CN116479153A - Nucleic acid sequence for detecting maize transformation event Zherui 8 and detection method thereof - Google Patents
Nucleic acid sequence for detecting maize transformation event Zherui 8 and detection method thereof Download PDFInfo
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Abstract
The invention discloses a nucleic acid sequence for detecting a transgenic corn event 'ZheRui 8' and a detection method thereof, wherein the nucleic acid sequence comprises SEQ ID NO.1 or a complementary sequence thereof and/or SEQ ID NO.2 or a complementary sequence thereof, the nucleic acid sequence is derived from the transgenic corn event 'ZheRui 8', and the transgenic corn event 'ZheRui 8' is preserved in the Chinese typical culture collection in the form of seeds, and the preservation number is as follows: CCTCC NO: P202202. "ZheRui 8" is resistant to the hazards of common lepidopteran pests of corn and has good tolerance to glyphosate. The nucleotide sequence provided by the invention is designed into a specific detection primer pair, the existence of a transgenic event of ZheRui 8 is accurately and stably identified by using a nucleic acid amplification method, and the research, production processing and application of ZheRui 8 can be traced and fully-flow supervision can be realized.
Description
Field of the art
The invention relates to a nucleic acid sequence of a transgenic corn event 'ZheRui 8' and a detection method thereof, in particular to a nucleic acid sequence of an insect-resistant herbicide-resistant transgenic corn event 'ZheRui 8' and a detection method thereof for detecting whether a biological sample contains a specific transgenic corn event 'ZheRui 8'.
(II) background art
Corn is an important grain crop, and is the grain crop with the widest global planting range and the largest yield. However, in the corn planting process, the pest damage often seriously reduces the corn yield and quality, increases the corn planting cost and seriously affects the corn production. The prevention and control of weeds in corn fields is also an important measure in corn production, and the control of weeds by using herbicides can reduce the influence of weeds on corn growth, stabilize and improve corn yield. The insect-resistant and herbicide-resistant characters are introduced into the corn by a genetic engineering method, so that the production efficiency of the corn can be improved, the production cost can be reduced, and the use amount of the pesticide can be reduced.
The transformation event is a molecular structure composed of the upstream and downstream flanking regions of the foreign gene at the genomic insertion site and the foreign gene. In genetic transformation, because the exogenous genes are randomly integrated into plant chromosomes, the obtained transgenic population contains a large number of independent transformation events, the chromosome positions of the exogenous genes inserted in different independent transformation events are different, the flanking sequences of the exogenous genes are also different, and each event is unique. Expression of a foreign gene in a plant is affected by the chromosomal location into which the foreign gene is inserted. This may result from the effects of regulatory elements near the chromatin structure or integration site. The same gene also has significant differences in expression levels, expression space and temporal patterns in different transformation events. Insertion of foreign genes may also affect expression of endogenous genes, thereby affecting agronomic traits of the plant. Therefore, a large number of events need to be screened in the development process, and transformation events of which the expression level and the expression pattern of the target gene can meet the requirements of production application need are screened out. The desired transformation event obtained by screening can be introduced into commercially planted varieties by conventional breeding methods of crossing. The progeny produced by the crossing retain the transgenic character of the original transformation event.
Specific detection of transformation events is valuable both in the production application of transformation events and in the regulation of laws and regulations. Conventional methods of polynucleotide and protein detection can effectively detect whether it is a transgene, but cannot distinguish between different transformation events, particularly those generated using the same gene or the same transformation vector. Only detection of the inserted gene and flanking sequences will allow accurate determination of the presence or absence of the transgene event of interest.
Summary of the invention
The invention provides a nucleic acid sequence for detecting a transgenic corn event 'ZheRui 8' and a specific detection method thereof, wherein the transgenic corn event 'ZheRui 8' has good resistance to corn pests and good tolerance to glyphosate herbicide, and the nucleic acid sequence can rapidly and accurately identify whether a biological sample contains DNA molecules of the specific transgenic corn event 'ZheRui 8'.
The invention adopts the technical scheme that:
the invention provides a nucleic acid sequence for detecting a transgenic corn event 'ZheRI8', which comprises SEQ ID NO.1 or a complementary sequence thereof and/or SEQ ID NO.2 or a complementary sequence thereof, wherein the nucleic acid sequence is derived from the transgenic corn event 'ZheRI8', and the transgenic corn event 'ZheRI8' is preserved in the form of seeds in China center for type culture Collection, with a preservation number: CCTCC NO: P202202, the preservation date 2022, 1 month 11, address university of Wuhan, china, post code 430072.
SEQ ID NO.1
GAAACCGGTCCATGCTTAGACAAC。
SEQ ID NO.2
AAGTTGTCTAAGCGTCAATTTGTT。
Further, it is preferred that the nucleic acid sequence is SEQ ID NO.3 or a complement thereof, and/or SEQ ID NO.4 or a complement thereof.
SEQ ID NO.3
SEQ ID NO.4
Still further, it is preferred that the nucleic acid sequence is SEQ ID NO.5 or a complement thereof.
The SEQ ID NO.1 or the complementary sequence thereof is a 24 nucleotide sequence with the length at the insertion joint part at the 5 '-end of the insertion sequence in the transgenic corn event' ZheRui 8 ', the SEQ ID NO.1 or the complementary sequence thereof spans the flanking genome sequence of the corn insertion site and the DNA sequence at the 5' -end of the insertion sequence, and the existence of the transgenic corn event 'ZheRui 8' can be identified by the SEQ ID NO.1 or the complementary sequence thereof.
The SEQ ID NO.2 or the complementary sequence thereof is a 24 nucleotide sequence with the length at the insertion joint part at the 3 '-end of the insertion sequence in the transgenic corn event' ZheRui 8 ', the SEQ ID NO.2 or the complementary sequence thereof spans the flanking genome sequence of the corn insertion site and the DNA sequence at the 3' -end of the insertion sequence, and the existence of the transgenic corn event 'ZheRui 8' can be identified by the SEQ ID NO.2 or the complementary sequence thereof.
The SEQ ID NO.3 or the complementary sequence thereof is a nucleotide sequence with the length of 1300 nucleotide sequences positioned at the 5' -end of the insertion sequence in an insertion junction region in a transgenic corn event ' ZheRui 8 ', 1-877bp is a corn genome DNA sequence flanking the insertion site, 878-1300bp is an insertion nucleotide sequence, and the existence of the transgenic corn event ' ZheRui 8 ' can be identified by comprising the SEQ ID NO.3 or the complementary sequence thereof.
The SEQ ID NO.4 or the complementary sequence thereof is a nucleotide sequence with 803bp of length positioned at the 3' -end of an insertion junction region in a transgenic corn event ' ZheRui 8 ', the 1 st to 300bp of SEQ ID NO.4 is an insertion gene nucleotide sequence, and 301 to 803bp is a corn flanking genome nucleotide sequence, and the existence of the transgenic corn event ' ZheRui 8 ' can be identified by comprising the SEQ ID NO.4 or the complementary sequence thereof.
The SEQ ID NO.5 or the complementary sequence thereof is a sequence with the length of 12701 nucleotide specific to transgenic corn event 'ZheRui 8', and the specific contained genome and genetic elements are shown in Table 1. The presence of the transgenic maize event "ZheRui 8" can be identified by the inclusion of said SEQ ID NO.5 or its complement.
Table 1. Genome and genetic elements comprised in SEQ ID NO.5
| Elements/genome | Length (bp) | At position on SEQ ID NO.5 |
| 5' genome | 877 | 1-877 |
| RB | 504 | 878-1381 |
| Action promoter | 1421 | 1382-2803 |
| Cry2Ab | 1902 | 2804-4705 |
| 35S terminator | 237 | 4706-4942 |
| pZmUbi-1 | 2017 | 4943-6959 |
| Cry1Ab | 1950 | 6970-8919 |
| PEPC terminator | 197 | 8920-9116 |
| 35S modified promoter | 1309 | 9117-10425 |
| G10evo-EPSPS | 1518 | 10426-11943 |
| 35S terminator | 190 | 11944-12133 |
| LB | 67 | 12132-12198 |
| 3' maize genome | 503 | 12199-12701 |
The invention provides a method for detecting the existence of DNA of transgenic corn event ZheRui 8 in a sample, which comprises the following steps: (1) Contacting a sample to be detected with a primer pair in a nucleic acid amplification reaction solution; (2) performing a nucleic acid amplification reaction; (3) detecting the presence of the amplified product; the amplification product comprises SEQ ID NO.1 or a complementary sequence thereof, and SEQ ID NO.2 or a complementary sequence thereof.
Preferably, the amplification product comprises at least 12 consecutive nucleotides of SEQ ID NO.3 or its complement, or at least 12 consecutive nucleotides of SEQ ID NO.4 or its complement.
Further, the amplified product comprises SEQ ID NO.6 or its complement, and/or SEQ ID NO.7 or its complement, identified as the presence of the transgenic maize event "ZheRui 8".
Further, the primer pair comprises a first primer and a second primer, wherein the first primer consists of at least 12 continuous nucleotides of the 1 st to 877 th nucleotide sequence of SEQ ID NO.3 or the complementary sequence thereof, or consists of at least 12 continuous nucleotides of the 1 st to 300 th nucleotide sequence of SEQ ID NO.4 or the complementary sequence thereof, and particularly the first primer is shown as SEQ ID NO.8 or shown as SEQ ID NO.10; the second primer consists of at least 12 continuous nucleotides of 878-1300 nucleotide sequence of SEQ ID NO.3 or complementary sequence thereof, or consists of at least 12 continuous nucleotides of 301-803 nucleotide sequence of SEQ ID NO.4 or complementary sequence thereof, and specifically, the second primer is shown as SEQ ID NO.9 or shown as SEQ ID NO.11.
