Rice RN85zN-eJ and detection method thereof

Abstract

The invention relates to a nucleic acid molecule of a rice transformation event RN85zN-eJ and a detection method. The nucleic acid molecule of the rice RN85zN-eJ comprises a sequence as shown in SEQ ID NO: 1 or a reverse complementary sequence of the sequence as shown in SEQ ID NO: 1, or a sequence as shown in SEQ ID NO: 2 or a reverse complementary sequence of the sequence as shown in SEQ ID NO: 2. The rice transformation event RN85zN-eJ has the characteristics of insect resistance and glufosinate-ammonium herbicide resistance, and the detection method can accurately and quickly identify whether a biological sample contains DNA molecules of the transgenic rice event RN85zN-eJ or not.

Description

Technical Field

[0001] This invention relates to the field of plant biotechnology. Specifically, it relates to a nucleic acid molecule and detection method for the rice transformation event RN85zN-eJ, particularly to a transgenic rice event RN85zN-eJ that is insect-resistant and tolerant to glufosinate-ammonia herbicide application, and a nucleic acid molecule and detection method for detecting whether a biological sample contains the specific transgenic rice event RN85zN-eJ. Background Technology

[0002] Rice is one of the most widely cultivated crops in my country. Throughout its growth cycle, rice is susceptible to various insect pests, among which the rice stem borer is a major pest, causing widespread damage and posing a serious threat to large rice paddies globally each year. Especially during critical growth seasons such as summer and autumn, rice stem borer infestations often lead to significant yield reductions. Developing insect-resistant and herbicide-tolerant genetically modified rice varieties and promoting genetically modified rice will be an effective means to increase yields and ensure food security.

[0003] Using a single Bt protein for pest control can easily lead to pesticide resistance in target pests. Therefore, it is necessary to develop new insecticidal proteins to improve insecticidal efficiency and prolong control effects. LC85-Ac protein is a novel insecticidal protein that can be used to control lepidopteran pests including fall armyworm, rice stem borer, armyworm, beet armyworm, cotton bollworm, cutworm, and two-spotted cutworm (patent application number 202410332785.6).

[0004] Weeds compete with crops for water, fertilizer, light, and growing space, directly impacting crop yield and quality. Many weeds also serve as intermediate hosts for crop pathogens and pests, making them a significant biological limiting factor for crop yield. Therefore, effectively controlling weeds is a crucial measure for promoting increased grain production. The widespread use of herbicides can significantly reduce the difficulty of field management and lower labor intensity. However, developing new, highly efficient, low-toxicity, and residue-free herbicides is costly, time-consuming, and challenging. Herbicide-tolerant rice, bred through transgenic technology, can overcome this challenge. Spraying herbicides once or twice during the rice's growing season can effectively solve the weed problem, reducing the amount of herbicide used and the associated costs. Therefore, herbicide-tolerant transgenic rice has vast application value and market potential.

[0005] This invention connects an insect-resistant gene expression cassette with a herbicide-tolerant gene expression cassette, enabling its efficient expression in transgenic rice, thus combining insect-resistant and herbicide-tolerant traits and further enhancing the product's application and economic value.

[0006] It is known that the expression of exogenous genes in plants is influenced by their chromosomal location, possibly due to the proximity of chromatin structures (such as heterochromatin) or transcriptional regulatory elements (such as enhancers) to the integration site. Therefore, screening a large number of events is often required to identify commercially viable events (i.e., events where the introduced target gene is optimally expressed). For example, significant differences in the expression levels of introduced genes have been observed between events in plants and other organisms; differences may also exist in spatial or temporal patterns of expression, such as the relative expression of transgenes in different plant tissues. This difference manifests as the actual expression pattern potentially differing from the expected expression pattern based on the transcriptional regulatory elements in the introduced gene construct, leading to variations in phenotypic expression of the transformation event. Therefore, it is often necessary to generate hundreds or thousands of different events and screen for a single event with the expected transgene expression levels and patterns for commercial purposes. Events with the expected transgene expression levels and patterns can be used to introduce transgenes into other genetic backgrounds through sexual crossbreeding using conventional breeding methods. Offspring produced through this crossbreeding retain the transgene expression characteristics of the original transformation event. Applying this strategy can ensure reliable gene expression in many varieties that are well adapted to local growing conditions. Therefore, more transformation events need to be identified and screened to obtain superior transformation events with excellent overall phenotypic performance and commercial potential.

