in bases 4624 to 6670 of SEQ ID NO: 1 was synthesized synthetically.
  • the maize codon preference was optimized using the Vector NTI software against the cp4 epsps coding region of the glyphosate gene, and the expression enhancing element ⁇ sequence1 was added at the 5' end.
  • the engineered DNA sequence was named cp4 epspsZM.
  • the glyphosate resistant herbicide gene as shown in bases 8954 to 10611 of SEQ ID NO: 1 was synthesized by artificial synthesis.
  • the bar gene sequence is shown in Gene bank Accession No. X17220.1, and the glufosinate-resistant herbicide gene shown in 957 to 1508 of SEQ ID NO: 1 was synthesized by artificial synthesis.
  • HindIII and PstI restriction sites were added to the 5' end of the synthesized cry1Ab/cry1AcZM, and a PmeI restriction site was added to the 3' end, and the synthesized sequence was cloned on the Puc57 simple vector and designated as pZZ01194.
  • the intermediate vector pZZ00005 containing the ubiquitin promoter (HindIII cleavage site at the 5' end and BamHI cleavage site at the 3' end) was digested with restriction enzymes HindIII and BamHI, and filled with T4 DNA polymerase. The sticky end of the ubiquitin promoter fragment was obtained.
  • the restriction enzyme PstI was used to treat pZZ01194, and the resulting sticky end was filled in with T4 DNA polymerase, and the ubiquitin promoter was ligated by blunt-end ligation to obtain a vector containing the ubiquitin promoter-cry1Ab/cry1AcZM fragment, which was named pZZ01201.
  • the restriction enzyme EcoRI was used for single digestion, and the T4 DNA polymerase was used to fill the resulting viscosity. At the end, the nos terminator sequence was obtained.
  • PmeI was treated with pZZ01201, and the nos terminator was ligated by blunt-end ligation to obtain a vector containing the ubiquitin promoter-cry1Ab/cry1AcZM-nos terminator fragment, which was named pZZ01205.
  • the ubiquitin promoter-egfp-nos terminator was removed by restriction enzymes HindIII and PmeI.
  • the pZZ01205 vector was treated with restriction enzymes HindIII and PmeI to obtain a ubiquitin promoter-cry1Ab/cry1AcZM-nos terminator fragment.
  • the two were ligated to obtain an expression vector containing two expression cassettes of ubiquitin promoter-cry1Ab/cry1AcZM-nos terminator and CaMV 35S promoter-bar-CaMV 35S terminator, designated pZHZH25017.
  • the intermediate vector pZZ01337 (CaMV 35S promoter-cp4 epspsZM-nos terminator) was used to excise CaMV 35S promoter using restriction enzymes HindIII and BamHI.
  • the ubiquitin4promoter was obtained using the restriction enzymes HindIII and BamHI for the intermediate vector pZZ00033. The two were ligated to obtain a vector containing the ubiquitin4 promoter-cp4 epspsZM-nos terminator fragment, designated pZZ01383.
  • the ubiquitin4 promoter-cp4 epspsZM-nos terminator fragment was obtained by double restriction enzyme digestion with restriction enzymes HindII and PmeI, and the resulting sticky ends were filled in with T4 DNA polymerase.
  • the pZHZH25017 was treated with the restriction enzyme PmeI, and the ubiquitin4 promoter-cp4 epspsZM-nos terminator was ligated by blunt-end ligation to obtain the ubiquitin promoter-cry1Ab/cry1AcZM-nos terminator, CaMV 35S promoter-bar-CaMV 35S terminator and ubiquitin4 promoter-cp4.
  • the plant expression vector of the three expression cassettes of epspsZM-nos terminator was named pZHZH35006, and its physical map is shown in Fig. 1 .
  • Transgenic maize is obtained using Agrobacterium-mediated genetic transformation methods.
  • the plasmid DNA of the vector pZHZH35006 was transformed into Agrobacterium EHA105 by electroporation and identified for use.
  • the maize inbred line Xiang 249 was self-crossed and the young embryos of about 1.5 mm in length were used for transformation.
  • the young embryos of about 200 ears were collected into one batch.
  • the suspension was aspirated in an EP tube, and the Agrobacterium liquid containing 200 ⁇ M acetosyringone was added for 5 min, and then the young embryos were transferred to the co-culture medium.
  • the young embryos after dark culture were placed on the callus induction medium, and after the callus grew, the culture medium containing 5 mg/L of bialaphos was placed, and the culture was screened and subcultured every two weeks.
  • the embryogenic callus with good condition is selected and transferred to the differentiation medium.
  • the culture condition is 26 ° C, 3000 Lux light intensity per day, light for 16 h, and regenerated seedlings appear two weeks later.
  • the regenerated plantlets were transferred to a rooting medium, and after the seedlings had grown secondary roots, they were transplanted in small pots mixed with nutrient soil and vermiculite (1:3).
  • the obtained transformed seedlings were subjected to transgenic positive detection according to the following procedure, and transgenic positive plants were selected.
  • the maize genomic DNA was extracted using the DNAsecure Plant Kit new plant genomic DNA extraction kit (centrifugal column type) from Tiangen Biotechnology Co., Ltd.
  • PCR buffer 10 times PCR buffer (Takara), deoxynucleotide mixture (10 mM, Sigma), including forward primer SEQ ID NO: 2 (CSP759): 5'-CACGCAGATTCCAGCGGTCAA- 3'; reverse primer SEQ ID NO: 3 (CSP760): 5'-GACGAGGTGAAGGCGTTAGCA-3') and corn leaf DNA template.
  • CSP759 forward primer SEQ ID NO: 5'-CACGCAGATTCCAGCGGTCAA- 3'
  • reverse primer SEQ ID NO: 3 CSP760
  • the mixture of the PCR reaction system was prepared, mixed, and centrifuged for several seconds.
  • PCR reaction system (20 ⁇ L): 2 ⁇ L of 10 ⁇ PCR buffer (Takara), 0.5 ⁇ L of deoxynucleotide mixture (10 mM, Sigma), 0.8 ⁇ L of forward and reverse primer mixture (5 ⁇ M), 0.2 ⁇ L of r-Taq (5 U, Takara) The rest is dd H 2 O. The mixture was dispensed into a 200 ⁇ L format PCR tube, and 1 ⁇ L of corn leaf DNA template was added, and the different samples were separately labeled for differentiation. The PCR reaction tube was placed in an ABI 9700 PCR instrument, and a predetermined PCR amplification program was selected to start the reaction.
  • the PCR reaction procedure was: pre-denaturation at 94 ° C for 2 min; 30 cycles: denaturation at 94 ° C for 30 sec, annealing at 58 ° C for 30 sec, extension at 72 ° C for 30 sec, and finally extension at 72 ° C for 5 min.
  • the material capable of amplifying a 333 bp band is a transgenic positive plant, and the material which cannot amplify the band size is a transgenic negative plant.
  • the transgenic positive plants were transplanted into large pots, which are T 0 plants.
  • T 0 generation plants were selfed and the resulting seeds were T 1 generation seeds.
  • the T 1 seeds were sown in a greenhouse to obtain T 1 progenies. Repeat the process until T 4 generation seeds.
  • Transgenic positive detection, herbicide tolerance analysis, insect resistance identification, and agronomic trait analysis were performed on T 1 to T 3 plants. Plants that are transgenic-positive, have insect-resistant and herbicide-tolerant properties, and have excellent agronomic traits are selected from each generation of plants for entry into the next generation of screening.
  • the positive plants detected by the step 1 were sown in a greenhouse, and the 6-8 leaf stage plants were subjected to herbicide resistance identification to remove the herbicide-tolerant plants.
  • the herbicide glufosinate herbicide "Basta" for spraying is produced by Bayer CropScience (China) Co., Ltd., and the active ingredient is 18% glufosinate solution.
  • the recommended dosage of the herbicide is 200-300 ml/mu. This application uses a recommended concentration of 250 ml/mu for spraying.
  • the herbicide tolerance performance was observed and recorded after 4-5 days. Maize plants with tolerance to glufosinate grew normally without any damage symptoms; corn plants sensitive to glufosinate showed obvious symptoms of phytotoxicity, including growth stagnation, chlorosis, blight, deformity, etc. until the whole plant died. .
  • Table 1 The expected separation ratio of each generation of transformants ZZM030
  • the glyphosate herbicide "Roundup" used in the spraying is produced by Monsanto, and the active ingredient is 41% isopropylamine salt.
  • the recommended dosage of the herbicide corn field is 150-250 ml/mu. This application uses a recommended concentration of 200 ml/mu for spraying, and observes and records the herbicide tolerance performance after one week.
  • the glyphosate-tolerant maize plants grew normally without any damage symptoms; the glyphosate-sensitive maize plants showed obvious symptoms of phytotoxicity, including growth inhibition, chlorosis, plaque, deformity, etc. until the whole plant died. .
  • the aphid resistance of the plants was identified in the field by the heart-leaf living organism.
  • Inoculation occurs in the growth and development of corn plants to the mid-heart stage (7-leaf stage).
  • the test insect was Ostrinia furnacalis. About 60 capsules of blackheads were placed in a centrifuge tube, and the nozzle was stoppered with absorbent cotton. Place the centrifuge tube in an incubator at 28 ° C and 80% humidity, or cover it with a wet towel at room temperature to moisturize it. After the egg is hatched, pull out the absorbent cotton and put it into the heart leaf plexus. Each plant receives 40-60 heads of insects.
  • the degree of damage of the heart of the plant was investigated on a plant-by-plant basis, and the damage level was classified according to the size and number of the wormholes on the damaged leaf, which was called the leaf level.
  • This application uses the 9-level grading standard developed by the International Corn Cob Team (Table 1). The leaf level was investigated on a plant-by-plant basis, and the average value of each plant was used as the leaf level for the identification line, and the level of resistance was determined according to the evaluation criteria of Table 3.
  • Figure 2 is a photograph of a plant after 4-5 days of spraying 250 ml/mu of glufosinate herbicide "Baozhida”.
  • Figure 2A shows ZZM030, the plant grows normally without any damage symptoms, and
  • Figure 2B shows the wild-type control Xiang 249.
  • the leaves are dry, chlorotic, plaque, and growth stagnation, showing obvious symptoms of phytotoxicity. This indicates that the event is highly resistant to glufosinate herbicides.
  • Fig. 3 is a photograph of a plant after one week of spraying the glyphosate herbicide "Nongda" at 200 ml/mu, wherein Fig. 3A is ZZM030, and the plant grows normally without any damage symptoms.
  • Fig. 3B shows the wild-type control Xiang 249, the leaves are dry, chlorotic, and the growth is stagnant, showing obvious symptoms of phytotoxicity. This indicates that the event showed high resistance to glyphosate herbicides.
  • Fig. 4 is a field experiment photograph of the identification of aphid resistance in plants, which was carried out by using the Asian corn borer by the heart-leaf living organism, and the white spots in the figure were wormholes.
  • Fig. 4A shows the ZZM030 leaf, pinhole wormhole, and the wormhole is sparse and dispersed.
  • Fig. 4B shows the non-transgenic negative control Xiang 249 leaves, mung bean size wormholes, and individual short-spotted mosaics. This indicates that the event has reached a high resistance level to Asian corn borers, and its average leaf level is 1.2-1.5 (see Table 4).
  • the specific identification methods are as follows: the aerial parts of the fresh corn plants grown to the 3-4 and 8-10 leaf stages are taken back indoors, the young heart leaves are taken, placed in a petri dish, and 10 heads of 1 day old insects are connected; One treatment per dish, each treatment was repeated 3 times; placed in an artificial climate chamber with a temperature of 28 ⁇ 1 ° C, a photoperiod of 14: 10 h (L: D), and a relative humidity of 70-80%. After 3 days, the number of surviving larvae was counted and the larval survival rate was calculated.
  • the survival rates of different species of maize armyworm were compared and analyzed at a significant level of 0.05.
  • the survival rate was subjected to a square root and an inverse sinusoidal transformation before statistical analysis, and the difference between the treatments was compared.
  • the resistance to the armyworm was qualitatively determined according to Table 5, and the identification results are shown in Table 6.
  • the Syngenta commercial transgenic maize variety Bt11 was used as a positive control.
  • the material contained the Cry1Ab gene, which has been reported to have resistance to armyworms.
  • the receptor material used in the construction of transgenic events was 249 and the Institute of Plant Protection of the Chinese Academy of Agricultural Sciences moved.
  • the conventional corn seedlings of the pest research group were raised as negative controls; the Z. 030 was tested for resistance to the armyworm; the tested armyworm was Mythimna separate (Walker).
