Corn event CSJ4351 and primer and method for detecting same

Abstract

The invention provides a corn transformation event CSJ4351 as well as a primer pair and a method for detecting the corn transformation event CSJ4351. The maize plant carrying the CSJ4351 transformation event comprises the event on a chromosome 3, namely the junction of an exogenous insertion DNA sequence and a maize genome DNA sequence. A detection primer can be designed by using an exogenous insertion DNA sequence and a DNA sequence of a junction region on a flanking corn genome, and is used for specific detection of a CSJ4351 event. The detection method aiming at the CSJ4351 event can provide a convenient means for tracking the insertion event of the specific gene for breeding by using the plant event, and the insertion event of the specific gene can be used as a molecular marker to improve the working efficiency of insect-resistant and herbicide-resistant breeding by using the transformation event.

Description

Technical Field

[0001] The present invention belongs to the field of plant molecular biology, especially the field of transgenic crop breeding in agricultural biotechnology research, and particularly relates to a transgenic maize event that is resistant to insects and tolerant to the application of glufosinate herbicide, as well as a nucleic acid sequence and method for detecting the presence of this transformation event. Background Art

[0002] Maize, as one of the world's three major staple foods and also the most important forage crop, occupies a crucial position in the global food system. However, the growth, yield, and quality of maize have long been severely threatened by insect pests such as the Asian corn borer and weeds. In the process of maize production, insect resistance and herbicide tolerance are two crucial agronomic traits. In terms of insect resistance, Lepidoptera insects, such as the Asian corn borer, cotton bollworm, armyworm, and fall armyworm, cause great harm to maize. By using transgenic technology to express resistance genes of Lepidoptera insects in maize plants, the resistance of maize to such pests can be effectively improved. In terms of herbicide tolerance, glufosinate is one of the commonly used herbicides in maize production. Biotechnology can introduce a glufosinate herbicide-tolerant gene (such as pat) into maize plants and make it express, thereby conferring maize tolerance to glufosinate herbicide. With these transgenic means, the agronomic traits and quality of maize can be significantly improved.

[0003] In the field of plant transgenic technology, the expression efficiency of foreign genes is closely related to the integration sites of chromosomes. The synergistic effect of chromatin structure characteristics (such as heterochromatin regions) and adjacent transcriptional regulatory elements (such as enhancers) leads to significant expression heterogeneity among different transformants. This site effect is specifically manifested as: (1) the expression levels of foreign genes vary by hundreds of times among different transformants; (2) the spatio-temporal expression patterns deviate from the designed expectations (such as tissue-specific disorders); (3) it may interfere with the normal expression of endogenous genes. Therefore, conventional technical means need to construct hundreds to thousands of independent transformation events and conduct multiple rounds of screening to obtain commercial transformants with both high-efficiency target trait expression and excellent agronomic characteristics.

[0004] In the international seed industry competition pattern, transformation events, due to their non-replicable molecular structure characteristics and unpredictable phenotypic advantages, have become the core objects of intellectual property protection. Mainstream commercial transformation events (such as MON810, Bt11, etc.) have all built technical barriers through patent layout, and important breakthroughs have also been made in the construction of the patent protection system for independent transformation events (such as Huahui 1). Establishing a transformation event-specific detection and verification method is not only a necessary condition for the compliant commercialization of varieties (meeting regulatory requirements such as GMO labeling), but also a key support for strengthening the exclusivity of patented technologies. Summary of the Invention

[0005] In one aspect, the present disclosure provides a maize transformation event, characterized in that a partial genomic sequence of maize is replaced with a T-DNA insertion sequence on chromosome 3 of the maize, wherein the T-DNA insertion sequence is the sequence of nucleotides 280-13210 of the sequence shown in SEQ ID NO: 13, and the partial genomic sequence is as shown in SEQ ID NO: 18.

[0006] In some embodiments, the upstream flanking maize genomic sequence of the T-DNA insertion sequence is: 1) the sequence of nucleotides 1-279 of the sequence shown in SEQ ID NO: 13; or 2) a 3' terminal sequence of the sequence of nucleotides 1-279 of the sequence shown in SEQ ID NO: 13 with a length of at least 100 bp.

[0007] In some embodiments, the downstream flanking maize genomic sequence of the T-DNA insertion sequence is: 1) the sequence of nucleotides 13211-13721 of the sequence shown in SEQ ID NO: 13; or 2) a 5' terminal sequence of the sequence of nucleotides 13211-13721 of the sequence shown in SEQ ID NO: 13 with a length of at least 100 bp.

[0008] In some embodiments, the maize transformation event results in the inclusion of a DNA sequence as shown in SEQ ID NO: 13 on chromosome 3 of the maize, the DNA sequence consisting of a T-DNA insertion sequence of nucleotides 280-13210, an upstream flanking maize genomic sequence of nucleotides 1-279, and a downstream flanking maize genomic sequence of nucleotides 13211-13721.

[0009] In some embodiments, the DNA fragment comprises, in order from the 5' end to the 3' end: 1) an upstream flanking maize genomic sequence: the sequence of nucleotides 1-279 of the sequence shown in SEQ ID NO: 13; or a 3' terminal sequence of the sequence of nucleotides 1-279 of the sequence shown in SEQ ID NO: 13 with a length of at least 100 bp; 2) a T-DNA insertion sequence: the sequence of nucleotides 280-13210 of the sequence shown in SEQ ID NO: 13; 3) a downstream flanking maize genomic sequence: the sequence of nucleotides 13211-13721 of the sequence shown in SEQ ID NO: 13; or a 5' terminal sequence of the sequence of nucleotides 13211-13721 of the sequence shown in SEQ ID NO: 13 with a length of at least 100 bp.

[0010] On the other hand, the present invention provides primer pairs for detecting the above-mentioned maize transformation event or the above-mentioned DNA fragment, wherein the primer pairs are composed of a first primer that specifically recognizes the upstream flanking maize genomic sequence or the downstream flanking maize genomic sequence and a second primer that specifically recognizes the T-DNA insertion sequence.

[0011] In some embodiments, the sequence of the first primer is as shown in SEQ ID NO: 14, and the sequence of the second primer is as shown in SEQ ID NO: 15; or the sequence of the first primer is as shown in SEQ ID NO: 16, and the sequence of the second primer is as shown in SEQ ID NO: 17.

[0012] On the other hand, the present invention provides a method for identifying whether the above-mentioned maize transformation event exists in a maize biological sample, comprising: (a) extracting a DNA sample from the maize biological sample to be identified; (b) using the above-mentioned primer pair for PCR amplification with the extracted DNA sample as a template; and (c) detecting the PCR amplification product. If the length of the PCR amplification product is the same as that obtained by amplifying a DNA template with the sequence shown in SEQ ID NO: 13, it indicates that the maize transformation event exists in the maize biological sample.

[0013] On the other hand, the present invention provides a method for obtaining a transgenic insect-resistant and herbicide-tolerant maize material, comprising: hybridizing a maize material containing the above-mentioned maize transformation event with other maize breeding materials, and then further performing backcrossing to obtain a new material containing the maize transformation event (such as obtaining pure-line plants); wherein during the hybridization and / or backcrossing process, the above-mentioned identification method is used to screen and identify in the offspring population to confirm the existence of the maize transformation event.

[0014] On the other hand, the present invention provides the use of the above-mentioned DNA fragment for improving the insect resistance and herbicide tolerance agronomic traits of maize, for maize breeding and / or as a molecular marker. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 It is a map of the plant expression vector pBioT004.

[0016] Figure 2 It is a schematic diagram of the sequence flanking the left border (LB).

[0017] Figure 3 It is a schematic diagram of the sequence flanking the right border (RB).

[0018] Figure 4Shows the result of amplifying an amplification product of about 387 bp using primer pairs with sequences SEQ ID NO:11 and SEQ ID NO:12 respectively with the receptor material C01 DNA as a template, and comparing the result with the flanking sequence of the CSJ4351 event after sequencing.

[0019] Figure 5 It is a schematic diagram of the inserted sequence and the flanking sequence.

[0020] Figure 6 Shows the results of amplifying maize samples of CSJ4351 (T1 generation), CSJ4351 (T2 generation), CSJ4351 (T3 generation), and C01 using the primer pairs CSJ4351LB5 / CSJ4351LB3 respectively. In the figure: M, marker, DL2000 plus; 1: CSJ4351-1 (the - after CSJ4351 indicates the generation, and the number indicates the plant number, the same below), 2: CSJ4351-2, 3: CSJ4351-1-1, 4: CSJ4351-1-2, 5: CSJ4351-1-1-1, 6: CSJ4351-1-1-1, 7: C01, the positive amplification band is about 854 bp.

[0021] Figure 7 Shows the results of amplifying maize samples of CSJ4351 (T1 generation), CSJ4351 (T2 generation), CSJ4351 (T3 generation), and C01 using the primer pairs CSJ4351RB5 / CSJ4351RB3 respectively. In the figure: M, marker, DL2000 plus; 1: CSJ4351-1, 2: CSJ4351-2, 3: CSJ4351-1-1, 4: CSJ4351-1-2, 5: CSJ4351-1-1-1, 6: CSJ4351-1-1-1, 7: C01, the positive amplification band is about 564 bp. Detailed implementation manners

[0022] Unless otherwise specified, all technical and scientific terms used herein have the meanings commonly understood by those of ordinary skill in the art.

[0023] The present invention provides a maize transformation event CSJ4351, as well as primer pairs and methods for its detection. Maize plants carrying the CSJ4351 transformation event contain the junction of the exogenous inserted DNA sequence and the maize genomic DNA sequence at chromosome 3. Using the exogenous inserted DNA sequence and the DNA sequences of the junction regions on its flanking maize genome, detection primers can be designed for specific detection of the CSJ4351 event. The detection method for the CSJ4351 event can provide a convenient means for tracking the specific gene insertion event for breeding applications using this plant event, and this specific gene insertion event can be used as a molecular marker to improve the efficiency of introgressive breeding for insect resistance and herbicide tolerance using this transformation event.

[0024] In the present invention, "transformation event" refers to the event in which an exogenous target gene is transformed into maize cells by the Agrobacterium-mediated genetic transformation method (well-known to those skilled in the art), and further the exogenous DNA sequence is inserted and integrated at a specific position in the genome of the transgenic maize plant obtained; the preferred maize transformation event CSJ4351 of the present invention has molecular characteristics with unique genetic information and insect resistance and herbicide tolerance characteristics, and is suitable for the cultivation and application of insect-resistant and herbicide-tolerant maize.