[0007] Being able to detect the presence of specific events to determine whether the offspring of sexual hybridization contain the target gene would be beneficial. Furthermore, methods for detecting specific events would help comply with relevant regulations, such as the requirement for formal approval and labeling of foods derived from recombinant crops before they can be placed on the market. Detecting the presence of transgenes using any well-known polynucleotide detection method is possible, such as polymerase chain reaction (PCR). These methods typically focus on commonly used genetic elements, such as promoters, terminators, and marker genes. Therefore, unless the sequence of the chromosomal DNA adjacent to the inserted transgene DNA (“flanking DNA”) is known, the above methods cannot be used to distinguish different events, especially those produced using the same DNA construct. Therefore, it is currently common to use a pair of primers spanning the junction of the inserted transgene and the flanking DNA via PCR to identify transgene-specific events; specifically, a first primer containing the flanking sequence and a second primer containing the inserted sequence. Summary of the Invention

[0008] The purpose of this invention is to provide a rice transformation event (also called a transformant) with excellent insect resistance and herbicide tolerance traits, as well as a nucleic acid molecule for detecting rice RN85zN-eJ and a detection method thereof. The transgenic rice event RN85zN-eJ exhibits excellent insect resistance and good tolerance to glufosinate-ammonium herbicide, and the detection method can accurately and rapidly identify whether a biological sample contains the DNA molecule of the specific transgenic rice event RN85zN-eJ.

[0009] To achieve the above objectives, this invention uses the pBWA(V)HS-bar-LC85AcA5 expression vector to transform Nipponbare rice via Agrobacterium-mediated transformation, obtaining a batch of positive transformation events. Identification of insect resistance and herbicide tolerance traits revealed that transformation event RN85zN-eJ exhibits excellent herbicide tolerance and insect resistance, and can be used to improve the insect resistance and herbicide tolerance traits of rice.

[0010] To characterize the identity of RN85zN-eJ, the present invention provides a nucleic acid molecule comprising the sequences shown in SEQ ID NO:1 and / or SEQ ID NO:2, or their reverse complementary sequences.

[0011] Furthermore, the nucleic acid molecule comprises the sequences shown in SEQ ID NO:3 and / or SEQ ID NO:4, or their reverse complementary sequences.

[0012] Furthermore, the nucleic acid molecule comprises the sequence shown in SEQ ID NO:6 and / or SEQ ID NO:7, or its reverse complementary sequence.

[0013] Furthermore, the nucleic acid molecule contains the sequence shown in SEQ ID NO:5 or its reverse complementary sequence.

[0014] The present invention also provides a probe for detecting rice transformation events, characterized in that it comprises the sequence shown in SEQ ID NO:1 or SEQ ID NO:2 or SEQ ID NO:3 or SEQ ID NO:4 or SEQ ID NO:6 or SEQ ID NO:7, a fragment thereof, a variant thereof, or an inverse complementary sequence thereof.

[0015] The present invention also provides primer pairs for detecting rice transformation events, characterized in that the amplification products of the primer pairs contain the sequence shown in SEQ ID NO:1 or SEQ ID NO:2 or SEQ ID NO:3 or SEQ ID NO:4 or SEQ ID NO:6 or SEQ ID NO:7, or a fragment thereof, or a variant thereof, or an inverse complementary sequence thereof.

[0016] In some embodiments, the primer pairs are the sequences shown in SEQ ID NO:8 and SEQ ID NO:9; or the sequences shown in SEQ ID NO:10 and SEQ ID NO:11.