  • the specific identification methods are as follows: the fresh leaves of the material 3-4 and 8-10 leaf stage are selected as the food for testworm; all the corn leaves are immersed in 0.1% sodium hypochlorite solution for 3 minutes, then rinsed with distilled water. Dry; put a whole corn into 750ml cans, each bottle is connected to 40 hatching larvae, each treatment is repeated 3 times; placed at temperature (24 ⁇ 1) ° C, humidity (70 ⁇ 5) %, photoperiod 14L: 10D in an artificial climate chamber; fresh correspondingly treated maize plants were replaced every 2 days, and larval deaths were examined on days 3, 6, and 9 after the start of the test.
  • Table 7 shows the effect of 3-4 leaf stage on the survival rate of the newly hatched armyworm.
  • the data in the table are the mean ⁇ standard error.
  • the same letter in the same column indicates that the difference is not significant at the P ⁇ 0.05 level by Tukey's HSD test.
  • the survival rate of the armyworm larvae of conventional corn seedlings was above 95% after treatment and on the 3rd, 6th and 9th day, indicating that the insects used in the experiment were healthy and the test operation was feasible.
  • the 3-4 leaf stage of different materials had a significant effect on the survival rate of newly hatched larvae.
  • the survival rate of larvae of all materials on the third day was higher.
  • the survival rate of larvae except Bt11 was significantly lower than other materials (P ⁇ 0.05). There was no significant difference (P>0.05).
  • Table 8 shows the effect of 8-10 leaf stage on the survival rate of the newly hatched armyworm.
  • the data in the table are the mean ⁇ standard error.
  • the same letter in the same column indicates that the difference is not significant at the P ⁇ 0.05 level by Tukey's HSD test.
  • From the larval survival rate of 3 days after the test the larval survival rate of Bt11 was significantly lower than that of Xiang 249 and conventional corn seedlings.
  • the difference in larval survival rate on day 6 after treatment was significantly increased: the survival rate of ZZM030 larvae was less than 45%, which was moderate resistance; Xiang 249 and conventional corn seedlings showed high sensation.
  • the larval survival rate of 9 days after treatment was high in xiang 249 and conventional corn seedlings, and was significantly higher than other materials; ZZM030 showed high resistance.
  • the method of inoculation is: 4-6 leaf stage insects, 40 strains per plot, each plant receives 40 hatching larvae; inoculation twice, interval 3 days; 14 days after the last insect, each plant is investigated
  • the community insect strain was affected by the armyworm level of the armyworm, and the resistance level of the material in the heart leaf stage was evaluated according to the standard.
  • the grading standards and the resistance level judgment standards shall be implemented in accordance with the provisions of the Ministry of Agriculture, Announcement No. 3-1-1-2007.
  • Table 9 The identification results are shown in Table 9 and Figure 5.
  • Table 9 the values are expressed as the mean ⁇ standard deviation of 3 replicates, and the difference in the same column of lowercase letters indicates that the difference between the different materials at the same place is significant (P ⁇ 0.01).
  • A is the transgenic event ZZM030 and B is the negative control 249.
  • the leaf edge and the missing part on the leaf are the wormholes after being eaten by the armyworm.
  • the average leaf level of the Gongzhuling test site ZZM030 was 1.2, the resistance type was high resistance, the average leaf level of the negative control receptor corn Xiang 249 was 5.8, the resistance type was medium resistance; the average leaf level of the Jinan test point ZZM030 The resistance type was 2.7, the average leaf level of the negative control receptor Ma Xiang 249 was 5.4, the resistance type was medium resistance; the average leaf level of the Jinghong test point ZZM030 was 1.5, and the resistance type was high resistance.
  • the average control leaf level of the negative control receptor, Ma Xiang 249 was 9.0, and the resistance type was high.
  • the probe was prepared using pZHZH35006 plasmid DNA as a template.
  • CSP759 SEQ ID NO 2
  • CSP760 SEQ ID NO 3
  • a probe for detecting cry1Ab/cry1AcZM was synthesized using a Roche PCR digoxigenin probe synthesis kit (Cat. No. 11636090910). It is 333 bp (the probe sequence is the nucleotide sequence shown by the Seq ID No: 1 position 6084-6416).
  • the amplification system contained: 5 ⁇ L (50 pg) of DNA template, 0.5 ⁇ L of each primer, 5 ⁇ L of PCR DIG mixture, 0.75 ⁇ L of DNA polymerase, 5 ⁇ L of PCR buffer (10 times), and ddH 2 O 33.25 ⁇ L.
  • the PCR reaction procedure was: pre-denaturation at 94 ° C for 5 min; 35 cycles: denaturation at 94 ° C for 30 sec, annealing at 55 ° C for 30 sec, extension at 72 ° C for 45 sec, and finally extension at 72 ° C for 7 min.
  • the label amplification effect was detected using a 1% agarose gel.
  • a specific probe for the bar gene was prepared using a primer pair (SEQ ID NO: FW-Csp73, SEQ ID NO: RV-Csp74), which was 408 bp in length (the probe sequence was the same) Seq ID No: 1 nucleotide sequence shown in positions 1035-1442).
  • a specific probe of the cp4 epspsZM gene was prepared using the primer pair (SEQ ID NO 6: FW-Csp1337, SEQ ID NO 7: RV-Csp1338) in the same manner as described in (1), and its length was 1138 bp (probe sequence). Same as the nucleotide sequence shown in Seq ID No: 1 at positions 9058-10195).
  • the total DNA of the leaf genome of the transgenic maize T 1 , T 2 or T 3 generation material is extracted, and the obtained DNA precipitate is dried and dissolved in ion-free water, and the concentration is determined and used.
  • cry1Ab/cry1AcZM probe When the cry1Ab/cry1AcZM probe is used, the maize genomic DNA is subjected to single digestion with HindIII or KpnI.
  • the maize genomic DNA is subjected to single digestion with HindIII or EcoRI.
  • the maize genomic DNA is subjected to single digestion with HindIII or KpnI.
  • a 200 ⁇ L digestion system containing 20 ⁇ g of maize genomic DNA, 20 ⁇ l of restriction enzyme, 20 ⁇ L of 10 ⁇ buffer, and ddH 2 O was added to 200 ⁇ L. After enzyme digestion for 16 hours, 20 ⁇ L was taken for electrophoresis detection to check whether the enzyme digestion effect was thorough.
  • the DNA was passed through a 0.8% gel and electrophoresed at 20 V for 16 h. Excess lanes and spotting wells were cut off, and the remaining gel was treated twice with denaturing solution for 15 min each time and gently shaken on a shaker. It was treated twice with neutralizing buffer for 15 min each time and gently shaken on a shaker. Wash with ultrapure water once. The film was transfected for 4 hours or more with the Whatman system for 10 min in a 20-fold SSC treatment.
  • the film was placed on Whatman 3MM filter paper impregnated with 10 times SSC, and crosslinked by an ultraviolet crosslinker for 3-5 min.
  • the membrane was simply washed with ddH 2 O and dried in air.
  • Hybridization and development procedures were performed according to the operating manual of Roche digoxin test kit I (article number: 11745832910) or Roche digoxigenin test kit II (article number: 11585614910).
  • Figure 6A shows the results of hybridization of the transformation event ZMM030 maize genomic DNA with HindIII and KpnI, respectively, to the cry1Ab/cry1AcZM specific probe molecule.
  • a positive band was displayed under the conditions of the two enzyme digestion conditions.
  • the band obtained by HindIII digestion was 10.9 kb, and the band obtained by KpnI digestion was 10.5 kb, indicating that the exogenous gene cry1Ab/cry1AcZM single copy was inserted.
  • a single copy conversion event is shown under the conditions of the two enzyme digestion conditions.
  • Figure 6B shows the results of a hybridization of the event ZZM030 maize genomic DNA by HindIII and EcoRI digestion with a bar-specific probe molecule, respectively.
  • a positive band was displayed under the conditions of two enzyme digestions.
  • the band obtained by HindIII digestion was 4.2 kb, and the band digested by EcoRI was 10.0 kb, which was in line with expectations, indicating that a single copy of the foreign gene was inserted.
  • the conversion event is a single copy conversion event.
  • Figure 6C shows the results of hybridization of the transformation event ZZM030 maize genomic DNA by HindIII and KpnI, respectively, to the cp4 epspsZM specific probe molecule.
  • a positive band was displayed under the conditions of two enzyme digestions.
  • the band obtained by HindIII digestion was 10.9 kb, and the band obtained by KpnI digestion was 10.5 kb, which was in line with expectations, indicating that the foreign gene cp4 epspsZM single copy was inserted.
  • the conversion event is a single copy conversion event.
  • transgenic procedures a large number of genetic transformations are typically performed using the same transformation vector, and a very small number of superior transformation events are screened from the many transformation events obtained. Therefore, the detection of the vector, the expression element, the foreign gene, and the like in the inserted foreign sequence can only prove that the test sample contains the transgenic component and cannot distinguish different transformation events.
  • Different transformation events are characterized by a combination of a flanking sequence of the insertion site and an inserted exogenous sequence. To this end, this example separates and identifies flanking sequences of corn transformation events.
  • the total DNA extracted from the leaves of the transformed event plants (T 2 generation or T 3 generation robust growth transgenic plants) was taken, and the FSNI-PCR method was used to amplify, clone and sequence the foreign gene flanking sequences inserted into the maize genome to obtain sequence results. .
  • Tail-PCR primer sequences are shown in Table 10.
  • the genomic DNA in the step (2) was used as a template for the first round of PCR reaction, and the reaction system is shown in Table 6 below.
  • the reaction procedure was: 95 ° C, 2.5 min; 2 cycles: 94 ° C, 10 sec, 62 ° C, 30 sec, 72 ° C, 2 min; 94 ° C, 10 sec; 25 ° C, 2 min; 72 ° C (5.1% ramp), 2 min; 5 cycles: 94 ° C, 10 sec; 62 ° C, 30 sec; 72 ° C, 2 min; 94 ° C, 10 sec; 62 ° C, 30 sec; 72 ° C, 2 min; 94 ° C, 10 sec; 44 ° C, 30 sec; 72 ° C, 2 min; 72 ° C, 5 min; 20 ° C, 10 min.
  • a third round of PCR amplification was carried out using the second round of PCR product (50-fold dilution of the mixture) as a template, and the reaction system is shown in Table 8 below.
  • the reaction procedure was: 94 ° C, 1.5 min; (94 ° C, 10 sec; 62 ° C, 30 sec; 72 ° C, 2 min) x 30 cycles; 72 ° C, 7 min; 20 ° C, 10 min.
  • sequencing primers are M13F and M13R primers; M13-F: 5'-TGTAAAACGACGGCCAGT-3', M13-R: 5'-CAGGAAACAGCTATGACC-3';
  • the total DNA extracted from the leaves of the transformed event plants (T 2 generation or T 3 generation robust transgenic plants) was extracted, and the flanking sequence of the foreign gene inserted into the maize genome was amplified, cloned and sequenced by the ligation PCR method to obtain sequence results.
  • connection joints the connection system is as follows:
  • the genomic DNA digestion-linker ligation product in the step (5) was used as a template for the first round of PCR reaction, and the reaction system is shown in Table 10 below.
  • the reaction procedure was: 94 ° C, 5 min; 7 cycles: 94 ° C 30 sec, 72 ° C 3 min; 32 cycles: 94 ° C 30 sec; 67 ° C 3 min; 67 ° C 7 min; 25 ° C 10 min.
  • a second round of PCR amplification was carried out using the first round of PCR product (40-fold dilution of the mixture) as a template, and the reaction system is shown in Table 11 below.
  • the reaction procedure was: 94 ° C, 5 min; 5 cycles: 94 ° C 30 sec; 72 ° C 3 min; 20 cycles: 94 ° C 30 sec, 67 ° C 3 min; 67 ° C 7 min; 25 ° C 10 min.
  • the sequencing primers are M13F and M13R primers; M13-F: 5'-TGTAAAACGACGGCCAGT-3', M13-R: 5'-CAGGAAACAGCTATGACC-3';
  • the single-copy insertion sequence of the exogenous gene of the present application includes a vector size of 10282 bp for the left and right border sequences.
  • the method of exogenous DNA sequencing by segmental PCR amplification confirmed that the actual size of the transformation event insertion sequence was 10235 bp (681-10915 of SEQ ID NO: 1), the T-DNA insert was deleted by 7 bp from the left end of the expression vector, and the right end was deleted by 40 bp. .
  • the amplification system contained: 2 ⁇ L (200 ng) of DNA template, 0.5 ⁇ L of each primer, 0.5 ⁇ L of DNA polymerase, 2 ⁇ L of PCR buffer (10 times), and 14.5 ⁇ L of ddH 2 O.
  • the PCR reaction procedure was: pre-denaturation at 94 ° C for 5 min; 35 cycles: denaturation at 94 ° C for 30 sec, annealing at 55 ° C for 30 sec, extension at 72 ° C for 3 min; and finally extension at 72 ° C for 7 min.
