Insect-resistant and herbicide-resistant transgenic maize event JZL22k-1 and detection method thereof

Abstract

The invention discloses an insect-resistant and herbicide-resistant transgenic maize event JZL22k-1 and a detection method of the insect-resistant and herbicide-resistant transgenic maize event JZL22k-1. The transgenic maize event JZL22k-1 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 transgenic maize plant JZL22k-1 has good resistance to lepidoptera insects and good tolerance to glyphosate herbicides and has no influence on the yield, and the detection method can accurately and stably identify the existence of a JZL22k-1 transgenic event by applying a nucleic acid amplification method. The method is of great significance in implementation of research of JZL22k-1, traceability of production processing and application and whole-process supervision.

Description

(I) Technical Field

[0001] This invention relates to an insect-resistant and herbicide-tolerant transgenic maize event and its detection method, particularly to the construction of a specific transgenic maize event JZL22k-1 by inserting a foreign gene into the maize cell genome and a nucleic acid sequence for detecting whether a biological sample contains the specific transgenic maize event JZL22k-1 and its detection method. (II) Background Technology

[0002] Corn is one of the world's most widely cultivated food crops, serving multiple purposes as food, feed, and energy. It is not only a major food crop but also a primary raw material for livestock and industrial production. Corn yield and quality play a vital role in agricultural production and global economic development. As an important food crop, corn is frequently attacked by various lepidopteran pests (such as the corn borer, fall armyworm, and beet armyworm), leading to significant yield reductions. While traditional chemical pesticides can control pests to some extent, they also cause environmental pollution and pesticide residues. Competition from weeds in farmland severely impacts corn growth and yield. Traditional weeding methods are labor-intensive and inefficient, and long-term use of chemical herbicides easily leads to increased weed resistance. Introducing insect-resistant and herbicide-tolerant genes into corn through genetic engineering can improve the efficiency of pest and weed control, reduce production costs, and bring significant economic benefits to agricultural production, thereby achieving positive social and ecological benefits.

[0003] Traditionally, chemical pesticides have been the primary means of controlling corn pests. However, long-term use of chemical pesticides has not only increased production costs but also led to environmental pollution and pesticide residues. With the development of biotechnology, transgenic technology has provided a new approach for insect-resistant corn breeding. By introducing insecticidal protein genes (such as the cry gene) from Bacillus thuringiensis, corn can produce its own insecticidal proteins, thereby effectively resisting pest infestations. Weed management in farmland is another major challenge in corn production. Traditional weeding methods are inefficient and labor-intensive, while the use of chemical herbicides easily leads to increased herbicide resistance in weeds. Through transgenic technology, introducing herbicide-resistant genes (such as cp4epsps) into the corn genome can make corn tolerant to specific herbicides (such as glyphosate), thereby simplifying weeding operations and improving weeding efficiency.

[0004] However, current insect-resistant transgenic maize varieties in my country mainly target a few lepidopteran pests such as the corn borer, with limited effectiveness against other important pests such as the fall armyworm and the beet armyworm. This presents the challenge of a limited insect resistance spectrum, and long-term use of the same or a few insecticidal protein genes can easily lead to pest resistance, thus reducing the insect-resistant effect of transgenic maize. Transgenic insect-resistant maize has been widely promoted in countries such as the United States, with good application results. The insect-resistant genes used in this invention are cry2Ab and cry1Fa, both Bacillus thuringiensis insecticides with high insecticidal activity against major maize pests such as the fall armyworm and the corn borer. Its core feature is the simultaneous introduction of the cry2Ab gene and the cry1Fa gene, which are highly resistant to lepidopteran pests such as the corn borer, achieving high-dose killing of major lepidopteran pests through dual action. It is worth noting that the cry1Fa gene has not yet been used in insect-resistant crops approved in my country. Furthermore, existing research indicates that the simultaneous expression of two different types of insect-resistant genes can effectively slow down the development of pest resistance. Therefore, the combined application of these two insect-resistant genes in maize has significant practical implications.

[0005] By employing a gene stacking strategy, simultaneously introducing multiple insect-resistant and herbicide-tolerant genes into the maize genome can significantly enhance the insect resistance and herbicide tolerance of transgenic maize, delaying the development of resistance to pests and weeds. Herbicide tolerance traits in crops can be obtained through genetic improvement techniques. Currently, a commonly used method is Agrobacterium-mediated transformation, which introduces the cp4epsps gene from Agrobacterium tumefaciens sp strain CP4 into crops. This gene expresses 5-enolpyruvate-3-phosphate synthase (EPSPS), enabling crops to acquire tolerance to the herbicide glyphosate.

[0006] A transformation event is a molecular structure consisting of the flanking regions upstream and downstream of the insertion site of a foreign DNA sequence in the genome and the foreign gene. During genetic transformation, because the foreign DNA sequence can randomly insert into any site on any chromosome of the plant genome, each resulting event is unique. The expression of foreign DNA in plants is influenced by the chromosomal location where the foreign DNA is inserted. Different chromosomal locations of the foreign DNA lead to significant differences in expression levels, spatial and temporal patterns, resulting in varying effects on agronomic traits. Different transformation events resulting from the same foreign gene often exhibit vastly different traits; therefore, screening a large number of transformation events is usually necessary to identify those that meet the requirements for production applications in terms of target gene expression levels, expression patterns, and functional traits. The ideal transformation events identified can be introduced into other genetic backgrounds using conventional breeding methods such as sexual hybridization. The offspring produced through hybridization retain the transgenic characteristics of the original event.

[0007] Identifying the integration site of exogenous genes in the genome is of significant value for hybridization breeding, production application, commercial registration, and legal regulation of transgenic crops. Information on the integration site of exogenous genes in transformation events is essential for detecting the presence of transformation events in plants using existing polynucleotide detection methods. While conventional polynucleotide and protein detection methods can detect whether a gene is transgenic, they cannot effectively distinguish between different transformation events, especially those using the same gene or transformation vector. Therefore, only detection of the inserted gene and flanking sequences can accurately determine the presence of the target transgenic event. (III) Summary of the Invention

[0008] This invention provides a transgenic maize event JZL22k-1 and its specific detection method and application. The transgenic maize event JZL22k-1 simultaneously expresses three proteins: cry2Ab, cry1Fa, and cp4epsps, with a single copy of the exogenous gene inserted. It exhibits excellent traits such as good genetic stability, high resistance to glyphosate, high resistance to fall armyworm, beet armyworm, and corn borer, without adverse effects on agronomic traits. The specific detection method can accurately and rapidly identify whether biological samples contain the DNA molecule of transgenic maize event JZL22k-1, improving breeding efficiency.

[0009] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0010] In a first aspect, the present invention provides a nucleic acid sequence of transgenic maize event JZL22k-1, the nucleic acid sequence comprising SEQ ID NO:1 or its complementary sequence, and / or SEQ ID NO:2 or its complementary sequence.

[0011] Furthermore, the nucleic acid sequence includes one of the sequences SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7+SEQ ID NO.8 connected end to end, or their complementary sequence; since the sequence is too long, a nucleic acid sequence is represented in the form of a sequence SEQ ID NO.7+SEQ ID NO.8 connected end to end.

[0012] The SEQ ID NO.1 or its complementary sequence is a 26-nucleotide sequence located at the 5' end of the insertion site of the transgenic maize event JZL22k-1, which includes 13 nucleotides from the maize gene and 13 nucleotides from the inserted T-DNA. Therefore, the SEQ ID NO.1 or its complementary sequence spans the flanking genomic DNA sequence of the foreign DNA molecule insertion site and the 5' end sequence of the foreign DNA molecule in the transgenic maize event JZL22k-1. The presence of the transgenic maize event JZL22k-1 can be identified by the presence of the SEQ ID NO.1 or its complementary sequence.