[0017] The present invention also provides a kit or microarray for detecting rice transformation events, characterized in that it comprises the above-described probe and / or primer pairs.

[0018] The present invention also provides a method for detecting rice transformation events, characterized in that it includes using the above-mentioned probe or primer pair or probe and primer pair or kit or microarray to detect whether the transformation event exists in the sample to be tested.

[0019] The present invention also provides a method for breeding rice, characterized in that the method includes the following steps:

[0020] 1) Obtain rice containing the above-mentioned nucleic acid molecules;

[0021] 2) The rice obtained in step 1) is subjected to pollen culture, unfertilized embryo culture, doubling culture, cell culture, tissue culture, self-pollination or hybridization, or a combination thereof, to obtain rice plants, seeds, plant cells, progeny plants, or plant parts; and optionally,

[0022] 3) Identify the insect resistance and / or herbicide resistance of the progeny plants obtained in step 2), and use the methods described above to detect whether the transformation event exists.

[0023] Furthermore, the present invention also provides articles made from rice plants, seeds, plant cells, progeny plants or plant parts obtained by the above method, including food, feed or industrial raw materials.

[0024] SEQ ID NO:1 is a 22-nucleotide sequence located near the insertion junction at the 5' end of the inserted sequence in the transgenic rice event RN85zN-eJ. SEQ ID NO:1 spans the left-hand genomic DNA sequence of the rice insertion site and the DNA sequence at the 5' end of the left boundary of the inserted sequence. The presence of SEQ ID NO:1 or its reverse complementary sequence is sufficient to identify the transgenic rice event RN85zN-eJ. SEQ ID NO:2 is a 22-nucleotide sequence located near the insertion junction at the 3' end of the inserted sequence in the transgenic rice event RN85zN-eJ. SEQ ID NO:2 spans the DNA sequence at the 3' end of the right boundary of the inserted sequence and the right-hand genomic DNA sequence of the rice insertion site. The presence of SEQ ID NO:2 or its reverse complementary sequence is sufficient to identify the transgenic rice event RN85zN-eJ.

[0025] In this invention, the nucleic acid sequence can be at least 11 or more consecutive polynucleotides (first nucleic acid sequence) of any portion of the transgenic insertion sequence in SEQ ID NO:3 or its reverse complementary sequence, or at least 11 or more consecutive polynucleotides (second nucleic acid sequence) of any portion of the 5' left-wing rice genomic DNA region in SEQ ID NO:3 or its reverse complementary sequence. The nucleic acid sequence can further be homologous to or reverse complementary to a portion of SEQ ID NO:3 comprising the complete SEQ ID NO:1 or SEQ ID NO:6. When the first and second nucleic acid sequences are used together, these nucleic acid sequences include a DNA primer pair in the DNA amplification method for generating the amplification product. When the amplification product generated in the DNA amplification method using the DNA primer pair is an amplification product comprising SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:6 or their reverse complementary sequence, the presence of the transgenic rice event RN85zN-eJ or its progeny can be diagnosed.

[0026] SEQ ID NO:3 is a 1351-nucleotide sequence located near the insertion junction at the 5' end of the inserted sequence in the transgenic rice event RN85zN-eJ. SEQ ID NO:3 consists of a 342-nucleotide rice left-wing genomic DNA sequence (nucleotides 1-342 of SEQ ID NO:3), a 102-nucleotide left-bound DNA sequence of the pBWA(V)HS-bar-LC85AcA5 construct (nucleotides 343-444 of SEQ ID NO:3), and a 907-nucleotide 5' end DNA sequence of the first expression cassette of the glufosinate-tolerant gene (nucleotides 445-1351 of SEQ ID NO:3). The presence of the transgenic rice event RN85zN-eJ can be identified by the presence of SEQ ID NO:3 or its reverse complementary sequence.