The present invention also provides a method of protecting corn from insect damage, the method comprising providing a transgenic corn plant, the target insect feeding the transgenic corn being inhibited from further feeding the transgenic corn plant; the genome of the transgenic corn plant comprises a nucleic acid sequence of a specific region from a transgenic corn event 'Zherui 8', and the nucleic acid sequence of the specific region sequentially comprises a nucleic acid sequence of SEQ ID NO.1, a nucleic acid sequence of SEQ ID NO.5 2804-4705, a nucleic acid sequence of SEQ ID NO.5 6970-8919 and a nucleic acid sequence of SEQ ID NO.2; the target insects include corn borers that develop resistance to Cry1Ab and Cry1 Ah. Wherein SEQ ID NO.1 may also be SEQ ID NO.3 and SEQ ID NO.2 may also be SEQ ID NO.4.
The present invention provides a method of increasing glyphosate tolerance in corn comprising applying an effective dose of a glyphosate herbicide to a field in which a transgenic corn plant is grown, said transgenic corn plant being tolerant to glyphosate; the genome of the transgenic corn plant comprises a nucleic acid sequence from a specific region of transgenic corn event Zherui 8, wherein the nucleic acid sequence of the specific region sequentially comprises a nucleic acid sequence from 10426 th to 11943 th positions in SEQ ID NO.1 and SEQ ID NO.5 and SEQ ID NO.2; or the nucleic acid sequence of the specific region comprises SEQ ID NO.5. Wherein SEQ ID NO.1 may also be SEQ ID NO.3 and SEQ ID NO.2 may also be SEQ ID NO.4.
The present invention provides a method of controlling weeds in a corn field, the method comprising applying a glyphosate herbicide to a field in which transgenic corn is grown, the transgenic corn being tolerant to glyphosate and the weeds in the corn field being killed by the glyphosate; the transgenic corn comprises in its genome a nucleic acid sequence from a specific region of transgenic corn event "Zheju 8", said specific region comprising in sequence the nucleic acid sequences of SEQ ID NO.1, SEQ ID NO.5, positions 10426-11943 and SEQ ID NO.2, or said specific region comprising SEQ ID NO.5.
The present invention also provides a method of growing a maize plant that is resistant to insects, the method comprising: planting a maize seed comprising a nucleic acid sequence of a specific region in the genome of the maize seed; the corn plants are inoculated with target insects, and plants with better insect-resistant effect compared with other plants without the specific region nucleic acid sequence are harvested; the genome of the corn seed comprises a nucleic acid sequence of a specific region from a transgenic corn event 'Zherui 8', wherein the nucleic acid sequence of the specific region sequentially comprises SEQ ID NO.1, SEQ ID NO.5 2804-4705, SEQ ID NO.5 nucleotide sequence 6970-8919 and SEQ ID NO.2, or the nucleic acid sequence of the specific region is SEQ ID NO.5; target insects feeding on the transgenic corn plants are inhibited from further feeding on the corn plants.
The present invention also provides a method of growing a corn plant tolerant to a glyphosate herbicide, the method comprising: planting transgenic corn, growing the corn into a corn plant, spraying the corn plant with a glyphosate herbicide, and harvesting a plant that is not damaged by glyphosate as compared to other plants that do not contain the specific region nucleic acid sequence; the genome of the corn seed comprises a nucleic acid sequence from a specific region of transgenic corn event "Zherui 8", which in turn comprises the nucleic acid sequences of SEQ ID NO.1, SEQ ID NO.5, positions 10426-11943 and SEQ ID NO.2, or the nucleic acid sequence of the specific region comprises SEQ ID NO.5.
The invention also provides a method of growing an insect-resistant and glyphosate-tolerant corn plant, the method comprising: planting transgenic corn, spraying glyphosate herbicide, and harvesting plants with better glyphosate resistance and insect resistance compared with other plants without specific region nucleic acid sequence; the genome of the transgenic corn seed comprises a nucleic acid sequence from a specific region of transgenic corn event "Zherui 8"; the nucleic acid sequence of the specific region sequentially comprises SEQ ID NO.1, 2804-4705, 6970-8919, 10426-11943 nucleic acid sequences and SEQ ID NO.2; or the nucleic acid sequence of the specific region comprises SEQ ID NO.5 in sequence. The method comprises the steps of carrying out a first treatment on the surface of the The method specifically comprises the following steps: crossing the pest-resistant glyphosate-tolerant transgenic corn event 'ZheRui 8' serving as a first parent and a second parent lacking pest-resistant and glyphosate-tolerant traits to generate a filial generation; spraying glyphosate on the filial generation; insect-resistant and glyphosate-tolerant plants were selected.
The present invention provides a method of producing a maize plant that is resistant to insects, the method comprising: crossing the transgenic maize plant with another maize plant, thereby producing a progeny plant; selecting plants having a better pest-resistant effect than other plants not having the specific region nucleic acid sequence; the genome of the transgenic corn plant comprises a nucleic acid sequence from a specific region of transgenic corn event 'Zherui 8', which comprises SEQ ID NO.1, SEQ ID NO.5, 2804-4705, 6970-8919 and SEQ ID NO.2 in sequence, or the nucleic acid sequence of the specific region comprises SEQ ID NO.5.
The present invention provides a method of producing a maize plant that is tolerant to glyphosate, the method comprising crossing a transgenic maize plant with another maize plant, thereby producing a progeny plant; harvesting plants that are not damaged by glyphosate as compared to other plants that do not contain the specific region nucleic acid sequence; the genome of the transgenic corn plant comprises a nucleic acid sequence from a specific region of transgenic corn event Zheju 8, wherein the nucleic acid sequence of the specific region comprises SEQ ID NO.1, the nucleic acid sequences of SEQ ID NO.5 10426-11943 and SEQ ID NO.2 in sequence, or the nucleic acid sequence of the specific region comprises SEQ ID NO.5.
The present invention provides a method of producing a corn plant that is resistant to insects and tolerant to glyphosate herbicide, the method comprising: crossing the transgenic corn with another corn plant, thereby producing a progeny plant; harvesting plants that are resistant to insects and are not damaged by glyphosate as compared to other plants not comprising the specific region nucleic acid sequence; the genome of the transgenic corn plant comprises a nucleic acid sequence from a specific region of transgenic corn event Zheju 8, wherein the nucleic acid sequence of the specific region comprises SEQ ID NO.1, SEQ ID NO.5 nucleotide sequences 2804-4705, SEQ ID NO.5 nucleotide sequences 6970-8919, SEQ ID NO.5 nucleotide sequences 10426-11943 and SEQ ID NO.2 in sequence, or the nucleic acid sequence of the specific region comprises SEQ ID NO.5.
The invention also provides a composition for generating transgenic corn event ZheRui 8, which is corn flour, corn oil, corn cob and corn starch; the transgenic corn event "ZheRui 8" is deposited in the form of seeds with China center for type culture Collection, accession number: CCTCC NO: P202202.
The invention also provides an agricultural or commodity product resulting from the transgenic corn event "Zherui 8", said agricultural or commodity product comprising corn starch, corn oil, corn silk, corn meal.
The term maize as used herein refers to maize (Zea mays) and includes all plant varieties that mate with maize, including wild maize varieties.
The inclusion refers to "including but not limited to". The plants include whole plants, plant cells, plant organs, plant protoplasts, plant cell tissue cultures from which plants can regenerate, plant calli, clumps, and whole plant cells in plants or plant parts such as embryos, pollen, ovules, seeds, leaves, flowers, branches, fruits, stalks, roots, root tips, anthers, and the like. The transgenic plant is derived from a transgenic plant transformed with a DNA molecule of the invention and thus at least partially consisting of transgenic cells or progeny thereof.
The maize transgenic event "ZheRui 8" as described herein is the maize plant "ZheRui 8", including plants and seeds of the maize transgenic event and plant cells or regenerable parts thereof, including but not limited to cells, pollen, ovules, flowers, buds, roots, stems, silks, floc, ears, leaves and products from the maize plant "ZheRui 8", such as corn flour, corn meal, corn steep liquor, corn cobs, corn starch and biomass left in the field of the maize crop.
The maize transgenic event "ZheRui 8" of the invention comprises a DNA construct that, when expressed in plant cells, confers resistance to insects and tolerance to glyphosate herbicide. The T-DNA construct comprises three expression cassettes in tandem, the first expression cassette comprising a suitable promoter for expression in a plant operably linked to nucleotide sequences of two insecticidal proteins, cry1Ab and Cry2Ab, the Cry1Ab protein being predominantly resistant to lepidopteran pests and the Cry2Ab protein being predominantly resistant to corn borer, peach borer, armyworm and cotton bollworm, among other corn common lepidopteran pests. The second expression cassette comprises a suitable promoter for expression in plants operably linked to a gene encoding 5-enol-pyruvylshikimate-3-phosphate synthase (EPSPS) that is tolerant to glyphosate herbicide and a suitable terminator. The promoter comprises an improved CaMV 35S promoter. The terminator includes a CaMV 35S terminator.
The DNA constructs are introduced into plants using transformation methods, including Agrobacterium-mediated transformation, gene gun transformation, and pollen tube channel transformation. The corresponding maize transgenic event "thunberg 8" was deposited as seeds (maize Zea mays l.) in chinese typical culture collection under accession number: CCTCC NO: P202202, 1 month 11 days of the preservation date 2022, university of Wuhan preservation center, post code 430072.