[0013] The SEQ ID NO.2 or its complementary sequence is a 26-nucleotide sequence located at the 3' end of the insertion site of the transgenic maize event JZL22k-1, which includes 13 nucleotides from the inserted T-DNA and 13 nucleotides from the maize gene. Therefore, the SEQ ID NO.2 or its complementary sequence spans the 3' end sequence and the flanking genomic DNA sequence of the exogenous DNA molecule at the insertion site of the exogenous DNA molecule in the transgenic maize event JZL22k-1. The presence of the SEQ ID NO.2 or its complementary sequence is sufficient to identify the transgenic maize event JZL22k-1.

[0014] The SEQ ID NO.3 or its complementary sequence is a 371 bp nucleotide sequence located at the 5' end of the insertion junction region of the transgenic maize event JZL22k-1. The first 138 bp of SEQ ID NO.3 is the flanking maize genomic DNA sequence near the insertion junction region, and the last 139-371 bp is the 5' end sequence of the T-DNA nucleotides near the insertion junction region. The presence of the transgenic maize event JZL22k-1 can be identified by the presence of the SEQ ID NO.3 or its complementary sequence.

[0015] The SEQ ID NO.4 or its complementary sequence is a 384 bp nucleotide sequence located at the 3' end of the insertion junction region of the transgenic maize event JZL22k-1. The first 1-241 bp of the SEQ ID NO.4 are the 3' end sequences of the T-DNA nucleotides near the insertion junction region, and the 242-384 bp are the flanking maize genomic DNA sequences near the insertion junction region. The presence of the SEQ ID NO.4 or its complementary sequence is sufficient to identify the transgenic maize event JZL22k-1.

[0016] The SEQ ID NO.5 or its complementary sequence is a 786 bp nucleotide sequence located at the 5' end of the insertion junction region of the transgenic maize event JZL22k-1. Bases 1-544 bp of SEQ ID NO.5 represent the flanking maize genomic DNA sequence near the insertion junction region, and bases 545-786 bp represent the 5' end sequence of the T-DNA nucleotides near the insertion junction region. The presence of the SEQ ID NO.5 or its complementary sequence is sufficient to identify the transgenic maize event JZL22k-1.

[0017] The SEQ ID NO. 6 or its complementary sequence is a 632 bp nucleotide sequence located at the 3' end of the insertion junction region of the transgenic maize event JZL22k-1. The first 278 bp of SEQ ID NO. 6 are the 3' end sequences of the T-DNA nucleotides near the insertion junction region, and the 279-632 bp are the flanking maize genomic DNA sequences near the insertion junction region. The presence of the transgenic maize event JZL22k-1 can be identified by the presence of the SEQ ID NO. 6 or its complementary sequence.

[0018] The sequence consisting of SEQ ID NO.7 and SEQ ID NO.8, or their complementary sequence, is a 26,226-nucleotide sequence unique to the transgenic maize event JZL22k-1, comprising the entire T-DNA sequence and its flanking maize genotype sequences at the 5' and 3' ends. The specific genomic and genetic elements included are shown in Table 1. The presence of the transgenic maize event JZL22k-1 can be identified by the presence of the sequence consisting of SEQ ID NO.7 and SEQ ID NO.8, or their complementary sequence.

[0019] Table 1. Genome and genetic elements contained in the sequence of SEQ ID NO.7 + SEQ ID NO.8 (connected end-to-end)

[0020]

[0021] This invention provides a unique continuous nucleotide sequence of the transgenic maize event JZL22k-1, which can be used to characterize the transgenic maize event JZL22k-1 and thus to detect the presence of the transgenic maize event JZL22k-1 in a sample. Specifically, the presence of at least 13 consecutive nucleotides in one or more of the sequences represented by SEQ ID NO.1-6, SEQ ID NO.7+SEQ ID NO.8 in the sample indicates the presence of the transgenic maize event JZL22k-1 in the sample.

[0022] It will be readily understood by those skilled in the art that, similar to the above-described scheme, the presence of SEQ ID NO.1 or its complementary sequence and SEQ ID NO.4 or its complementary sequence in the sample can also indicate the presence of transgenic maize event JZL22k-1 in the sample; or, the presence of SEQ ID NO.2 or its complementary sequence and SEQ ID NO.3 or its complementary sequence in the sample can also indicate the presence of transgenic maize event JZL22k-1 in the sample; or, the presence of SEQ ID NO.2 or its complementary sequence and SEQ ID NO.5 or its complementary sequence in the sample can also indicate the presence of transgenic maize event JZL22k-1 in the sample; or, the presence of SEQ ID NO.3 or its complementary sequence and SEQ ID NO.6 or its complementary sequence in the sample can also indicate the presence of transgenic maize event JZL22k-1 in the sample; or, the presence of SEQ ID NO.4 or its complementary sequence and SEQ ID NO.5 or its complementary sequence in the sample can also indicate the presence of transgenic maize event JZL22k-1 in the sample; or, the presence of SEQ ID NO.4 or its complementary sequence and SEQ ID NO.6 or its complementary sequence in the sample can also indicate the presence of transgenic maize event JZL22k-1 in the sample. The presence of NO.5 or its complementary sequence can also indicate the presence of transgenic maize event JZL22k-1 in the sample.

[0023] The transgenic maize event JZL22k-1 described in this invention is a DNA molecule obtained by inserting a foreign DNA molecule (i.e., T-DNA) between the 3' end of SEQ ID NO.23 and the 5' end of SEQ ID NO.24 on chromosome 4 of the maize genome; the foreign DNA molecule includes cry1Fa、cry2Ab Gene expression cassettes and cp4epsps Gene expression cassette; cry1Fa、cry2Ab Gene expression cassettes include: used as cry1Fa The gene is promoted by the ZmUbi promoter derived from the maize polyubiquitin-1 gene. cry1Fa Gene coding frames, used as cry1Fa The Tnos terminator for gene termination, used as cry2Ab The promoter pFMV of Scrophularia mosaic virus, cry2AbGene coding frames, used as cry2Ab The cp4epsps gene expression cassette includes: a ZmUbi promoter for initiating the cp4epsps gene, a cp4epsps gene coding frame, and a CaMV35S terminator for terminating the cp4epsps gene.

[0024] Preferably, the DNA molecule nucleic acid sequence of the transgenic maize event JZL22k-1 is shown as the sequence of SEQ ID NO.7 + SEQ ID NO.8 linked end to end.

[0025] Preferably, the herbicide refers to glyphosate; the insecticide refers to major lepidopteran pests of crops (corn borer, fall armyworm, oriental armyworm, cotton bollworm, etc.).

[0026] It is particularly important to note that the transgenic maize event JZL22k-1 described in this invention references the Maize Genome Database (Zm-B73-REFERENCE-NAM-5.0). Researchers in the art know that the maize genome contains a large number of active transposon sequences, and sequence shifts may occur in maize genomes under different genetic backgrounds. Maize events in which the flanking sequences of exogenous T-DNA in the genome of any event obtained by researchers in the art through hybridization or other methods are SEQ ID NO.23 and SEQ ID NO.24 should be considered part of this invention.

[0027] In a second aspect, the present invention provides a method for detecting the presence of DNA molecules of transgenic maize event JZL22k-1 in a sample, comprising: (1) contacting the sample to be tested with a first primer and a second primer in a nucleic acid amplification reaction solution; the first primer being one of SEQ ID NO.9, SEQ ID NO.11, SEQ ID NO.13 or SEQ ID NO.15; the second primer being one of SEQ ID NO.10, SEQ ID NO.12, SEQ ID NO.14 or SEQ ID NO.16; (2) performing a nucleic acid amplification reaction; and (3) detecting the presence of amplification products; wherein the amplification products include SEQ ID NO.1 or its complementary sequence, SEQ ID NO.2 or its complementary sequence.