[0027] The nucleic acid sequence may be at least 11 or more consecutive polynucleotides (third nucleic acid sequence) of any portion of the transgenic insertion sequence in SEQ ID NO:4 or its reverse complementary sequence, or at least 11 or more consecutive polynucleotides (fourth nucleic acid sequence) of any portion of the 3' right wing rice genomic DNA region in SEQ ID NO:4 or its reverse complementary sequence. The nucleic acid sequence may further be homologous to or reverse complementary to a portion of SEQ ID NO:4 containing the complete SEQ ID NO:2 or SEQ ID NO:7. When the third and fourth nucleic acid sequences are used together, these nucleic acid sequences include a DNA primer set in the DNA amplification method that generates the amplification product. When the amplification product generated in the DNA amplification method using the DNA primer pair is an amplification product including SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:7 or their reverse complementary sequences, the presence of the transgenic rice event RN85zN-eJ or its progeny can be diagnosed.

[0028] SEQ ID NO:4 is a 1164-nucleotide sequence located near the insertion junction at the 3' end of the inserted sequence in the transgenic rice event RN85zN-eJ. SEQ ID NO:4 consists of the 3' end DNA sequence of the second expression cassette of the insect-resistant gene (nucleotides 1-533 of SEQ ID NO:4), the right boundary DNA sequence of the pBWA(V)HS-bar-LC85AcA5 construct (nucleotides 534-822 of SEQ ID NO:4), and the right wing genomic DNA sequence of the rice integration site (nucleotides 823-1164 of SEQ ID NO:4), which contains SEQ ID NO:4 or its reverse complementary sequence. The presence of the transgenic rice event RN85zN-eJ can be identified by the presence of SEQ ID NO:4 or its reverse complementary sequence.

[0029] SEQ ID NO:5 is a 6705-nucleotide sequence characterizing the transgenic rice event RN85zN-eJ. The presence of the transgenic rice event RN85zN-eJ can be identified by the presence of SEQ ID NO:5 or its reverse complementary sequence.

[0030] The present invention also provides a method for protecting rice plants from damage caused by herbicides, characterized in that it includes applying an effective dose of glufosinate herbicide to a field in which at least one transgenic rice plant is planted, wherein the transgenic rice plant contains, in sequence, the nucleic acid sequences of SEQ ID NO:1, SEQ ID NO:5 positions 343-6363, and SEQ ID NO:2 in its genome, or the genome of the transgenic rice plant contains SEQ ID NO:5; the transgenic rice plant is tolerant to glufosinate herbicide.

[0031] The present invention also provides a method for protecting rice plants from insect infestation, characterized in that it includes providing at least one transgenic rice plant cell in the diet of target insects, wherein the transgenic rice plant cell contains, in sequence, the nucleic acid sequences of SEQ ID NO:1, SEQ ID NO:5 positions 343-6363 and SEQ ID NO:2 in its genome, or the genome of the transgenic rice plant cell contains SEQ ID NO:5; the target insects that ingest the transgenic rice plant cell are inhibited from further ingesting the rice plant.

[0032] In the present invention for detecting nucleic acid molecules in rice plants and the detection method thereof, the following definitions and methods are intended to better define the present invention and guide those skilled in the art to implement it. Unless otherwise stated, the terms shall be understood according to their conventional usage by those skilled in the art.

[0033] This invention provides a method for detecting nucleic acid molecules in rice plants, specifically the transgenic rice event RN85zN-eJ, which exhibits insect resistance and tolerance to glufosinate herbicide. Rice plants with this trait express LC85-Ac protein and phosphinic acid acetyltransferase (PAT) protein, conferring insect resistance and tolerance to glufosinate. Furthermore, the detection method of this invention utilizes SEQ ID NO:1 or its reverse complementary sequence, SEQ ID NO:2 or its reverse complementary sequence, SEQ ID NO:3 or its reverse complementary sequence, SEQ ID NO:4 or its reverse complementary sequence, SEQ ID NO:6 or its reverse complementary sequence, or SEQ ID NO:7 or its reverse complementary sequence as DNA primers or probes to generate amplification products diagnosed as transgenic rice event RN85zN-eJ or its progeny, enabling rapid, accurate, and stable identification of plant material derived from the transgenic rice event RN85zN-eJ.