The 5' transgene insertion site in the transgenic maize event "Zherui 8" of SEQ ID No.1 and 12 nucleotides each side of the flanking maize genome; the 3' transgene insertion site in the transgenic maize event "Zherui 8" of SEQ ID No.2 and 12 nucleotides each side of the flanking maize genome; 1197 nucleotide sequences near the junction of the 5' transgene insertion site in SEQ ID NO.3 transgenic maize event "ZheRI 8"; 803 nucleotide sequences of the 3' -end transgene insertion site junction region in SEQ ID NO.4 transgenic corn event "ZheRI 8"; SEQ ID NO.5 transgenic maize event "ZheRui 8" inserted into the T-DNA sequence and the maize genomic sequences at the 5 'and 3' ends; SEQ ID NO.6 is located within SEQ ID NO.3, spanning the "ZheRI 8" insertion site junction; SEQ ID NO.7 is located within SEQ ID NO.4, spanning the "ZheRI 8" insertion site junction; SEQ ID NO.8 detects the first primer of SEQ ID NO. 3; SEQ ID NO.9 detects the second primer of SEQ ID NO. 3; SEQ ID NO.10 detects the first primer of SEQ ID NO. 4; SEQ ID NO.11 detects the second primer of SEQ ID NO.4.
The corn transgenic event 'ZheRui 8' expresses two insecticidal proteins Cry1Ab and Cry2Ab, can improve the insecticidal efficiency, expand the insecticidal spectrum, particularly can effectively control corn pests with resistance to the Cry1Ab insecticidal protein, can highly resist corn lepidoptera pests and can resist glyphosate herbicide. 'ZheRui 8' has high tolerance to glyphosate herbicide, and can be used for transgenic screening in the process of crossbreeding and weed control in the process of corn planting.
Compared with the prior art, the invention has the beneficial effects that:
the invention provides a nucleotide sequence for detecting a maize transgenic event 'Zherui 8' and a specific detection method thereof. The transgenic event 'ZheRui 8' provided by the invention is preserved in the China center for type culture Collection with the preservation number: CCTCC NO: P202202. The ZheRui 8 can be highly resistant to corn lepidoptera pests and resistant to glyphosate herbicide, and the application of the ZheRui 8 can reduce the use area of shelter in the pest-resistant transgenic corn planting process, improve the corn production efficiency and reduce the corn production cost. 'ZheRui 8' has high tolerance to glyphosate herbicide, and can be used for transgenic screening in the process of crossbreeding and weed control in the process of corn planting. The SEQ ID NO.1, the SEQ ID NO.2, the SEQ ID NO.3 and the SEQ ID NO.4 provided by the detection method are unique sequences of ZheR 8, a specific detection primer pair can be designed aiming at the nucleotide sequences provided by the invention, the existence of a transgenic event of ZheR 8 can be accurately and stably identified by using a nucleic acid amplification method, and the research, the production processing and the application of ZheR 8 can be traced and the whole flow supervision can be realized.
(IV) description of the drawings:
FIG. 1 is a schematic diagram of exogenous insert genes and maize genome structure.
FIG. 2 is a map of transformation vectors.
Fig. 3 is a picture of Zhejiang 8 and Zhengdan 958 heart leaf anti corn borer.
Fig. 4 is a photograph of Zhey 8 and control heart leaf resistant to Oriental myxoplasma.
Fig. 5 is a photograph of Zhey 8 and a control highly resistant corn borer.
Fig. 6 is a photograph of the tolerance of Zheril 8 and controls to glyphosate.
FIG. 7 shows an electrophoresis pattern of maize genome-specific assays (M: marker;1: zhejiang 8;2: ruifeng 125;3: MON810;4: bt11;5: MON89034;6: conventional maize; 7: conventional soybean; 8: conventional rice).
FIG. 8 shows an electrophoresis pattern of ZheRui 8 5' end specificity detection (1: zheRui 8;2: ruifeng 125;3: MON810;4: bt11;5: MON89034;6: conventional corn; 7: conventional soybean; 8: conventional rice).
FIG. 9 shows an electrophoresis pattern of ZheRui 8 3' end specificity detection (1: zheRui 8;2: ruifeng 125;3: MON810;4: bt11;5: MON89034;6: conventional corn; 7: conventional soybean; 8: conventional rice).
(fifth) detailed description of the invention
Example 1: obtaining plasmid vector containing exogenous Gene
The vector map for corn transformation is shown in figure 2, the transformation plasmid vector takes pCambia1300 (GenBank: AF 234296.1) as a plant transformation vector frame, and T-DNA containing a complete glyphosate-resistant expression frame and an insect-resistant expression frame is added into a multi-cloning site region of the transformation plasmid vector, and the transformation plasmid vector specifically comprises the following parts: the 35S promoter of the CaMV after optimization is connected with a glyphosate-resistant gene G10evo-EPSPS which codes for an AHAS gene chloroplast signal peptide, and the terminator is the 35S gene terminator of the CaMV; insect-resistant expression cassette: the promoters of Cry1Ab and Cry2Ab drive Cry1Ab are promoters of a corn-derived polyglutin-1 gene (pZmUbi-1), a terminator is a PEP carboxylase gene (pepc) terminator derived from corn, a promoter of a target gene Cry2Ab is an action promoter, a terminator is a 35S gene terminator of CaMV, and the T-DNA sequence is 878-12198 nucleotides in SEQ ID NO: 5.
The obtained transformant plasmid was introduced into Agrobacterium LBA4404 by the electric shock method (2500V) to obtain Agrobacterium containing the transformant vector.
Example 2: transformant obtaining
Genetic transformation of maize with Agrobacterium is carried out, in particular according to the method and the medium formulation reported by Frame et al (Plant Physiol,2002, 129:13-22), using glyphosate as screening agent, the following steps are followed: collecting the corncob 8-10 days after pollination, and collecting immature embryo with the size of 1.0-1.5 mm. The agrobacterium containing the transformation vector is co-cultured with the immature embryo at 22 ℃ for 2-3 days. The immature embryos after cultivation are transferred to callus induction medium containing 200mg/L of timentin antibiotic (ghatti, usa) and dark-cultured at 28 ℃ for 10-14 days to kill agrobacterium. All calli after induction culture were transferred to screening medium containing final concentration of 2mM glyphosate and dark cultured at 28℃for 2-3 weeks. After induction, all calli were transferred to fresh (2 mM) glyphosate-containing screening medium and dark cultured at 28℃for 2-3 weeks. Transferring the surviving embryogenic tissue to a regeneration culture medium, culturing in dark at 28 ℃ for 10-14 days, transferring to a fresh regeneration culture medium, and culturing in light at 26 ℃ for 10-14 days. And (3) picking the plants with complete growth on a rooting medium, carrying out illumination culture at 26 ℃ until the roots are complete in growth, and transplanting the rooted regenerated seedlings into a greenhouse for seed reproduction for screening analysis.
A total of 350 independent transgenic individuals were generated. Through the influence on Cry1Ab, cry2Ab, G10evo protein content, insert copy number, genetic stability, insect resistance effect, glyphosate tolerance and corn agronomic characters, a transformation event 'ZheRI8' with good insect resistance and glyphosate resistance performance, single copy insertion of exogenous genes, excellent agronomic characters and stable inheritance of insect resistance and glyphosate resistance characters is selected.
Example 3: corn transgenic event "Zherui 8" detection
1. Analysis of flanking DNA sequences
The sequence of the region flanking the foreign transgene DNA insertion site of the elite transformation event "ZheRIP 8" screened in example 2 was determined using the TAIL-PCR (Thermal asymmetric interlaced PCR) method reported by Liu et al (Liu, plant Journal 1995,8 (3): 457-463). The method carries out continuous PCR amplification by 3 nested specific primers and degenerate primer combinations respectively, and selectively amplifies target fragments by using different annealing temperatures. Designing three nest-type specific PCR primers LB-SP1, LB-SP2 and LB-SP3 according to the left and right boundary areas of the T-DNA respectively; and carrying out PCR amplification on the RB-SP1, the RB-SP2 and the RB-SP3 sequentially and with a degenerate primer AD4L group, wherein the primer sequences are shown in table 2, and the PCR reaction conditions are shown in table 3.
TABLE 2 TAIL-PCR primer sequences
| Primer(s) | Sequence(s) |
| LB-SP1 | TTTCTCCATAATAATGTGTGAGTAGTTCCC |
| LB-SP2 | CTCATGTGTTGAGCATATAAGAAACCCTTAG |
| LB-SP3 | CTAAAACCAAAATCCAGTACTAAAATCC |
| RB-SP1 | CTTGGCACTGGCCGTCGTTT |
| RB-SP2 | GACTGGGAAAACCCTGGCGTT |
| RB-SP3 | AGCTGGCGTAATAGCGAAGAGG |
| AD4L | AGGTTATGCTANTCAGSTWTSGWGWT |
TABLE 3 TAIL-PCR reaction conditions
Table 4, PCR reaction system:
first round reaction: LB-SP1+ RB-SP1 and AD4L are used as primers, and a ZheRIE 8 genome is used as a template;
second round reaction: LB-SP2+ RB-SP2 and AD4L are used as primers, and the first round of products are used as templates;
third round of reaction: LB-SP3+ RB-SP3 and AD4L were used as primers and the second round of products as templates.
The PCR product recovery kit of Axygen company is used for recovering the PCR amplification product of the 3 rd round, the PCR amplification product is connected to a PMD20-T cloning vector (TaKaRa, code: D107A), escherichia coli is transformed, and the obtained positive clone is sequenced by Shanghai Boshang sequencing company. The sequence information obtained was analyzed by comparison with a database on the maize network (http:// www.maizegdb.org) to retrieve similar maize genomic sequences.
2. T-DNA right flanking region
The fragment identified as comprising the 5' flanking region obtained by the TAIL-PCR method was sequenced, the sequence of 1-877bp corresponding to maize genomic DNA and the sequence of 878-1300bp corresponding to exogenous DNA, as a result of the sequencing SEQ ID NO. 3.
3. T-DNA left flanking region
The fragment identified as comprising the 3' flanking region was sequenced, the sequencing result being SEQ ID NO.4, 1-300bp as the insert nucleotide sequence and 301-803bp as the maize flanking genomic nucleotide sequence.