  • the label amplification effect was detected using a 1% agarose gel.
  • the specifically amplified amplification product, ie, the fragment of interest, is recovered.
  • the above 47 bp nucleotide is located in the border sequence of the non-coding region. Its deletion did not affect the integrity of the insect resistance gene cry1Ab/cry1AcZM and the selection marker bar and cp4 epspsZm genes. DNA sequence analysis alignment indicated that the actual insertion nucleotide sequence of the transformation event was identical to the vector sequence without any base mutation.
  • the maize genome at the insertion site is a repeat, and the 47 bp nucleotide deletion does not destroy any known maize endogenous functional genes.
  • a pair of primers (SEQ ID NO. 8: FW-csp3758 and SEQ ID NO. 9: RV-csp2344) were designed using the sequence of the left wing genome of the maize transformation event and the sequence of the 35S polyA terminator in the exogenous fragment. A qualitative PCR identification method for this transformation event product.
  • the primers designed for the 5' end of the left border (LB) T-DNA of the integration site of the external DNA fragment of ZMM030 are:
  • Seq ID No. 8 5'-TGATGGTTAATGAGGCAAGA-3' (corn genomic region);
  • Seq ID No. 9 (RV-csp2344): 5'-TATAGGGTTTCGCTCATGTG-3' (35S PolyA region).
  • the above specific primers were used to amplify the DNA fragments by temperature gradient PCR at 50-60 ° C to determine the optimal annealing temperature.
  • PCR reaction conditions and procedures were 95 ° C for 5 min, 35 cycles: 95 ° C for 30 s, 58 ° C for 30 s, 72 ° C for 1 min; 72 ° C for 7 min.
  • the results indicate that only the DNA of this transformation event can have a positive result, and other transformation events or negative control maize varieties are negative results, see Figure 7A.
  • lanes 1-4 were sterile water, Xiang 249 DNA, ZZM030 DNA, and other DNAs of the same vector transformation event; only the ZZM030 genomic DNA lane 3 was clearly visible, and the DNA fragment size was 898 bp as expected, and the DNA fragment was cloned and sequenced. The results were also in line with expectations.
  • a pair of primers (SEQ ID NO. 10: FW-csp3879 and SEQ ID NO. 11: RV-csp3889) were designed using the cp4 epspsZM sequence in the exogenous fragment and the right-wing genomic sequence of the maize transformation event to establish the transformation event.
  • Qualitative PCR identification method were designed using the cp4 epspsZM sequence in the exogenous fragment and the right-wing genomic sequence of the maize transformation event to establish the transformation event.
  • Seq ID No. 10 (FW-csp3879): 5'-AAGATTGAGCTGTCGGATAC-3' (cp4epspsZM region),
  • Seq ID No. 11 (RV-csp3889): 5'-TTTGATCATGTGAGGAACGT-3' (maize genomic region).
  • the above specific primers were used to amplify the DNA fragments by temperature gradient PCR at 46-61 ° C to determine the optimal annealing temperature.
  • PCR reaction conditions and procedures were 95 ° C for 5 min; 35 cycles: 95 ° C for 30 s, 58 ° C for 30 s, 72 ° C for 1 min cycle; 72 ° C for 7 min.
  • the results indicate that only the DNA of this transformation event can have a positive result, and other transformation events or negative control maize varieties are negative results, see Figure 7B.
  • lanes 1-4 were sterile water, Xiang 249 DNA, ZZM030 DNA, and other DNAs of the same vector transformation event; only the ZZM030 genomic DNA lane 3 was clearly visible, and the DNA fragment size was 796 bp as expected, and the DNA fragment was cloned and sequenced. The results were also in line with expectations.

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Abstract

抗虫耐除草剂玉米转化事件，及相关的创制方法、检测方法和应用。以玉米自交系祥249为受体，经过农杆菌介导的遗传转化，得到在特定基因组位点插入外源基因插入物的玉米植株，其中外源基因插入物包含以下三个基因：抗虫基因、抗草铵膦基因和抗草甘膦基因。所获得的转化事件中，所插入的外源基因位于玉米基因组的非功能位点，不影响受体植物自身其他基因的表达，在使转基因玉米植物获得抗虫和耐除草剂特性的同时，保持了其良好的农艺性状。

Description

抗虫耐除草剂玉米转化事件 技术领域
本申请涉及植物生物技术领域，具体涉及一种抗虫耐除草剂玉米转化事件的创制方法、检测方法和应用。
背景技术
玉米是重要的饲料和工业原料作物，其作为中国种植面积最大的农作物，长期自给有余，但自2010年以来，进口量逐年增加。
玉米螟俗称玉米钻心虫，其危害是造成玉米常年减产的重要生物灾害之一，严重影响玉米的产量和质量，包括亚洲玉米螟(Ostrinia furnacalis)和欧洲玉米螟(Ostrinia nubilalis)。中国是亚洲玉米螟(Ostrinia furnacalis)的多发区和重发区，几乎每两年就大规模发生一次。一般年份玉米受玉米螟危害减产10％-15％，大发生年减产可达30％以上，甚至绝收。因玉米螟的危害，每年损失的玉米就达600-900万吨。玉米螟不仅直接造成玉米产量损失，而且还诱发和加重玉米穗腐病的发生，使玉米的品质下降。目前防治玉米螟的主要方式仍然以农药防治为主。农药的大量使用，既增加种植成本，又破坏生态环境。田间杂草与作物竞争水、肥、光能及生长空间，同时又是危害作物病菌及害虫的中间寄主，是作物增产的重要生物限制因子之一。我国常年受杂草严重危害的农作物面积高达12亿亩，其中玉米1.9亿亩。当前，广泛采用的选择性除草剂施用量大，且残留期长、容易影响下茬作物的正常生长。草铵膦等灭生性除草剂具有高效、低毒、易降解、无残留等特点，但它们除草没有选择性，不能直接用于作物的生长期。植物转基因育种技术具有目的性强、周期短、效率高，能够实现不同物种间优良基因的转移等优点。自1996年第一个转基因作物商业化以来，这项技术已经为全球农业带来了巨大的改变。
就转基因玉米而言，以防治鳞翅目玉米螟虫的Cry1Ac、Cry1F等基因为主，除此外，亦有多个具有鞘翅目抗性的品系如MON88017等，以及利用Cry3类基因获得抗玉米根虫品系如MON863等投入商业化生产。
世界范围内，1996年至今已有40余种转Bt基因抗虫玉米被26个国家批准投入商业化生产或饲料食品加工，如美国孟山都公司的MON810、MON89034等。
发明内容
一方面，本申请提供了核酸分子，其包含：i)包含SEQ ID NO:1的第381-780位核苷酸和/或第10815-11214位核苷酸所示序列，或其 片段或其变体或其互补序列；ii)包含SEQ ID NO:1的第381-780位核苷酸和第6239-6338位核苷酸所示序列，或其片段或其变体或其互补序列；iii)包含SEQ ID NO:1的第6239-6338位核苷酸和第10815-11214位核苷酸所示序列，或其片段或其变体或其互补序列；或者iv)包含SEQ ID NO:1的第381-780位核苷酸，第6239-6338位核苷酸和第10815-11214位核苷酸所示序列，或其片段或其变体或其互补序列。
在一实施方案中，本申请所提供的核酸分子包含SEQ ID NO:1所示序列，或其片段或其变体或其互补序列。
在另一实施方案中，本申请所提供的核酸分子，其包含以下表达盒：表达抗草铵膦基因的第一表达盒，如SEQ ID NO:1的第748-2288位核苷酸所示序列；表达抗虫基因的第二表达盒，如SEQ ID NO:1的第2620-6959位核苷酸所示序列；和表达抗草甘膦基因的第三表达盒，如SEQ ID NO:1的第6968-10892位核苷酸所示序列。
在另一实施方案中，本申请所提供的核酸分子，其通过将以下表达盒导入玉米的基因组中获得：表达抗草铵膦基因的第一表达盒，如SEQ ID NO:1的第748-2288位核苷酸所示序列；表达抗虫基因的第二表达盒，如SEQ ID NO:1的第2620-6959位核苷酸所示序列；和表达抗草甘膦基因的第三表达盒，如SEQ ID NO:1的第6968-10892位核苷酸所示序列。
本申请所提供的核酸分子存在于玉米植物、种子、植物细胞、后代植物或植物部分中。
另一方面，本申请提供了用于检测玉米转化事件的探针，其包含SEQ ID NO:1的第381-780位核苷酸或第10815-11214位核苷酸所示序列，或其片段或其变体或其互补序列。
本申请还提供了用于检测玉米转化事件的引物对，其能够特异性扩增产生包含SEQ ID NO:1的第381-780位核苷酸或第10815-11214位核苷酸所示序列，或其片段或其变体或其互补序列。