[0025] "Transformation event" is not a plant cell or plant. A plant cell or plant is merely a carrier for the existence of the transformation event; the core feature of the transformation event is a characteristic DNA sequence formed by the connection of the exogenous inserted sequence and a specific maize genomic sequence resulting from the insertion of the exogenous gene at a specific locus in the plant genome, and it exhibits specific application attributes. Although the exogenous target gene vectors are the same, different transformation events exhibit different genetic traits due to different insertion positions of the exogenous gene vectors. This difference comes from two aspects. On the one hand, the exogenous gene has different expression patterns due to the position effect of insertion; on the other hand, the insertion of the exogenous gene changes the gene structure of the recipient plant, resulting in changes in the genetic traits of the recipient plant. Seeking intellectual property protection for transgenic crops through patents related to transformation events is a new trend internationally. Because a transformation event entering commercialization is a unique biological creation obtained by the inventor through screening and identification, the integration of the exogenous gene with a specific position in the genome of the recipient crop not only endows the transformation event with unique molecular characteristics but also has a unique exogenous gene expression pattern, different from other transformation events obtained during the research process, and has characteristics more suitable for commercial application, thus having obvious novelty, practicality, and creativity. Currently, there are already many transformation events internationally that have obtained intellectual property protection, all owned by some multinational companies, and some have also applied for patents in China, such as the Chinese patent ZL02802047.2.

[0026] "T-DNA insertion sequence" refers to an exogenous DNA sequence (including the iaaM protein coding sequence) that can be transferred into a plant with the aid of the T-DNA sequence of Agrobacterium. Naming this insertion sequence as the T-DNA insertion sequence only indicates that the method of the present invention achieves plant transformation with the aid of Agrobacterium, and does not limit the maize transformation event of the present invention to only being achievable through the T-DNA sequence.

[0027] "Upstream flanking maize genomic sequence" refers to a sequence in the maize genome itself. In the plant genome including the maize transformation event of the present invention, it is located upstream (5' end) of the T-DNA insertion sequence and adjacent to the 5' end of the T-DNA insertion sequence. In this way, the insertion position of the T-DNA insertion sequence in the maize genome can be determined. In some embodiments, the upstream flanking maize genomic sequence is the 1-279 bp sequence of the sequence shown in SEQ ID NO: 13. In other embodiments, the upstream flanking maize genomic sequence is the 3' end sequence of the 1-279 bp sequence of the sequence shown in SEQ ID NO: 13 with a length of not less than 100 bp (such as 120 bp, 140 bp, 160 bp, 180 bp, etc.). Those skilled in the art can understand that as long as the length of this 3' end sequence is sufficient to determine the genomic position in the maize genome, especially on chromosome 3 (and thus determine the insertion position of the T-DNA insertion sequence), therefore, its length may even be less than 100 bp.

[0028] "Downstream flanking maize genomic sequence" refers to a sequence in the maize genome itself. In the plant genome including the maize transformation event of the present invention, it is located downstream (3' end) of the T-DNA insertion sequence and adjacent to the 3' end of the T-DNA insertion sequence. In this way, the insertion position of the T-DNA insertion sequence in the maize genome can also be determined. In some embodiments, the downstream flanking maize genomic sequence is the 13211-13721 bp sequence of the sequence shown in SEQ ID NO: 13. In other embodiments, the upstream flanking maize genomic sequence is the 5' end sequence of the 13211-13721 bp sequence of the sequence shown in SEQ ID NO: 13 with a length of not less than 100 bp (such as 200 bp, 300 bp, 400 bp, 500 bp, etc.). Those skilled in the art can understand that as long as the length of this 5' end sequence is sufficient to determine the genomic position in the maize genome, especially on chromosome 3 (and thus determine the insertion position of the T-DNA insertion sequence), therefore, its length may even be less than 100 bp.

[0029] When referring to a primer pair, the "specific recognition" used herein means that the first primer in the primer pair is complementary or identical to a partial upstream sequence of the given template, and the second primer in the primer pair is identical or complementary to a partial downstream sequence of the given template, so as to perform PCR amplification to obtain an amplicon including the partial upstream sequence, the partial downstream sequence, and the sequence therebetween.

[0030] The inventors obtained a maize plant transformation event that confers insect resistance and herbicide tolerance in maize through a transgenic method, named CSJ4351. The representative seeds of this plant transformation event maize ( Zea mays ) were deposited with the General Microbiology Center of the China Committee for Culture Collection of Microorganisms (CGMCC, address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing) on March 21, 2025, and the deposit number is: CGMCC No. 30704.

[0031] In the first aspect of the present invention, there is provided a maize transformation event CSJ4351, characterized in that a DNA sequence on chromosome 2 of group D is as shown in SEQ ID NO: 13, which is composed of a T-DNA insertion sequence from 280 - 13210 bp, an upstream flanking maize genomic sequence from 1 - 279 bp, and a downstream flanking maize genomic sequence from 13211 - 13721 bp.

[0032] In the second aspect of the present invention, there is provided a DNA sequence, which at least includes part or all of the T-DNA insertion sequence and part or all of the flanking maize genomic sequence. This characteristic DNA sequence fragment can be detected by the method provided in the fourth aspect of the present invention.

[0033] In the third aspect of the present invention, there is provided a primer pair for detecting the maize transformation event described in the first aspect of the present invention and the characteristic DNA sequence fragment described in the second aspect of the present invention, which is composed of a first primer that specifically recognizes the flanking sequence on any side described in the first aspect of the present invention and a second primer that specifically recognizes the T-DNA insertion sequence described in the first aspect of the present invention. In some embodiments, the sequence of the first primer is SEQ ID NO: 14, and the sequence of the second primer is SEQ ID NO: 15; or the sequence of the first primer is SEQ ID NO: 16, and the sequence of the second primer is SEQ ID NO: 17.

[0034] In the fourth aspect of the present invention, there is provided a method for identifying the transformation event CSJ4351 in a maize biological sample, which includes: (a) Extracting a DNA sample from the maize biological sample to be identified; (b) Using the primer pair described in the third aspect of the present invention for PCR amplification with the extracted DNA sample as a template; (c) Detect the PCR amplification product. If the length of the amplification product is consistent with the theoretical length between the sequences of the PCR primer pair described in SEQ ID NO: 13, it indicates the presence of the CSJ4351 transformation event in the maize biological sample.

[0035] The fifth aspect of the present invention provides a method for obtaining insect-resistant and herbicide-tolerant transgenic maize materials, including: After hybridizing the maize material containing the maize transformation event described in the first aspect of the present invention with other maize breeding materials, preferably further backcrossing, a new material containing the transformation event described in the first aspect of the present invention is obtained; During the hybridization or backcrossing process, use the method described in the fourth aspect of the present invention to screen and identify in the offspring population to confirm the presence of the transformation event described in the first aspect of the present invention.

[0036] The sixth aspect of the present invention provides the use of the transformation event described in the first aspect of the present invention, the fragment of the characteristic DNA sequence of the transformation event described in the second aspect of the present invention, and the methods described in the fourth and fifth aspects of the present invention for improving insect resistance and herbicide tolerance traits, for plant breeding, and as molecular markers.

[0037] This new transformation event of the present invention that enhances insect resistance and herbicide tolerance can express insect-resistant and herbicide-tolerant proteins and can be used to significantly improve the agronomic traits and quality of maize.

[0038] The present invention will be further described below in conjunction with non-limiting examples.

[0039] Example 1. Genetic transformation of maize The method used to obtain the maize transformation event in this study is the Agrobacterium-mediated method, and the transformation is carried out according to the method and culture medium formula reported by Frame et al. (Frame BR et.al ., Plant Physiol, 2002, 129: 13-22), and glufosinate is used as the screening reagent.

[0040] The specific steps are as follows: (1) Take the parental maize ears 10-12 days after pollination and collect immature embryos with a size of 1.0-1.5 mm. Co-culture the Agrobacterium containing the transformation vector with the immature embryos at 22 °C for 2-3 days.

[0041] (2) Transfer the immature embryos cultured in step (1) to a callus induction medium containing a final concentration of 200 mg / L ticarcillin antibiotic (GlaxoSmithKline, USA), and incubate in the dark at 28 °C for 10-14 days to kill Agrobacterium.

[0042] (3) Transfer all the calli after the induction culture in step (2) to a selection medium containing glufosinate-ammonium with a final concentration of 3 mg / L, and culture them in the dark at 28 °C for 2 - 3 weeks.

[0043] (4) Transfer all the calli in step (3) to a fresh selection medium containing 3 mg / L glufosinate-ammonium, and culture them in the dark at 28 °C for 2 - 3 weeks.

[0044] (5) Then, transfer the surviving embryogenic tissues in step (4) to a regeneration medium, and culture them in the dark at 28 °C for 10 - 14 days, with one strain per petri dish.

[0045] (6) Transfer the embryogenic tissues in step (5) to a fresh regeneration medium, and culture them under light at 26 °C for 10 - 14 days.

[0046] (7) Transfer all the fully developed plants in step (6) to a rooting medium, and culture them under light at 26 °C until the roots are fully developed. Transplant the regenerated seedlings with roots to the greenhouse for growth and propagation for screening and analysis.

[0047] Example 2. Field experiment of transgenic maize According to the method of Example 1, 1028 independent transformants of insect-resistant and glufosinate-ammonium-tolerant maize were obtained through Agrobacterium-mediated transformation, numbered CSJ4001 - CSJ41028. Screen for the presence of the glufosinate-ammonium tolerance gene using the ELISA method pat , insect-resistant gene cry2Ab and insect-resistant gene cry1FAt the same time, maize transformants with relatively high expression were preliminarily tested for insect resistance and glufosinate tolerance in the greenhouse: by spraying 2000 PPM of glufosinate, the transformants with poor glufosinate resistance were removed, and the remaining transformants were inoculated with Ostrinia furnacalis, and the transformants without damage by Ostrinia furnacalis were screened. A total of 28 maize transformants with strong insect resistance and glufosinate tolerance containing transformation event numbers "CSJ4008", "CSJ4048", "CSJ4051", "CSJ4104", "CSJ4134", "CSJ4152", "CSJ4253", "CSJ4301", "CSJ4351", "CSJ4357", "CSJ4359", "CSJ4402", "CSJ4404", "CSJ4405", "CSJ4406", "CSJ4408", "CSJ4415", "CSJ4445", "CSJ4447", "CSJ4449", "CSJ4490", "CSJ4531", "CSJ4553", "CSJ4694", "CSJ4703", "CSJ4758", "CSJ4840", "CSJ4966" were obtained. The transformants containing independent transformation events of insect resistance and herbicide tolerance were respectively reserved for seed and subjected to subsequent screening and analysis.