4. Integration of "ZheRui 8" into genomic sequence information
The sequence of the upstream and downstream side sequences of the insertion site, the exogenous insect-resistant gene expression frame and the herbicide-resistant gene expression frame which are subjected to sequencing comparison and verification are spliced to form the transformation event, and the nucleotide sequence is SEQ ID NO.5. Corresponding to the maize transgenic event "ZheRui 8" was deposited in the China center for type culture Collection with accession number: CCTCC NO: P202202.
Example 3: "Zherui 8" insect resistance determination
The corn transformation event Zhey 8 and the conventional corn plants (Zhengdan 958, xueyu 335) are respectively subjected to bioassay on Asian corn borer, asian corn borer Cry1Ab resistant strain, asian corn borer Cry1Ah resistant strain, oriental mythimna separata and cotton bollworm by adopting an indoor bioassay method, and the test insects are provided by plant protection of China national academy of agricultural science.
1. Corn borer
Respectively taking undeployed tender heart leaves, cutting into 2-3cm size by using a pair of sterilizing scissors, placing into a culture dish, and inoculating 10 head of first hatched larvae (corn borer, asian corn borer Cry1Ab resistant strain and Asian corn borer Cry1Ah resistant strain), wherein each culture dish is repeated for 6 times. Placing the mixture in an artificial climate incubator with the temperature of 28+/-1 ℃ and the photoperiod of 16h to 8h (L: D) and the relative humidity of 70% -80% for culture. The larval mortality was calculated from the larval mortality of each treatment for 24h, 48h and 72h, respectively, and the resistance of transgenic maize "Zherui 8" to corn borers was determined.
When the indoor insect resistance is identified, the death rate of the larvae in the control group is more than 20 percent, the test result is abandoned, and the death rate of the larvae in the control group is less than 20 percent, so that the corrected death rate is calculated. Corrected mortality (%) = (treatment group larval mortality-control group larval mortality)/(1-control group average larval mortality) ×100. In this experiment, control larval mortality was the average of the mortality of non-transgenic corn Zhengdan 958 larvae.
Statistical results show that all corn borers fed "Zherun 8" die after 72h compared to the control, while corn borers on control Zhengdan 958 are mostly as long as 2 instar, indicating that "Zherun 8" has good insect resistance (fig. 3), and "Zherun 8" also has good resistance to corn borer anti-Cry 1Ab lines and Cry1Ah lines, and mortality reaches 100% after 72h of insect inoculation (Table 5).
Table 5 results of indoor bioassays of transgenic corn "ZheRui 8" on the target pest corn borer
Note that: data in the table are mean ± standard deviation, "x" indicates significant differences in the test compared to the control.
2. Bollworm (Bowls)
As with the corn borer detection method, the cotton bollworm biological detection result shows that the larva mortality of transgenic insect-resistant corn Zherui 8 is 69.8% after 24 hours of insect inoculation. After 72h, the larval mortality rate of "ZheRI 8" reached 100%, which is significantly higher than that of the non-transgenic control treatment, "ZheRI 8" was highly resistant to bollworms, as shown in Table 6.
TABLE 6 identification of indoor resistance to bollworms (percent mortality)
Data are mean ± standard error, and the letter after the data represents the significance of the difference between the two treatments at the 5% level. Corrected mortality (%) = (treatment group larval mortality-control group larval mortality)/(1-control group average larval mortality) ×100, control group larval mortality is the average of non-transgenic maize zhengdan 958 and zebra 335 larval mortality. The following is the same.
3. Oriental armyworm
The corn borer detection method is similar to that of corn borer, and common corn is used as a control, and the oriental myxoma biological detection result shows that the larva mortality of transgenic insect-resistant corn Zherui 8 is 53.6% after 24 hours of insect inoculation. After 72h, the larval mortality of "ZheRui 8" reached 100%. The mortality of the larvae of armyworm fed "ZheRui 8" was significantly higher than that of the normal corn (Zhengdan 958 and Yu 335) treatment, and "ZheRui 8" was highly resistant to Oriental armyworm (Table 7, FIG. 4).
TABLE 7 identification of indoor resistance to biological assay in the myxomycete section (% mortality)
Example 4: identification of field resistance of ZheRui 8 to Lepidoptera pests
And carrying out field resistance identification on Asiatic corn borer, armyworm and cotton bollworm which are 3 lepidoptera pests in total. The insect-receiving period and the method are as follows:
asian corn borer: artificial inoculation of the initially hatched larvae in the corn leaf stage (6-8 leaf stage) and the silking stage, and the cell area of each cell (cell area 25 m) 2 (5 m.times.5 m)) artificial grafting 40 plants of ZheRui 8, 40 heads per plant. Two times of insect inoculation are carried out at each period, and the interval is 3d. And (5) investigating the pest situation of the pest-receiving strain after the last pest-receiving 14 d. The investigation method was performed as specified in NY/T1248.5. The insect resistance evaluation in the corn cob stage is carried out according to the specification of NY/T1248.5, and the average value of the insect resistance injury level of corn borers in the corn cob stage on female ears in each cell is calculated according to the damage condition of female ears, the number of boreholes, the length of tunnels, the number of surviving larva ages and the like. The classification criteria and the resistance evaluation criteria were carried out as prescribed in agricultural division 953 bulletin-10.1-2007. Common corn (Zhengdan 958 and Xueyu 335) is used as a control.
And (3) sticking insects: 4-6 leaf period insect-catching, per cell (cell area 25m 2 (5 m.times.5 m)) were artificially inoculated with 40 strains of "ZheRui 8", each strain was inoculated with 40 heads of the initially hatched larvae. Two times of insect inoculation are carried out at intervals of 3d. And (5) investigating the pest situation of the pest-receiving strain after the last pest-receiving 14 d. The classification criteria and the resistance level judgment criteria were carried out in accordance with the specifications of the Ministry of agriculture 953 bulletin-10.1-2007. Common corn (Zhengdan 958 and Xueyu 335) is used as a control.
Cotton bollworm: during the laying period, each cell (cell area 25m 2 (5 m×5m)) and 40 strains of ZheRui 8 are artificially inoculated, each strain is inoculated with 20-30 initially hatched larvae, inoculated on maize filaments, inoculated again by the same method after 3d, investigated for 14d, and classified and resistance judged according to the pest rate of female ears and the survival number of larvae and the pest length of female ears. The classification criteria and the resistance level judgment criteria were carried out in accordance with the specifications of the Ministry of agriculture 953 bulletin-10.1-2007. Common corn (zhengdan 958) was used as a control.
The field insect-resistant test results show that ZheRui 8 has high resistance to Asian corn borers (figure 5) in heart leaves, filaments, stems and female ears, and ZheRui 8 has high resistance to armyworms and cotton bollworms (Table 8).
Table 8. Results of "Zherui 8" field resistance identification
Data are mean ± standard error, and the letters after the data represent the significance of differences between treatments at the 5% level.
Example 5: 'Zherui 8' glyphosate tolerance test
Adopting random block design, repeating for 3 times, 24 cells, each cell having an area of 4mX6m, double sowing, plant spacing of 25cm, row spacing of 50cm, 1m interval between cells, and spraying glyphosate at 5-7 leaf stage, and processing as follows: 1) Spraying is not performed; 2) Spraying glyphosate in a medium dosage, and effectively dosing 80 g/mu; 3) 2 times of the medium dose of glyphosate and 160 g/mu of effective dose; 4) The medium dose is 4 times of glyphosate, and the effective dose is 320 g/mu. The seedling rate, plant height (selecting the highest 5 plants), and phytotoxicity symptoms (selecting the least 5 plants) were investigated 1 week, 2 weeks, and 4 weeks after the administration, and the classification of phytotoxicity symptoms was performed according to GB/T17980.42-2000. Herbicide damage rate(X-damage rate in% N-peer damage number; S-grade number; T-total number; M-highest grade). The variance analysis method is used for comparing the differences of the transgenic herbicide-resistant corn and the non-transgenic corn which are treated differently in the aspects of emergence rate, seedling rate and damage rate. And judging the tolerance level of the transgenic herbicide-resistant corn to the herbicide. Common corn (zhengdan 958) was used as a control.
Glyphosate field test results the first transgenic corn "thunberg 8" was highly tolerant to glyphosate (table 9, fig. 6).
Table 9 "ZheRui 8" questionnaire for glyphosate tolerance
Example 6: 'Zherui 8' foreign matter detection
The 5 'and 3' flanking sequences of the transformation event 'ZheRui 8' are respectively shown as nucleotide numbers 1-796 and 12118-12620 of SEQ ID NO.5, and primers are respectively designed aiming at the 5 'end and 3' end insertion site sequences of the corn transformation event 'ZheRui 8', and PCR reactions are carried out, wherein the reaction conditions and the reaction systems are shown in the following tables 10 and 11.
TABLE 10 primer information
Table 11, PCR reaction system:
| reagent(s) | Volume of |
| Genomic DNA | 2μl |
| Forward primer (10. Mu.M) | 1μl |
| Reverse primer (10. Mu.M) | 1μl |
| 2×PrimeSTAR GC Buffer(Mg2 + plus) | 25μl |
| dNTP Mixture | 4μl |
| PrimeSTAR HS DNA Polymerase | 0.5μl |
| ddH 2 O | 16.5μl |
| Total volume of | 50μl |
The PCR conditions were: 30 cycles, each cycle 98℃for 10 seconds; 58 ℃,30 seconds; 72℃for 40 seconds.
PCR reaction conditions: denaturation at 94℃for 5min, denaturation at 94℃for 30s, annealing at 58℃for 30s, extension at 72℃for 30s for 35 cycles; extending at 72℃for 7min.