[0034] The transgenic rice RN85zN-eJ exhibits strong tolerance to glufosinate and outstanding insect resistance. These characteristics make the RN85zN-eJ transformant suitable for improving glufosinate herbicide tolerance and insect resistance in rice, thereby enabling the breeding of new insect-resistant and herbicide-tolerant rice varieties.

[0035] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0036] Figure 1 Physical map of the recombinant expression vector pBWA(V)HS-bar-LC85AcA5.

[0037] Figure 2 Indoor bioassay results for rice stem borer.

[0038] A, D: Nipponbare (leaves); B, C, E, F: RN85N-eJ (leaves).

[0039] Figure 3 PCR validation results specific to the RN85zN-eJ transformation event.

[0040] M: DNA Marker, size indicated next to it (unit: bp);

[0041] N: Blank control water;

[0042] C: Recipient control Nipponbare genomic DNA;

[0043] 1: Genomic DNA of the transformant RN85zN-eJ;

[0044] 2: Samples containing RN85zN-eJ.

[0045] A: Expected size of the PCR fragment at the left boundary is 929 bp; B: Expected size of the PCR fragment at the right boundary is 753 bp. Detailed Implementation

[0046] The transformation event RN85zN-eJ involved in this application refers to rice plants obtained by genetic transformation of Nipponbare rice as the recipient, resulting in the insertion of a foreign gene insert (T-DNA insert) between specific genomic sequences. In a specific embodiment, the expression vector used for the transgenic process has... Figure 1 The physical map shown indicates that the obtained T-DNA insert has the sequence shown in nucleotides 343-6363 of SEQ ID NO:5. The transformation event RN85zN-eJ can refer to this transgenic process, the T-DNA insert within the genome obtained by this process, a combination of the T-DNA insert and flanking sequences, or the rice plant obtained by this transgenic process. In specific examples, this event also applies to plants obtained by transforming other recipient varieties with the same expression vector, thereby inserting the T-DNA insert into the same genomic location. The transformation event RN85zN-eJ can also refer to offspring plants obtained by asexual reproduction, sexual reproduction, doubling or multiplication, or a combination thereof, from the aforementioned plants.

[0047] Example 1: Acquisition of Transformation Events and Identification of Traits

[0048] The LC85-AcA5 gene encodes the protein LC85-Ac, which has a significant control effect on lepidopteran pests such as the rice stem borer; the bar gene encodes phosphatidylinosin acetyltransferase, which can improve the plant's tolerance to glufosinate herbicide. This invention uses the pBWA(V)HS-bar-LC85AcA5 expression vector (vector physical map shown in...). Figure 1 The gene cassette (containing the LC85-AcA5 gene expression cassette and the bar gene expression cassette) was transformed into the recipient Nipponbare using Agrobacterium-mediated transformation, resulting in 39 positive transformation events. The herbicide tolerance and insect resistance of the T1 generation plants from these transformed seedlings were then identified and screened.

[0049] 1. Herbicide resistance screening

[0050] Using Nipponbare as a reference, herbicide-tolerant transformants were screened by spraying glufosinate at twice the recommended concentration in the field during the seedling stage. The results showed that 10 transformation events showed significantly higher tolerance to glufosinate than the control (Table 1), and they were named RN85N-eA~RN85N-eJ.

[0051] Table 1 Herbicide tolerance performance

[0052]

[0053] The values ​​are derived from the mean ± standard deviation of three biological replicates. Statistical analysis was performed using LSD multiple comparisons (α = 0.05), where different letters indicate statistical significance of differences in data within the same column at the same herbicide concentration.