[0048] (1) Glufosinate resistance screening Twenty-eight T1 and T2 generations of transgenic maize containing transformation events were selected for comparison of glufosinate tolerance. Twenty to thirty days after the germination of transgenic maize and parental non-transgenic control seeds, when they grew to the 4-5 leaf stage, glufosinate with a final concentration of 4000 PPM was sprayed, and the application rate was 35 L / mu. After 10 days, the growth and development and mortality of maize were recorded. The results of the glufosinate spraying test are shown in Table 1. The transgenic lines all had obvious glufosinate tolerance. Among them, the tolerance levels of those containing transformation events "CSJ4008", "CSJ4051", "CSJ4253", "CSJ4301", "CSJ1031", "CSJ4357", "CSJ4402", "CSJ4404", "CSJ4405", "CSJ4406", "CSJ4408", "CSJ4415", "CSJ4445", "CSJ4449", "CSJ4531", "CSJ4553", "CSJ4694", "CSJ4758", "CSJ4966" were high. Table 1. Maize glufosinate tolerance test (2) Insect resistance test The T1 and T2 generations of transgenic maize with 28 transformation events such as "CSJ4008" were respectively selected for insect resistance analysis. After the transgenic maize and the parental non-transgenic control germinated in the greenhouse, when it was determined to be transgenic by spraying glufosinate ammonium with a final concentration of 4000 PPM 20 days after germination, 10 plants were taken from each, and 10 first-instar corn armyworms were inoculated on each plant. The corn armyworms were from the Corn Pest Group of the Institute of Plant Protection, Chinese Academy of Agricultural Sciences. The egg masses were incubated under the conditions of 28±1°C, RH 70±5%, 16h:8h (L:D), and the larvae within 12 hours after hatching were selected for bioassay experiments. The damage situation of corn armyworms was investigated 7 days after inoculation, and insect resistance grading was carried out. The insect resistance grading adopted a 9-level standard (Announcement No. 953-10.1-2007 Detection of Environmental Safety of Transgenic Plants and Their Products Insect-Resistant Maize Part 1). The results are shown in Table 2. "CSJ4051", "CSJ4351", "CSJ4406", "CSJ4408", "CSJ4415", "CSJ4445", "CSJ4449" and "CSJ4758" have high insect resistance performance.

[0049] Table 2: Identification of Resistance Levels of Different Transformation Events to Corn Armyworms Example 3. Flanking Sequence Analysis Sample Preparation: The genomic DNA of the maize material containing the CSJ4351 transformation event was extracted by the plant DNA extraction method well-known to those skilled in the art. 2.5 µg of DNA was taken and digested with Pvu II for 6-8 hours. After purification by alcohol precipitation, an appropriate amount of water was added to dissolve it.

[0050] Ligation of Adaptors: According to the analysis of the vector restriction sites, two pairs of adaptors were respectively designed and synthesized: GenomeWalker Adaptor +, GenomeWalker Adaptor – (the sequences are shown in SEQ ID NO: 1 and SEQ ID NO: 2 respectively), in which the 5' end of SEQ ID NO: 2 was phosphorylated and the 3' end was added with an amino group.

[0051] Equally mix GenomeWalker Adaptor + and GenomeWalker Adaptor –, incubate at 70°C for 10 minutes, and then slowly cool to room temperature. Take 4 μl of the digested and purified DNA and add it to a tube containing 1.9 μl of GenomeWalker Adaptor (25 μM), 1.6 μl of 10X ligation buffer, and 0.5 μl of T4 DNA ligase (6 units / μl). Incubate for ligation overnight at 16°C, stop the reaction, incubate at 70°C for 5 min. In each tube, add 72 μl of TE (10 / 1, pH 7.5), and shake at low speed for 5 - 10 s.

[0052] Analysis of the left border (LB) end sequence: Use the Clontech GenomeWalker TM Universal kit. Using primers AP1 and GSP1 (sequences are shown as SEQ ID NO:3 and SEQ ID NO:4 respectively), with the ligation product as the template, perform the first round of amplification: 7 cycles: 94°C for 25 s, 72°C for 4 min; 32 cycles: 94°C for 25 s, 67°C for 4 min; after the last cycle, incubate at 67°C for 7 minutes. After diluting the PCR product 50 - fold, use primers AP2 and GSP2 (sequences are shown as SEQ ID NO: 5 and SEQ ID NO: 6 respectively) for the second round of PCR amplification. The PCR program is as follows: 5 cycles: 94°C for 25 s, 72°C for 5 min; 20 cycles: 94°C for 25 s, 67°C for 4 min; after the last cycle, incubate at 67°C for 10 minutes. Recover the product for sequencing.

[0053] SEQ ID NO: 1 GTAATACGACTCACTATAGGGCACGCGTGGTCGACGGCCCGGGCTGGT SEQ ID NO: 2 ACCAGCCC SEQ ID NO: 3 GTAATACGACTCACTATAGGGC SEQ ID NO: 4 TAGAGCTTTCGTTCGTATCATCGGT SEQ ID NO: 5 ACTATAGGGCACGCGTGGT SEQ ID NO: 6 CATCAGTTTCATTGCGCACACACC Analysis of the left border (LB) end sequence of the CSJ4351 event is as Figure 2As shown, a nucleotide sequence of 1204 bp was obtained (the sequence is as shown in SEQ ID NO:7). From the 1st bp to the 595th bp is the partial sequence of the E9 terminator, from the 596th bp to the 620th bp is the loxP site, from the 621st bp to the 693rd bp is the intercalated sequence (a non-specific DNA sequence required for genetic manipulation, from T-DNA), and from the 694th bp to the 1204th bp is the maize DNA sequence.

[0054] Sequence description of SEQ ID NO: 7: 1-595 Partial sequence of E9 terminator 596-620 loxP 621-693 Intercalated sequence 694-1204 Maize DNA 1 CATCAGTTTC ATTGCGCACA CACCAGAATC CTACTGAGTT TGAGTATTAT GGCATTGGGA 61 AAACTGTTTT TCTTGTACCA TTTGTTGTGC TTGTAATTTA CTGTGTTTTT TATTCGGTTT 121 TCGCTATCGA ACTGTGAAAT GGAAATGGAT GGAGAAGAGT TAATGAATGA TATGGTCCTT 181 TTGTTCATTC TCAAATTAAT ATTATTTGTT TTTTCTCTTA TTTGTTGTGT GTTGAATTTG 241 AAATTATAAG AGATATGCAA ACATTTTGTT TTGAGTAAAA ATGTGTCAAA TCGTGGCCTC 301 TAATGACCGA AGTTAATATG AGGAGTAAAA CACTTGTAGT TGTACCATTA TGCTTATTCA 361 CTAGGCAACA AATATATTTT CAGACCTAGA AAAGCTGCAA ATGTTACTGA ATACAAGTAT 421 GTCCTCTTGT GTTTTAGACA TTTATGAACT TTCCTTTATG TAATTTTCCA GAATCCTTGT 481 CAGATTCTAA TCATTGCTTT ATAATTATAG TTATACTCAT GGATTTGTAG TTGAGTATGA 541 AAATATTTTT TAATGCATTT TATGACTTGC CAATTGATTG ACAACATGCA TCAATATAAC 601 TTCGTATAAT GTATGCTATA CGAAGTTATG AATTCCCCCG AATTAATTCG GCGTTAATTC 661 AGTACATTAA AAACGTCCGC AATGTGTTAT TAA GTTTGCG TCTCCCACCG TACAGATCAG 721 CTAGCTTTTA ATTACCTGGT GGTACGTACA GACACAGTCC TCGTCGTCCT CCTCCACCGC 781 CCGCCCCCCG TGGATCTTCA CGCCTCCTGG GCGGTGCACG GTGGGCGGTG GCACTTTTAG 841 TTAGTGCCGG CTGCATCTTT AGCGCAAAAG TTTGCAACTA CCTGATACAG AGCCCCGGCT 901 TCACCGCCAT GTTCATCCAA ACGCCTGCTT TTTCGCGAAT CCGGCTCCCA ACTTTTTCAA 961 TAACTGCGAG CCTGTATAGC ATCAGATTCT GGTGGATTCT GCGTTCCGTA GGCTGTAGCA 1021 ATTGCATGCG GGAAGATTTT CAGAACAATT TCTTTCCGCG ATGCAACTTG AGAACCAGTA 1081 GCTGAGCCGG GCCGCTTTGT TGTATATTAT TGCGTGCAGT GACGGAGCTG GTGGAAGGGG 1141 ACTCGTCGCG GAAGATCGTG GAGATCATCT GCCGGACGAG CCTGCTCAAG TCGGAGAGCA 1201 GCTG (SEQ ID NO: 7) Right border (RB) end sequence analysis: According to the obtained maize flanking sequences, comparative analysis was carried out using the published maize genome sequence (https: / / phytozome-next.jgi.doe.gov / blast-search), and the upstream primer CSJ4351RBF (SEQ ID NO: 8) was designed. Using CSJ4351 genomic DNA as a template, PCR amplification was performed with CSJ4351RBM and the downstream primer (shown in SEQ ID NO: 9). The PCR program was as follows: 35 cycles: 94°C for 25 s, 62°C for 25 s, 72°C for 1 min; after the last cycle, incubate at 72°C for 5 minutes. The product was recovered and sequenced.

[0055] SEQ ID NO: 8 GCACGCTCCG CCCTGGCACG C SEQ ID NO: 9 GGAGCTCAGAGCCAATCAGCGTCA The right boundary (RB) end sequence analysis of CSJ4351 event is as follows Figure 3 As shown, a nucleotide sequence of 4093 bp was obtained (the sequence is as shown in SEQ ID NO: 10), including a maize DNA sequence from 1 bp to 279 bp, an E9 terminator sequence from 280 bp to 829 bp, a pat gene sequence from 830 bp to 1384 bp, an intercalated sequence from 1385 bp to 1396 bp, a 35S promoter sequence from 1397 bp to 1689 bp, an intercalated sequence from 1690 bp to 1729 bp, an Ara5 terminator sequence from 1730 bp to 1942 bp, a cry1F partial sequence from 1942 bp to 2489 bp, a Ubit10 promoter sequence from 2490 bp to 3900 bp, and a CTP2 leader peptide sequence from 3901 bp to 4093 bp.