在一实施方案中，所述引物对为：i)特异性识别包含SEQ ID NO:1的第381-780位核苷酸所示序列的引物对；ii)特异性识别包含SEQ ID NO:1的第10815-11214位核苷酸所示序列的引物对；iii)特异性识别包含SEQ ID NO:1的第381-780位核苷酸所示序列的正向引物，和特异性识别包含SEQ ID NO:1的第681-10915位核苷酸所示序列的反向引物；iv)特异性识别包含SEQ ID NO:1的第681-10915位核苷酸所示序列的正向引物，和特异性识别包含SEQ ID NO:1的第10815-11214位核苷酸所示序列的反向引物。
在一实施方案中，本申请所提供的引物对为SEQ ID No:8和SEQ ID No:9所示的核苷酸序列或其互补序列；或者SEQ ID No:10和SEQ  ID No:11所示的核苷酸序列或其互补序列。
此外，本申请还提供了用于检测玉米转化事件的试剂盒或微阵列，其包含上述的探针和/或上述的引物对。
还一方面，本申请提供了检测玉米转化事件的方法，其包括利用以下来检测待测样品中是否存在所述转化事件：上述的探针；上述的引物对；上述的探针和引物对；或者上述的试剂盒或微阵列。
本申请还提供了对玉米进行育种的方法，所述方法包括以下步骤：1)获得包含上述的核酸分子的玉米；2)将步骤1)所获得的玉米通过花粉培养、未受精胚培养、加倍培养、细胞培养、组织培养、自交或杂交或以上的组合得到后代植物、种子、植物细胞、后代植物或植物部分；以及任选地，3)对步骤2)所获得的后代植物进行除草剂草铵膦和草甘膦以及螟虫和/或粘虫的抗性鉴定，并利用上述的方法来检测其中是否存在所述转化事件。
进一步地，本申请还提供了由上述的方法获得的玉米植物、种子、植物细胞、后代植物或植物部分等，以及由这些玉米植物、种子、植物细胞、后代植物或植物部分等制成的制品，包括食品、饲料或工业原料等。
此外，本申请提供了控制鳞翅目害虫群体的方法，包括使所述鳞翅目害虫群体接触由上述的方法获得的玉米植物、种子、植物细胞、后代植物或植物部分。
本申请还提供了杀死鳞翅目害虫的方法，包括使所述鳞翅目害虫接触杀虫有效量的由上述的方法获得的玉米植物、种子、植物细胞、后代植物或植物部分。
本申请还提供了减轻鳞翅目害虫对玉米的伤害的方法，包括将以下表达盒导入玉米的基因组中：表达抗草铵膦基因的第一表达盒，如SEQ ID NO:1的第748-2288位核苷酸所示序列；表达抗虫基因的第二表达盒，如SEQ ID NO:1的第2620-6959位核苷酸所示序列；和表达抗草甘膦基因的第三表达盒，如SEQ ID NO:1的第6968-10892位核苷酸所示序列。
在具体实施方案中，上述方法中所述的鳞翅目害虫为亚洲玉米螟(Ostrinia furnacalis)、欧洲玉米螟(Ostrinia nubilalis)或东方粘虫(Mythimna separate(Walker))。
附图说明
图1为载体pZHZH35006的结构示意图，其中：
ubiquitin promoter     来自玉米的泛素基因启动子
Ωsequence             源自烟草花叶病毒基因表达增强元件
Kozak sequence         存在于真核生物mRNA的一段序列，用于 翻译起始
cry1Ab/cry1AcZM      优化的Bt基因序列
polyA                多聚腺苷酸序列
nos terminator       农杆菌胭脂碱合成酶基因终止子
T-Border(right)      T-DNA右边界序列
CaMV 35S promoter    花椰菜花叶病毒35S启动子
bar                  抗草铵膦基因序列
CaMV 35S terminator  花椰菜花叶病毒35S终止子
T-Border(left)       T-DNA左边界序列
Kanamycin (R)        卡那霉素抗性序列
pBR322 ori           pBR322起始区序列
pBR322 bom           pBR322骨架区序列
pVS1 rep             pVS1复制子
pVS1 sta             pVS1转录起始区
cp4 epspsZM          编码EPSPS蛋白的抗草甘膦基因序列
Ω sequence1         源自烟草蚀纹病毒基因表达增强元件
ubiquitin4 promoter  来自甘蔗的泛素基因启动子
bp                   碱基对
图2为250毫升/亩喷施草铵膦除草剂“保试达”4-5天后植株照片，其中：
A为ZZM030，植株生长正常，无任何受害症状；
B为非转基因阴性对照野生型祥249，叶片干枯、失绿、枯斑、生长停滞，表现明显药害症状。
图3为200毫升/亩喷施草甘膦除草剂“农达”一周后植株照片，其中：
A为ZZM030，植株生长正常，无任何受害症状；
B为非转基因阴性对照野生型照祥249，叶片干枯、褪绿、生长停滞，表现明显药害症状。
图4为植株螟虫抗性鉴定的田间实验照片，图中白色斑点为虫孔,其中：
A为ZZM030叶片，针孔状虫孔，虫孔稀少、分散；
B为非转基因阴性对照野生型祥249叶片，绿豆大小虫孔，个别呈短条孔花叶。
图5为植株粘虫抗性鉴定的田间实验照片，图中叶片边缘及叶片上缺失部分为被粘虫蚕食后的虫孔，其中：
A为ZZM030叶片；
B为非转基因阴性对照野生型祥249叶片。
图6为ZZM030 Southern杂交拷贝数检测结果，其中:
图6A为cry1Ab/cry1AcZM基因插入拷贝数检测结果，其中：泳道1，DNA分子标记；泳道2，空白；泳道3，HindIII酶切的ZZM030基因组DNA与cry1Ab/cry1AcZM特异性探针杂交；泳道4，HindIII酶切的野生型祥249基因组DNA与cry1Ab/cry1AcZM特异性探针杂交,作为阴性对照；泳道5，KpnI酶切的ZZM030基因组DNA与cry1Ab/cry1AcZM特异性探针杂交；泳道6，KpnI酶切的野生型祥249基因组DNA与cry1Ab/cry1AcZM特异性探针杂交，作为阴性对照；泳道7，EcoRI酶切的ZZM030基因组DNA与cry1Ab/cry1AcZM特异性探针杂交，作为阳性对照；泳道8，EcoRI酶切的野生型祥249基因组DNA与cry1Ab/cry1AcZM特异性探针杂交，作为阴性对照；泳道9，EcoRI酶切的质粒及野生型祥249基因组DNA与cry1Ab/cry1AcZM特异性探针杂交，作为阳性对照；
图6B为bar基因插入拷贝数检测结果，其中：泳道1，DNA分子标记；泳道2，空白；泳道3，HindIII酶切的ZZM030基因组DNA与bar特异性探针杂交；泳道4，HindIII酶切的野生型祥249基因组DNA与bar特异性探针杂交,作为阴性对照；泳道5，EcoRI酶切的ZZM030基因组DNA与bar特异性探针杂交；泳道6，EcoRI酶切的野生型祥249基因组DNA与bar特异性探针杂交，作为阴性对照；泳道7，KpnI酶切的ZZM030基因组DNA与bar特异性探针杂交，作为阳性对照；泳道8，KpnI酶切的野生型祥249基因组DNA与bar特异性探针杂交，作为阴性对照；泳道9，KpnI酶切的质粒及野生型祥249基因组DNA与bar特异性探针杂交，作为阳性对照；
图6C为cp4 epspsZM基因插入拷贝数检测结果，其中：泳道1，DNA分子标记；泳道2，空白；泳道3，HindIII酶切的ZZM030基因组DNA与cp4 epspsZM特异性探针杂交；泳道4，HindIII酶切的野生型祥249基因组DNA与cp4 epspsZM特异性探针杂交,作为阴性对照；泳道5，KpnI酶切的ZZM030基因组DNA与cp4 epspsZM特异性探针杂交；泳道6，KpnI酶切的野生型祥249基因组DNA与cp4 epspsZM特异性探针杂交，作为阴性对照；泳道7，EcoRI酶切的ZZM030基因组DNA与cp4epspsZM特异性探针杂交，作为阳性对照；泳道8，EcoRI酶切的野生型祥249基因组DNA与cp4 epspsZM特异性探针杂交，作为阴性对照；泳道9，EcoRI酶切的质粒及野生型祥249基因组DNA与cp4 epspsZM特异性探针杂交，作为阳性对照。
图7为ZZM030事件特异性PCR检测结果，其中：
图7A为左边界检测结果；
图7B为右边界检测结果；
其中泳道1-4分别为：无菌水、祥249基因组DNA、ZZM030基因组DNA、相同载体转化后获得的非ZZM030事件基因组DNA。
具体实施方式
提供以下定义和方法用以更好地界定本申请以及在本申请实践中指导本领域普通技术人员。除非另作说明，术语按照相关领域普通技术人员的常规用法理解。本文所引用的所有专利文献、学术论文、行业标准及其他公开出版物等，其中的全部内容整体并入本文作为参考。
如本文所用，“玉米”是任何玉米植物并包括可以与玉米育种的所有植物品种，包括整株植物、植物细胞、植物器官、植物原生质体、植物可以从中再生的植物细胞组织培养物、植物愈伤组织、和植物或植物部分中完整的植物细胞，所述植物部分例如胚、花粉、胚珠、种子、叶、花、枝、果实、茎杆、根、根尖、花药等。
本技术领域人员公知，外源基因在植物体内的表达具有位置效应，即受到插入染色体位置的影响，这种影响可能是由于染色体结构或整合位点附近的转录调节元件造成的。因此，通常需要生产数百个不同的转化事件，并从这些事件中筛选出外源基因表达水平及模式符合预期要求的优异转化事件，以达到商业化生产应用的目的。
优异的转化事件可通过常规育种方法即有性杂交的方式将外源基因转育到其它遗传背景的种质中，其后代保持了原始转化体的转基因表达特性。本申请涉及通过从诸多转化事件中筛选出的优异转化事件ZZM030。
在本申请中，“转化事件ZZM030”是指以玉米自交系祥249为受体经过农杆菌介导的遗传转化，得到在特定基因组位点插入外源基因插入物(T-DNA插入物)的玉米植株，其中外源基因插入物包含以下三个基因：抗虫基因、抗草铵膦基因和抗草甘膦基因。本申请所获得转化事件ZZM030，所插入的外源基因位于玉米基因组的非功能位点，不影响受体植物自身其他基因的表达，在使转基因玉米植物获得抗虫和耐除草剂特性的同时，保持了其良好的农艺性状。
在具体实例中，转基因后所得到的T-DNA插入物具有SEQ ID NO:1的第681-10915位核苷酸所示序列。转化事件ZZM030可以指这一转基因过程，也可以指由这一过程所得到的基因组内的T-DNA插入物，或T-DNA插入物与侧翼序列的组合，或可以指由这一转基因过程得到的玉米植株。转化事件ZZM030还可以指由上述植物进行无性繁殖、有性繁殖、减倍或加倍繁殖或以上的组合而得到的后代植物。
在其他实施方案中，该事件也适用于同样的外源基因(SEQ ID NO:1的第681-10915位核苷酸所示序列)转化其他植物受体品种，从而将T-DNA插入物插入到同样基因组位置而获得的植物。适用的植物包括单子叶植物，如水稻、小麦、燕麦、大麦、青稞、粟、高粱和甘蔗等。
在本申请中，获得了以SEQ ID NO:1的第1-680位核苷酸为左侧侧翼序列和SEQ ID NO:1的第10916-11375位核苷酸为右侧侧翼序列 的T-DNA插入物(第681-10915位核苷酸)。侧翼序列并非仅限于SEQ ID NO:1的第1-680位核苷酸和第10916-11375位核苷酸，因为列出的侧翼序列仅是用于表示T-DNA插入物在基因组中的位置，即T-DNA插入物左侧插入点位于第4号染色体40636901bp；T-DNA插入物右侧插入点位于第4号染色体40636883bp。因此本申请的侧翼序列可以根据基因组序列而向两侧延伸，即左侧侧翼序列可向第4号染色体40636901bp下游延伸，右侧侧翼序列可向第4号染色体40636883bp上游延伸。
由于转化事件ZZM030产生向基因组中特定位点插入的T-DNA插入物，因此其插入位点是特异性的，可以用于检测生物样品中是否包含转化事件ZZM030。在具体实施方案中，包含转化事件ZZM030的T-DNA插入物与侧翼序列的接合位点的任何序列，均可用于检测本申请的转化事件ZZM030，包括但不限于包含上游插入位点(左侧翼序列与T-DNA插入物的接合位点)或下游插入位点(右侧翼序列与T-DNA插入物的接合位点)的以下序列或其片段或其变体或其互补序列中的一种或多种：i)包含SEQ ID NO:1的第381-780位核苷酸所示序列；ii)包含SEQ ID NO:1的第1-898位核苷酸所示序列；iii)包含SEQ ID NO:1的第6239-6338位核苷酸所示序列；iv)包含SEQ ID NO:1的第10815-11214位核苷酸所示序列；v)包含SEQ ID NO:1的第10578-11373位核苷酸所示序列；vi)包含SEQ ID NO:1的第381-11241位核苷酸所示序列；vii)包含SEQ ID NO:1所示的序列。
在具体实例中，可用于检测本申请的转化事件ZZM030的序列为包含上游插入位点的序列或其片段或其变体或其互补序列，如包含SEQ ID NO:1的第381-780位核苷酸所示序列或包含SEQ ID NO:1的第1-898位核苷酸所示序列，或者为包含下游插入位点的序列，例如，包含SEQ ID NO:1的第10815-11214位核苷酸所示序列或包含SEQ ID NO:1的第10578-11373位核苷酸所示序列，或者为包含上游插入位点的序列与包含下游插入位点的序列的组合。