Genomic DNA is extracted from transgenic corn ZheRui 8, and grain powder of industrially applied transgenic corn Ruifeng 125, MON810, bt11, MON89034, conventional corn, conventional soybean and conventional rice, and nucleic acid specific detection is carried out, and detection primer pairs GF (SEQ ID NO: 10) and ZG-R (SEQ ID NO: 11), and ZG-F (SEQ ID NO: 8) and GR (SEQ ID NO: 9) and internal reference primers (zSSIIb-1F and zSSIIb-1R) are respectively adopted for amplification. The agarose electrophoresis results of the amplified products showed that samples containing corn genome (ZheRui 8, ruifeng 125, MON810, bt11, MON89034 and conventional corn) were able to detect bands of about 150bp (FIG. 7), consistent with the expected band size, indicating that the quality of the corn genome used for the test was satisfactory. No bands were detected in the genomic samples amplified from conventional rice and conventional soybean. The electrophoresis result of the amplified products of GF and ZG-R using the primer pair shows that only the sample of ZheR 8 can detect the band with the size of 500bp, the size of the band is consistent with that of the expected band, no specific band can be detected by other transformation event of the genome of ZheR 8, and the sample of the genome of conventional corn, soybean and rice, and the primer pair provided by the invention can specifically detect the existence of ZheR 8 (figure 8).
PCR amplification is carried out by using the primer pair ZG-F and GR, and an electrophoresis result of an amplification product shows that only a sample of ZheR 8 can detect a band with the size of 550bp, the size of the band is consistent with that of expectations (figure 9), other corn transformation events without ZheR 8 genome and samples without conventional corn, soybean and rice genome are not detected, and the primer pair provided by the invention can specifically detect the existence of ZheR 8. Therefore, the test primer pair provided by the invention can specifically detect the existence of a sample containing ZheRui 8.
Sequence listing
<110> Hangzhou Ruifeng biotechnology Co Ltd
<120> nucleic acid sequence for detecting maize transformation event "Zherui 8" and detection method thereof
<160> 11
<170> SIPOSequenceListing 1.0
<210> 1
<211> 24
<212> DNA
<213> Unknown (Unknown)
<400> 1
gaaaccggtc catgcttaga caac 24
<210> 2
<211> 24
<212> DNA
<213> Unknown (Unknown)
<400> 2
aagttgtcta agcgtcaatt tgtt 24
<210> 3
<211> 1300
<212> DNA
<213> Unknown (Unknown)
<400> 3
gattcggcct gcacaggttc atgactccaa gtggcgccgc cgagcagata ctgaacctag 60
gagcgctagg gtaccgagca ccggagctgg cgaacgcggg gaaggcggca ccgacgttca 120
aggcggacgt gtacgcgttc ggggtggtgg tcatggagat gctgacgcgg aggagcgcgg 180
gagacatcat ctcggggcag tccggcgcgg tcgacctgac cgactgggtc cagatgtgca 240
gcagggaagg gcggggagcc gactgcttcg accgcgacat cgccggcctg gaagagcgcc 300
ccggggtgat ggaggagctg ctggcggtct cgctcaggtg tatcctccct gtaaatgaga 360
ggcctaacat gaagacagtc tgcgatgacc tgtgctccat cataacagcg tgagtttagt 420
ttccccttcc ttgccttgcc ttgccttgcc ccagtgtaca tgtaaccgat cggttttccc 480
gtccgttggt tggtccctat tttttctttc tttctcccgg tgggttttcc ggagcccgtg 540
tagccagggt gggttcgttt gattggtaac tgctagtgta aagaggattc gattcgatcc 600
acccccattg attgattgat atcccaacca ttgccgtctt gatgagttgg cgtctctctg 660
ttcgctggtc aagttcttca cactgagcat tttcccagct gcccgatact gtagctgcaa 720
cacctaccct gcagctgatc tgctgctcgt gcgaaagtta ctggcctcac atgcggctct 780
ggagccgctg cagcagcatg tgttgggtat tcagtcctga tcctgggcac aaggatttat 840
tgaacgaaac acaggggtcg ttactgaaac cggtccatgc ttagacaact taataacaca 900
ttgcggacgt ttttaatgta ctgaattaac gccgaattaa ttcgggggat ctggatttta 960
gtactggatt ttggttttag gaattagaaa ttttattgat agaagtattt tacaaataca 1020
aatacatact aagggtttct tatatgctca acacatgagc gaaaccctat aggaacccta 1080
attcccttat ctgggaacta ctcacacaat attatggaga aactcgagct cctattaggc 1140
ggtggcctca gcgtactctg atagtttaaa ctgaaggcgg gaaacgacaa tctgatccaa 1200
gctcaagctg ctctagcatt cgccattcag gctgcgcaac tgttgggaag ggcgatcggt 1260
gcgggcctct tcgctattac gccagctggc gaaaggggga 1300
<210> 4
<211> 803
<212> DNA
<213> Unknown (Unknown)
<400> 4
gaagccctcg gcgctcgctt cgagtacgct gaggccaccg cctaatagga gctcgagttt 60
ctccataata atgtgtgagt agttcccaga taagggaatt agggttccta tagggtttcg 120
ctcatgtgtt gagcatataa gaaaccctta gtatgtattt gtatttgtaa aatacttcta 180
tcaataaaat ttctaattcc taaaaccaaa atccagtact aaaatccaga tcccccgaat 240
taattcggcg ttaattcagt acattaaaaa cgtccgcaat gtgttattaa gttgtctaag 300
cgtcaatttg tttacaccac aatgttccca cgaatccgat cgtatcttca tctcagccgt 360
cgacaccctg tgtcgccagt tggcactggg acttgccagg tgggcccgcg atgtcgccat 420
ggtgagagtg ctctggttct tggactcatg ggccgcaatg gaatggttct gacttctgat 480
gagtgaaaga cactgtgctg tggtggcctg tgggcggtct atggtctttt tgctgcaaca 540
gttgcgtatg tattggcact cttgtcggcc gacgcgcatc atcagttcgc aaatgcacct 600
gtccttgccc atgattaccg ctgattctga gcatctttga gtctgccgga tcaatgcgat 660
gtgcagcctt ttatactcct ttctagggca gcacagcaac tcagagtatt ttcttaggtg 720
tcaagcaagc tcatcatgat ccaagtatcc aaacccagtc atttaggaac ggccaaatgc 780
cagtgccaag gttcaggtcc tac 803
<210> 5
<211> 12701
<212> DNA
<213> Unknown (Unknown)
<400> 5
gattcggcct gcacaggttc atgactccaa gtggcgccgc cgagcagata ctgaacctag 60
gagcgctagg gtaccgagca ccggagctgg cgaacgcggg gaaggcggca ccgacgttca 120
aggcggacgt gtacgcgttc ggggtggtgg tcatggagat gctgacgcgg aggagcgcgg 180
gagacatcat ctcggggcag tccggcgcgg tcgacctgac cgactgggtc cagatgtgca 240
gcagggaagg gcggggagcc gactgcttcg accgcgacat cgccggcctg gaagagcgcc 300
ccggggtgat ggaggagctg ctggcggtct cgctcaggtg tatcctccct gtaaatgaga 360
ggcctaacat gaagacagtc tgcgatgacc tgtgctccat cataacagcg tgagtttagt 420
ttccccttcc ttgccttgcc ttgccttgcc ccagtgtaca tgtaaccgat cggttttccc 480
gtccgttggt tggtccctat tttttctttc tttctcccgg tgggttttcc ggagcccgtg 540
tagccagggt gggttcgttt gattggtaac tgctagtgta aagaggattc gattcgatcc 600
acccccattg attgattgat atcccaacca ttgccgtctt gatgagttgg cgtctctctg 660
ttcgctggtc aagttcttca cactgagcat tttcccagct gcccgatact gtagctgcaa 720
cacctaccct gcagctgatc tgctgctcgt gcgaaagtta ctggcctcac atgcggctct 780
ggagccgctg cagcagcatg tgttgggtat tcagtcctga tcctgggcac aaggatttat 840
tgaacgaaac acaggggtcg ttactgaaac cggtccatgc ttagacaact taataacaca 900
ttgcggacgt ttttaatgta ctgaattaac gccgaattaa ttcgggggat ctggatttta 960
gtactggatt ttggttttag gaattagaaa ttttattgat agaagtattt tacaaataca 1020
aatacatact aagggtttct tatatgctca acacatgagc gaaaccctat aggaacccta 1080
attcccttat ctgggaacta ctcacacaat attatggaga aactcgagct cctattaggc 1140
ggtggcctca gcgtactctg atagtttaaa ctgaaggcgg gaaacgacaa tctgatccaa 1200
gctcaagctg ctctagcatt cgccattcag gctgcgcaac tgttgggaag ggcgatcggt 1260
gcgggcctct tcgctattac gccagctggc gaaaggggga tgtgctgcaa ggcgattaag 1320
ttgggtaacg ccagggtttt cccagtcacg acgttgtaaa acgacggcca gtgccaagct 1380
taggtcattc atatgcttga gaagagtcgg gatagtccaa aataaaacaa aggtaagatt 1440
acctggtcaa aagtgaaaac atcagttaaa aggtggtatg aagtaaaata tcggtaataa 1500
aaggtggccc aaagtgaaat ttactctttt ctactattat aaaaattgag gatgtttttg 1560
tcggtacttt gatacgtcat ttttgtatga attggttttt aagtttattc gcttttggaa 1620
atgcatatct gtatttgagt cgggttttaa gttcgtctgc ttttgtaaat acagagggat 1680
ttgtataaga aatatcttta aaaaaaccca tatgctaatt tgacataatt tttgagaaaa 1740
atatatattc aggcgaattc tcacaatgaa caataataag attaaaatag ctttcccccg 1800