[0056] Description of SEQ ID NO:10 sequence: 1 - 279 Maize DNA sequence 280 - 829 E9 terminator 830 - 1384 pat gene 1385 - 1396 Intercalated sequence 1397 - 1689 35S promoter 1690 - 1729 Intercalated sequence 1730 - 1942 Ara5 terminator 1943 - 2489 cry1F partial sequence 2490 - 3900 Ubit10 promoter 3901 - 4093 CTP2 leader peptide 1 GCACGCTCCG CCCTGGCACG CCGGGCCCAG CGTGGGCCGG ACACGGACTG CCGTACAGCG 61 GCTCCGTGCG GGGCAGCGTG CGCTGCACGC CGCCGAGGTC GCCGAACGTG ATGCTCGAGA 121 GGAATGGCTC AGTGGTGAAC GGCCACCGCG CGTCATGCGA CGAAACCGCT AAAAACGGCG 181 CCAGCAAGGG CTCCGGTGGG CTCAGCTGCC ACAAGTGCGG CGAGCAGTTC AGCAAGTGGG 241 AGGCGCTGGA GGCGCACCAC CTCTCCAAAC ACGCAGGTG A TAAAGCAATG ATTAGAATCT 301 GACAAGGATT CTGGAAAATT ACATAAAGGA AAGTTCATAA ATGTCTAAAA CACAAGAGGA 361 CATACTTGTA TTCAGTAACA TTTGCAGCTT TTCTAGGTCT GAAAATATAT TTGTTGCCTA 421 GTGAATAAGC ATAATGGTAC AACTACAAGT GTTTTACTCC TCATATTAAC TTCGGTCATT 481 AGAGGCCACG ATTTGACACA TTTTTACTCA AAACAAAATG TTTGCATATC TCTTATAATT 541 TCAAATTCAA CACACAACAA ATAAGAGAAA AAACAAATAA TATTAATTTG AGAATGAACA 601 AAAGGACCAT ATCATTCATT AACTCTTCTC CATCCATTTC CATTTCACAG TTCGATAGCG 661 AAAACCGAAT AAAAAACACA GTAAATTACA AGCACAACAA ATGGTACAAG AAAAACAGTT 721 TTCCCAATGC CATAATACTC AAACTCAGTA GGATTCTGGT GTGTGCGCAA TGAAACTGAT 781 GCATTGAACT TGACGAACGT TGTCGAAACC GATGATACGA ACGAAAGCTC TATCAAATCT 841 GGGTCACGGG GCGAACCGGG CGCGGAGGCG CGGGCAGCTC GAAGTCCCTC TGCCAGAACC 901 CGACGTCGTG CCAGCCACCG TGCTTGTAGC CGGCTGCGCG CAGCGTGCCC CGAGCGGTGT 961 AGCCCAGGGC TTCGTGGAGC CTCACGGAGG GATCGTTCGG GAGGCCGATG ACGGCGACCA 1021 CGCTCTTGAA GCCTTGCGCC TCCATCGACT TGAGCAGGTG CGTGTACAGCGTGGAGCCGA 1081 GGCCCAGGCG CTGGTGGCGG TGGGACACGT AGACGGTGCT CTCGACGGTCCAGTCGTAGG 1141 CGTTGCGAGC CTTCCACGGC CCGGCGTAGG CGATGCCGGC CACGACGCCTTCGACCTCCG 1201 CGACGAGCCA GGGGTAGCGG TCCTGGAGGC GCTCAAGGTC GTCGATCCACTCCTGCGGCG 1261 TCTGCGGCTC GGTGCGGAAG TTCACGGTGG ACGTTTCGAT GTAGTGGTTGACGATGTCGC 1321 ACACCGCAGC CATGTCGGCT GCTGTGGCCG GGCGGATCTC GACGGGCCTGCGCTCGGGCG 1381 ACATGGTTCA GCGTGTCCTC TCCAAATGAA ATGAACTTCC TTATATAGAGGAAGGGTCTT 1441 GCGAAGGATA GTGGGATTGT GCGTCATCCC TTACGTCAGT GGAAGTATCACATCAATCCA 1501 CTTGCTTTGA AGACGTGGTT GGAACGTCTT CTTTTTCCAC GATGCTCCTCGTGGGTGGGG 1561 GTCCATCTTT GGGACCACTG TCGGCAGAGG CATCTTGAAT GATAGCCTTTCCTTTATCGC 1621 AATGATGGCA TTTGTAGGTG CCACCTTCCT TTTCTACTGT CCTTTTGATGAAGTGACAGG 1681 TAGGATCGGA TAACTTCGTA TAGCATACAT TATACGAAGT TATGAGCTCGGCACCCTCCT 1741 TAGCATGACA TTCCATTTTT CAATCTCATC ATCAAATCAA AGAACAACGACGTACGGATA 1801 GCTAGCGAAT CATCAAGATT TATAGAGAGG AACATCGGAG TTCATCATTTATTCACCACA 1861 CATATGCTTC CAAACAAAAG GAAAACACTA ATTATTTTAG TAACCACACCACATACATGC 1921 ACAGCTAGCT ACAGTAGCCT AGTCATTACT CCTCCATCAG GAGCAGCTCCACGCTGTCCA 1981 CAATGAACGT GCCCTCGGTT TCGCCGATCT CGATCCACAC TTTGTCGGTCTCAGGGAAGT 2041 ACTCAAGCTC CTTGGTGACG TAGCCCGCTG GAAGCGGTGT GTAGTCCCCGTAGCCCCGGT 2101 TCGACTCGCA GGGGTTGTCG CGGCGGCCAT CGGTGTAGGA CTTCTCTTCATAGACGGAGG 2161 CGTAGTCCGC GGGGACGGAG GAGTTCGAGC CGTAGGTTTC GTCGTAGCCGCGGTTACGGC 2221 TCGTGTAGGC CCCGCCGTAC TCTTCCTGGT TCGCGGTGTA GTCGTTGCACGTAACGGTGT 2281 TGTTGGGGTA GACTTCTTCC TCGACGCAGT TGGAGAACTT CAGCTCGTCGGTGTTGTTCT 2341 CGATCTCGTG GATGGTGACG CAACCCTCGC CGTACCCCTC CTTGTAGGCGGTCACGCGGA 2401 GGATGTAGCC GCGCCCTGGG CAGACGCGGA CCTCTTGGGA CACTTCGGCTTCCCACTCGG 2461 GGACCACCAG GACGGAGCGG TGGTTGTTCC AGCCTGTTAA ACGCCGTCGACGAGTCTAAC 2521 GGACACCAAC CAGCGAACCA GCAGCGTCGC GTCGGGCCAA GCGAAGCAGACGGCACGGCA 2581 TCTCTGTCGC TGCCTCTGGA CCCCTCTCGA GAGTTCCGCT CCACCGTTGGACTTGCTCCG 2641 CTGTCGGCAT CCAGAAATTG CGTGGCGGAG CGGCAGACGT GAGCCGGCACGGCAGGCGGC 2701 CTCCTCCTCC TCTCACGGCA CCGGCAGCTA CGGGGGATTC CTTTCCCACCGCTCCTTCGC 2761 TTTCCCTTCC TCGCCCGCCG TAATAAATAG ACACCCCCTC CACACCCTCTTTCCCCAACC 2821 TCGTGTTGTT CGGAGCGCAC ACACACACAA CCAGATCTCC CCCAAATCCACCCGTCGGCA 2881 CCTCCGCTTC AAGGTACGCC GCTCGTCCTC CCCCCCCCCT CTCTACCTTCTCTAGATCGG 2941 CGTTCCGGTC CATGGTTAGG GCCCGGTAGT TCTACTTCTG TTCATGTTTGTGTTAGATCC 3001 GTGTTTGTGT TAGATCCGTG CTGCTAGCGT TCGTACACGG ATGCGACCTGTACGTCAGAC 3061 ACGTTCTGAT TGCTAACTTG CCAGTGTTTC TCTTTGGGGA ATCCTGGGATGGCTCTAGCC 3121 GTTCCGCAGA CGGGATCGAT TTCATGATTT TTTTTGTTTC GTTGCATAGGGTTTGGTTTG 3181 CCCTTTTCCT TTATTTCAAT ATATGCCGTG CACTTGTTTG TCGGGTCATCTTTTCATGCT 3241 TTTTTTTGTC TTGGTTGTGA TGATGTGGTC TGGTTGGGCG GTCGTTCTAGATCGGAGTAG 3301 AATTCTGTTT CAGACTACCT GGTGGATTTA TTAATTTTGG ATCTGTATGTGTGTGCCATA 3361 CATATTCATA GTTACGAATT GAAGATGATG GATGGAAATA TCGATCTAGGATAGGTATAC 3421 ATGTTGATGC GGGTTTTACT GATGCATATA CAGAGATGCT TTTTGTTCGCTTGGTTGTGA 3481 TGATGTGGTG TGGTTGGGCG GTCGTTCATT CGTTCTAGAT CGGAGTAGAATACTGTTTCA 3541 AACTACCTGG TGTATTTATT AATTTTGGAA CTGTATGTGT GTGTCATACATCTTCATAGT 3601 TACGAGTTTA AGATGGATGG AAATATCGAT CTAGGATAGG TATACATGTTGATGTGGGTT 3661 TTACTGATGC ATATACATGA TGGCATATGC AGCATCTATT CATATGCTCTAACCTTGAGT 3721 ACCTATCTAT TATAATAAAC AAGTATGTTT TATAATTATT TTGATCTTGATATACTTGGA 3781 TGATGGCATA TGCAGCAGCT ATATGTGGAT TTTTTTAGCC CTGCCTTCATACGCTATTTA 3841 TTTGCTTGGT ACTGTTTCTT TTGTCGATGC TCACCCTGTT GTTTGGTGTTACTTCTGCAG 3901 ACCATGGCGC AAGTTAGCAG GATCTGCAAC GGCGTGCAGA ACCCGTCGCTCATCTCCAAC 3961 CTCTCGAAAT CCAGTCAACG CAAATCGCCC TTGTCGGTCT CCCTGAAGACGCAGCAGCAC 4021 CCGCGAGCCT ACCCGATCTC GTCGTCGTGG GGCTTGAAGAGCGGGATGACGCTGATT 4081 GGCTCTGAGCTCC (SEQ ID NO:10) Using the receptor material C01 DNA as a template, primers CSJ4351J14F (sequence as SEQ ID NO: 11) and CSJ4351J14R (sequence as SEQ ID NO: 12) were designed on the LB and RB ends of the inserted sequence, respectively, to obtain an amplified product of about 387 bp and sequence it. Comparison with the flanking sequence of the CSJ4351 event revealed that the event was obtained by replacing 19 bp of the original genome sequence (SEQ ID NO: 18 AGACTGCTGCATCCGCGTT) with the inserted sequence. A screenshot of the alignment result is shown in Figure 4 .