在另一实例中，可用于检测本申请的转化事件ZZM030的序列为包含上游插入位点的序列或其片段或其变体或其互补序列与包含T-DNA插入物的序列或其片段或其变体或其互补序列的组合，例如，包含SEQ ID NO:1的第381-780位核苷酸所示序列或包含SEQ ID NO:1的第1-898位核苷酸所示序列，与包含SEQ ID NO:1的第6239-6338位核苷酸所示序列或包含SEQ ID NO:1的第681-10915位核苷酸所示序列的组合。
在另一实例中，可用于检测本申请的转化事件ZZM030的序列为包含下游插入位点的序列或其片段或其变体或其互补序列与包含T-DNA插入物的序列或其片段或其变体或其互补序列的组合，例如， 包含SEQ ID NO:1的第10815-11214位核苷酸所示序列或包含SEQ ID NO:1的第10578-11373位核苷酸所示序列，与包含SEQ ID NO:1的第6239-6338位核苷酸所示序列或包含SEQ ID NO:1的第681-10915位核苷酸所示序列的组合。
在另一实例中，可用于检测本申请的转化事件ZZM030的序列为包含SEQ ID NO:1的第381-11241位核苷酸所示序列或其片段或其变体或其互补序列，或者为包含SEQ ID NO:1所示的序列或其片段或其变体或其互补序列。
因此，能够特异性检测转化事件ZZM030的T-DNA插入物与侧翼序列的接合位点的引物对、探针、以及引物对和探针的组合均可用于检测本申请的转化事件ZZM030。
如本文所用，“核苷酸序列”包括涉及单链或双链形式的脱氧核糖核苷酸或核糖核苷酸多聚物，并且除非另有限制，核苷酸序列以5’至3’方向从左向右书写。
在一些实施方案中，本申请还涉及核酸序列的片段，其是指完整部分中的不完整的更小片段的部分。例如，SEQ ID NO：1的片段包括SEQ ID NO：1的完整序列的至少约10个核苷酸、至少约20个核苷酸、至少约30个核苷酸、至少约40个核苷酸、或至少约50个核苷酸的序列或更多。
在一些实施方案中，可以对本申请的核酸序列进行改变，以进行保守氨基酸替换。在某些实施方案中，可以依照单子叶密码子偏好性对本申请的核苷酸序列进行不改变氨基酸序列的替换，例如可以用单子叶植物偏好的密码子替换编码同一氨基酸序列的密码子，而不改变该核苷酸序列所编码的氨基酸序列。在一些实施方案中，本申请还涉及核酸序列的变体。一般来讲，特定核酸片段的变体将与该特定核苷酸序列具有至少约70％、75％、80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、99.5％或99.9％或更高的序列同一性，或以上的互补序列。这样的变体序列包括一个或多个核酸残基的添加、缺失或替换，从而可以导致相应的氨基酸残基的添加、移除或替换。通过本领域内已知的序列比对程序包括杂交技术确定序列同一性。实施方案的核苷酸序列变体与本申请的序列的差异可以少至1-15个核苷酸、少至1-10个(例如6-10个)，少至5个，少至4、3、2或甚至1个核苷酸。
如本文所用，“探针”是附加了常规可检测的标记或报告分子例如放射性同位素、配体、化学发光剂或酶的分离的多核苷酸，其与靶多核苷酸的链是互补的。
在具体实施方案中，本申请所提供的用于检测转化事件ZZM030的DNA探针，包括包含SEQ ID NO：1的足够长度的连续核苷酸的序列或 其完全互补序列，该DNA探针在严格杂交条件下与包含上游插入位点或下游插入位点的核苷酸序列杂交并且在严格杂交条件下不与不含上游插入位点或下游插入位点的核苷酸序列杂交。
在具体实例中，本申请所提供的探针包含SEQ ID NO:1的第381-780位核苷酸或第10815-11214位核苷酸所示序列，或其片段或其变体或其互补序列。如本文所用，“引物”是分离的多核苷酸，其通过核酸杂交形成引物和靶DNA链之间的杂交体而与互补靶DNA链退火，然后凭借例如DNA聚合酶沿着靶DNA链伸展。引物对涉及其靶多核苷酸扩增用途，例如通过聚合酶链反应(PCR)或其他常规核酸扩增方法。
在具体实施方案中，本申请所提供的用于检测转化事件ZZM030的引物对包括第一DNA分子以及不同于第一DNA分子的第二DNA分子，其中所述第一DNA分子和第二DNA分子各自包含SEQ ID NO：1的足够长度的连续核苷酸的核苷酸序列或其完全互补序列，且其中所述第一DNA分子存在于SEQ ID NO：1的T-DNA插入物中和所述第二DNA分子存在于SEQ ID NO：1的侧翼序列中，当与来自转化事件ZZM030的DNA在扩增反应中共同使用时，该DNA引物产生用于检测样品中转化事件ZZM030DNA的扩增子，且其中所述扩增子包含SEQ ID NO:1的第381-780位核苷酸或第10815-11214位核苷酸所示序列，或其片段或其变体或其互补序列。
在具体实施方案中，本申请所提供的引物对为特异性识别包含SEQ ID NO:1的第381-780位或第1-898位核苷酸所示序列的引物对。
在具体实施方案中，本申请所提供的引物对为特异性识别包含SEQ ID NO:1的第10815-11214位或第10578-11373位核苷酸所示序列的引物对。
在具体实施方案中，本申请所提供的引物对为：i)特异性识别包含SEQ ID NO:1的第381-780位或第1-898位核苷酸所示序列的引物对；和ii)特异性识别包含SEQ ID NO:1的第10815-11214位或第10578-11373位核苷酸所示序列的引物对；或者，所述引物对包含：特异性识别包含SEQ ID NO:1的第381-780位或第1-898位核苷酸所示序列的正向引物，和特异性识别包含SEQ ID NO:1的第10815-11214位或第10578-11373位核苷酸所示序列的反向引物。
在另一具体实施方案中，本申请所提供的引物对为：i)特异性识别包含SEQ ID NO:1的第381-780位或第1-898位核苷酸所示序列的引物对；和ii)特异性识别包含SEQ ID NO:1的第6239-6338位或第681-10915位核苷酸所示序列的引物对；或者，所述引物对包含：特异性识别包含SEQ ID NO:1的第381-780位或第1-898位核苷酸所示序列的正向引物，和特异性识别包含SEQ ID NO:1的第6239-6338位或第681-10915位核苷酸所示序列的反向引物。
在另一具体实施方案中，本申请所提供的引物对为：i)特异性识别包含SEQ ID NO:1的第6239-6338位或第681-10915位核苷酸所示序列的引物对；和ii)特异性识别包含SEQ ID NO:1的第10815-11214位或第10578-11373位核苷酸所示序列的引物对；或者，所述引物对包含：特异性识别包含SEQ ID NO:1的第6239-6338位或第681-10915位核苷酸所示序列的正向引物，和特异性识别包含SEQ ID NO:1的第10815-11214位或第10578-11373位核苷酸所示序列的反向引物。
在另一具体实施方案中，本申请所提供的引物对为：i)特异性识别包含SEQ ID NO:1的第381-780位或第1-898位核苷酸所示序列的引物对，ii)特异性识别包含SEQ ID NO:1的第6239-6338位或第681-10915位核苷酸所示序列的引物对，和iii)特异性识别包含SEQ ID NO:1的第10815-11214位或第10578-11373位核苷酸所示序列的引物对。
在另一具体实施方案中，本申请所提供的引物对为特异性识别包含SEQ ID NO:1所示序列的引物对。
在具体实例中，所述引物对为SEQ ID No:8和SEQ ID No:9所示的核苷酸序列或其互补序列；或者SEQ ID No:10和SEQ ID No:11所示的核苷酸序列或其互补序列。
设计和使用引物和探针的方法是本领域熟知的，例如Sambrook等人的《分子克隆实验手册》(Sambrook J&Russell DW,Molecular cloning:a laboratory manual,2001)和Wiley-Blackwell出版的《分子生物学实验室指南》(Current Protocols in Molecular Biology)中描述。
如本文所用，“试剂盒”或“微阵列”是指用于生物样品中玉米转化事件ZZM030的鉴定和/或检测目的的试剂组或芯片。为质量控制(例如种子批次的纯度)、植物材料中或包含植物材料或来源于植物材料的材料例如但不限于食品或饲料产品中事件ZZM030的检测的目的，可以使用试剂盒或芯片，并且其组分可以具体地调整。
在具体实施方案中，本申请所提供的试剂盒或探针包括本申请所提供的任一种探针或任一种引物对。在另一具体实施方案中，本申请所提供的试剂盒或探针包括本申请所提供的任一种探针或任一种引物对的组合。
此外，本申请还提供了转基因玉米植物、后代、种子、植物细胞或植物部分及其制品，包括但不限于食品、饲料或工业原料。这些植物、后代、种子、植物细胞、植物部分及其制品中均包含可检测的本申请所提供的T-DNA插入物与侧翼序列的接合位点的核酸分子序列。
进一步地，本申请还提供了对玉米进行育种的方法，包括以下步骤：1)获得包含本申请所提供的T-DNA插入物与侧翼序列的接合位点的核酸分子序列的玉米；2)将步骤1)所获得的玉米通过花粉培养、未受 精胚培养、加倍培养、细胞培养、组织培养、自交或杂交或以上的组合得到后代植物、种子、植物细胞、后代植物或植物部分；以及任选地步骤3)，对步骤2)所获得的玉米植物进行抗除草剂草铵膦和草甘膦以及抗虫鉴定，并利用本申请所提供的探针、引物对、试剂盒或阵列来检测其中是否存在转化事件ZZM030。
此外，本申请提供了在田间控制杂草的方法，以及控制或杀死鳞翅目害虫的方法。
在具体实施方案中，本申请所提供的在田间控制杂草的方法，包括在田间种植包含转化事件ZZM030的玉米植物，以及在所述田间应用有效剂量的能够在不伤害所述包含事件ZZM030的转基因玉米植物的情况下控制杂草的草甘膦和草铵膦除草剂。
在具体实施方案中，本申请所提供的控制或杀死鳞翅目害虫的方法，包括使所述鳞翅目害虫接触有效量的转化事件ZZM030的玉米植物，或者向所述鳞翅目害虫喂食有效量的转化事件ZZM030的玉米植物，或者使所述鳞翅目害虫进食有效量的转化事件ZZM030的玉米植物。所述鳞翅目害虫，包括但不限于亚洲玉米螟(Ostrinia furnacalis)、欧洲玉米螟(Ostrinia nubilalis)、东方粘虫(Mythimna separate(Walker))等。
如本文所用，“有效量”或“杀虫有效量”是指存在于害虫环境的具有杀虫活性的物质或生物体的量。
实施例
以下实施例用于说明本发明，但不用来限制本发明的范围。在不背离本发明精神和实质的情况下，对本发明方法、步骤或条件所作的修改或替换，均属于本申请的范围。
若无特别指明，实施例按照常规实验条件，如Sambrook等人的《分子克隆实验手册》(Sambrook J&Russell DW,Molecular cloning:a laboratory manual,2001)，或按照制造厂商说明书建议的条件。
若未特别指明，实施例中所用的化学试剂均为常规市售试剂，实施例中所用的技术手段为本领域技术人员所熟知的常规手段。
以下实施例所涉及的玉米品种材料均由中国种子集团有限公司提供，其中玉米自交系祥249为玉米品种长城799的母本，是以国外引进的玉米种质资源为材料，用系谱法自交分离和严格选择，经过10个世代，于1996年选育而成。
实施例1.载体构建
1.抗虫基因的合成
利用本申请人在PCT国际申请WO2017012577A1中公开的在植物中表达并产生抗虫效果的抗虫基因cry1Ab/cry1AcZM。该基因以经过融合与改造的Cry1Ab和Cry1Ac N端608个氨基酸序列为基础，使用植物偏爱的密码子置换编码序列，并通过置换密码子的方法对DNA序列中存在的造成植物转录不稳定的富含AT序列以及常用限制性酶切位点进行改正消除。同时，在5’端增加了一段67个核苷酸的Ωsequence和3个核苷酸(ACC)的Kozak sequence用来增强真核基因的翻译效率。3’端添加135bp的polyA序列。该基因编码的蛋白质含有3个功能区段，其中N端两个功能区高度同源于Cry1Ab对应部分，C端的功能区高度同源于Cry1Ac。以人工合成的方式合成如SEQ ID NO:1第4624至6670位碱基所示的抗虫基因cry1Ab/cry1AcZM。
2.抗除草剂基因外源基因的合成
利用Vector NTI软件对抗草甘膦基因cp4 epsps编码区序列进行玉米密码子偏好性优化，并在5’端添加表达增强元件Ωsequence1，改造后的DNA序列命名为cp4 epspsZM。以人工合的方式合成如SEQ ID NO:1第8954至10611位碱基所示的抗草甘膦除草剂基因。
bar基因序列参见Gene bank登录号为X17220.1所示的序列，以人工合的方式合成如SEQ ID NO:1第957至1508位碱基所示的抗草铵膦除草剂基因。
3.载体构建
在合成的cry1Ab/cry1AcZM的5’端添加HindIII和PstI酶切位点，3’端添加PmeI酶切位点，并将合成的序列克隆在Puc57 simple载体上，命名为pZZ01194。
对已有含ubiquitin promoter(5’端有HindIII酶切位点，3’端有BamHI酶切位点)的中间载体pZZ00005，使用限制酶HindIII和BamHI进行双酶切，用T4DNA聚合酶补平产生的粘末端，获得ubiquitin promoter片段。