[0054] 2. Insect resistance identification

[0055] Using Nipponbare as a reference, transformants with better insect resistance were screened from the above 10 transformation events through indoor leaf bioassay. The insect resistance of the materials was evaluated by feeding newly hatched larvae of the rice stem borer with detached rice leaves.

[0056] The results of the indoor bioassay are shown in Table 2. Among these 10 transformants, RN85zN-eH and RN85zN-eJ showed high resistance to the rice stem borer, while the others showed resistance, moderate resistance, low resistance, or susceptibility.

[0057] Figure 2 The effects of the leaflets of the transformant RN85zN-eJ on the rice stem borer were demonstrated in an indoor bioassay.

[0058] Table 2 Indoor life measurement

[0059]

[0060]

[0061] Numerical values ​​are expressed as mean ± standard deviation of four biological replicates. Significance of differences in data within the same column was analyzed using the LSD method (α = 0.05).

[0062] In summary, RN85zN-eH and RN85zN-eJ are transformants with excellent herbicide tolerance and insect resistance. They can be used to improve the glufosinate herbicide tolerance and insect resistance traits of rice, thereby breeding new insect-resistant and herbicide-tolerant rice varieties.

[0063] Example 2: Molecular Characterization of the Transformation Event RN85zN-eJ

[0064] To further clarify the identity characteristics of the transformation event RN85zN-eJ, this invention analyzed the insertion site of the exogenous sequence of RN85zN-eJ in the rice genome and the insertion structure of the exogenous sequence.

[0065] 1. Analysis of flanking sequences at insertion sites of exogenous sequences in the rice genome

[0066] 100 mg of plant leaves were rapidly ground with liquid nitrogen, and total DNA was extracted using the CTAB method. The concentration of genomic DNA was determined to ensure a total DNA volume >1 μg. Genome resequencing was performed, with each read being 150 bp in length, yielding at least 10 Gb of data, ensuring a data quality index Q30 ≥ 80% (i.e., the proportion of bases with a sequencing error rate greater than 0.1% is less than 20%). Based on the genome resequencing results, sequence homology comparison was performed using BWA software (http: / / bio-bwa.sourceforge.net / , default settings) with the transgenic vector T-DNA sequence as a template. The selected sequences were further assembled and screened, removing reads that were entirely vector sequences, resulting in a final class of reads characterized by half being genomic sequences and the other half being vector sequences. The obtained genomic sequences were compared with the Nipponbare MSU 7.0 reference genome.

[0067] Homology alignment analysis was performed at (http: / / rice.uga.edu / pub / data / Eukaryotic_Projects / o_sativa / annotation_dbs / pseudomolecles / version_7.0 / all.dir / ) to obtain the specific location of the genome sequence on the genome, which is the possible insertion site.

[0068] The sequencing results were compared with the reference genome and the exogenous T-DNA sequence to obtain the insertion location of the exogenous insert fragment, which was found to be at position 26846963-26846988 bp in the rice genome (Chr2). Subsequently, forward and reverse primers were designed using the Primerblast software from the NCBI website (https: / / blast.ncbi.nlm.nih.gov / Blast) on the flanking sequences of the genome at the left and right boundaries of the insertion site and on the exogenous insert sequence, respectively.

[0069] The forward primer sequence for amplifying the left boundary is shown in SEQ ID NO:8, and the reverse primer sequence is shown in SEQ ID NO:9; the forward primer sequence for amplifying the right boundary is shown in SEQ ID NO:10, and the reverse primer sequence is shown in SEQ ID NO:11.

[0070] Using transgenic rice line genomic DNA as a template, the insertion site was verified by PCR amplification. The amplification product fused a portion of the rice genome sequence and a portion of the T-DNA sequence, and the PCR product was sequenced and analyzed.

[0071] The PCR reaction system specifically includes 1.0 μL of genomic DNA, 10.0 μL of 2×PCR Mixture, 0.5 μL of forward primer (10 μM) and 0.5 μL of reverse primer (10 μM), and water is added to bring the volume to 20 μL.