[0057] SEQ ID NO:11 CTCAGCTGCC ACAAGTGCGGCG SEQ ID NO:12 GATGCTATAC AGGCTCGCAG T Based on the above results, a person skilled in the art can easily derive the characteristic DNA sequence of the CSJ4351 event (SEQ ID NO: 13), as shown below. The underlined portion shows the T-DNA insertion sequence, and the ununderlined portion shows the flanking maize genomic DNA sequence of the insertion sequence. The schematic diagram of the insertion sequence and the flanking sequence is shown in FIG. Figure 5 shown.

[0058] SEQ ID NO:13: 1 GCACGCTCCG CCGTGGCACG CCGGGCCCAG CGTGGGCCGG ACACGGACTG CCGTACAGCG 61 GCTCCGTGCG GGGCAGCGTG CGCTGCACGC CGCCGAGGTC GCCGAACGTG ATGCTCGAGA 121 GGAATGGCTC AGTGGTGAAC GGCCACCGCG CGTCATGCGA CGAAACCGCT AAAAACGGCG 181 CCAGCAAGGG CTCCGGTGGG CTCAGCTGCC ACAAGTGCGG CGAGCAGTTC AGCAAGTGGG 241 AGGCGCTGGA GGCGCACCAC CTCTCCAAAC ACGCAGGTG A TAAAGCAATG ATTAGAATCT 301 GACAAGGATT CTGGAAAATT ACATAAAGGA AAGTTCATAA ATGTCTAAAA CACAAGAGGA 361 CATACTTGTA TTCAGTAACA TTTGCAGCTT TTCTAGGTCT GAAAATATAT TTGTTGCCTA 421 GTGAATAAGC ATAATGGTAC AACTACAAGT GTTTTACTCC TCATATTAAC TTCGGTCATT 481 AGAGGCCACG ATTTGACACA TTTTTACTCA AAACAAAATG TTTGCATATC TCTTATAATT 541 TCAAATTCAA CACACAACAA ATAAGAGAAA AAACAAATAA TATTAATTTG AGAATGAACA 601 AAAGGACCAT ATCATTCATT AACTCTTCTC CATCCATTTC CATTTCACAG TTCGATAGCG 661 AAAACCGAAT AAAAAACACA GTAAATTACA AGCACAACAA ATGGTACAAG AAAAACAGTT 721 TTCCCAATGC CATAATACTC AAACTCAGTA GGATTCTGGT GTGTGCGCAA TGAAACTGAT 781 GCATTGAACT TGACGAACGT TGTCGAAACC GATGATACGA ACGAAAGCTC TATCAAATCT 841 GGGTCACGGG GCGAACCGGG CGCGGAGGCG CGGGCAGCTC GAAGTCCCTC TGCCAGAACC 901 CGACGTCGTG CCAGCCACCG TGCTTGTAGC CGGCTGCGCG CAGCGTGCCC CGAGCGGTGT 961 AGCCCAGGGC TTCGTGGAGC CTCACGGAGG GATCGTTCGG GAGGCCGATG ACGGCGACCA 1021 CGCTCTTGAA GCCTTGCGCC TCCATCGACT TGAGCAGGTG CGTGTACAGC GTGGAGCCGA 1081 GGCCCAGGCG CTGGTGGCGG TGGGACACGT AGACGGTGCT CTCGACGGTC CAGTCGTAGG 1141 CGTTGCGAGC CTTCCACGGC CCGGCGTAGG CGATGCCGGC CACGACGCCT TCGACCTCCG 1201 CGACGAGCCA GGGGTAGCGG TCCTGGAGGC GCTCAAGGTC GTCGATCCAC TCCTGCGGCG 1261 TCTGCGGCTC GGTGCGGAAG TTCACGGTGG ACGTTTCGAT GTAGTGGTTG ACGATGTCGC 1321 ACACCGCAGC CATGTCGGCT GCTGTGGCCG GGCGGATCTC GACGGGCCTG CGCTCGGGCG 1381 ACATGGTTCA GCGTGTCCTC TCCAAATGAA ATGAACTTCC TTATATAGAG GAAGGGTCTT 1441 GCGAAGGATA GTGGGATTGT GCGTCATCCC TTACGTCAGT GGAAGTATCA CATCAATCCA 1501 CTTGCTTTGA AGACGTGGTT GGAACGTCTT CTTTTTCCAC GATGCTCCTC GTGGGTGGGG 1561 GTCCATCTTT GGGACCACTG TCGGCAGAGG CATCTTGAAT GATAGCCTTT CCTTTATCGC 1621 AATGATGGCA TTTGTAGGTG CCACCTTCCT TTTCTACTGT CCTTTTGATG AAGTGACAGG 1681 TAGGATCGGA TAACTTCGTA TAGCATACAT TATACGAAGT TATGAGCTCG GCACCCTCCT 1741 TAGCATGACA TTCCATTTTT CAATCTCATC ATCAAATCAA AGAACAACGA CGTACGGATA 1801 GCTAGCGAAT CATCAAGATT TATAGAGAGG AACATCGGAG TTCATCATTT ATTCACCACA 1861 CATATGCTTC CAAACAAAAG GAAAACACTA ATTATTTTAG TAACCACACC ACATACATGC 1921 ACAGCTAGCT ACAGTAGCCT AGTCATTACT CCTCCATCAG GAGCAGCTCC ACGCTGTCCA 1981 CAATGAACGT GCCCTCGGTT TCGCCGATCT CGATCCACAC TTTGTCGGTC TCAGGGAAGT 2041 ACTCAAGCTC CTTGGTGACG TAGCCCGCTG GAAGCGGTGT GTAGTCCCCG TAGCCCCGGT 2101 TCGACTCGCA GGGGTTGTCG CGGCGGCCAT CGGTGTAGGA CTTCTCTTCA TAGACGGAGG 2161 CGTAGTCCGC GGGGACGGAG GAGTTCGAGC CGTAGGTTTC GTCGTAGCCG CGGTTACGGC 2221 TCGTGTAGGC CCCGCCGTAC TCTTCCTGGT TCGCGGTGTA GTCGTTGCAC GTAACGGTGT 2281 TGTTGGGGTA GACTTCTTCC TCGACGCAGT TGGAGAACTT CAGCTCGTCG GTGTTGTTCT 2341 CGATCTCGTG GATGGTGACG CAACCCTCGC CGTACCCCTC CTTGTAGGCG GTCACGCGGA 2401 GGATGTAGCC GCGCCCTGGG CAGACGCGGA CCTCTTGGGA CACTTCGGCT TCCCACTCGG 2461 GGACCACCAG GACGGAGCGG TGGTTGTTCC AGCCTGTTAA ACGCCGTCGA CGAGTCTAAC 2521 GGACACCAAC CAGCGAACCA GCAGCGTCGC GTCGGGCCAA GCGAAGCAGA CGGCACGGCA 2581 TCTCTGTCGC TGCCTCTGGA CCCCTCTCGA GAGTTCCGCT CCACCGTTGG ACTTGCTCCG 2641 CTGTCGGCAT CCAGAAATTG CGTGGCGGAG CGGCAGACGT GAGCCGGCAC GGCAGGCGGC 2701 CTCCTCCTCC TCTCACGGCA CCGGCAGCTA CGGGGGATTC CTTTCCCACC GCTCCTTCGC 2761 TTTCCCTTCC TCGCCCGCCG TAATAAATAG ACACCCCCTC CACACCCTCT TTCCCCAACC 2821 TCGTGTTGTT CGGAGCGCAC ACACACACAA CCAGATCTCC CCCAAATCCA CCCGTCGGCA 2881 CCTCCGCTTC AAGGTACGCC GCTCGTCCTC CCCCCCCCCT CTCTACCTTC TCTAGATCGG 2941 CGTTCCGGTC CATGGTTAGG GCCCGGTAGT TCTACTTCTG TTCATGTTTG TGTTAGATCC 3001 GTGTTTGTGT TAGATCCGTG CTGCTAGCGT TCGTACACGG ATGCGACCTG TACGTCAGAC 3061 ACGTTCTGAT TGCTAACTTG CCAGTGTTTC TCTTTGGGGA ATCCTGGGAT GGCTCTAGCC 3121 GTTCCGCAGA CGGGATCGAT TTCATGATTT TTTTTGTTTC GTTGCATAGG GTTTGGTTTG 3181 CCCTTTTCCT TTATTTCAAT ATATGCCGTG CACTTGTTTG TCGGGTCATC TTTTCATGCT 3241 TTTTTTTGTC TTGGTTGTGA TGATGTGGTC TGGTTGGGCG GTCGTTCTAG ATCGGAGTAG 3301 AATTCTGTTT CAGACTACCT GGTGGATTTA TTAATTTTGG ATCTGTATGT GTGTGCCATA 3361 CATATTCATA GTTACGAATT GAAGATGATG GATGGAAATA TCGATCTAGG ATAGGTATAC 3421 ATGTTGATGC GGGTTTTACT GATGCATATA CAGAGATGCT TTTTGTTCGC TTGGTTGTGA 3481 TGATGTGGTG TGGTTGGGCG GTCGTTCATT CGTTCTAGAT CGGAGTAGAATACTGTTTCA 3541 AACTACCTGG TGTATTTATT AATTTTGGAA CTGTATGTGT GTGTCATACA TCTTCATAGT 3601 TACGAGTTTA AGATGGATGG AAATATCGAT CTAGGATAGG TATACATGTT GATGTGGGTT 3661 TTACTGATGC ATATACATGA TGGCATATGC AGCATCTATT CATATGCTCT AACCTTGAGT 3721 ACCTATCTAT TATAATAAAC AAGTATGTTT TATAATTATT TTGATCTTGA TATACTTGGA 3781 TGATGGCATA TGCAGCAGCT ATATGTGGAT TTTTTTAGCC CTGCCTTCAT ACGCTATTTA 3841 TTTGCTTGGT ACTGTTTCTT TTGTCGATGC TCACCCTGTT GTTTGGTGTT ACTTCTGCAG 3901 ACCATGGCGC AAGTTAGCAG GATCTGCAAC GGCGTGCAGA ACCCGTCGCT CATCTCCAAC 3961 CTCTCGAAAT CCAGTCAACG CAAATCGCCC TTGTCGGTCT CCCTGAAGAC GCAGCAGCAC 4021 CCGCGAGCCT ACCCGATCTC GTCGTCGTGG GGCTTGAAGA AGAGCGGGAT GACGCTGATT 4081 GGCTCTGAGC TCCGTCCCCT CAAGGTCATG TCTTCTGTGT CCACGGCGTG CATGGACAAC 4141 TCCGTGCTCA ACTCTGGTCG CACTACCATT TGCGATGCGT ACAACGTGGC TGCCCATGAC 4201 CCATTCAGCT TCCAGCACAA GAGCCTGGAC ACTGTTCAAA AGGAATGGAC CGAGTGGAAG 4261 AAGAACAACC ACAGCCTCTA CCTGGACCCC ATCGTCGGGA CGGTGGCCAG CTTCCTCCTC 4321 AAGAAGGTCG GCTCTCTCGT CGGCAAGCGC ATCCTGTCGG AGCTTAGGAA CCTCATCTTC 4381 CCATCTGGTT CCACCAACCT GATGCAGGAC ATCCTGAGGG AAACCGAGAA GTTCCTGAAC 4441 CAGCGTCTCA ACACTGACAC GCTCGCGAGA GTGAATGCTG AGCTGACGGG CCTCCAGGCG 4501 AACGTCGAGG AGTTCAATCG CCAAGTGGAC AACTTCCTGA ACCCGAACCG CAACGCAGTA 4561 CCGCTCTCCA TCACCTCTTC