限制酶PstI处理pZZ01194，用T4DNA聚合酶补平产生的粘末端，将ubiquitin promoter通过平末端连接方式连入，获得含有ubiquitin promoter-cry1Ab/cry1AcZM片段的载体，命名为pZZ01201。
对已有含有nos terminator(5’端有EcoRI酶切位点，3’端有PmeI，EcoRI位点)的中间载体pZZ01188，使用限制酶EcoRI进行单酶切，用T4DNA聚合酶补平产生的粘末端，获得nos terminator序列。
PmeI处理pZZ01201，将nos terminator通过平末端连接方式连入，获得含有ubiquitin promoter-cry1Ab/cry1AcZM-nos terminator片段的载体，命名为pZZ01205。
对中间载体pZZ00015(含有CaMV 35S promoter-bar-CaMV35S terminator和ubiquitin promoter–egfp-nos terminator表达元件)， 通过限制酶HindIII和PmeI去除ubiquitin promoter-egfp-nos terminator。使用限制酶HindIII和PmeI处理pZZ01205载体，获得ubiquitin promoter-cry1Ab/cry1AcZM-nos terminator片段。将两者进行连接，获得含有ubiquitin promoter-cry1Ab/cry1AcZM-nos terminator和CaMV 35S promoter-bar-CaMV 35S terminator两个表达盒的表达载体,命名为pZHZH25017。
对中间载体pZZ01337(CaMV 35S promoter-cp4 epspsZM-nos terminator)使用限制酶HindIII和BamHI切除CaMV 35S promoter。对中间载体pZZ00033使用限制酶HindIII和BamHI获得ubiquitin4promoter。将两者进行连接，获得含有ubiquitin4 promoter-cp4 epspsZM-nos terminator片段的载体，命名为pZZ01383。
使用限制酶HindII和PmeI双酶切处理pZZ01383获得ubiquitin4 promoter-cp4 epspsZM-nos terminator片段，用T4DNA聚合酶补平产生的粘末端。
使用限制酶PmeI处理pZHZH25017，将ubiquitin4 promoter-cp4 epspsZM-nos terminator通过平末端连接方式连入，获得含有ubiquitin promoter-cry1Ab/cry1AcZM-nos terminator,CaMV 35S promoter–bar-CaMV 35S terminator和ubiquitin4 promoter-cp4 epspsZM-nos terminator三个表达盒的植物表达载体，命名为pZHZH35006，其物理图谱见(图1)。
实施例2.转基因玉米的获得
使用农杆菌介导的遗传转化方法获得转基因玉米。
将载体pZHZH35006的质粒DNA通过电击法转化到农杆菌EHA105中，鉴定后备用。
利用本申请人在中国发明专利申请CN104745622A中公开的玉米骨干自交系的高效转基因方法进行转化。
具体地，玉米自交系祥249自交后取长度1.5mm左右的幼胚用于转化。收集约200个果穗的幼胚为一批次，置于EP管中把悬浮液吸出，加入含有200μM乙酰丁香酮的农杆菌菌液，共培养5min，然后把幼胚转移到共培养基上，暗培养3天。将暗培养后的幼胚放置在愈伤诱导培养基上，待有愈伤组织长出后，放置含5mg/L双丙氨膦的筛选培养基上，筛选培养，每两周继代一次。当抗性愈伤长出时，挑选出状态良好的胚性愈伤转到分化培养基上，培养条件为26℃，每日3000Lux光强，光照16h，两周后出现再生小苗。将再生的小植株移到生根培养基中，待小苗长出二级次根后，移栽于混有营养土和蛭石(1∶3)的小盆中。
按照下述步骤对获得的转化苗进行转基因阳性检测，挑选出转基 因阳性植株。
(1)DNA抽提
采用天根生化技术公司的DNAsecure Plant Kit新型植物基因组DNA提取试剂盒(离心柱型)抽提玉米基因组DNA。
(2)PCR
将下列试剂从-20℃冰箱中取出解冻：10倍PCR缓冲液(Takara)、脱氧核苷酸混合物(10mM，Sigma)、包括正向引物SEQ ID NO:2(CSP759)：5’-CACGCAGATTCCAGCGGTCAA-3’；反向引物SEQ ID NO:3(CSP760)：5’-GACGAGGTGAAGGCGTTAGCA-3’)以及玉米叶片DNA模板。所有试剂解冻完毕后，离心数秒，置于冰上待用。配制PCR反应体系的混合液，混匀，离心数秒。PCR反应体系(20μL)：2μL10倍PCR缓冲液(Takara)，0.5μL脱氧核苷酸混合物(10mM，Sigma)，0.8μL正反向引物混合物(5μM)，0.2μL r-Taq(5U，Takara)，其余为dd H 2O。将混合液分装至200μL规格的PCR管中，再加入1μL玉米叶片DNA模板，对于不同样品分别做好标记以便区分。将PCR反应管放入ABI 9700型PCR扩增仪，选择预设PCR扩增程序，开始运行反应。PCR反应程序为：94℃预变性2min；30个循环：94℃变性30sec，58℃退火30sec，72℃延伸30sec；最后72℃延伸5min。
(3)琼脂糖凝胶电泳检测
PCR结束后，取5μL PCR产物进行琼脂糖凝胶电泳检测。配制1.5％的琼脂糖凝胶，150V电泳25min后在溴化乙啶(EB)中染色10min，在紫外凝胶成像系统中拍照。
(4)结果的判断
能扩增出333bp大小条带的材料为转基因阳性植株，不能扩增出该条带大小的材料为转基因阴性植株。
将上述转基因阳性植株移栽至大花盆中，即为T 0代植株。
实施例3.转基因玉米的抗性鉴定
T 0代植株自交，所得种子为T 1代种子。将T 1代种子播种于温室大棚中，得到T 1代植株。重复上述过程，直至获得T 4代种子。
对T 1至T 3代植株进行转基因阳性检测、耐除草剂分析、抗虫鉴定、农艺性状分析。从每代植株中挑选出转基因阳性，具有抗虫、耐除草剂特性，并且农艺性状表现优异的植株进入下一代筛选。
1.阳性检测
检测方法及步骤同实施例2所述。
2.耐除草剂性状鉴定
将经步骤1检测的阳性植株自交种子播种于温室大棚中，对6-8叶片期植株进行除草剂抗性鉴定，去除不耐受除草剂的植株。
(1)草铵膦除草剂耐受鉴定
喷施所用草铵膦除草剂“保试达”(Basta)为拜耳作物科学(中国)有限公司生产，有效成分为18％草铵膦可溶液剂。该除草剂的推荐用量为200-300毫升/亩，本申请采用推荐浓度中量250毫升/亩的用药量进行喷施。4-5天后观察并记录除草剂耐性表现。对草铵膦具有耐性的玉米植株生长正常，无任何受害症状；对草铵膦敏感的玉米植株表现出明显的药害症状，包括生长停滞、失绿、枯斑、畸形等，直至全株死亡。
在T 1、T 2和T 3代植株群体中，根据观测到草铵膦抗性的实际分离比与根据孟德尔遗传定律计算的预期分离比(表1)，按以下公式进行Chi平方检验：χ 2＝Σ[(|o–e|–0.5) 2/e]；其中，“o”为阳性株数或阴性株数观测值，“e”为阳性株数或阴性株数期望值，“0.5”为自由度为1时Yates分析校正因子。
表1 转化体ZZM030各世代预期分离比
Figure PCTCN2019074611-appb-000001
*：在单一位点单拷贝插入的情况下，按照孟德尔遗传定律计算的预期分离比。
表2 转化体ZZM030各世代分离分析-χ 2检验
Figure PCTCN2019074611-appb-000002
表2中，“观测值”为喷施草铵膦后观测到的实际阳性植株数和阴性植株数；“期望值”为根据孟德尔遗传定律按照表1计算的理论转基因阳性植株数与转基因阴性植株数；“χ 2”为根据Chi平方检验公式计算得到的Chi平方值；“χ 2 0.05,1”为在显著性水平α＝0.05，自由度为1时，查χ 2界值表得到的值；“概率”为χ 2与χ 2 0.05,1两者比较后的结果，当χ 2<χ 2 0.05,1时，则P>0.05，说明观测值与期望值之间无显著性差异。表2中χ 2检验分析表明，在T 1-T 3调查世代中，观察到的遗传分离比和预期遗传分离比之间没有显著性的差异(P>0.05)，说明ZZM030在不同世代之间按照孟德尔遗传规律稳定遗传，且在T 3代达到纯合。
(2)草甘膦除草剂耐受鉴定
喷施所用草甘膦除草剂“农达”(Roundup)为孟山都公司生产，有效成分为41％异丙胺盐。该除草剂玉米田推荐用药量为150-250毫升/亩，本申请采用推荐浓度中量200毫升/亩的用药量进行喷施，一周后观察并记录除草剂耐性表现。对草甘膦具有耐性的玉米植株生长正常，无任何受害症状；对草甘膦敏感的玉米植株表现出明显的药害症状， 包括生长抑制、褪绿、枯斑、畸形等，直至全株死亡。
3.转基因玉米植株螟虫抗性鉴定
采用心叶期活体接虫法在田间对植株的螟虫抗性进行了鉴定。
在玉米植株生长发育至心叶中期(7叶片期)进行接虫。供试昆虫为亚洲玉米螟(Ostrinia furnacalis)，将黑头期卵约60粒放入离心管中，用脱脂棉塞住管口。将离心管放入28℃、湿度80％的培养箱中，或置于室温条件下盖上湿毛巾保湿，待卵块孵化后将脱脂棉拔掉，投放入心叶丛中。每个植株接虫40-60头。接虫2-3周后逐株调查植株心叶的被害程度，根据被害叶片上的虫孔大小和多少划分受害等级，称为食叶级别。本申请采用国际玉米螟协作组制定的9级分级标准(表1)。逐株调查食叶级别，以各株平均值作为供鉴定品系的食叶级别，并根据表3的评价标准确定其抗螟性级别。
表3.玉米抗螟虫性田间鉴定评价标准
Figure PCTCN2019074611-appb-000003
注：*蛀茎评估时隧道2.5cm为1个孔。
**HR：高抗；R：抗；MR：中抗；S：感；HS：高感
4.玉米转化事件ZZM030
经过上述过程，最终筛选出玉米转化事件ZZM030。
图2为250毫升/亩喷施草铵膦除草剂“保试达”4-5天后植株照片，其中图2A为ZZM030，植株生长正常，无任何受害症状，图2B为野生型对照祥249，叶片干枯、失绿、枯斑、生长停滞，表现明显药害症状。表明该事件对草铵膦除草剂表现为高抗。
图3为200毫升/亩喷施草甘膦除草剂“农达”一周后植株照片，其中图3A为ZZM030，植株生长正常，无任何受害症状。图3B为野生型对照祥249，叶片干枯、褪绿、生长停滞，表现明显药害症状。表明该事件对草甘膦除草剂均表现为高抗。
图4为植株螟虫抗性鉴定的田间实验照片，利用亚洲玉米螟，采用心叶期活体接虫法进行，图中白色斑点为虫孔。图4A为ZZM030叶片，针孔状虫孔，虫孔稀少、分散。图4B为非转基因阴性对照祥249叶片，绿豆大小虫孔，个别呈短条孔花叶。表明该事件对亚洲玉米螟虫达到高抗级别，其叶片平均食叶级别为1.2-1.5(见表4)。
表4.转化体不同世代螟虫食叶级别生测结果
Figure PCTCN2019074611-appb-000004
备注：调查株数n＝10；“*”代表与阴性对照相比差异显著
实施例4.玉米转化事件ZZM030的粘虫抗性鉴定
1)粘虫抗性离体生测实验I以先正达商业化转基因玉米品种Bt11为阳性对照，该材料含有Cry1Ab基因，已报道具有粘虫抗性；以构建转基因事件所使用的受体材料祥249为阴性对照；对转化事件ZZM030进行粘虫抗性的离体生测；供试粘虫为东方粘虫(Mythimna separate(Walker))。
具体的鉴定方法如下：分别取生长至3-4和8-10叶期的新鲜玉米植株地上部分带回室内，取幼嫩心叶，放入培养皿内，接入10头1日龄虫；每皿一个处理，每个处理重复3次；放置在温度28±1℃、光周期14:10h(L:D)、相对湿度70-80％的人工气候箱中培养。3d后统计存活幼虫数并计算幼虫存活率。
对不同品种玉米粘虫存活率进行多重比较分析，显著水平为0.05，存活率在统计分析前进行一次开方和反正弦转化，比较各处理间差异显著性。根据取食转基因玉米和对照玉米组织幼虫存活率差异显著性，参考表5定性判定其对粘虫的抗性，鉴定结果如表6所示。
表5.东方粘虫离体生测评价标准
Figure PCTCN2019074611-appb-000005
表6.不同玉米材料粘虫抗性离体生测结果
Figure PCTCN2019074611-appb-000006
2)粘虫抗性离体生测实验II
以先正达商业化转基因玉米品种Bt11为阳性对照，该材料含有Cry1Ab基因，已报道具有粘虫抗性；以构建转基因事件所使用的受体材料祥249和中国农业科学院植物保护研究所迁飞害虫研究组累代饲 养粘虫的常规玉米苗为阴性对照；对ZZM030进行粘虫抗性的离体生测；供试粘虫为东方粘虫(Mythimna separate(Walker))。
具体的鉴定方法如下：选择材料3-4和8-10叶期的新鲜叶片为供试粘虫的食物；所有的玉米叶片用0.1％的次氯酸纳溶液浸泡3min消毒，然后用蒸馏水冲洗干净晾干；将l整株玉米放入750ml的罐头瓶中，每瓶接入初孵幼虫40头，每个处理做3次重复；置于温度(24±1)℃，湿度(70±5)％，光周期14L:10D的人工气候箱中饲养；每隔2天更换新鲜的相应处理的玉米植株，分别于试验开始后的第3、6、9天检查幼虫的死亡情况。