[0028] Preferably, the amplification product comprises at least 13 consecutive nucleotides in SEQ ID NO.3 or its complementary sequence, and / or at least 13 consecutive nucleotides in SEQ ID NO.4 or its complementary sequence.

[0029] More preferably, the amplification product comprises at least 13 consecutive nucleotides in SEQ ID NO.5 or its complementary sequence, and / or at least 13 consecutive nucleotides in SEQ ID NO.6 or its complementary sequence.

[0030] Furthermore, the presence of transgenic maize event JZL22k-1 can be identified by the amplification product comprising at least 13 consecutive nucleotides in SEQ ID NO.1 or its complementary sequence, SEQ ID NO.2 or its complementary sequence, SEQ ID NO.3 or its complementary sequence, SEQ ID NO.4 or its complementary sequence, SEQ ID NO.5 or its complementary sequence, SEQ ID NO.6 or its complementary sequence, and / or the sequence of SEQ ID NO.7+SEQ ID NO.8 joined end-to-end or its complementary sequence.

[0031] Thirdly, the present invention provides a primer pair for detecting DNA molecules of transgenic maize event JZL22k-1 in a sample, the primer pair comprising a first primer and a second primer, wherein the first primer is one of SEQ ID NO. 9, SEQ ID NO. 11, SEQ ID NO. 13 or SEQ ID NO. 15; and the second primer is one of SEQ ID NO. 10, SEQ ID NO. 12, SEQ ID NO. 14 or SEQ ID NO. 16.

[0032] In the above technical solution, the primers comprise at least one of the nucleotide sequences. Specifically, the first primers are designed from SEQ ID NO.3 (primer name: RB-F1, corresponding number: SEQ ID NO.9), SEQ ID NO.4 (primer name: LB-F1, corresponding number: SEQ ID NO.11), SEQ ID NO.5 (primer name: RB-F2, corresponding number: SEQ ID NO.13), and SEQ ID NO.6 (primer name: LB-F2, corresponding number: SEQ ID NO.15); the second primers are designed from SEQ ID NO.3 (primer name: RB-R1, corresponding number: SEQ ID NO.10), SEQ ID NO.4 (primer name: LB-R1, corresponding number: SEQ ID NO.12), SEQ ID NO.5 (primer name: RB-R2, corresponding number: SEQ ID NO.14), and SEQ ID NO.6 (primer name: LB-R2, corresponding number: SEQ ID NO.16).

[0033] Fourthly, the present invention also provides a method for cultivating herbicide-resistant maize plants containing the transgenic maize event JZL22k-1, the method comprising: planting maize seeds containing a specific region nucleic acid sequence, allowing the maize to grow into maize plants, spraying the maize plants with a herbicide, and harvesting plants whose herbicide tolerance is significantly improved compared to other maize plants that do not contain the specific region nucleic acid sequence; the specific region nucleic acid sequence is derived from the transgenic maize event JZL22k-1, and the specific region nucleic acid sequence comprises one of the nucleotide sequences shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, or SEQ ID NO.7+SEQ ID NO.8 connected end-to-end, or their complementary sequences; the herbicide is glyphosate.

[0034] Fifthly, the present invention also provides a method for obtaining herbicide-tolerant maize plants containing the transgenic maize event JZL22k-1, the method comprising crossing a maize plant containing a specific region nucleic acid sequence with another maize plant to produce progeny plants; harvesting plants with significantly improved herbicide tolerance compared to other plants not containing the specific region nucleic acid sequence; the specific region nucleic acid sequence is derived from the transgenic maize event JZL22k-1, and the specific region nucleic acid sequence comprises one of the nucleotide sequences shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, or SEQ ID NO.7+SEQ ID NO.8 connected end-to-end, or their complementary sequences; the herbicide is glyphosate.

[0035] In a sixth aspect, the present invention provides a method for controlling field weeds in transgenic maize containing the transgenic maize event JZL22k-1, the method comprising spraying a herbicide onto a field planted with transgenic maize, thereby killing the field weeds; the transgenic maize genome contains a specific region nucleic acid sequence from the transgenic maize event JZL22k-1, the specific region nucleic acid sequence comprising one of the nucleotide sequences shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6 or SEQ ID NO.7+SEQ ID NO.8 connected end-to-end, or their complementary sequence; the herbicide is glyphosate.

[0036] Seventhly, the present invention also provides a method for cultivating insect-resistant maize plants containing the transgenic maize event JZL22k-1, the method comprising: planting maize seeds containing a specific region nucleic acid sequence, allowing the maize to grow into maize plants, placing the unfolded tender heart leaves into a petri dish containing target pests, the larvae feeding on the transgenic maize heart leaves turning black, and all or most of them dying within 72 hours, and harvesting plants whose insect resistance is significantly improved compared to other maize plants that do not contain the specific region nucleic acid sequence; the specific region nucleic acid sequence is derived from the transgenic maize event JZL22k-1, and the specific region nucleic acid sequence includes one of the nucleotide sequences shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6 or SEQ ID NO.7+SEQ ID NO.8 connected end-to-end, or their complementary sequences; the target pests are major crop lepidopteran pests (corn borer, fall armyworm, oriental armyworm, cotton bollworm, etc.).

[0037] Eighthly, the present invention also provides a method for obtaining insect-resistant maize plants containing the transgenic maize event JZL22k-1, the method comprising hybridizing a maize plant containing a specific region nucleic acid sequence with another maize plant to produce progeny plants; harvesting plants that exhibit significantly increased resistance to target pests compared to other plants that do not contain the specific region nucleic acid sequence; the specific region nucleic acid sequence is derived from the transgenic maize event JZL22k-1, and the specific region nucleic acid sequence comprises one of the nucleotide sequences shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, or SEQ ID NO.7+SEQ ID NO.8 connected end-to-end, or their complementary sequences; the target pest is a major crop lepidopteran pest (corn borer, fall armyworm, oriental armyworm, cotton bollworm, etc.).

[0038] In a ninth aspect, the present invention also provides an agricultural product or commodity arising from the genetically modified corn event JZL22k-1, said agricultural product or commodity including corn flour, cornmeal, corn oil, corn starch, corn gluten, corn cakes, cosmetics containing corn ingredients, or excipients containing corn ingredients.

[0039] The present invention relates to a transgenic maize event JZL22k-1, which comprises a DNA construct that, when expressed in plant cells, imparts tolerance to glyphosate herbicides and resistance to major lepidopteran pests. The T-DNA construct comprises two tandem expression cassettes, the first of which contains components for expression in plants. cry2Ab , cry1Fa A suitable promoter and a suitable terminator for the protein, wherein the promoter is operatively connected to... cry2Ab , cry1Fa The nucleotide sequence of the protein, the cry2Ab , cry1Fa The first expression cassette contains a promoter for expressing the EPSPS protein in plants against major crop lepidopteran pests. The promoter includes the Scrophularia mosaic virus 35S promoter (pFMV) and the maize polyubiquitin-1 gene promoter (ZmUbi). The terminator includes the transcription termination sequence (Tnos) at the 3' end of the Agrobacterium nicotinate synthase (nos) encoding gene. The second expression cassette contains a suitable promoter and a suitable terminator for expressing the EPSPS protein in plants. The promoter is operatively linked to the gene encoding 5-enol-pyruvylshikimate-3-phosphate synthase (EPSPS), which is resistant to glyphosate herbicides. The promoter includes the maize polyubiquitin-1 gene promoter (ZmUbi), and the terminator includes the CaMV 35S terminator.

[0040] The DNA constructs were introduced into plants using transformation methods, including Agrobacterium-mediated transformation, gene gun transformation, and pollen tube pathway transformation.