CGTGAACACC ATGCAGCAAC TGTTCCTCAA CCGCTTGCCA 4621 CAGTTCCAAA TGCAGGGCTA CCAGCTCCTG CTCCTGCCTC TCTTCGCGCA AGCCGCTAAC 4681 CTGCACCTCT CCTTCATTCG CGACGTGATC CTGAACGCCG ACGAGTGGGG CATCTCTGCC 4741 GCAACGCTGC GCACCTACAG GGACTACCTC AAGAACTACA CGCGCGACTA CTCCAACTAT 4801 TGCATCAACA CCTACCAGTC GGCCTTCAAG GGCCTCAATA CCAGGCTTCA CGACATGCTG 4861 GAGTTCCGCA CCTACATGTT CCTGAACGTC TTTGAATACG TGAGCATCTG GTCGCTCTTC 4921 AAGTATCAGA GCTTGCTTGT CTCCTCCGGC GCCAACCTCT ACGCTTCCGG CTCTGGTCCG 4981 CAGCAAACCC AGAGCTTCAC CAGCCAGGAT TGGCCGTTCC TGTACTCGCT GTTCCAAGTC 5041 AACTCCAACT ACGTCCTCAA CGGTTTCTCT GGCGCGCGTC TCTCCAACAC CTTTCCCAAC 5101 ATCGTTGGCC TGCCCGGCTC CACAACTACC CATGCTCTTC TGGCCGCTCG CGTGAACTAC 5161 TCCGGAGGCA TCAGCAGCGG CGACATCGGC GCGTCGCCCT TCAATCAGAA CTTCAACTGC 5221 TCCACCTTCC TGCCACCGCT CCTGACTCCG TTCGTCCGCT CCTGGCTTGA CAGCGGCTCC 5281 GACCGCGAGG GCGTCGCCAC CGTGACCAAC TGGCAGACCG AGTCCTTCGA GACGACCCTC 5341 GGCCTTCGTT CCGGCGCGTT CACCGCACGC GGCAACTCTA ACTACTTCCC CGACTACTTC 5401 ATCAGGAACA TTTCTGGCGT GCCTCTGGTG GTCCGCAACG AGGACCTGCG CCGCCCGCTG 5461 CACTACAACG AGATCCGCAA CATTGCCTCT CCCTCCGGGA CGCCAGGTGG AGCAAGGGCC 5521 TACATGGTCA GCGTGCATAA CAGGAAGAAC AACATCCACG CCGTGCATGA GAACGGCTCC 5581 ATGATCCACC TGGCGCCCAA CGATTACACC GGCTTCACCA TCTCTCCAAT CCACGCCACC 5641 CAAGTGAACA ACCAGACGCG CACCTTCATC TCCGAGAAGT TCGGCAACCA GGGCGACTCC 5701 CTGCGCTTCG AGCAGAACAA CACGACCGCT CGCTACACCC TGAGAGGCAA CGGCAACAGC 5761 TACAACCTCT ACCTGCGCGT GTCCTCCATT GGCAACTCCA CCATCAGGGT CACCATCAAC 5821 GGGCGCGTGT ACACCGCCAC AAACGTCAAC ACAACCACGA ATAACGATGG CGTCAACGAC 5881 AATGGCGCCA GGTTCAGCGA CATCAACATC GGCAACGTCG TGGCCAGCAG CAACTCCGAC 5941 GTGCCGCTCG ACATCAACGT CACCCTGAAC TCTGGCACCC AGTTCGACCT GATGAACATC 6001 ATGCTGGTGC CCACTAACAT CTCGCCGCTG TACTGATAGC AGGGTTCTTG CCTGGTGCCT 6061 TGGCAATGCT TGATTACTGC TGCTATCCTA TGATCTGTCC GTGTGGGCTT CTATCTATCA 6121 GTTTGTGTGT CTGGTTTTGA AAAACATTTG CTTTTCGATT ATGTAGGGTT TGCTTGTAGC 6181 TTTCGCTGCT GTGACCTGTG TTGTTTATGT GAACCTTCTT TGTGGCATCT TTAATATCCA 6241 AGTTCGTGGT TTGTCGTAAA ACGAAGCCTC TACTTCGTAA AGTTGTGTCT ATAGCATTGA 6301 AATCGTTTTT TTGCTCGAGA ATAATTGTGA CCTTTAGTTG GCGTGAAACT AGTTTTGGAT 6361 ATCTGATTCT CTGGTTCGCA ATCTTGAGAT CGTCGCTGCT TAGGTGAGCT AAGTGATGTT 6421 CCTAAGTAAA TGCTCCTCAC CAGAATACGT AGCTGTGTGA AAAGAGAACG CGTGAATACG 6481 TAGCTGTGTA AAGATTGTGT CCCAAGTAAA CCTCAGTGAT TTTTGTTTGG ATTTTTAATT 6541 TAGAAACATT CGACTGGGAG CGGCTAGAGC CACACCCAAG TTCCTAACTA TGATAAAGTT 6601 GCTCTGTAAC AGAAAACACC AGGTACCAGA TCTTGAGCCA ATCAAAGAGG AGTGATGTAG 6661 ACCTAAAGCA ATAATGGAGC CATGACGTAA GGGCTTACGC CCATACGAAA TAATTAAAGG 6721 CTGATGTGAC CTGTCGGTCT CTCAGAACCT TTACTTTTTA TGTTTGGCGT GTATTTTTAA 6781 ATTTCCACGG CAATGACGAT GTGACCCAAC GAGATCTTGA GCCAATCAAA GAGGAGTGAT 6841 GTAGACCTAA AGCAATAATG GAGCCATGAC GTAAGGGCTT ACGCCCATAC GAAATAATTA 6901 AAGGCTGATG TGACCTGTCG GTCTCTCAGA ACCTTTACTT TTTATATTTG GCGTGTATTT 6961 TTAAATTTCC ACGGCAATGA CGATGTGACC TGTGCATCCG CTTTGCCTAT AAATAAGTTT 7021 TAGTTTGTAT TGATCGACAC GGTCGAGAAG ACACGGCCAT AACCATCTTC CACACACTCA 7081 AGCCACACTA TTGGAGAACA CACAGGGACA ACACACCATA ACCTCCGCCG CCGCCGCGCC 7141 GGTAACCACC CCGCCCCTCT CCTCTTTCTT TCTCCGTTTT TTTTTCCGTC TCGGTCTCGA 7201 TCTTTGGCCT TGGTAGTTTG GGTGGGCGAG AGGCGGCTTC GTGCGCGCCC AGATCGGTGC 7261 GCGGGAGGGG CGGGATCTCG CGGCTGGGGC TCTCGCCGGC GTGGATCCGG CCCGGATCTC 7321 GCGGGGAATG GGGCTCTCGG ATGTAGATCT GCGATCCGCC GTTGTTGGGG GAGATGATGG 7381 GGGGTTTAAA ATTTCCGCCG TGCTAAACAA GATCAGGAAG AGGGGAAAAG GGCACTATGG 7441 TTTATATTTT TATATATTTC TGCTGCTTCG TCAGGCTTAG ATGTGCTAGA TCTTTCTTTC 7501 TTCTTTTTGT GGGTAGAATT TGAATCCCTC AGCATTGTTC ATCGGTAGTT TTTCTTTTCA 7561 TGATTTGTGA CAAATGCAGC CTCGTGCGGA GCTTTTTTGT AGGTAGAAGA CCATGGCACA 7621 GATCAACAAC ATGGCTCAGG GCATCCAAAC CCTCAATCCC AATTCCAATT TCCACAAGCC 7681 CCAGGTGCCT AAGTCCAGCA GCTTCCTCGT CTTCGGCTCC AAGAAACTGA AGAATAGCGC 7741 CAATTCAATG TTGGTCCTGA AGAAGGACTC CATCTTCATG CAGAAGTTCT GCTCCTTCAG 7801 GATCTCCGCC TCAGTGGCCA CCGCGTGCAT GGAGAACAAC ATCCAGAATC AATGCGTCCC 7861 CTACAACTGC CTCAACAATC CGGAGGTCGA GATCCTCAAC GAAGAGAGGA GCACGGGCCG 7921 CCTGCCGCTG GACATCTCCC TGTCCCTGAC CCGCTTCCTG CTGTCCGAGT TCGTGCCCGG 7981 CGTCGGAGTC GCGTTCGGCC TCTTCGACCT CATCTGGGGG TTCATCACCC CATCTGACTG 8041 GAGCCTCTTC CTGCTCCAAA TCGAGCAGCT GATCGAGCAA AGGATTGAGA CCTTGGAACG 8101 CAATCGGGCC ATCACGACCC TCAGGGGCCT GGCAGACAGC TATGAGATCTACATTGAAGC 8161 GCTGCGCGAG TGGGAAGCCA ACCCTAACAA TGCCCAGCTG CGCGAGGATG TGCGTATCCG 8221 CTTCGCTAAC ACCGACGACG CTCTGATCAC GGCCATCAAC AACTTCACCC TTACCAGCTT 8281 CGAGATCCCT CTGCTCTCGG TCTACGTGCA GGCCGCTAAC CTGCACCTGT CGCTGCTGCG 8341 CGACGCGGTG TCCTTTGGCC AGGGTTGGGG CCTGGACATC GCCACCGTCA ACAATCACTA 8401 CAACCGCCTC ATCAATCTGA TCCACCGCTA CACGAAACAC TGCTTGGACA CCTACAATCA 8461 GGGGCTTGAG AACCTGCGCG GGACCAACAC GCGCCAGTGG GCCAGGTTCA ATCAGTTCCG 8521 CCGCGACCTT ACGCTGACCG TCCTCGATAT CGTCGCTCTC TTCCCGAACT ACGATGTTCG 8581 CACCTACCCG ATTCAGACCT CCTCCCAACT GACGAGGGAG ATCTACACCA GCTCCGTCAT 8641 TGAGGACTCC CCGGTGTCTG CGAACATCCC CAATGGCTTC AACCGCGCCG AGTTCGGCGT 8701 CCGCCCGCCC CACCTCATGG ACTTCATGAA CTCTCTGTTC GTCACCGCGG AGACCGTCAG 8761 GTCCCAGACC GTCTGGGGGG GCCACCTGGT CAGCTCGCGC AACACGGCTG GCAACCGCAT 8821 CAACTTCCCC AGCTACGGCG TGTTCAACCC CGGCGGTGCC ATCTGGATCG CGGACGAAGA 8881 CCCAAGGCCG TTCTACCGGA CCCTGTCGGA CCCCGTCTTC GTGCGCGGCG GTTTCGGCAA 8941 TCCGCACTAC GTCCTCGGTC TGAGGGGCGT CGCCTTCCAG CAAACCGGCA CGAATCACAC 9001 CCGCACGTTC AGGAACTCCG GGACCATCGA CTCCCTGGAT GAGATCCCGC CTCAAGACAA 9061 CAGCGGCGCA CCCTGGAACG ACTACTCCCA TGTGCTGAAC CACGTTACCT TCGTGCGGTG 9121 GCCGGGCGAG ATCTCGGGCT CCGACTCGTG GCGCGCGCCG ATGTTCTCCT GGACGCACAG 9181 GAGCGCTACC CCCACGAACA CCATCGACCC GGAGCGCATC ACGCAGATCC CCCTGGTGAA 9241 GGCACACACA CTCCAGTCGG GGACCACGGT TGTCCGCGGG CCGGGCTTCA CGGGCGGAGA 9301 CATCCTGCGG CGCACCAGCG GCGGCCCGTT CGCGTACACC ATCGTCAACA TCAATGGGCA 9361 ACTCCCCCAG CGCTACCGCG CCAGGATTCG CTATGCCTCT ACGACCAACC TGAGAATCTA 9421 CGTTACGGTC GCGGGCGAAC GCATTTTCGC CGGGCAGTTC AACAAGACGA TGGATACCGG 9481 CGACCCGCTG ACGTTCCAGT CCTTCTCCTA CGCCACGATC AACACCGCGT TCACCTTCCC 9541 GATGAGCCAG AGCAGCTTCA CCGTGGGCGC CGACACCTTC TCCTCGGGCA ACGAGGTCTA 