[0072] The PCR amplification program was as follows: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, annealing for 30 s, extension at 72℃ for a certain time (set at 60 s / kb), 30 cycles; extension at 72℃ for 7 min.

[0073] PCR amplification results are shown below Figure 3 The PCR amplification products at the left and right boundaries were sequenced to obtain the T-DNA insertion boundary sequence and flanking sequences of the RN85zN-eJ transformant, which were assembled as follows (uppercase letters represent rice genome sequences, lowercase letters represent T-DNA sequences, and shading indicates the positions of transformant-specific PCR primers):

[0074] Left boundary:

[0075]

[0076] Right boundary:

[0077]

[0078]

[0079] Sequencing analysis results showed that the exogenous RN85zN-eJ fragment was inserted in reverse from the LB to RB ends into the rice genome at the position Chr2:26846963-26846988bp.

[0080] Display of the recipient rice genome sequence at the insertion site of the transformant (strikethrough lines indicate genomic deletions caused by the insertion of the transformant T-DNA):

[0081]

[0082] Analysis of the left and right boundary sequences revealed that the insertion of the foreign sequence caused a 24 bp deletion mutation in the recipient rice genome; simultaneously, a 3 bp deletion occurred at the LB end and a 190 bp deletion occurred at the RB end of the T-DNA region. Analysis of the genome sequences upstream and downstream of the insertion site indicated that the insertion site was located in the intergenic space of the rice genome, and no known functional genes were present.

[0083] 2. Analysis of in vitro full-length insertion sequences in transformed organisms

[0084] Using SEQ ID NO:8 and SEQ ID NO:11 as primers and the genomic DNA of the transformant RN85zN-eJ as a template, PCR amplification was performed, and the obtained PCR product was the exogenous full-length insert sequence. Next-generation sequencing analysis and sequence assembly were performed on the PCR product to obtain the actual insert sequence of the RN85zN-eJ transformant and the left and right flanking rice genome sequences, as shown in SEQ ID NO:5.

[0085] Example 3: Detection method for the transformation event RN85zN-eJ

[0086] New varieties can be bred from the transgenic rice event RN85zN-eJ and used to produce agricultural products or commodities. If a sufficient amount is detected in the agricultural product or commodity, it is expected to contain a nucleotide sequence capable of diagnosing the presence of the transgenic rice event RN85zN-eJ material in the agricultural product or commodity. The agricultural product or commodity includes, but is not limited to, rice flour, rice oil, rice bran, rice germ, rice protein, rice starch, rice bran nutrient oil or rice bran polysaccharide, and any other food intended for animal consumption, or cosmetics, industrial products, etc. Nucleic acid detection methods and / or kits based on probe or primer pairs can be developed to detect the transgenic rice event RN85zN-eJ nucleotide sequence, such as that shown in SEQ ID NO:1 or SEQ ID NO:2, wherein the probe sequence or primer amplification sequence is selected from 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, and SEQ ID NO:7, to diagnose the presence of the transgenic rice event RN85zN-eJ.

[0087] One of the detection methods is to use PCR to detect samples containing RN85zN-eJ, and the PCR primer pairs used are SEQ ID NO:8 (forward primer), SEQ ID NO:9 (reverse primer) or SEQ ID NO:10 (forward primer), SEQ ID NO:11 (reverse primer).

[0088] PCR reaction system:

[0089]

[0090] The reaction procedure is as follows:

[0091] 95℃, 5min; (95℃, 30sec; 58℃, 30sec; 72℃, 60sec)×30 cycles; 72℃, 7min.

[0092] The PCR products were electrophoresed on a 1% (w / v) 1×TAE agarose gel, and the results are as follows. Figure 3 As shown.