[0041] Description of the nucleotide sequence of this invention:

[0042] SEQ ID NO.1 represents 13 nucleotides on each side of the flanking maize genome and the 5' transgenic insertion site in transgenic maize event JZL22k-1;

[0043] SEQ ID NO.2 represents the 3' end transgenic insertion site and 13 nucleotides on each side of the flanking maize genome in transgenic maize event JZL22k-1;

[0044] SEQ ID NO.3 represents the 371 nucleotide sequence near the 5' transgenic insertion site junction region in transgenic maize event JZL22k-1;

[0045] SEQ ID NO.4 represents the 384 nucleotide sequence near the junction of the 3' transgenic insertion site in transgenic maize event JZL22k-1;

[0046] SEQ ID NO.5 represents the 786 nucleotide sequence near the 5' transgenic insertion site junction region in transgenic maize event JZL22k-1;

[0047] SEQ ID NO.6 represents a 632-nucleotide sequence near the junction of the 3' transgenic insertion site in transgenic maize event JZL22k-1;

[0048] The sequence SEQ ID NO.7+SEQ ID NO.8, linked end to end, represents the transgenic maize event JZL22k-1 insertion T-DNA sequence and its 5' and 3' maize genome sequences;

[0049] SEQ ID NO.9 indicates the detection of the first primer (RB-F1) of SEQ ID NO.3;

[0050] SEQ ID NO.10 indicates the detection of the second primer (RB-R1) of SEQ ID NO.3;

[0051] SEQ ID NO.11 indicates the detection of the first primer (LB-F1) of SEQ ID NO.4;

[0052] SEQ ID NO.12 indicates the detection of the second primer (LB-R1) of SEQ ID NO.4;

[0053] SEQ ID NO.13 indicates the detection of the first primer (RB-F2) of SEQ ID NO.5;

[0054] SEQ ID NO.14 indicates the detection of the second primer (RB-R2) of SEQ ID NO.5;

[0055] SEQ ID NO.15 indicates the detection of the first primer (LB-F2) of SEQ ID NO.6;

[0056] SEQ ID NO.16 indicates the detection of the second primer (LB-R2) of SEQ ID NO.6.

[0057] The transgenic maize strain JZL22k-1 of this invention expresses three proteins: Cry1Fa, Cry2Ab, and CP4 EPSPS. It exhibits high tolerance to glyphosate herbicides and high resistance to major lepidopteran pests of crops. Glyphosate herbicides can be used for transgenic screening during hybridization breeding, and glyphosate herbicides can be used for weed control during transgenic maize planting, which can reduce the damage caused by major lepidopteran pests of crops.

[0058] Compared with the prior art, the beneficial effects of the present invention are mainly reflected in:

[0059] (1) This invention provides an insect-resistant and herbicide-tolerant transgenic maize event JZL22k-1, which will target the gene cry2Ab , cry1Fa and cp4 epspsThe single copy insertion at a specific site in the maize genome ensures stable integration, stable expression, and stable herbicide tolerance and pest resistance in different generations of the transgenic maize event JZL22k-1 and in maize materials containing the transgenic maize event JZL22k-1.

[0060] (2) The nucleotide sequence for specifically detecting transgenic maize event JZL22k-1 provided by the present invention, the sequence of SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6 or SEQ ID NO.7+SEQ ID NO.8 connected end to end, can specifically detect transgenic maize event JZL22k-1.

[0061] (3) In the detection method of the present invention for detecting the transgenic maize event JZL22k-1, specific detection primer pairs are designed for the JZL22k-1 specific sequence SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6 or SEQ ID NO.7+SEQ ID NO.8 connected end to end. The presence of the JZL22k-1 transgenic event can be accurately and stably identified by nucleic acid amplification method, and the traceability and full-process supervision of the research, production, processing and application of JZL22k-1 can be realized.

[0062] (4) The transgenic maize event JZL22k-1 described in this invention can be screened for transgenics using glyphosate herbicide during hybridization breeding, and can be used for weed control during maize planting. This can effectively improve weed control efficiency, reduce the risk of herbicide-resistant weeds, reduce weed control costs, and also prevent the infestation of major lepidopteran pests of crops, reduce the risk of herbicide-resistant pests, and reduce pest control costs.

[0063] (5) Agricultural products or commodities containing the genetically modified corn event JZL22k-1 obtained by the method of the present invention. (iv) Description of the attached drawings

[0064] Figure 1Map of the transformation vector; Tnos: terminator of the NOS gene; cry2Ab: gene encoding the insecticidal protein cry2Ab; pFMV: promoter of Scrophularia mosaic virus; pZmUbi: promoter of the maize Ubiqutin-1 gene; cry1Fa: gene encoding the insecticidal protein cry1Fa; cp4epsps: gene encoding CP4EPSPS; t35s: terminator of the CaMV 35S gene; Left border, Right border: left and right borders of the T-DNA.

[0065] Figure 2 The results of chromosome localization of the JZL22k-1 insertion sequence in plant cells.

[0066] Figure 3 This is a schematic diagram of the exogenous inserted gene and the structure of the maize genome.

[0067] Figure 4 Electrophoresis image of JZL22k-1 left-wing specific PCR detection. M, DNA molecular weight standard; B, blank control; P, positive control (PCR product verified by sequencing); 1, 2: JZL22k-1; 3, 4: Ruifeng 125; 5, 6: Zhejiang University Ruifeng 8; 7, 8: CAL16; 9, 10: non-GMO maize Ruifeng-1; The amplified target fragment size was 656 bp, which is consistent with the expected size.

[0068] Figure 5 Electrophoresis diagram of JZL22k-1 right wing specific PCR detection. M, DNA molecular weight standard; B, blank control; P, positive control (PCR product verified by sequencing); 1, 2: JZL22k-1; 3, 4: Ruifeng 125; 5, 6: Zhejiang University Ruifeng 8; 7, 8: CAL16; 9, 10: non-GMO maize Ruifeng-1; The amplified target fragment size was 782 bp, which is consistent with the expected size. (V) Detailed Implementation Methods

[0069] The invention will now be described more fully with reference to the accompanying drawings, which show some, but not all, embodiments of the invention. In fact, the invention can be practiced in many different forms and should not be construed as limited to the embodiments listed herein. The following definitions and methods will better define the invention and will enable those skilled in the art to practice it. Many modifications and other embodiments of the invention can be made by those skilled in the art based on the information provided in the description and drawings herein. Therefore, it should be understood that the invention is not limited to the specific embodiments disclosed, and modifications and other embodiments based on the teachings of the invention are also included within the scope of the appended claims.

[0070] Unless otherwise stated, the terminology used in this invention should be understood in accordance with the common usage of those skilled in the art.

[0071] The "corn" mentioned in this invention refers to maize (Corn). Zea mays L.), which includes all plant species that mate with maize, including wild maize species.

[0072] The term "comprising" is synonymous with "including" and "contains," and refers to "including but not limited to." The plant includes the whole plant, plant cells, plant organs, plant protoplasts, plant cell tissue cultures from which the plant can regenerate, plant callus, plant clusters, and complete plant cells from plants or plant parts, such as embryos, pollen, ovules, seeds, leaves, flowers, branches, fruits, stems, roots, root tips, anthers, etc. The transgenic plant is derived from a transgenic plant or its progeny that has been transformed from the DNA molecules of this invention and is therefore at least partially composed of transgenic cells.

[0073] The transgenic “event” is obtained by transforming plant cells with a construct containing exogenous DNA (e.g., an expression cassette including at least one containing the target gene), inserting it into the plant genome via transgenic methods to generate a plant population, regenerating the plant population, and selecting specific plants with characteristics of the insertion at a specific genomic site. The term “event” refers to the original event including the exogenous DNA and / or the offspring of that event. The term “event” also refers to the offspring obtained by sexually crossing an event with other varietal individuals containing exogenous DNA, where, even after repeated backcrossing with backcrossing parents, the inserted DNA and flanking genomic DNA from the event parent are present at the same chromosomal location in the hybrid offspring. The term “event” also refers to a DNA sequence from the original event containing the inserted DNA and flanking genomic sequences closely adjacent to the inserted DNA, which is intended to be transferred to progeny produced by sexually crossing a parental line containing the inserted DNA (e.g., the original event and its self-crossed progeny) with a parental line not containing the inserted DNA, and the progeny receiving the inserted DNA containing the target gene.