ttgcagcgca tgggtatttt ttctagtaaa aataaaagat agacttagac tcaaaacatt 1860
tacaaaaaca acccctaaag ttcctaaagc ccaaagtgct atccacgatc catagcaagc 1920
ccagcccaac ccaacccaac ccaacccacc ccagtccagc caactggaca atagtctcca 1980
caccccccca ctatcaccgt gagttgttcg cacgcaccgc acgtctcgca gccaaaaaaa 2040
aaaaaaagaa agaaaaaaaa gaaaaagaaa aaacagcagg tgggtccggg tcgtgggggc 2100
cggaaacgcg aggaggatcg cgagccagcg acgaggccgg ccctccctcc gcttccaaag 2160
aaacgccccc catcgccact atatacatac ccccccctct cctcccatcc ccccaaccct 2220
accaccacca ccaccaccac ctccacctcc tcccccctcg ctgccggacg acgagctcct 2280
cccccctccc cctccgccgc cgccgcgccg gtaaccaccc cgcccctctc ctctttcttt 2340
ctccgttttt tttttccgtc tcggtctcga tctttggcct tggtagtttg ggtgggcgag 2400
aggcggcttc gtgcgcgccc agatcggtgc gcgggagggg cgggatctcg cggctggggc 2460
tctcgccggc gtgagtcggc ccgaatcctc gcggggaatg gggctctcgg atgtagatct 2520
gcgatccgcc gttgttgggg gagatgatgg ggggtttaaa atttccgcca tgctaaacaa 2580
gatcaggaag aggggaaaag ggcactatgg tttatatttt tatatatttc tgctgcctcg 2640
tcaggcttag atctgctaga tctttctttc ttctttttgt gggtagaatt tgaatccctc 2700
agcattgttc atcggtagtt tttcttttca tgatttgtga caaatgcagc ctcgtgcgga 2760
gcttttttgt aggtagaaga tggctgacgc cgaggatcca acaatggctt ccgtgctcaa 2820
ctccggccgc accaccatct gcgacgccta caacgtggcc gctcacgacc ctttctcctt 2880
ccagcacaag tccctcgaca ccgtgcagaa ggagtggaca gagtggaaga agaacaacca 2940
ctctctctac ctcgacccaa tcgtgggcac cgtggcctcc ttcctcctca agaaggtggg 3000
ctccctcgtg ggaaagcgca tcctctccga gcttcgcaac ctcatcttcc cgtccggctc 3060
cactaacctc atgcaggaca tcctccgcga gaccgagaag ttcctcaacc agcgcctcaa 3120
caccgacact ctcgctcgcg tgaacgccga gctaaccggc ctccaggcca acgtggagga 3180
gttcaaccgt caggtggaca acttcctcaa cccgaaccgc aacgccgtgc cgctctccat 3240
cacctcttcc gtgaacacca tgcagcagct cttcctcaac cgccttccgc agttccagat 3300
gcagggctac cagcttctcc tactccctct cttcgcccag gctgccaacc tccacctctc 3360
tttcatccgc gacgtgatcc tcaacgccga cgagtggggc atctccgctg ccaccctccg 3420
cacctaccgc gactacctca agaactacac ccgcgactac tccaactact gcatcaacac 3480
ctaccagtcc gccttcaagg gcctcaacac ccgcctccac gacatgctcg aattccgcac 3540
ctacatgttc ctcaacgtgt tcgagtacgt gtccatctgg tccctcttca agtaccagtc 3600
cctcctcgtg tcctccggcg ccaacctcta cgcctccggc tccggcccgc agcagaccca 3660
gtccttcacc tcccaggact ggccgttcct ctactccctc ttccaggtga actccaacta 3720
cgtgctcaac ggcttctccg gcgcccgcct ctccaacacc ttcccgaaca tcgtgggcct 3780
ccctggctcc acaactaccc acgccctcct cgccgctcgc gtgaactact ccggtggcat 3840
ctcttccggc gacatcggcg cttccccgtt caaccagaac ttcaactgct ctaccttcct 3900
ccctccgctc ctcacaccgt tcgtgcgctc ctggctcgac tccggctccg accgcgaggg 3960
cgtggctacc gtgaccaact ggcagaccga gtccttcgag accaccctcg gcctccgctc 4020
cggtgccttc accgcacgcg gcaactccaa ctacttcccg gactacttca tccgcaacat 4080
ctccggcgtg ccgctcgtgg tgcgcaacga ggacctccgt cgcccactcc actacaacga 4140
gatccgcaac atcgcctccc cgtccggcac ccctggaggt gcacgcgctt acatggtgtc 4200
cgtgcacaac cgcaagaaca acatccacgc tgtgcacgag aacggctcca tgatccacct 4260
cgctccaaac gactacaccg gcttcaccat ctccccgatc cacgctaccc aggtgaacaa 4320
ccagacccgc accttcatct ccgagaagtt cggcaaccag ggcgactctc tccgcttcga 4380
gcagaacaac accaccgctc gttacaccct ccgcggtaac ggcaactcct acaacctcta 4440
cctccgcgtg tcttccatcg gcaactccac catccgcgtg accatcaacg gccgtgtgta 4500
caccgccaca aacgtgaaca ccaccacaaa caacgacggc gtgaacgaca acggtgctcg 4560
cttctccgac atcaacatcg gcaacgtggt ggcctcctcc aactccgacg tgccgctcga 4620
catcaacgtg accctcaact ctggcacaca gttcgacctc atgaacatca tgctcgtgcc 4680
gaccaacatc tccccgctct actaatagat gccgaccgga tctgtcgatc gacaaggagc 4740
tcgagtttct ccataataat gtgtgagtag ttcccagata agggaattag ggttcctata 4800
gggtttcgct catgtgttga gcatataaga aacccttagt atgtatttgt atttgtaaaa 4860
tacttctatc aataaaattt ctaattccta aaaccaaaat ccagtactaa aatccagatc 4920
ccccgaatta atttgaggta ccaagcttgc atgcctgcag tgcagcgtga cccggtcgtg 4980
cccctctcta gagataatga gcattgcatg tctaagttat aaaaaattac cacatatttt 5040
ttttgtcaca cttgtttgaa gtgcagttta tctatcttta tacatatatt taaactttac 5100
tctacgaata atataatcta tagtactaca ataatatcag tgttttagag aatcatataa 5160
atgaacagtt agacatggtc taaaggacaa ttgagtattt tgacaacagg actctacagt 5220
tttatctttt tagtgtgcat gtgttctcct ttttttttgc aaatagcttc acctatataa 5280
tacttcatcc attttattag tacatccatt tagggtttag ggttaatggt ttttatagac 5340
taattttttt agtacatcta ttttattcta ttttagcctc taaattaaga aaactaaaac 5400
tctattttag tttttttatt taataattta gatataaaat agaataaaat aaagtgacta 5460
aaaattaaac aaataccctt taagaaatta aaaaaactaa ggaaacattt ttcttgtttc 5520
gagtagataa tgccagcctg ttaaacgccg tcgacgagtc taacggacac caaccagcga 5580
accagcagcg tcgcgtcggg ccaagcgaag cagacggcac ggcatctctg tcgctgcctc 5640
tggacccctc tcgagagttc cgctccaccg ttggacttgc tccgctgtcg gcatccagaa 5700
attgcgtggc ggagcggcag acgtgagccg gcacggcagg cggcctcctc ctcctctcac 5760
ggcaccggca gctacggggg attcctttcc caccgctcct tcgctttccc ttcctcgccc 5820
gccgtaataa atagacaccc cctccacacc ctctttcccc aacctcgtgt tgttcggagc 5880
gcacacacac acaaccagat ctcccccaaa tccacccgtc ggcacctccg cttcaaggta 5940
cgccgctcgt cctccccccc cccccctctc taccttctct agatcggcgt tccggtccat 6000
ggttagggcc cggtagttct acttctgttc atgtttgtgt tagatccgtg tttgtgttag 6060
atccgtgctg ctagcgttcg tacacggatg cgacctgtac gtcagacacg ttctgattgc 6120
taacttgcca gtgtttctct ttggggaatc ctgggatggc tctagccgtt ccgcagacgg 6180
gatcgatttc atgatttttt ttgtttcgtt gcatagggtt tggtttgccc ttttccttta 6240
tttcaatata tgccgtgcac ttgtttgtcg ggtcatcttt tcatgctttt ttttgtcttg 6300
gttgtgatga tgtggtctgg ttgggcggtc gttctagatc ggagtagaat tctgtttcaa 6360
actacctggt ggatttatta attttggatc tgtatgtgtg tgccatacat attcatagtt 6420
acgaattgaa gatgatggat ggaaatatcg atctaggata ggtatacatg ttgatgcggg 6480
ttttactgat gcatatacag agatgctttt tgttcgcttg gttgtgatga tgtggtgtgg 6540
ttgggcggtc gttcattcgt tctagatcgg agtagaatac tgtttcaaac tacctggtgt 6600
atttattaat tttggaactg tatgtgtgtg tcatacatct tcatagttac gagtttaaga 6660
tggatggaaa tatcgatcta ggataggtat acatgttgat gtgggtttta ctgatgcata 6720
tacatgatgg catatgcagc atctattcat atgctctaac cttgagtacc tatctattat 6780
aataaacaag tatgttttat aattattttg atcttgatat acttggatga tggcatatgc 6840
agcagctata tgtggatttt tttagccctg ccttcatacg ctatttattt gcttggtact 6900
gtttcttttg tcgatgctca ccctgttgtt tggtgttact tctgcaggtc gactctagag 6960
gatccaacaa tggacaacaa ccccaacatc aacgagtgca tcccctacaa ctgcctgagc 7020
aaccccgagg tggaggtgct gggcggcgag cgcatcgaga ccggctacac ccccatcgac 7080
atcagcctga gcctgaccca gttcctgctg agcgagttcg tgcccggcgc cggcttcgtg 7140
ctgggcctgg tggacatcat ctggggcatc ttcggcccca gccagtggga cgccttcctg 7200
gtgcagatcg agcagctgat caaccagcgc atcgaggagt tcgcccgcaa ccaggccatc 7260
agccgcctgg agggcctgag caacctgtac caaatctacg ccgagagctt ccgcgagtgg 7320
gaggccgacc ccaccaaccc cgccctgcgc gaggagatgc gcatccagtt caacgacatg 7380
aacagcgccc tgaccaccgc catccccctg ttcgccgtgc agaactacca ggtgcccctg 7440
ctgagcgtgt acgtgcaggc cgccaacctg cacctgagcg tgctgcgcga cgtcagcgtg 7500
ttcggccagc gttggggctt cgacgccgcc accatcaaca gccgctacaa cgacctgacc 7560
cgcctgatcg gcaactacac cgaccacgcc gtgcgctggt acaacaccgg cctggagcgc 7620
gtgtggggtc ccgacagccg cgactggatc aggtacaacc agttccgccg cgagctgacc 7680
ctgaccgtgc tggacatcgt gagcctgttc cccaactacg acagccgcac ctaccccatc 7740
cgcaccgtga gccagctgac ccgcgagatt tacaccaacc ccgtgctgga gaacttcgac 7800
ggcagcttcc gcggcagcgc ccagggcatc gagggcagca tccgcagccc ccacctgatg 7860
gacatcctga acagcatcac catctacacc gacgcccacc gcggcgagta ctactggagc 7920