9601 CATTGACCGC TTCGAGCTGA TCCCAGTGAC CGCCACGTTC GAAGCTGAGT ACGACCTTGA 9661 GCGCGCCCAG AAGGCCGTGA ACGCCCTCTT CACCTCCATC AATCAGATCG GCATCAAGAC 9721 TGACGTCACC GACTACCACA TCGACCAGGT GTCCAACCTG GTGGACTGCC TCAGCGATGA 9781 GTTCTGCCTC GACGAGAAGC GCGAACTCTC CGAGAAGGTT AAGCACGCCA AGCGTCTCAG 9841 CGACGAGAGG AATCTGCTCC AGGACCCGAA CTTCAAGGGC ATCAACAGGC AGCTGGACCG 9901 TGGTTGGCGC GGCAGCACCG ACATCACCAT CCAGCGCGGC GACGATGTCT TCAAGGAGAA 9961 CTACGTCACC CTCCCCGGCA CCTTCGACGA GTGCTACCCC ACCTACCTGT ACCAGAAGAT 10021 CGATGAGTCC AAGCTCAAGC CCTACACCCG CTATCAGCTG CGCGGCTACA TCGAAGACAG 10081 CCAGGACCTT GAAATCTACC TCATCCGCTA CAACGCCAAG CACGAGACCG TCAACGTCCT 10141 CGGCACTGGT TCCCTCTGGC CGCTGTCCGT CCAGTCTCCC ATCCGCAAGT GCGGCGAGCC 10201 TAACCGTTGC GCGCCGCACC TTGAGTGGAA CCCCGACCTG GACTGCTCGT GCAGGGACGG 10261 CGAGAAGTGC GCCCACCACT CCCATCACTT CTCCCTGGAC ATCGATGTGG GCTGCACCGA 10321 CCTGAACGAG GACCTCGACG TCTGGGTCAT CTTCAAGATC AAGACCCAGG ATGGCCACGC 10381 CCGCCTCGGC AACCTGGAGT TCCTCGAAGA GAAGCCGCTG GTCGGCGAAG CCCTCGCTCG 10441 CGTCAAGCGC GCAGAGAAGA AGTGGCGCGA CAAGCGCGAG AAGCTCGAAC TCGAAACGAA 10501 CATCGTCTAC AAGGAGGCCA AAGAGTCCGT CGATGCTCTG TTCGTCAACT CCCAGTACGA 10561 CCAGTTGCAA GCCGACACCA ACATCGCCAT GATCCACGCC GCGGACAAGC GTGTGCACCG 10621 CATTCGTGAG GCATACCTGC CTGAGCTGTC CGTGATCCCC GGTGTCAACG TGGACATCTT 10681 TGAAGAGCTT AAGGGCCGCA TCTTCACCGC GTTCTTCCTG TACGATGCCC GCAACGTCAT 10741 CAAGAACGGC GACTTCAACA ACGGCCTCAG CTGCTGGAAC GTGAAGGGCC ACGTGGACGT 10801 CGAGGAACAG AACAACCACC GCTCCGTCCT GGTGGTCCCC GAGTGGGAAG CCGAAGTGTC 10861 CCAAGAGGTC CGCGTCTGCC CAGGGCGCGG CTACATCCTC CGCGTGACCG CCTACAAGGA 10921 GGGGTACGGC GAGGGTTGCG TCACCATCCA CGAGATCGAG AACAACACCG ACGAGCTGAA 10981 GTTCTCCAAC TGCGTCGAGG AAGAAGTCTA CCCCAACAAC ACCGTTACGT GCAACGACTA 11041 CACCGCGAAC CAGGAAGAGT ACGGCGGGGC CTACACGAGC CGTAACCGCG GCTACGACGA 11101 AACCTACGGC TCGAACTCCT CCGTCCCCGC GGACTACGCC TCCGTCTATG AAGAGAAGTC 11161 CTACACCGAT GGCCGCCGCG ACAACCCCTG CGAGTCGAAC CGGGGCTACG GGGACTACAC 11221 ACCGCTTCCA GCGGGCTACG TCACCAAGGA GCTTGAGTAC TTCCCTGAGA CCGACAAAGT 11281 GTGGATCGAG ATCGGCGAAA CCGAGGGCAC GTTCATTGTG GACAGCGTGG AGCTGCTCCT 11341 GATGGAGGAG TAATGACTAG GCTACTGTAG CTAGCTGTGC ATGTATGTGG TGTGGTTACT 11401 AAAATAATTA GTGTTTTCCT TTTGTTTGGA AGCATATGTG TGGTGAATAA ATGATGAACT 11461 CCGATGTTCC TCTCTATAAA TCTTGATGAT TCGCTAGCTA TCCGTACGTC GTTGTTCTTT 11521 GATTTGATGA TGAGATTGAA AAATGGAATG TCATGCTAAG GAGGGTGCCG AGCTCATAAC 11581 TTCGTATAAT GTATGCTATA CGAAGTTATC CGATCCTACC TGTCACTTCA TCAAAAGGAC 11641 AGTAGAAAAG GAAGGTGGCA CCTACAAATG CCATCATTGC GATAAAGGAA AGGCTATCAT 11701 TCAAGATGCC TCTGCCGACA GTGGTCCCAA AGATGGACCC CCACCCACGA GGAGCATCGT 11761 GGAAAAAGAA GACGTTCCAA CCACGTCTTC AAAGCAAGTG GATTGATGTG ATACTTCCAC 11821 TGACGTAAGG GATGACGCAC AATCCCACTA TCCTTCGCAA GACCCTTCCT CTATATAAGG 11881 AAGTTCATTT CATTTGGAGA GGACACGCTG AACCATGTCG CCCGAGCGCA GGCCCGTCGA 11941 GATCCGCCCG GCCACAGCAG CCGACATGGC TGCGGTGTGC GACATCGTCA ACCACTACAT 12001 CGAAACGTCC ACCGTGAACT TCCGCACCGA GCCGCAGACG CCGCAGGAGT GGATCGACGA 12061 CCTTGAGCGC CTCCAGGACC GCTACCCCTG GCTCGTCGCG GAGGTCGAAG GCGTCGTGGC 12121 CGGCATCGCC TACGCCGGGC CGTGGAAGGC TCGCAACGCC TACGACTGGA CCGTCGAGAG 12181 CACCGTCTAC GTGTCCCACC GCCACCAGCG CCTGGGCCTC GGCTCCACGC TGTACACGCA 12241 CCTGCTCAAG TCGATGGAGG CGCAAGGCTT CAAGAGCGTG GTCGCCGTCA TCGGCCTCCC 12301 GAACGATCCC TCCGTGAGGC TCCACGAAGC CCTGGGCTAC ACCGCTCGGG GCACGCTGCG 12361 CGCAGCCGGC TACAAGCACG GTGGCTGGCA CGACGTCGGG TTCTGGCAGA GGGACTTCGA 12421 GCTGCCCGCG CCTCCGCGCC CGGTTCGCCC CGTGACCCAG ATTTGATAGA GCTTTCGTTC 12481 GTATCATCGG TTTCGACAAC GTTCGTCAAG TTCAATGCAT CAGTTTCATT GCGCACACAC 12541 CAGAATCCTA CTGAGTTTGA GTATTATGGC ATTGGGAAAA CTGTTTTTCT TGTACCATTT 12601 GTTGTGCTTG TAATTTACTG TGTTTTTTAT TCGGTTTTCG CTATCGAACT GTGAAATGGA 12661 AATGGATGGA GAAGAGTTAA TGAATGATAT GGTCCTTTTG TTCATTCTCA AATTAATATT 12721 ATTTGTTTTT TCTCTTATTT GTTGTGTGTT GAATTTGAAA TTATAAGAGATATGCAAACA 12781 TTTTGTTTTG AGTAAAAATG TGTCAAATCG TGGCCTCTAA TGACCGAAGT TAATATGAGG 12841 AGTAAAACAC TTGTAGTTGT ACCATTATGC TTATTCACTA GGCAACAAAT ATATTTTCAG 12901 ACCTAGAAAA GCTGCAAATG TTACTGAATA CAAGTATGTC CTCTTGTGTT TTAGACATTT 12961 ATGAACTTTC CTTTATGTAA TTTTCCAGAA TCCTTGTCAG ATTCTAATCA TTGCTTTATA 13021 ATTATAGTTA TACTCATGGA TTTGTAGTTG AGTATGAAAA TATTTTTTAA TGCATTTTAT 13081 GACTTGCCAA TTGATTGACA ACATGCATCA ATATAACTTC GTATAATGTA TGCTATACGA 13141 AGTTATGAAT TCCCCCGAAT TAATTCGGCG TTAATTCAGT ACATTAAAAA CGTCCGCAAT 13201 GTGTTATTAA GTTTGCGTCT CCCACCGTAC AGATCAGCTA GCTTTTAATTACCTGGTGGT 13261 ACGTACAGAC ACAGTCCTCG TCGTCCTCCT CCACCGCCCG CCCCCCGTGGATCTTCACGC 13321 CTCCTGGGCG GTGCACGGTG GGCGGTGGCA CTTTTAGTTA GTGCCGGCTGCATCTTTAGC 13381 GCAAAAGTTT GCAACTACCT GATACAGAGC CCCGGCTTCA CCGCCATGTTCATCCAAACG 13441 CCTGCTTTTT CGCGAATCCG GCTCCCAACT TTTTCAATAA CTGCGAGCCTGTATAGCATC 13501 AGATTCTGGT GGATTCTGCG TTCCGTAGGC TGTAGCAATT GCATGCGGGAAGATTTTCAG 13561 AACAATTTCT TTCCGCGATG CAACTTGAGA ACCAGTAGCT GAGCCGGGCCGCTTTGTTGT 13621 ATATTATTGC GTGCAGTGAC GGAGCTGGTG GAAGGGGACT CGTCGCGGAAGATCGTGGAG 13681 ATCATCTGCC GGACGAGCCT GCTCAAGTCG GAGAGCAGCT G Example 4. Detection of Transformation Event Whether the unique signature DNA information of the CSJ4351 transformation event is present in the maize material can identify whether the maize material was obtained using the technology of the present invention. Therefore, specific DNA primer pairs can be used for PCR amplification to detect the CSJ4351 event. The primer pairs consist of a first primer that specifically recognizes either flanking sequence of the inserted sequence and a second primer that specifically recognizes the T-DNA insertion sequence of the present invention. For example, when the first primer is CSJ4351LB5 (SEQ ID NO:14), the second primer can be CSJ4351LB3 (SEQ ID NO:15); when the first primer is CSJ4351RB5 (SEQ ID NO:16), the second primer can be CSJ4351RB3 (SEQ ID NO:17). The results of amplifying maize samples of the CSJ4351 event and C01 using the primer pairs CSJ4351LB5 / CSJ4351LB3 and CSJ4351RB5 / CSJ4351RB3 are respectively as Figure 6 and Figure 7 shown.