[0074] The term "transgenic" includes any cell, cell line, callus, tissue, plant part, or plant whose genotype has been altered due to the presence of exogenous nucleic acid. "Transgenic" includes the original transgenic organism that was so altered, as well as offspring individuals generated from the original transgenic organism through sexual hybridization or asexual reproduction. In this invention, the term "transgenic" does not include genomic (chromosomal or extrachromosomal) alterations achieved through conventional plant breeding methods or naturally occurring events such as random allogeneic fertilization, non-recombinant viral infection, non-recombinant bacterial transformation, non-recombinant transposition, or spontaneous mutation.

[0075] The “transgenic maize event JZL22k-1” refers to the DNA molecule obtained by inserting a foreign DNA molecule into the chr4:212344935 site of the maize genome. It also includes the plant JZL22k-1 containing the transgenic maize event JZL22k-1, seeds, and plant cells or their regenerable parts. The plant parts include, but are not limited to, cells, pollen, ovules, flowers, buds, roots, stems, silks, inflorescences, ear-like clusters, leaves, and products from the maize plant JZL22k-1, such as corn flour, cornmeal, corn steep liquor, corn silks, corn starch, and biomass remaining in the maize crop field.

[0076] The "primer" is a segment of isolated nucleic acid molecule that binds to a complementary target DNA strand through nucleic acid hybridization and annealing, forming a hybrid between the primer and the target DNA strand. This hybrid then extends along the target DNA strand under the action of a polymerase (e.g., DNA polymerase). The primer pairs of this invention relate to their application in the amplification of target nucleic acid sequences, for example, by polymerase chain reaction (PCR) or other conventional nucleic acid amplification methods.

[0077] The technical solution of the present invention is further illustrated below through specific embodiments.

[0078] Example 1: Obtaining a plasmid vector containing a foreign gene

[0079] This invention utilizes the pCambia1300 vector (Genbank accession NO. AF234296) as the genetic transformation vector. T-DNA containing the complete expression cassettes of CP4 EPSPS and Cry1Fa and Cry2Ab proteins is inserted into its multiple cloning site region. The main construction process involves removing the hygromycin resistance gene (XhoI digestion) and replacing it with the glyphosate tolerance gene cp4epsps. The remaining selection gene, the kanamycin resistance gene kanR, is not present in the T-DNA and therefore generally cannot be inserted into the plant genome; it serves only as a selection marker in E. coli during vector construction. Some pathogenic genes in the Agrobacterium T-DNA have been removed, retaining only the boundary sequences. Currently, the pCambia1300 vector is a commonly used vector in Agrobacterium-mediated transgenic methods, and its pathogenicity has not been reported for a long time, nor is it theoretically possible for it to evolve into a pathogenic gene. The structure of the plasmid vector containing the foreign gene is shown below. Figure 1 As shown, the exogenous DNA molecule includes cry1Fa、cry2Ab Gene expression cassettes and cp4epsps Gene expression cassette; cry1Fa、cry2Ab Gene expression cassettes include: used as cry1Fa The gene is promoted by the ZmUbi promoter derived from the maize polyubiquitin-1 gene. cry1Fa Gene coding frames, used as cry1Fa The Tnos terminator for gene termination, used as cry2Ab The promoter pFMV of Scrophularia mosaic virus, cry2Ab Gene coding frames, used as cry2Ab The cp4epsps gene expression cassette includes: a ZmUbi promoter for initiating the cp4epsps gene, a cp4epsps gene coding frame, and a CaMV35S terminator for terminating the cp4epsps gene.

[0080] Example 2: Obtaining JZL22k-1 from plants containing transgenic maize event JZL22k-1

[0081] Agrobacterium-mediated genetic transformation of maize was performed according to the method and culture medium formulation reported by Frame et al. (Plant Physiol, 2002, 129:13-22), using glyphosate as the screening reagent. The steps were as follows: Maize ears were collected 8-10 days after pollination, and immature embryos measuring 1.0-1.5 mm were collected. Agrobacterium containing the transformation vector was mixed into the infection medium, and the OD660 concentration was adjusted to 0.5-0.6. The collected immature embryos were placed in the infection solution containing Agrobacterium and allowed to stand at room temperature for 5 min. The embryos were then poured into a co-culture medium, the liquid was aspirated, and the embryos were placed face down on the co-culture medium and cultured at 22°C for 2-3 days. The cultured immature embryos were then transferred to callus induction medium containing a final concentration of 200 mg / L termethin antibiotic (GlaxoSmithKline, USA) and cultured in the dark at 28°C for 7-10 days to kill Agrobacterium. All callus tissues after induction culture were transferred to selection medium containing a final concentration of 2 mM glyphosate and cultured in the dark at 28 °C for 2-3 weeks. After induction culture, all callus tissues were transferred to fresh selection medium containing 2 mM glyphosate and cultured in the dark at 28 °C for 2-3 weeks. Viable embryogenic tissues were transferred to regeneration medium and cultured in the dark at 28 °C for 10-14 days, then transferred to fresh regeneration medium and cultured under light at 26 °C for 10-14 days. Fully developed plantlets were selected and cultured on rooting medium at 26 °C under light until roots were fully developed. The rooted regenerated seedlings were transplanted to a greenhouse for propagation, serving as the T0 generation event for screening and analysis.

[0082] Example 3: Screening of maize transformant JZL22k-1 containing transgenic maize event JZL22k-1

[0083] The 950 T0 generation events obtained in Example 2 were transplanted into a greenhouse after hardening off, and 801 seedlings survived in the greenhouse. When the T0 generation transgenic maize reached the 4-5 leaf stage, glyphosate (effective dose of glyphosate is 60 g / mu) was sprayed. 210 events showed no phytotoxicity, 640 events showed phytotoxicity, and 100 events resulted in death (Table 2).

[0084] Table 2 Tolerance of T0 generation events to glyphosate herbicides

[0085]

[0086] Quantitative PCR was performed on events without phytotoxicity to determine the content of exogenous genes in the events, thereby assessing the T-DNA insertion copy number and discarding events with two or more copies. Plants from the 210 surviving events were used to extract plant genomes using the CTAB method. PCR was then performed using the SYBR Green Real-Time PCR Kit (BIO RAD) on a Bio-Rad RadCFX96. TM The reaction was performed using a real-time PCR instrument. The copy number of the cp4 epsps gene was determined by comparing Ct values. zSSIIb from the maize genome was selected as an internal reference gene. A random maize event was chosen as a baseline, and the relative abundance of the target gene at the initial stage of the reaction was calculated.

[0087] Table 3 Primers for Quantitative PCR

[0088]

[0089] By analyzing the experimental results of the copy number of the cp4 epsps gene, it was confirmed that the exogenous gene had been integrated into the chromosome set of the maize plants tested, including 41 single-copy transgenic maize plants.

[0090] When glyphosate herbicide (effective dose of glyphosate 120 g / acre) was sprayed on the progeny of 41 selected single-copy events, the results showed that 7 events were tolerant to higher concentrations of herbicide (Table 4).

[0091] Table 4. Tolerance of T1 generation events to glyphosate herbicides

[0092]

[0093] The expression levels of seven events (denoted as JZL22k-1 to JZL22k-7) with good herbicide tolerance were determined.