ggccaccaga tcatggccag ccccgtcggc ttcagcggcc ccgagttcac cttccccctg 7980
tacggcacca tgggcaacgc tgcacctcag cagcgcatcg tggcacagct gggccaggga 8040
gtgtaccgca ccctgagcag caccctgtac cgtcgacctt tcaacatcgg catcaacaac 8100
cagcagctga gcgtgctgga cggcaccgag ttcgcctacg gcaccagcag caacctgccc 8160
agcgccgtgt accgcaagag cggcaccgtg gacagcctgg acgagatccc ccctcagaac 8220
aacaacgtgc cacctcgaca gggcttcagc caccgtctga gccacgtgag catgttccgc 8280
agtggcttca gcaacagcag cgtgagcatc atccgtgcac ctatgttcag ctggattcac 8340
cgcagtgccg agttcaacaa catcatcccc agcagccaga tcacccagat ccccctgacc 8400
aagagcacca acctgggcag cggcaccagc gtggtgaagg gccccggctt caccggcggc 8460
gacatcctgc gccgcaccag ccccggccag atcagcaccc tgcgcgtgaa catcaccgcc 8520
cccctgagcc agcgctaccg cgtccgcatc cgctacgcca gcaccaccaa cctgcagttc 8580
cacaccagca tcgacggccg ccccatcaac cagggcaact tcagcgccac catgagcagc 8640
ggcagcaacc tgcagagcgg cagcttccgc accgtgggct tcaccacccc cttcaacttc 8700
agcaacggca gcagcgtgtt caccctgagc gcccacgtgt tcaacagcgg caacgaggtg 8760
tacatcgacc gcatcgagtt cgtgcccgcc gaggtgacct tcgaggccga gtacgacctg 8820
gagagggctc agaaggccgt gaacgagctg ttcaccagca gcaaccagat cggcctgaag 8880
accgacgtga ccgactacca catcgatcag gtgcgataag agctctagat ctgttctgca 8940
caaagtggag tagtcagtca tcgatcagga accagacacc agacttttat tcatacagtg 9000
aagtgaagtg aagtgcagtg cagtgagttg ctggtttttg tacaacttag tatgtatttg 9060
tatttgtaaa atacttctat caataaaatt tctaattcct aaaaccaaaa tccaggggta 9120
cctccagctt atggtggagc acgacactct cgtctactcc aagaatatca aagatacagt 9180
ctcagaagac caaagggcta ttgagacttt tcaacaaagg gtaatatcgg gaaacctcct 9240
cggattccat tgcccagcta tctgtcactt catcaaaagg acagtagaaa aggaaggtgg 9300
cacctacaaa tgccatcatt gcgataaagg aaaggctatc gttcaagatg cctctgccga 9360
cagtggtccc aaagatggac ccccacccac gaggagcatc gtggaaaaag aagacgttcc 9420
aaccacgtct tcaaagcaag tggattgatg tgaacatggt ggagcacgac actctcgtct 9480
actccaagaa tatcaaagat acagtctcag aagaccaaag ggctattgag acttttcaac 9540
aaagggtaat atcgggaaac ctcctcggat tccattgccc agctatctgt cacttcatca 9600
aaaggacagt agaaaaggaa ggtggcacct acaaatgcca tcattgcgat aaaggaaagg 9660
ctatcgttca agatgcctct gccgacagtg gtcccaaaga tggaccccca cccacgagga 9720
gcatcgtgga aaaagaagac gttccaacca cgtcttcaaa gcaagtggat tgatgtgata 9780
tctccactga cgtaagggat gacgcacaat cccactatcc ttcgcaagac ccttcctcta 9840
tataaggaag ttcatttcat ttggagagga cacgctgaaa tcaccagtct ctctctacaa 9900
atctatctct ctcgagctcc tcccccctcc ccctccgccg ccgccgcgcc ggtaaccacc 9960
ccgcccctct cctctttctt tctccgtttt ttttttccgt ctcggtctcg atctttggcc 10020
ttggtagttt gggtgggcga gaggcggctt cgtgcgcgcc cagatcggtg cgcgggaggg 10080
gcgggatctc gcggctgggg ctctcgccgg cgtgagtcgg cccgaatcct cgcggggaat 10140
ggggctctcg gatgtagatc tgcgatccgc cgttgttggg ggagatgatg gggggtttaa 10200
aatttccgcc atgctaaaca agatcaggaa gaggggaaaa gggcactatg gtttatattt 10260
ttatatattt ctgctgcctc gtcaggctta gatctgctag atctttcttt cttctttttg 10320
tgggtagaat ttgaatccct cagcattgtt catcggtagt ttttcttttc atgatttgtg 10380
acaaatgcag cctcgtgcgg agcttttttg taggtaggat ctaccatggc caccgccgcc 10440
gccgcgtcta ccgcgctcac tggcgccact accgctgcgc ccaaggcgag gcgccgggcg 10500
cacctcctgg ccacccgccg cgccctcgcc gcgcccatca ggtgctcagc ggcgtcaccc 10560
gccatgccga tggctccccc ggccaccccg ctccggccgt ggggccccac cgatccccgc 10620
aagggatccg acgccctgcc cgccaccttc gacgtgatcg tgcatccagc tcgcgaactc 10680
cgcggcgagc ttcgcgctca gccatccaag aactacacca ctcgctacct cctcgccgct 10740
gccctcgctg agggcgagac ccgcgtggtg ggcgtggcta cctctgagga cgccgaggcc 10800
atgctccgct gcctccgcga ctggggcgct ggcgtggagc ttgtgggcga tgacgccgtg 10860
atccgcggtt tcggcgctcg cccacaggcc ggtgtgaccc tcaacccagg caacgctgcc 10920
gcggtggccc gcctcctcat gggcgtggcc gctctcacct ctggcaccac tttcgtgacc 10980
gactacccgg actccctcgg caagcgccct cagggcgacc tccttgaggc cctcgaacgc 11040
ctcggtgcct gggtgtcctc caacgacggt cgcctcccga tctccgtgtc cggcccagtg 11100
cgcggtggca ccgtggaggt gtccgccgag cgctcctccc agtacgcctc cgccctcatg 11160
ttcctcggcc ctctcctccc ggacggactc gaactccgcc tcaccggcga catcaagtcc 11220
cacgctccgc tccgccagac actcgacacc ctctctgact tcggcgtgcg cgccactgcc 11280
tccgacgacc tccgccgcat ctccatcccg ggtggccaga agtaccgccc aggccgcgtg 11340
ctcgtgccgg gcgactaccc gggctccgct gccatcctca ccgccgctgc cctcctccca 11400
ggcgaggtgc gcctctctaa cctccgcgag cacgacctcc agggcgagaa ggaggccgtg 11460
aacgtgctcc gcgagatggg cgctgacatc gtgcgcgagg gcgataccct caccgtgcgc 11520
ggtggccgcc ctctccacgc cgtgactcgc gacggcgatt ccttcaccga cgccgtgcaa 11580
gccctcaccg ccgctgctgc cttcgccgag ggcgacacca cctgggagaa cgtggccact 11640
ctccgcctca aggagtgcga ccgcatctct gacacccgcg ctgagcttga gcgcctcggc 11700
ctccgcgcac gcgagaccgc cgactctctc tccgtgactg gctctgctca cctcgctggt 11760
ggcatcaccg ccgacggcca cggcgaccac cgcatgatca tgctcctcac cctcctcggc 11820
ctccgcgcag acgctccact ccgcatcacc ggcgcacacc acatccgcaa gtcctaccct 11880
cagttcttcg ctcacctcga agccctcggc gctcgcttcg agtacgctga ggccaccgcc 11940
taataggagc tcgagtttct ccataataat gtgtgagtag ttcccagata agggaattag 12000
ggttcctata gggtttcgct catgtgttga gcatataaga aacccttagt atgtatttgt 12060
atttgtaaaa tacttctatc aataaaattt ctaattccta aaaccaaaat ccagtactaa 12120
aatccagatc ccccgaatta attcggcgtt aattcagtac attaaaaacg tccgcaatgt 12180
gttattaagt tgtctaagcg tcaatttgtt tacaccacaa tgttcccacg aatccgatcg 12240
tatcttcatc tcagccgtcg acaccctgtg tcgccagttg gcactgggac ttgccaggtg 12300
ggcccgcgat gtcgccatgg tgagagtgct ctggttcttg gactcatggg ccgcaatgga 12360
atggttctga cttctgatga gtgaaagaca ctgtgctgtg gtggcctgtg ggcggtctat 12420
ggtctttttg ctgcaacagt tgcgtatgta ttggcactct tgtcggccga cgcgcatcat 12480
cagttcgcaa atgcacctgt ccttgcccat gattaccgct gattctgagc atctttgagt 12540
ctgccggatc aatgcgatgt gcagcctttt atactccttt ctagggcagc acagcaactc 12600
agagtatttt cttaggtgtc aagcaagctc atcatgatcc aagtatccaa acccagtcat 12660
ttaggaacgg ccaaatgcca gtgccaaggt tcaggtccta c 12701
<210> 6
<211> 557
<212> DNA
<213> Unknown (Unknown)
<400> 6
ccgatactgt agctgcaaca cctaccctgc agctgatctg ctgctcgtgc gaaagttact 60
ggcctcacat gcggctctgg agccgctgca gcagcatgtg ttgggtattc agtcctgatc 120
ctgggcacaa ggatttattg aacgaaacac aggggtcgtt actgaaaccg gtccatgctt 180
agacaactta ataacacatt gcggacgttt ttaatgtact gaattaacgc cgaattaatt 240
cgggggatct ggattttagt actggatttt ggttttagga attagaaatt ttattgatag 300
aagtatttta caaatacaaa tacatactaa gggtttctta tatgctcaac acatgagcga 360
aaccctatag gaaccctaat tcccttatct gggaactact cacacaatat tatggagaaa 420
ctcgagctcc tattaggcgg tggcctcagc gtactctgat agtttaaact gaaggcggga 480
aacgacaatc tgatccaagc tcaagctgct ctagcattcg ccattcaggc tgcgcaactg 540
ttgggaaggg cgatcgg 557
<210> 7
<211> 509
<212> DNA
<213> Unknown (Unknown)
<400> 7
ccagtactaa aatccagatc ccccgaatta attcggcgtt aattcagtac attaaaaacg 60
tccgcaatgt gttattaagt tgtctaagcg tcaatttgtt tacaccacaa tgttcccacg 120
aatccgatcg tatcttcatc tcagccgtcg acaccctgtg tcgccagttg gcactgggac 180
ttgccaggtg ggcccgcgat gtcgccatgg tgagagtgct ctggttcttg gactcatggg 240
ccgcaatgga atggttctga cttctgatga gtgaaagaca ctgtgctgtg gtggcctgtg 300
ggcggtctat ggtctttttg ctgcaacagt tgcgtatgta ttggcactct tgtcggccga 360
cgcgcatcat cagttcgcaa atgcacctgt ccttgcccat gattaccgct gattctgagc 420
atctttgagt ctgccggatc aatgcgatgt gcagcctttt atactccttt ctagggcagc 480
acagcaactc agagtatttt cttaggtgt 509
<210> 8
<211> 24
<212> DNA
<213> Unknown (Unknown)
<400> 8
ccgatactgt agctgcaaca ccta 24
<210> 9
<211> 24
<212> DNA