试验得到不同处理的第3、6、9天幼虫的存活数据用Excel整理后，采用美国SPSS Inc公司的SPSS软件(SPSS 16.0)对不同处理的粘虫存活率进行方差分析，差异显著后采用Tukey's HSD进行多重比较。存活率数据在统计分析前进行一次开方和反正弦转化。
表7为不同材料3-4叶期对初孵粘虫存活率的影响，表中数据为平均数±标准误，同一列中相同字母表示经Tukey's HSD法检验在P<0.05水平差异不显著。常规玉米苗的粘虫幼虫存活率在处理后和第3、6、9天的存活率均在95％以上，表明试验所用虫体健康，试验操作可行。不同材料3-4叶期对初孵幼虫存活率有显著影响，第3天的各材料幼虫存活率均较高，除Bt11的幼虫存活率显著低于其他材料外(P<0.05)，其余处理间无显著差异(P>0.05)。第6天各处理间幼虫存活率差异更加明显，特别是Bt11的幼虫存活率显著下降，仅为3l.7％，根据表5的评价标准，抗虫水平为中抗。第9天各处理间幼虫存活率差异进一步加剧。
表7.不同材料3-4叶期对初孵粘虫存活率的影响
Figure PCTCN2019074611-appb-000007
表8为不同材料8-10叶期对初孵粘虫存活率的影响，表中数据为平均数±标准误，同一列中相同字母表示经Tukey's HSD法检验在P<0.05水平差异不显著。从试验后3天的幼虫存活率来看，Bt11的幼虫存活率显著低于祥249和常规玉米苗。处理后第6天的幼虫存活率差异显著增大：其中ZZM030幼虫存活率低于45％，表现为中抗；祥249和常规玉米苗表现为高感。处理后9天的幼虫存活率以祥249和常规玉米苗表现为高感，并显著高于其它材料；ZZM030表现为高抗。
表8.不同材料8-10叶期对初孵粘虫存活率的影响
Figure PCTCN2019074611-appb-000008
Figure PCTCN2019074611-appb-000009
3)田间粘虫抗性鉴定
在吉林公主岭、济南饮马泉、云南景洪分别设立三个试验地点，以活体接虫的方式分别对ZZM030进行了田间粘虫抗性鉴定，以祥249为对照，供试粘虫为东方粘虫(Mythimna separate(Walker))。
接虫方法为：4-6叶期接虫，每小区人工接40株，每株接初孵幼虫40头；接虫两次，间隔3天；最后一次接虫14天后，逐株调查每个小区接虫株被粘虫为害的食叶级别，并根据标准评价材料心叶期抗性水平。分级标准及抗性级别判定标准按照农业部953号公告-10.1-2007的规定执行。
鉴定结果见表9和图5。表9中，数值以3次重复的平均值±标准差表示，同列小写字母不同表示相同地点不同材料之间差异显著(P<0.01)。图5中，A为转基因事件ZZM030，B为阴性对照249。叶片边缘及叶片上缺失部分为被粘虫蚕食后的虫孔。公主岭试验点ZZM030的平均食叶级别为1.2，抗性类型为高抗，阴性对照受体玉米祥249的平均食叶级别为5.8，抗性类型为中抗；济南试验点ZZM030的平均食叶级别为2.7，抗性类型为抗，阴性对照受体玉米祥249的平均食叶级别为5.4，抗性类型为中抗；景洪试验点ZZM030的平均食叶级别为1.5，抗性类型为高抗，阴性对照受体玉米祥249的平均食叶级别为9.0，抗性类型为高感。三个试验点转化事件ZZM030与阴性对照(受体玉米祥249)对粘虫的抗性差异显著，表明田间条件下转化事件ZZM030在4-6叶期对粘虫的抗性较好。
表9.不同材料田间抗性鉴定结果
Figure PCTCN2019074611-appb-000010
实施例5.玉米转化事件ZZM030Southern印迹鉴定
1.探针的制备
(1)cry1Ab/cry1AcZM探针的制备
以pZHZH35006质粒DNA为模板制备探针。以CSP759(SEQ ID NO 2)、CSP760(SEQ ID NO 3)为引物，采用罗氏公司PCR地高辛探 针合成试剂盒(货号：11636090910)合成用于检测cry1Ab/cry1AcZM的探针，探针大小为333bp(探针序列同Seq ID No：1第6084-6416位所示核苷酸序列)。扩增体系包含：DNA模板5μL(50pg)，引物各0.5μL，PCR DIG混合物5μL，DNA聚合酶0.75μL，PCR缓冲液(10倍)5μL，ddH 2O 33.25μL。PCR反应程序为：94℃预变性5min；35个循环：94℃变性30sec，55℃退火30sec，72℃延伸45sec；最后72℃延伸7min。使用1％琼脂糖凝胶检测标记扩增效果。将特异性扩增的扩增产物，即用于检测cry1Ab/cry1AcZM的探针，于-20℃保存。
(2)bar探针的制备
使用如(1)所述相同方法，利用引物对(SEQ ID NO 4:FW-Csp73，SEQ ID NO 5:RV–Csp74)制备了bar基因的特异探针，其长度为408bp(探针序列同Seq ID No：1第1035-1442位所示核苷酸序列)。
(3)cp4 epspsZM探针的制备
使用如(1)所述相同方法，利用引物对(SEQ ID NO 6:FW-Csp1337，SEQ ID NO 7:RV-Csp1338)制备了cp4 epspsZM基因的特异探针，其长度为1138bp(探针序列同Seq ID No：1第9058-10195位所示核苷酸序列)。
2.DNA提取
提取转基因玉米T 1、T 2或者T 3代材料的叶片基因组总DNA，获得的DNA沉淀干燥后溶于无离子水，测定浓度后备用。
3.酶切、电泳、转膜、显影
当使用cry1Ab/cry1AcZM探针时，玉米基因组DNA需用HindIII或KpnI进行单酶切。
当使用bar探针时，玉米基因组DNA需用HindIII或EcoRI进行单酶切。
当使用cp4 epspsZM探针时，玉米基因组DNA需用HindIII或KpnI进行单酶切。
使用200μL酶切体系，其中含有20μg玉米基因组DNA、20μl限制性内切酶、20μL10倍缓冲液，加ddH 2O补齐至200μL。酶切16h后取20μL进行电泳检测，检测酶切效果是否彻底。
酶切产物加ddH 2O补充至400μl，加入1/10体积的3M醋酸钠溶液(pH 5.2)，加入4μL的TaKaRa Dr.GenTLE Precipitation Carrier，加入2.5倍体积的无水乙醇，充分混匀，12,000rpm 4℃离心15min。将沉淀用50μL ddH 2O溶解，加5μL 6倍上样缓冲液。
将DNA通过0.8％凝胶，20V电泳16h。切掉多余泳道及点样孔，剩余凝胶用变性溶液处理2次，每次15min，在摇床上轻摇。再用中和缓冲液处理2次，每次15min，在摇床上轻摇。超纯水清洗一次。 20倍SSC处理10min中，用Whatman系统进行转膜4小时以上。
转膜结束后，将膜放置在用10倍SSC浸润的Whatman 3MM滤纸上，紫外交联仪交联3-5min。用ddH 2O简单洗膜，于空气中干燥。杂交及显影过程均按照罗氏地高辛检测试剂盒I(货号：11745832910)或者罗氏地高辛检测试剂盒II(货号：11585614910)的操作手册进行。
4.结果分析
图6A示出了转化事件ZZM030玉米基因组DNA分别由HindIII和KpnI酶切与cry1Ab/cry1AcZM特异探针分子杂交结果。两种酶切条件下分别显示一条阳性带，经HindIII酶切得到的条带为10.9kb，经KpnI酶切得到的条带为10.5kb，表明外源基因cry1Ab/cry1AcZM单拷贝插入，该转化事件为单拷贝转化事件。
图6B示出了转化事件ZZM030玉米基因组DNA分别由HindIII和EcoRI酶切与bar特异探针分子杂交结果。两种酶切条件下分别显示一条阳性带，经HindIII酶切得到的条带为4.2kb，经EcoRI酶切得到的条带为10.0kb，均符合预期，表明外源基因bar单拷贝插入，该转化事件为单拷贝转化事件。
图6C示出了转化事件ZZM030玉米基因组DNA分别由HindIII和KpnI酶切与cp4 epspsZM特异探针分子杂交结果。两种酶切条件下分别显示一条阳性带，经HindIII酶切得到的条带为10.9kb，经KpnI酶切得到的条带为10.5kb，均符合预期，表明外源基因cp4 epspsZM单拷贝插入，该转化事件为单拷贝转化事件。
实施例6.玉米转化事件ZZM030序列分析
在转基因操作中，通常使用相同的转化载体进行大量的遗传转化，从获得的诸多转化事件中筛选极少数优异的转化事件。因此，对插入的外源序列中的载体、表达元件、外源基因等的检测，仅能证明检测样品含有该转基因成份，无法区分不同的转化事件。而不同的转化事件，是以其插入位点的侧翼序列与插入的外源序列的组合为特征。为此，本实施例分离并鉴定了玉米转化事件的侧翼序列。
1.左侧翼序列分析
取待测转化事件植株叶片提取总DNA(T 2代或者T 3代生长健壮的转基因植株)，利用FPNI-PCR方法进行插入玉米基因组的外源基因侧翼序列扩增、克隆、测序，得到序列结果。
(1)Tail-PCR引物序列如表10所示。
表10.引物序列
Figure PCTCN2019074611-appb-000011
Figure PCTCN2019074611-appb-000012
其中W＝A/T，N＝A/G/C/T。
(2)制备高质量玉米基因组DNA备用，稀释至100ng/μL后备用；
(3)以步骤(2)中的基因组DNA作为第一轮PCR反应的模板，反应体系如下表6。反应程序为：95℃，2.5min；2个以下循环：94℃，10sec，62℃，30sec，72℃，2min；94℃，10sec；25℃，2min；72℃(5.1％ramp)，2min；5个以下循环：94℃，10sec；62℃，30sec；72℃，2min；94℃，10sec；62℃，30sec；72℃，2min；94℃，10sec；44℃，30sec；72℃，2min；72℃，5min；20℃，10min。
表11.第一轮PCR反应体系
Figure PCTCN2019074611-appb-000013
(4)以第一轮PCR产物(混合液母液)为模板进行第二轮PCR扩增，反应体系如下表7。反应程序为：94℃，1.5min；(94℃，10sec；62℃，30sec；72℃，2min)×30cycles；72℃，7min；20℃，10min。
表12.第二轮PCR反应体系
Figure PCTCN2019074611-appb-000014
(5)以第二轮PCR产物(混合液稀释50倍)为模板进行第三轮PCR扩增，反应体系如下表8。反应程序为：94℃，1.5min；(94℃，10sec；62℃，30sec；72℃，2min)×30cycles；72℃，7min；20℃，10min。
表13.第三轮PCR反应体系
Figure PCTCN2019074611-appb-000015
(6)取第三轮PCR的产物于1％(w/v)1×TAE琼脂糖凝胶中电泳检测，回收300bp-2kb之间的DNA片段；
(7)将回收的片段连接T载体，16℃过夜连接；
(8)转化(7)的连接产物；
(9)用M13F：5’-TGTAAAACGACGGCCAGT-3’和M13R：5’-CAGGAAACAGCTATGACC-3’引物扩增(8)中的转化产物，并挑取阳性克隆摇床培养菌液，利用TIANprep Rapid Mini Plasmid Kit快速质粒小提试剂盒(离心柱型)提取质粒DNA；
(10)用测序引物对(9)中的质粒DNA进行测序，利用
Figure PCTCN2019074611-appb-000016
Terminator v3.1Cycle Sequencing Kit对质粒DNA进行测序PCR。测序引物为M13F和M13R引物；M13-F：5’-TGTAAAACGACGGCCAGT-3’，M13-R：5’-CAGGAAACAGCTATGACC-3’；
(11)用NaAc和无水乙醇纯化、甲酰胺变性(10)中的PCR产物
(12)将(11)中纯化变性好的PCR产物利用ABI DNA测序仪3730开机测序并读出测序结果。
(13)测序结果在PlantGDB数据库中用BLASTN工具与玉米基因组序列进行同源搜索，以最好的匹配结果为插入位点的染色体号和碱基对位置号，通常序列一致性为90-100％。
(14)通过本实验检测出插入T-DNA的左侧翼序列680bp，见序列SEQ ID NO：1的核苷酸1-680。以玉米B73全基因组序列为参考(http://www.plantgdb.org/ZmGDB/cgi-bin/blastGDB.pl)，分析比较确定本申请转化事件左侧插入点位于第4号染色体40636901bp。
2.右侧翼序列分析
取待测转化事件植株叶片提取总DNA(T 2代或者T 3代生长健壮的转基因植株)，利用接头PCR方法进行插入玉米基因组的外源基因侧翼序列扩增、克隆、测序，得到序列结果。
(1)人工合成接头PCR所需引物，稀释后备用；引物序列如表14所示。
表14.引物序列
Figure PCTCN2019074611-appb-000017
(2)制备高质量玉米基因组DNA备用，稀释后备用；
(3)用ddH 2O将接头引物AD-L和AD-S分别稀释到100μmol/L，等体积混合，94℃水浴变性4min，自然冷却至室温后即为50μmol/L 的接头；
(4)酶切玉米基因组DNA，酶切体系如下：
Figure PCTCN2019074611-appb-000018
37℃酶切3h。
(5)连接接头，连接体系如下：
Figure PCTCN2019074611-appb-000019
16℃过夜连接。