[0094] The detection kits for Cry2Ab and CP4EPSPS proteins used in this study were purchased from Shanghai Youlong Biotechnology Co., Ltd., with catalog numbers AA0741 and AA0841, respectively. The Cry1Fa protein kit was ordered from Nanjing Zhongding Biotechnology Co., Ltd. by our unit. The experimental methods were performed in accordance with the kit instructions, and the specific steps are as follows.

[0095] CP4 EPSPS and Cry2Ab kit operation steps:

[0096] (1) Remove the kit from the refrigerated environment and place it at room temperature (20-25 ℃) for more than 30 min to allow all reagents and the required strips to return to room temperature. Shake each liquid reagent well before use.

[0097] (2) Take 40-50 mg of corn tissue, place it in a 2 mL centrifuge tube, add steel beads, freeze in liquid nitrogen, and grind it using a grinder. Add 600 µL of sample extract, vortex to mix for 5 min, let stand at room temperature for 5 min, and centrifuge at 12000 rpm for 10 min. Dilute the sample with sample extract as needed before use for determination. 450 The value is controlled within the measurable range.

[0098] (3) Dilute the standard concentrations to 48 ppb, 24 ppb, 12 ppb, 6 ppb and 3 ppb. Dilute the 20× concentrated washing solution with deionized water to make a 1× washing working solution. Dilute the 11× concentrated enzyme standard solution with enzyme diluent at a volume ratio of 1:10.

[0099] (4) Add 100 µL of sample extraction solution (blank control) / standard / sample to each well of the ELISA plate, gently shake to mix, seal the ELISA plate with Parafilm membrane, and shake on a horizontal shaker in the dark for 45 min at room temperature.

[0100] (5) After the reaction is complete, pour out the liquid in the plate, add 250 µL of washing working solution to each well and wash thoroughly 4-5 times. Pat the plate dry on absorbent paper.

[0101] (6) Add 100 µL of enzyme-labeled working solution to each well, gently shake to mix, seal the ELISA plate with Parafilm membrane, and shake on a horizontal shaker in the dark for 30 min at room temperature. Repeat step 5.

[0102] (7) Add 100 µL of colorimetric reagent to each well, gently shake to mix, seal the ELISA plate with Parafilm membrane, and shake on a horizontal shaker in the dark for 15 min at room temperature.

[0103] (8) Add 100 µL of stop solution to each well, gently vortex to mix, and measure OD in a microplate reader within 5 min.450 .

[0104] (9) Based on the OD of the standard sample 450 A standard curve can be plotted using these values. To eliminate systematic errors between measurements, a standard curve is prepared for each sample measurement. The formula for the standard curve of one CP4 EPSPS measurement is: y = 0.0475x + 0.0232(R² + π / 2)². 2 = 0.9998), where the standard curve formula for one Cry2Ab determination is: y = 0.0337x + 0.0923 (R = 0.9998). 2 = 0.996).

[0105] (10) The OD of the sample 450 Substitute the value into the standard curve, read the corresponding concentration of the sample from the standard curve, and you can calculate the CP4 EPSPS protein content (µg / g) = sample concentration (ppb). Dilution factor Sample extraction volume (µL) / leaf weight (mg) / 1000, Cry2Ab protein content (µg / g) = sample concentration (ppb) Dilution factor Sample extraction liquid volume (µL) / leaf weight (mg) / 1000.

[0106] Cry1Fa reagent kit operation steps

[0107] (1) Dilute the goat anti-Cry1Fa antibody to a protein concentration of 10 μg / mL with 0.05 M pH 9.0 carbonate coating buffer. Add 0.1 mL to each well of the polystyrene plate and incubate overnight at 4°C. The next day, discard the solution in the wells and wash three times with washing buffer for 3 minutes each time (hereinafter referred to as washing).

[0108] (2) Add 0.1 mL of a certain amount of diluted test sample to the above-mentioned coated reaction wells and incubate at 37°C for 1 hour. Then wash (simultaneously prepare blank wells, negative control wells and positive control wells);

[0109] (3) Add 0.1 mL of freshly diluted rabbit anti-Cry1Fa antibody (titrated dilution of 1:5000) to each well. Incubate at 37°C for 1 hour, then wash.

[0110] (4) Add 0.1 mL of freshly diluted enzyme-labeled goat anti-rabbit IgG antibody (titrated to a dilution of 1:10000) to each reaction well. Incubate at 37°C for 1 hour, then wash.

[0111] (5) Add 0.1 mL of the prepared TMB substrate solution to each well and incubate at 37°C for 20 minutes. Then add 0.05 mL of 2 M sulfuric acid to each well to terminate the reaction.

[0112] (6) On the ELISA instrument, measure the OD value of each well at 450 nm after zeroing with the blank control well;

[0113] (7) Standard Curve: After experiments with a wide range of different concentrations, the linear region (relationship between antigen concentration and OD) of Cry1Fa assay was determined. The standard curve formula for one Cry1Fa assay is: y = 0.0475x + 0.0496(R) 2 = 0.9985).

[0114] (8) The OD of the sample 450 Substitute the value into the standard curve, read the corresponding concentration of the sample from the standard curve, and you can calculate the Cry1Fa protein content (µg / g) = sample concentration (ppb). Dilution factor Sample extraction liquid volume (µL) / leaf weight (mg) / 1000.

[0115] Table 5. Expression levels of exogenous proteins in different events

[0116]

[0117] Events with moderate expression levels of CP4 EPSPS, Cry2Ab, and Cry1Fa proteins were selected, and combined with field agronomic traits, the superior event JZL22k-1 was finally selected through screening. This event has the characteristics of a single copy insertion site of exogenous gene, strong glyphosate tolerance, good insect resistance, stable inheritance of exogenous gene and traits, and outstanding agronomic traits.

[0118] Example 4: Detection of the JZL22k-1 transgenic event in maize

[0119] 1. Extraction of maize genome

[0120] Genomic DNA of event JZL22k-1, screened in Example 3, was extracted using the CTAB (hexadecyltrimethylammonium bromide) method. 1000 mg of young leaves of the JZL22k-1 cultivar were ground into powder in liquid nitrogen. 0.8 mL of preheated CTAB buffer (20 g / L CTAB, 1.4 M NaCl, 100 mM Tris-HCl, 20 mM EDTA, pH 8.0) was added and thoroughly mixed. The mixture was then incubated at 65°C for 60 min. An equal volume of chloroform was added, and the mixture was inverted and centrifuged at 12000 rpm for 10 min. The supernatant was transferred to a new centrifuge tube. 0.7 volumes of isopropanol were added, and the tube was gently shaken. The tube was centrifuged at 12000 rpm for 1 min, and the DNA was collected at the bottom of the tube. The supernatant was discarded, and 1 mL of 75% ethanol was added to wash the precipitate. The precipitate was centrifuged at 12000 rpm for 1 min, and the washing was repeated once. The precipitate was then dried in a clean bench. The DNA precipitate was dissolved in an appropriate amount of TE buffer (10... The concentration of DNA was determined by Nanodrop in mM Tris-HCl, 1mM EDTA, pH 8.0, and stored for later use.

[0121] 2. Analysis of flanking DNA sequences

[0122] The hiTAIL-PCR (High-efficiency thermal asymmetric interlaced PCR) method reported by Liu et al. (Liu, Yao Guang, and Yuanling Chen. 2007. High-efficiency thermal asymmetric interlaced PCR for amplification of unknown flanking sequences. Biotechniques, 43: 649-650.) was used to determine the region flanking the insertion site of the exogenous transgenic DNA of the superior event JZL22k-1 screened in Example 3. This method involves sequential PCR amplification using three nested specific primers combined with degenerate primers, selectively amplifying the target fragment using different annealing temperatures. Primer sequences are shown in Table 6, PCR reaction systems in Table 7, and PCR reaction conditions in Table 8.