<213> Unknown (Unknown)
<400> 9
ccgatcgccc ttcccaacag ttgc 24
<210> 10
<211> 24
<212> DNA
<213> Unknown (Unknown)
<400> 10
ccagtactaa aatccagatc cccc 24
<210> 11
<211> 24
<212> DNA
<213> Unknown (Unknown)
<400> 11
acacctaaga aaatactctg agtt 24
Claims (15)
1. A nucleic acid sequence comprising SEQ ID No.1 or its complement, and/or SEQ ID No.2 or its complement, said nucleic acid sequence being derived from a transgenic maize event "thunberg 8", said transgenic maize event "thunberg 8" being deposited as seeds in chinese typical culture collection under accession No.: CCTCC NO: P202202, the preservation date 2022, 1 month 11, address university of Wuhan, china, post code 430072.
2. The nucleic acid sequence according to claim 1, characterized in that the nucleic acid sequence comprises SEQ ID No.3 or the complement thereof and/or SEQ ID No.4 or the complement thereof.
3. The nucleic acid sequence according to claim 2, characterized in that the nucleic acid sequence comprises SEQ ID No.5 or the complement thereof.
4. A method of detecting the presence of DNA from a transgenic maize event "thunberg 8" in a sample, the method comprising: contacting a sample to be detected with a primer pair in a nucleic acid amplification solution, performing a nucleic acid amplification reaction, and detecting an amplification product; the amplified product comprises SEQ ID NO.1 or its complement, and/or SEQ ID NO.2 or its complement.
5. The method of claim 4, wherein the amplification product comprises SEQ ID No.6 or its complement and/or SEQ ID No.7 or its complement.
6. The method of claim 4 or 5, wherein the primer pair comprises a first primer and a second primer, the first primer being SEQ ID No.8 or SEQ ID No.10; the second primer is SEQ ID NO.9 or SEQ ID NO.11.
7. A method of protecting corn from insect damage, the method comprising providing a transgenic corn plant, wherein target insects feeding on the transgenic corn plant are inhibited from further feeding on the transgenic corn plant; the genome of the transgenic corn plant comprises a nucleic acid sequence from a specific region of transgenic corn event Zheju 8, which comprises, in sequence, SEQ ID NO.1, SEQ ID NO.5 nucleic acids 2804-4705, SEQ ID NO.5 nucleic acids 6970-8919, and SEQ ID NO.2.
8. A method of increasing glyphosate tolerance in corn comprising spraying a glyphosate herbicide into a field in which a transgenic corn plant is grown, said transgenic corn plant having glyphosate tolerance; the genome of the transgenic corn plant comprises a nucleic acid sequence from a specific region of transgenic corn event "Zheju 8", which comprises, in sequence, the nucleic acid sequences of SEQ ID NO.1, SEQ ID NO.5, positions 10426-11943, and SEQ ID NO.2, or the nucleic acid sequence of the specific region comprises SEQ ID NO.5.
9. A method of controlling weeds in a corn field, the method comprising spraying a glyphosate herbicide into a field in which transgenic corn is grown, the transgenic corn being tolerant to glyphosate and the weeds in the corn field being killed by the glyphosate; the transgenic corn genome comprises a nucleic acid sequence from a specific region of transgenic corn event "Zherui 8", which comprises, in sequence, the nucleic acid sequences of SEQ ID NO.1, SEQ ID NO.5, positions 10426-11943, and SEQ ID NO.2, or the nucleic acid sequence of the specific region comprises SEQ ID NO.5.
10. A method of growing a maize plant that is resistant to insects, said method comprising: planting corn seeds, grafting target insects on corn plants, and harvesting plants with better insect-resistant effect compared with other plants without specific region nucleic acid sequences; the genome of the corn seed comprises a nucleic acid sequence from a specific region of transgenic corn event "Zherui 8", which comprises, in sequence, SEQ ID NO.1, SEQ ID NO.5 nucleic acids 2804-4705, SEQ ID NO.5 nucleic acid sequence 6970-8919, and SEQ ID NO.2, or the nucleic acid sequence of the specific region comprises SEQ ID NO.5.
11. A method of growing a corn plant tolerant to a glyphosate herbicide, the method comprising: planting corn seeds, growing the corn into corn plants, spraying the corn plants with a glyphosate herbicide, and harvesting plants that are not damaged by glyphosate as compared to other plants that do not contain the specific region nucleic acid sequence; the genome of the corn seed comprises a nucleic acid sequence from a specific region of transgenic corn event "Zherui 8", which in turn comprises the nucleic acid sequences of SEQ ID NO.1, SEQ ID NO.5, positions 10426-11943 and SEQ ID NO.2, or the nucleic acid sequence of the specific region comprises SEQ ID NO.5.
12. A method of producing a maize plant resistant to insects, comprising: crossing the transgenic maize plant with another maize plant, thereby producing a progeny plant; selecting plants having a better pest-resistant effect than other plants not having the specific region nucleic acid sequence; the genome of the transgenic corn plant comprises a nucleic acid sequence from a specific region of transgenic corn event 'Zherui 8', which comprises, in sequence, SEQ ID NO.1, SEQ ID NO.5 nucleotide sequences 2804-4705, 6970-8919 and SEQ ID NO.2, or the nucleic acid sequence of the specific region comprises SEQ ID NO.5.
13. A method of producing a maize plant that is tolerant to glyphosate, the method comprising crossing a transgenic maize plant with another maize plant, thereby producing a progeny plant; harvesting plants that are not damaged by glyphosate as compared to other plants that do not contain the specific region nucleic acid sequence; the genome of the transgenic corn plant comprises a nucleic acid sequence from a specific region of transgenic corn event Zheju 8, wherein the nucleic acid sequence of the specific region comprises SEQ ID NO.1, the nucleic acid sequences of SEQ ID NO.5 10426-11943 and SEQ ID NO.2 in sequence, or the nucleic acid sequence of the specific region comprises SEQ ID NO.5.
14. A method of producing a corn plant resistant to insects and tolerant to a glyphosate herbicide, the method comprising: crossing the transgenic maize plant with another maize plant, thereby producing a progeny plant; harvesting plants that are resistant to insects and are not damaged by glyphosate as compared to other plants not comprising the specific region nucleic acid sequence; the genome of the transgenic corn plant comprises a nucleic acid sequence from a specific region of transgenic corn event Zheju 8, wherein the nucleic acid sequence of the specific region comprises SEQ ID NO.1, SEQ ID NO.5 nucleotide sequences 2804-4705, SEQ ID NO.5 nucleotide sequences 6970-8919, SEQ ID NO.5 nucleotide sequences 10426-11943 and SEQ ID NO.2 in sequence, or the nucleic acid sequence of the specific region comprises SEQ ID NO.5.
15. A composition for generating a transgenic corn event "thunberg 8", wherein said composition is corn meal, corn flour, corn oil, corn starch, or corn cobs; the transgenic corn event "ZheRui 8" is deposited in the form of seeds with China center for type culture Collection, accession number: CCTCC NO: P202202.
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