(6)以步骤(5)中的基因组DNA酶切-接头连接产物作为第一轮PCR反应的模板，反应体系如下表10。反应程序为：94℃，5min；7个以下循环：94℃ 30sec，72℃ 3min；32个以下循环：94℃ 30sec；67℃ 3min；67℃ 7min；25℃ 10min。
表15.第一轮PCR反应体系
Figure PCTCN2019074611-appb-000020
(7)以第一轮PCR产物(混合液稀释40倍)为模板进行第二轮PCR扩增，反应体系如下表11。反应程序为：94℃，5min；5个以下循环：94℃ 30sec；72℃ 3min；20个以下循环：94℃ 30sec，67℃ 3min；67℃ 7min；25℃ 10min。
表16.第二轮PCR反应体系
Figure PCTCN2019074611-appb-000021
(8)取第二轮PCR的产物于1％(w/v)1×TAE琼脂糖凝胶中电泳检测，回收300bp-2kb之间的DNA片段；
(9)将回收的片段连接T载体，16℃过夜连接；
(10)转化(9)的连接产物；
(11)用M13F和M13R引物扩增(10)中的转化产物，并挑取阳性克隆摇床培养菌液，利用TIANprep Rapid Mini Plasmid Kit快速质粒小提试剂盒(离心柱型)提取质粒DNA；
(12)用测序引物对(11)中的质粒DNA进行测序，利用
Figure PCTCN2019074611-appb-000022
Terminator v3.1Cycle Sequencing Kit对质粒DNA进行测序PCR。测序引物为M13F和M13R引物；M13-F：5’-TGTAAAACGACGGCCAGT-3’，M13-R：5’-CAGGAAACAGCTATGACC-3’；
(13)用NaAc和无水乙醇纯化、甲酰胺变性(12)中的PCR产物。
(14)将(13)中纯化变性好的PCR产物利用ABI DNA测序仪3730开机测序并读出测序结果。
(15)测序结果在Plant GDB数据库中用BLASTN工具与玉米基因组序列进行同源搜索，以最好的匹配结果为插入位点的染色体号和碱基对位置号，通常序列一致性为90-100％。
(16)通过本实验检测出插入T-DNA的右侧翼序列460bp，见序列SEQ ID NO：1的核苷酸10915-11374。以玉米B73全基因组序列为参考(http://www.plantgdb.org/ZmGDB/cgi-bin/blastGDB.pl)，分析比较确定本申请转化事件右侧插入点位于第4号染色体40636883bp。
3.插入序列的大小和对玉米原内源基因组的影响
本申请外源基因单拷贝插入序列包括左右边界序列的载体大小为10282bp。
通过分段PCR扩增外源DNA测序的方法，确定转化事件插入序列实际大小为10235bp(SEQ ID NO:1的681-10915)，T-DNA插入物相对于表达载体左端缺失7bp，右端缺失40bp。
扩增体系包含：DNA模板2μL(200ng)，引物各0.5μL，DNA聚合酶0.5μL，PCR缓冲液(10倍)2μL，ddH 2O 14.5μL。PCR反应程序为：94℃预变性5min；35个循环：94℃变性30sec，55℃退火30sec，72℃延伸3min；最后72℃延伸7min。使用1％琼脂糖凝胶检测标记扩增效果。回收特异性扩增的扩增产物，即目的片段。
上述47bp核苷酸位于非编码区的边界序列。其缺失不影响抗虫基因cry1Ab/cry1AcZM和筛选标记bar和cp4 epspsZm基因的完整性。DNA序列分析比对表明，转化事件实际插入核苷酸序列与载体序列完全一致，没有发生任何碱基突变。
另外，插入位点处的玉米基因组为重复序列，47bp核苷酸缺失没有破坏任何已知的玉米内源功能基因。
实施例7.玉米转化事件ZZM030的检测方法
1.左侧翼DNA序列检测：
利用玉米转化事件的左侧翼基因组序列和外源片段中的35S polyA终止子的序列设计了一对引物(SEQ ID NO.8:FW-csp3758和SEQ ID NO.9:RV-csp2344)，建立该转化事件产品的定性PCR鉴定方法。
依据玉米转化事件ZZM030外源DNA片段整合位点左边界(LB)T-DNA 5’端设计的引物为：
Seq ID No.8(FW-csp3758):5’-TGATGGTTAATGAGGCAAGA-3’(玉米基因组区)；
Seq ID No.9(RV-csp2344):5’-TATAGGGTTTCGCTCATGTG-3’(35S PolyA区域)。
上述特异引物在50-60℃条件下利用温度梯度PCR扩增DNA片段确定了最佳退火温度。结果证实用58℃为最佳扩增温度；PCR反应程序为95℃ 5min，(95℃ 30s，50-60℃ 30s，72℃ 1min)35个循环，72℃ 7min。
为测试上述引物(FW-csp3758；RV-csp2344)特异扩增本转化事件特性，不同来源的玉米DNA被用来进行PCR扩增。
PCR反应条件和程序为95℃ 5min，35个以下循环：95℃ 30s，58℃ 30s，72℃ 1min；72℃ 7min。结果表明只有本转化事件DNA能够有阳性结果，其他转化事件或者阴性对照玉米品种均为阴性结果，见图7A。其中，泳道1-4分别为无菌水、祥249DNA、ZZM030DNA、其他相同载体转化事件DNA；仅有ZZM030基因组DNA泳道3有带清晰可见，其DNA片段大小898bp与预期一致，DNA片段克隆测序得到结果亦与预期一致。
2.右侧翼DNA序列检测：
利用外源片段中的cp4 epspsZM序列和玉米转化事件的右侧翼基因组序列设计了一对引物(SEQ ID NO.10:FW-csp3879和SEQ ID NO.11:RV-csp3889)，建立该转化事件的定性PCR鉴定方法。
依据ZZM030转化事件外源DNA片段整合位点右边界(RB)T-DNA 5’端设计的引物为：
Seq ID No.10(FW-csp3879):5’-AAGATTGAGCTGTCGGATAC-3’(cp4epspsZM区域),
Seq ID No.11(RV-csp3889):5’-TTTGATCATGTGAGGAACGT-3’(玉米基因组区)。
上述特异引物在46-61℃条件下利用温度梯度PCR扩增DNA片段确定了最佳退火温度。结果证实用58℃为最佳扩增温度；PCR反应程序为95℃ 5min；35个以下循环：95℃ 30s，46-61℃ 30s，72℃ 1min；72℃ 7min。
为测试上述引物(FW-csp3879；RV-csp3889)特异扩增本转化事件 特性，不同来源的玉米DNA被用来进行PCR扩增。
PCR反应条件和程序为95℃ 5min；35个以下循环：95℃ 30s，58℃ 30s，72℃ 1min循环；72℃ 7min。结果表明只有本转化事件DNA能够有阳性结果，其他转化事件或者阴性对照玉米品种均为阴性结果，见图7B。其中，泳道1-4分别为无菌水、祥249DNA、ZZM030 DNA、其他相同载体转化事件DNA；仅有ZZM030基因组DNA泳道3有带清晰可见，其DNA片段大小796bp与预期一致，DNA片段克隆测序得到结果亦与预期一致。
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明技术原理的前提下，还可以做出若干改进和修改，这些改进和修改也应视为本发明的保护范围。

Claims (12)

  1. 核酸分子，其为：
    i)包含SEQ ID NO:1的第381-780位核苷酸和/或第10815-11214位核苷酸所示序列，或其片段或其变体或其互补序列；
    ii)包含SEQ ID NO:1的第381-780位核苷酸和第6239-6338位核苷酸所示序列，或其片段或其变体或其互补序列；
    iii)包含SEQ ID NO:1的第6239-6338位核苷酸和第10815-11214位核苷酸所示序列，或其片段或其变体或其互补序列；
    iv)包含SEQ ID NO:1的第381-780位核苷酸，第6239-6338位核苷酸和第10815-11214位核苷酸所示序列，或其片段或其变体或其互补序列；或者
    v)包含SEQ ID NO:1所示序列，或其片段或其变体或其互补序列。
  2. 如权利要求1所述的核酸分子，其包含以下表达盒：
    表达抗草铵膦基因的第一表达盒，如SEQ ID NO:1的第748-2288位核苷酸所示序列；
    表达抗虫基因的第二表达盒，如SEQ ID NO:1的第2620-6959位核苷酸所示序列；和
    表达抗草甘膦基因的第三表达盒，如SEQ ID NO:1的第6968-10892位核苷酸所示序列。
  3. 如权利要求1所述的核酸分子，其通过将以下表达盒导入玉米的基因组中获得：
    表达抗草铵膦基因的第一表达盒，如SEQ ID NO:1的第748-2288位核苷酸所示序列；
    表达抗虫基因的第二表达盒，如SEQ ID NO:1的第2620-6959位核苷酸所示序列；和
    表达抗草甘膦基因的第三表达盒，如SEQ ID NO:1的第6968-10892位核苷酸所示序列；
    任选地，所述的核酸分子，其存在于玉米植物、种子、植物细胞、后代植物或植物部分中。
  4. 用于检测玉米转化事件的探针，其包含SEQ ID NO:1的第381-780位核苷酸或第10815-11214位核苷酸所示序列，或其片段或其变体或其互补序列。
  5. 用于检测玉米转化事件的引物对，其能够特异性扩增产生包含SEQ ID NO:1的第381-780位核苷酸或第10815-11214位核苷酸所示序列，或其片段或其变体或其互补序列；
    任选地，所述引物对为：
    i)特异性识别包含SEQ ID NO:1的第381-780位核苷酸所示序列的引物对；
    ii)特异性识别包含SEQ ID NO:1的第10815-11214位核苷酸所示序列的引物对；
    iii)特异性识别包含SEQ ID NO:1的第381-780位核苷酸所示序列的正向引物，和特异性识别包含SEQ ID NO:1的第681-10915位核苷酸所示序列的反向引物；或者
    iv)特异性识别包含SEQ ID NO:1的第681-10915位核苷酸所示序列的正向引物，和特异性识别包含SEQ ID NO:1的第10815-11214位核苷酸所示序列的反向引物；
    任选地，所述引物对为SEQ ID No:8和SEQ ID No:9所示的核苷酸序列或其互补序列；或者SEQ ID No:10和SEQ ID No:11所示的核苷酸序列或其互补序列。
  6. 用于检测玉米转化事件的试剂盒或微阵列，其包含权利要求4所述的探针和/或权利要求5所述的引物对。
  7. 检测玉米转化事件的方法，其包括利用以下来检测待测样品中是否存在所述转化事件：
    -权利要求4所述的探针；
    -权利要求5所述的引物对；
    -权利要求4所述的探针和权利要求5所述的引物对；或者
    -权利要求6所述的试剂盒或微阵列。
  8. 对玉米进行育种的方法，所述方法包括以下步骤：
    1)获得包含权利要求1所述的核酸分子的玉米；
    2)将步骤1)所获得的玉米通过花粉培养、未受精胚培养、加倍培养、细胞培养、组织培养、自交或杂交或以上的组合得到玉米植物、种子、植物细胞、后代植物或植物部分；以及任选地，
    3)对步骤2)所获得的后代植物进行除草剂草铵膦和草甘膦以及螟虫和/或粘虫的抗性鉴定，并利用权利要求7所述的方法来检测其中是否存在所述转化事件。
  9. 由权利要求8的方法获得的玉米植物、种子、植物细胞、后代 植物或植物部分制成的制品，包括食品、饲料或工业原料。
  10. 控制鳞翅目害虫群体的方法，包括使所述鳞翅目害虫群体接触由权利要求8的方法获得的玉米植物、种子、植物细胞、后代植物或植物部分；优选地，其中所述鳞翅目害虫为亚洲玉米螟(Ostrinia furnacalis)、欧洲玉米螟(Ostrinia nubilalis)或东方粘虫(Mythimna separate(Walker))。
  11. 杀死鳞翅目害虫的方法，包括使所述鳞翅目害虫接触杀虫有效量的由权利要求8的方法获得的玉米植物、种子、植物细胞、后代植物或植物部分；优选地，其中所述鳞翅目害虫为亚洲玉米螟(Ostrinia furnacalis)、欧洲玉米螟(Ostrinia nubilalis)或东方粘虫(Mythimna separate(Walker))。
  12. 减轻鳞翅目害虫对玉米的伤害的方法，包括将以下表达盒导入玉米的基因组中：
    表达抗草铵膦基因的第一表达盒，如SEQ ID NO:1的第748-2288位核苷酸所示序列；
    表达抗虫基因的第二表达盒，如SEQ ID NO:1的第2620-6959位核苷酸所示序列；和
    表达抗草甘膦基因的第三表达盒，如SEQ ID NO:1的第6968-10892位核苷酸所示序列；
    优选地，其中所述鳞翅目害虫为亚洲玉米螟(Ostrinia furnacalis)、欧洲玉米螟(Ostrinia nubilalis)或东方粘虫(Mythimna separate(Walker))。
PCT/CN2019/074611 2018-02-11 2019-02-02 抗虫耐除草剂玉米转化事件 WO2019154373A1 (zh)

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