[0123] Table 6. hiTAIL-PCR primer sequences

[0124]

[0125] Table 7 hiTAIL-PCR reaction system

[0126]

[0127] Table 8. HiTAIL-PCR Reaction Conditions

[0128]

[0129] The third-round PCR amplification products were recovered using an Axygen PCR product recovery kit, ligated into the PMD19-T cloning vector (TaKaRa, Code: D102A), transformed into *E. coli*, and the resulting positive clones were sequenced by Hangzhou Youkang Biotechnology Co., Ltd. The obtained sequence information was compared with the maize online database (http: / / www.maizegdb.org) to search for similar maize genome sequences.

[0130] 3. Right flank region of T-DNA

[0131] The fragment identified by hiTAIL-PCR as containing the 5' flanking region was sequenced, and the sequencing result is SEQ ID NO.5. The sequence from 1 to 544 bp corresponds to maize genomic DNA, and the sequence from 545 to 786 bp corresponds to exogenous DNA.

[0132] 4. Left wing region of T-DNA

[0133] The fragment identified as containing the 3' flanking region was sequenced, and the sequencing result is SEQ ID NO.6. The first 1-354 bp is the nucleotide sequence of the maize flanking genome, and the second 355-632 bp is the nucleotide sequence of the inserted gene.

[0134] 4. JZL22k-1 integrates into the genome sequence information

[0135] The above-mentioned flanking sequences upstream and downstream of the insertion site, which have been sequenced, compared, and verified, and the herbicide resistance gene expression cassette sequence (including T-DNA expressing the complete CP4 EPSPS protein expression cassette and the Cry1Fa and Cry2Ab protein expression cassettes) are spliced ​​together to form the transgenic maize event JZL22k-1 described in this invention. The nucleotide sequence is the sequence of SEQ ID NO.7 + SEQ ID NO.8 linked end to end. The gene structure diagram is as follows. Figure 3 As shown.

[0136] Example 5: Specificity detection of transgenic maize event JZL22k-1

[0137] The 5' and 3' flanking sequences of the transgenic maize event JZL22k-1 are shown as nucleotides 1-6953 and 18890-26226 of the sequence connected end-to-end in SEQ ID NO.7+SEQ ID NO.8, respectively. Primers were designed for the 5' and 3' insertion sites of the transgenic maize event JZL22k-1, and PCR reactions were performed. Primer information is shown in Table 9, and the reaction system is shown in Table 10.

[0138] Table 9 Primer Information

[0139]

[0140] Table 10 PCR Reaction System

[0141]

[0142] The PCR reaction program was as follows: denaturation at 94℃ for 5 min, denaturation at 94℃ for 30 s, annealing at 58℃ for 30 s, extension at 72℃ for 30 s, for a total of 35 cycles, with a final extension at 72℃ for 7 min.

[0143] Non-GMO corn, 1% GMO corn JZL22k-1, 1% GMO corn Ruifeng 125, 1% GMO corn Zheda Ruifeng 8, and 1% GMO soybean CAL16 were used as test subjects.

[0144] Similarly, primer pairs RB-F2 (SEQ ID NO.13) and RB-R2 (SEQ ID NO.14) were used to specifically detect the nucleic acid in non-GMO maize, 1% GMO maize JZL22k-1, 1% GMO maize Ruifeng 125, 1% GMO maize Zheda Ruifeng 8, and 1% GMO soybean CAL16, respectively. Electrophoresis results showed that only the JZL22k-1 sample showed a band of approximately 780 bp, consistent with expectations. Other samples lacking the JZL22k-1 genome did not show any specific bands. The primer pairs provided in this invention can specifically detect the presence of JZL22k-1. Figure 4 ).

[0145] Primers LB-F1 (SEQ ID NO.11) and LB-R1 (SEQ ID NO.12) were used to perform PCR amplification on non-GMO maize, 1% GMO maize JZL22k-1, 1% GMO maize Ruifeng 125, 1% GMO maize Zheda Ruifeng 8, and 1% GMO soybean CAL16, respectively. Electrophoresis results showed that only the JZL22k-1 sample showed a band of approximately 650 bp, consistent with the expected band size. Figure 5 Other samples lacking the JZL22k-1 genome showed no bands detected, but the primer pair provided by this invention can specifically detect the presence of JZL22k-1. Therefore, the test primer pair provided by this invention can specifically detect the presence of samples containing JZL22k-1.

[0146] Example 7: Determination of resistance to lepidopteran pests in major crops

[0147] 1. Indoor bioassay

[0148] B through indoor bioassay The resistance levels of the whorls, silks, and kernels of transgenic maize JZL22k-1, non-transgenic control maize Ruifeng-1, and the local control variety Jundan 58 to the target pests fall armyworm, beet armyworm, and corn borer were determined. The determination methods are as follows:

[0149] During the heart leaf stage, the above-ground parts of corn plants with 5-8 leaves were brought indoors. Unopened, tender heart leaves were cut into 2-3 cm pieces with sterilized scissors and placed in petri dishes. Ten newly hatched larvae were inoculated into each dish, with each dish constituting a replicate, repeated 6 times. The dishes were placed in an artificial climate incubator at 28±1℃, a photoperiod of 16 h:8 h (L:D), and a relative humidity of 70%-80%. Based on tissue feeding and consumption, fresh tissue from the same source was replaced every other day, and the number of surviving larvae was recorded.

[0150] During the silking stage of maize, newly silking (unpollinated) maize female ears were collected from the field and brought indoors. The husks were removed, the silks were gently removed, and placed in petri dishes, each inoculated with 10 newly hatched larvae. Each petri dish served as a replicate, with 6 replicates. The dishes were cultured in an artificial climate incubator at 28±1℃, a photoperiod of 16 h:8 h (L:D), and a relative humidity of 70%-80%. Based on tissue consumption, new tissue from the same source was replaced every other day, and the number of surviving larvae was recorded.

[0151] During the corn grain-filling stage, fresh female ears were collected from the field, their husks removed, and a suitable amount of kernels were placed in a petri dish. Ten newly hatched larvae were inoculated into each dish, with each dish constituting a replicate, repeated six times. The dishes were then placed in an artificial climate incubator at a temperature of 28±1℃, a photoperiod of 16 h:8 h (L:D), and a relative humidity of 70%-80%. Based on kernel consumption, the tissue was replaced with fresh tissue from the same source every other day, and the number of surviving larvae was recorded.

[0152] Statistical analysis was performed, and larval mortality rates were calculated based on the number of larvae killed in each treatment at 24 h, 48 h, and 72 h. The resistance levels of transgenic maize JZL22k-1, non-transgenic control maize Ruifeng-1, and the local control variety Jundan 58 to fall armyworm, beet armyworm, and corn borer were determined. During indoor insect resistance assessment, results were discarded if the larval mortality rate in the control group was >20%, and corrected mortality rates were calculated if the larval mortality rate in the control group was <20%. In this experiment, the larval mortality rate in the control group was the average of the larval mortality rates of non-transgenic maize Ruifeng-1 and Jundan 58. Corrected mortality rate (%) = (Larval mortality rate in treatment groups - Larval mortality rate in control groups) / (1 - Average larval mortality rate in control groups) × 100.

[0153] The results showed that the young leaves of JZL22k-1 exhibited high resistance to newly hatched larvae of the fall armyworm, beet armyworm, and corn borer. Larvae feeding on the whorls of transgenic corn turned black and all died within 72 hours (Table 11); while non-transgenic corn was severely fed on by insect larvae, but the larvae survived and grew normally. The silks of JZL22k-1 also exhibited high resistance to newly hatched larvae of the fall armyworm, beet armyworm, and corn borer. Larvae feeding on the silks of JZL22k-1 turned black and all died within 72 hours, while the silks of corn were almost undamaged. Non-transgenic corn was severely fed on by insect larvae, but the larvae survived and grew normally (Table 11). The kernels of JZL22k-1 also exhibited high resistance to newly hatched larvae of the fall armyworm, beet armyworm, and corn borer. All larvae that fed on JZL22k-1 kernels died within 72 hours, and the corn kernels were almost undamaged; while the non-genetically modified corn kernels were severely fed on by insect larvae, and the larvae survived and grew normally (Table 13).