[0059] The PCR system is as follows: 10×Ex buffer 5.0 μl dNTP (both 2.5 mmol / L) 1.0 μl 5’ primer (10 μmol / L) 2.0 μl 3’ primer (10 μmol / L) 2.0 μl Template DNA 100 ng Ex Taq 1 U Add sterile water to a total volume of 50 μl For CSJ4351LB5 / CSJ4351LB3 and CSJ4351RB5 / CSJ4351RB3, the PCR program: 94.0°C for 5 min; 35 cycles: 94.0°C for 30 s, 62.0°C for 30 s, 72°C for 30 s; 72.0°C for 5 min.

[0060] SEQ ID NO:14 CAGTTTCATTGCGCACACACC SEQ ID NO:15 GATGCAGCCGGCACTAACTAAA SEQ ID NO:16 AAGTGCGGCGAGCAGTTCAGCA SEQ ID NO:17 GTTTCATTGCGCACACACCAG Example 5. Chromosomal Location According to the obtained maize flanking sequences, by using the published maize genome sequence (https: / / phytozome-next.jgi.doe.gov / blast-search) for comparative analysis, it can be known that in the CSJ4351 event, the maize flanking DNA sequence joined to the inserted sequence is highly homologous to the sequence on chromosome 3. Therefore, it can be known that the integration site of the foreign DNA inserted sequence in the CSJ4351 event is located on chromosome 3 of the recipient maize. The representative seeds of the maize of this plant transformation event were deposited on March 21, 2025 at the General Microbiology Center of the China Committee for Culture Collection of Microorganisms (CGMCC, Address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing), and the deposit number is: CGMCC No. 30704.

Claims

1. A maize conversion event, characterized in that, The partial genomic sequence of maize is replaced with a T-DNA insertion sequence on chromosome 3 of the maize, wherein the T-DNA insertion sequence is the 280-13210 bp sequence of the sequence shown in SEQ ID NO: 13, and the partial genomic sequence is as shown in SEQ ID NO:

18.

2. The maize conversion event according to claim 1, wherein The upstream flanking maize genomic sequence of the T-DNA insertion sequence is: 1) The 1-279 bp sequence of the sequence shown in SEQ ID NO: 13; or 2) The 3' end sequence of the 1-279 bp sequence of the sequence shown in SEQ ID NO: 13 with a length of not less than 100 bp.

3. The maize conversion event according to claim 1 or 2, characterized in that, The downstream flanking maize genomic sequence of the T-DNA insertion sequence is: 1) The 13211-13721 bp sequence of the sequence shown in SEQ ID NO: 13; or 2) The 5' end sequence of the 13211-13721 bp sequence of the sequence shown in SEQ ID NO: 13 with a length of not less than 100 bp.

4. The maize conversion event according to any one of claims 1-3, characterized in that, The DNA sequence shown in SEQ ID NO: 13 is included on chromosome 3 of maize, and the DNA sequence is composed of a T-DNA insertion sequence of 280-13210 bp, an upstream flanking maize genomic sequence of 1-279 bp, and a downstream flanking maize genomic sequence of 13211-13721 bp.

5. A DNA fragment, characterized in that, The DNA fragment sequentially includes from the 5' end to the 3' end: 1) Upstream flanking maize genomic sequence: the 1-279 bp sequence of the sequence shown in SEQ ID NO: 13; or the 3' end sequence of the 1-279 bp sequence of the sequence shown in SEQ ID NO: 13 with a length of not less than 100 bp; 2) T-DNA insertion sequence: the 280-13210 bp sequence of the sequence shown in SEQ ID NO: 13; 3) Downstream flanking maize genomic sequence: the 13211-13721 bp sequence of the sequence shown in SEQ ID NO: 13; or the 5' end sequence of the 13211-13721 bp sequence of the sequence shown in SEQ ID NO: 13 with a length of not less than 100 bp.

6. A primer pair for detecting the maize conversion event according to any one of claims 1-4 or the DNA fragment according to claim 5, characterized in that, It consists of a first primer that specifically recognizes the upstream flanking maize genomic sequence or the downstream flanking maize genomic sequence and a second primer that specifically recognizes the T-DNA insertion sequence.

7. The primer pair according to claim 6, wherein the sequence of the first primer is as shown in SEQ ID NO: 14, and the sequence of the second primer is as shown in SEQ ID NO: 15; or the sequence of the first primer is as shown in SEQ ID NO: 16, and the sequence of the second primer is as shown in SEQ ID NO:

17.

8. A method for identifying whether the maize transformation event recited in claim 1 is present in a maize biological sample, characterized in that, Includes: (a) Extract a DNA sample from the maize biological sample to be identified; (b) Using the extracted DNA sample as a template, perform PCR amplification with the primer pair according to claim 6 or 7; and (c) Detect the PCR amplification product. If the length of the PCR amplification product is consistent with the length obtained by amplifying using the DNA template of the sequence shown in SEQ ID NO: 13, it indicates the presence of the maize transformation event in the maize biological sample.

9. A method for obtaining a transgenic insect-resistant and herbicide-tolerant maize material, characterized in that, Comprising: After hybridizing the maize material containing the maize transformation event according to any one of claims 1-4 with other maize breeding materials, further backcrossing is carried out to obtain a new material containing the maize transformation event; wherein during the hybridization and / or backcrossing process, the method described in claim 8 is used to screen and identify in the offspring population to confirm the presence of the maize transformation event.

10. Use of the DNA fragment according to claim 5 for improving the agronomic traits of maize in terms of insect resistance and herbicide tolerance, for maize breeding and / or as a molecular marker.

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