[0093] The expected target bands (SEQ ID NO:6 and SEQ ID NO:7, respectively) can be amplified during the RN85zN-eJ transformation event. Furthermore, this PCR method can track the existence of the transformation event, thus enabling its application in breeding work. In summary, the transgenic rice event RN85zN-eJ of this invention can improve plant insect resistance and exhibit high tolerance to glufosinate herbicide, and can also be used to improve other rice germplasm and create new rice hybrid combinations. Its detection method can accurately and rapidly identify whether biological samples contain the DNA molecules of the transgenic rice event RN85zN-eJ.

[0094] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.

Claims

1. A nucleic acid molecule, characterized in that, Including any of the following: i) Contains the sequence shown in SEQ ID NO:1 and / or SEQ ID NO:2, or the reverse complementary sequence thereof; ii) Contains the sequence shown in SEQ ID NO:3 and / or SEQ ID NO:4, or its reverse complementary sequence; iii) Contains the sequence shown in SEQ ID NO:6 and / or SEQ ID NO:7, or its reverse complementary sequence; iv) Contains the sequence shown in SEQ ID NO:5, or its reverse complementary sequence.

2. A probe for detecting rice transformation events, characterized in that, Includes the sequence shown in SEQ ID NO:1 or SEQ ID NO:2 or SEQ ID NO:3 or SEQ ID NO:4 or SEQ ID NO:6 or SEQ ID NO:7, or a fragment thereof or a variant thereof or its inverse complementary sequence.

3. A primer pair for detecting rice transformation events, characterized in that, The amplification product of the primer pair contains the sequence described in claim 2.

4. The primer pair according to claim 3, characterized in that, The primer pair is the sequence shown in SEQ ID NO:8 and SEQ ID NO:9; or the sequence shown in SEQ ID NO:10 and SEQ ID NO:

11.

5. A reagent kit or microarray for detecting rice transformation events, characterized in that, It comprises the probe of claim 2 and / or the primer pair of any one of claims 3-4.

6. A method for detecting rice transformation events, characterized in that, This includes using any of the following to detect the presence of the transformation event in the sample to be tested: i) The probe according to claim 2; ii) The primer pair according to any one of claims 3-4; iii) The probe of claim 2 and the primer pair of any one of claims 3-4; iv) The kit or microarray according to claim 5.

7. A method for breeding rice, characterized in that, The method includes the following steps: 1) Obtain rice containing the nucleic acid molecule of claim 1; 2) The rice obtained in step 1) is subjected to pollen culture, unfertilized embryo culture, doubling culture, cell culture, tissue culture, self-pollination or hybridization, or a combination thereof, to obtain rice plants, seeds, plant cells, progeny plants, or plant parts; optionally, 3) Evaluate the insect resistance traits and / or herbicide resistance of the progeny plants obtained in step 2), and use the method of claim 6 to detect whether a transformation event exists therein.

8. The article obtained by the method of claim 7, characterized in that, The product is made from rice plants, seeds, plant cells, offspring plants, or plant parts, including food, feed, or industrial raw materials.

9. A method for protecting rice plants from damage caused by herbicides, characterized in that, The invention includes applying an effective dose of glufosinate herbicide to a field in which at least one transgenic rice plant is planted, wherein the transgenic rice plant contains, in sequence, the nucleic acid sequences of SEQ ID NO:1, SEQ ID NO:5 positions 343-6363, and SEQ ID NO:2 in its genome, or wherein the genome of the transgenic rice plant contains SEQ ID NO:5; wherein the transgenic rice plant is tolerant to glufosinate herbicide.

10. A method for protecting rice plants from insect infestation, characterized in that, The invention includes providing at least one transgenic rice plant cell in the diet of target insects, said transgenic rice plant cell containing, in its genome, the nucleic acid sequences of SEQ ID NO:1, SEQ ID NO:5 positions 343-6363 and SEQ ID NO:2, or the genome of said transgenic rice plant cell contains SEQ ID NO:5; the target insects that ingest said transgenic rice plant cell are inhibited from further ingesting said rice plant.

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