[0154] Table 11 Results of indoor bioactivity assays of JZL22k-1 transgenic maize whorls against insects

[0155]

[0156] Table 12 Results of indoor bioactivity assays of JZL22k-1 transgenic maize silks against insects

[0157]

[0158] Table 13 Results of indoor bioactivity assays of JZL22k-1 transgenic maize kernels against insects

[0159]

[0160] 2. Field infestation detection

[0161] Field resistance tests against the beet armyworm showed that JZL22k-1 at the whorl and silking stages exhibited high resistance to the beet armyworm, while the non-GMO control maize Ruifeng-1 and the local control variety Jundan 58 showed susceptibility. The leaf damage level of JZL22k-1 was significantly lower than that of the non-GMO maize control Ruifeng-1 and the local control Jundan 58 (Table 14).

[0162] Field resistance tests on corn borers showed that JZL22k-1 at the whorl and silking stages exhibited high resistance to corn borers. The non-GMO control corn variety Ruifeng-1 and the local control variety Jundan 58 showed susceptibility to corn borers. The leaf-eating and ear-damage levels of JZL22k-1 were significantly lower than those of the non-GMO controls Jundan 58 and Ruifeng-1 (Table 15). Stem dissection results showed that the number of borer holes, tunnel length, and larvae per plant were significantly lower in JZL22k-1 than in the non-GMO control and the local control variety Jundan 58. No corn borer damage was observed in JZL22k-1, while the non-GMO control variety Ruifeng-1 and the local control variety showed more severe damage (Table 16).

[0163] Table 14. Field resistance identification results of JZL22k-1 against Spodoptera litura.

[0164]

[0165] Numerical values ​​are expressed as mean ± SD; different lowercase letters in the same column indicate significant differences at the 5% level, and the same applies to the following tables.

[0166] Table 15. Field resistance identification results of JZL22k-1 against corn borer.

[0167]

[0168] Table 16. Stem section survey results of JZL22k-1 infested with corn borer.

[0169]

[0170] Example 8: Glyphosate Tolerance Test of JZL22k-1

[0171] Seeds from the transgenic maize event JZL22k-1 (Example 4) and common maize Zhengdan 958 (as a control) were used in a randomized block design with three replicates, resulting in 24 plots. Each plot was 4m × 6m in size, with double seeds sown. Plant spacing was 25 cm, row spacing was 50 cm, and plots were spaced 1m apart. Glyphosate was sprayed at the 3-5 leaf stage under the following treatments: 1) no spraying; 2) spraying with a medium dose of glyphosate (effective dose of 60 g / mu); 3) spraying with twice the medium dose of glyphosate (effective dose of 120 g / mu); 4) spraying with four times the medium dose of glyphosate (effective dose of 240 g / mu). Seedling survival rate, plant height (selecting the 5 tallest plants), and phytotoxicity symptoms (selecting the 5 plants with the mildest symptoms) were assessed at 1, 2, and 4 weeks after application. Phytotoxicity symptom grading was performed according to GB / T 17980.42-2000.

[0172] Formula for calculating herbicide damage rate:

[0173]

[0174] In the formula, X: damage rate, in percentage (%); N: number of affected plants at a certain level; S: level value; Z: total number of plants; M: highest level.

[0175] Analysis of variance was used to compare the differences in emergence rate, seedling survival rate, and damage rate between transgenic herbicide-resistant maize and non-transgenic maize under different treatments. The tolerance level of transgenic herbicide-resistant maize to herbicides was determined, and the results are shown in Table 17.

[0176] Table 17. Survey of Glyphosate Tolerance of JZL22k-1

[0177]

Claims

1. A nucleic acid sequence of a transgenic maize event JZL22k-1, characterized in that, The nucleic acid sequence comprises SEQ ID NO:1 or its complementary sequence, and / or SEQ ID NO:2 or its complementary sequence.

2. The nucleic acid sequence as described in claim 1, characterized in that, The nucleic acid sequence includes one of the sequences of SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7+SEQ ID NO.8 connected end to end, or a complementary sequence thereof.

3. A method for detecting the transgenic maize event JZL22k-1 as described in claim 1, characterized in that, The method includes: (1) Contact the sample to be tested with the first primer and the second primer in a nucleic acid amplification reaction solution; the first primer is one of SEQ ID NO.9, SEQ ID NO.11, SEQ ID NO.13 or SEQ ID NO.15; the second primer is one of SEQ ID NO.10, SEQ ID NO.12, SEQ ID NO.14 or SEQ ID NO.16; (2) Perform nucleic acid amplification reaction; (3) Detect the presence of amplification products; the amplification products include one of the sequences of SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7+SEQ ID NO.8 connected end to end or their complementary sequences.

4. A primer pair for detecting the transgenic maize event JZL22k-1 as described in claim 1, characterized in that, The primer pair includes a first primer and a second primer; the first primer is one of SEQ ID NO. 9, SEQ ID NO. 11, SEQ ID NO. 13 or SEQ ID NO. 15; the second primer is one of SEQ ID NO. 10, SEQ ID NO. 12, SEQ ID NO. 14 or SEQ ID NO.

16.

5. A method for obtaining herbicide-resistant maize plants containing the transgenic maize strain JZL22k-1 as described in claim 1, characterized in that, Maize plants containing a specific region of nucleic acid sequence are crossed with another type of maize plant to produce offspring plants. Compared to other plants that do not contain the specific region of nucleic acid sequence, the offspring plants exhibit significantly improved tolerance to herbicides. The specific region of nucleic acid sequence originates from the transgenic maize event JZL22k-1, and comprises one or a complementary sequence of the nucleotide sequences shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, or SEQ ID NO.7+SEQ ID NO.

8. The herbicide is glyphosate.

6. A method for cultivating insect-resistant maize plants containing the transgenic maize strain JZL22k-1 as described in claim 1, characterized in that, The method includes: planting corn seeds containing a specific region nucleic acid sequence, allowing the corn to grow into corn plants, obtaining the unfolded tender heart leaves, placing them in a petri dish containing a target pest, and harvesting plants that show significantly improved pest resistance compared to other corn plants that do not contain the specific region nucleic acid sequence; the specific region nucleic acid sequence is from the transgenic corn event JZL22k-1, and the specific region nucleic acid sequence includes one of the nucleotide sequences shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, or SEQ ID NO.7+SEQ ID NO.8 connected end-to-end, or their complementary sequences; the target pest is a major lepidopteran pest of crops.

7. A method for obtaining insect-resistant maize plants containing the transgenic maize strain JZL22k-1 as described in claim 1, characterized in that, The method involves hybridizing a maize plant containing a specific region of nucleic acid sequence with another maize plant to produce offspring plants; the offspring plants harvested show significantly increased resistance to target pests compared to other plants that do not contain the specific region of nucleic acid sequence. The specific region nucleic acid sequence is from the transgenic maize event JZL22k-1, and the specific region nucleic acid sequence includes one of the nucleotide sequences shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6 or SEQ ID NO.7+SEQ ID NO.8 connected end to end, or their complementary sequence; the target pest is a major crop lepidopteran pest.

8. An agricultural product or commodity derived from the transgenic maize event JZL22k-1 as described in claim 1, characterized in that, The agricultural products or commodities mentioned include corn flour, cornmeal, corn oil, corn starch, corn gluten, corn cakes, cosmetics containing corn ingredients, or auxiliary materials containing corn ingredients.

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