Insect-resistant and herbicide-resistant transgenic rice RN85N-e10 and detection method thereof

Abstract

The invention relates to a nucleic acid molecule of a rice transformation event RN85N-e10 and a detection method. The nucleic acid molecule of the rice transformation event RN85N-e10 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 RN85N-e10 disclosed by the invention 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 RN85N-e10 or not.

Description

Technical Field

[0001] The present invention relates to the field of plant biotechnology. Specifically, it relates to a nucleic acid molecule and a detection method of a rice transformation event RN85N-e10, and particularly to a transgenic rice event RN85N-e10 with insect resistance and tolerance to glufosinate herbicide application, and a nucleic acid molecule for detecting whether a biological sample contains the specific transgenic rice event RN85N-e10 and its detection method. Background Art

[0002] Rice is one of the most widely planted crops in China. The entire growth period of rice is affected by various insect pests. Among them, Chilo suppressalis is one of the main pests of rice, and its damaged area is extensive, posing a serious threat to a large number of rice fields globally every year. Especially during the critical seasons of rice growth, such as summer and autumn, the rampant occurrence of Chilo suppressalis often leads to a significant decrease in rice yield. Cultivating insect-resistant and herbicide-tolerant transgenic rice varieties and promoting transgenic rice will be an effective means for us to increase per-unit yield and ensure food security.

[0003] Using a single Bt protein control strategy is likely to cause target pests to develop resistance. Therefore, it is necessary to develop new insecticidal proteins to improve insecticidal efficiency and extend the control effect. The LC85-Ac protein is a new insecticidal protein that can be used to control Lepidoptera pests including Spodoptera frugiperda, Chilo suppressalis, Mythimna separata, Spodoptera exigua, Prodenia litura, Agrotis ypsilon, and Athetis lepigone (Patent Application No. 202410332785.6).

[0004] Field weeds compete with crops for water, fertilizer, light energy and growing space, directly affecting the yield and quality of crops. At the same time, many weeds are intermediate hosts of crop pathogens and pests, and are one of the important biological limiting factors for crop yield increase. According to the statistics of the Food and Agriculture Organization of the United Nations, the global food production loss caused by weeds reaches up to $95 billion annually, equivalent to a loss of 380 million tons of wheat, accounting for more than half of the global wheat production in 2009. Among the economic losses of $95 billion, poor developing countries bear approximately $70 billion (FAO. The lurking menace of weeds[J / OL]. (http: / / www.fao.org / news / story / en / item / 29402 / icode / ),2009-08-11.). Therefore, effectively controlling field weeds is one of the important measures to promote food production increase. In addition, with the accelerating speed of rural population migration to cities in China, the large-scale and mechanization of agricultural planting is a foreseeable trend, which makes the traditional manual weeding method unrealistic. The popularization and use of herbicides can greatly reduce the difficulty of field management and lower the labor intensity. Developing new herbicide products with high efficiency, low toxicity and no residue is costly, time-consuming and difficult. Cultivating herbicide-tolerant rice through transgenic technology can overcome this problem. Spraying 1-2 times during the rice growth period can effectively solve the weed problem, reducing the dosage and input cost of herbicides. Therefore, herbicide-tolerant transgenic rice has very broad application value and market potential.

[0005] In the present invention, an insect-resistant gene expression cassette and a herbicide-tolerant gene expression cassette are connected in series to enable their highly efficient expression in transgenic rice, with both insect-resistant and herbicide-tolerant traits, further enhancing the application and economic value of the product.

[0006] It is known that the expression of foreign genes in plants is affected by their chromosomal positions, possibly due to the proximity of chromatin structures (such as heterochromatin) or transcriptional regulatory elements (such as enhancers) to the integration site. Therefore, it is usually necessary to screen a large number of events to identify events that can be commercialized (i.e., events in which the introduced target gene is optimally expressed). For example, it has been observed in plants and other organisms that the expression levels of introduced genes can vary widely among events; there may also be differences in the spatial or temporal patterns of expression, such as differences in the relative expression of transgenes between different plant tissues. This difference is manifested in that the actual expression pattern may not be consistent with the expression pattern expected based on the transcriptional regulatory elements in the introduced gene construct, resulting in differences in the trait performance of transformation events. Therefore, it is usually necessary to generate hundreds or thousands of different events and screen from these events a single event with the expected transgene expression level and expression pattern for commercial purposes. Events with the expected transgene expression level and expression pattern can be used to introgress the transgene into other genetic backgrounds through sexual outcrossing using conventional breeding methods. The offspring produced by this hybridization method retain the transgene expression characteristics of the original transformation event. Applying this strategic model can ensure reliable gene expression in many varieties that are well adapted to local growth conditions. Therefore, more transformation events need to be characterized and screened to obtain excellent transformation events with excellent comprehensive trait performance and commercial prospects.

[0007] It would be beneficial to be able to detect the presence of a specific event to determine whether the offspring of a sexual cross contain the gene of interest. In addition, methods for detecting specific events will also help to comply with relevant regulations. For example, foods derived from recombinant crops require official approval and labeling before being put on the market. It is possible to detect the presence of transgenes by any well-known polynucleotide detection method, such as polymerase chain reaction (PCR). These detection methods usually focus on commonly used genetic elements, such as promoters, terminators, marker genes, etc. Therefore, unless the sequence of the chromosomal DNA ("flanking DNA") adjacent to the inserted transgenic DNA is known, these methods cannot be used to distinguish different events, especially those generated with the same DNA construct. Therefore, currently, a pair of primers spanning the junction of the inserted transgene and the flanking DNA is often used by PCR to identify specific transgene events, specifically, a first primer containing the flanking sequence and a second primer containing the inserted sequence. Summary of the Invention

[0008] The object of the present invention is to provide a rice transformation event (also called transformant) with excellent insect resistance and herbicide tolerance, as well as a nucleic acid molecule for detecting RN85N-e10 in rice and its detection method. The transgenic rice event RN85N-e10 has excellent insect resistance and good tolerance to glufosinate herbicide, and the detection method can accurately and rapidly identify whether a biological sample contains the DNA molecule of a specific transgenic rice event RN85N-e10.

[0009] To achieve the above object, the present invention uses the pBWA(V)HS-bar-LC85AcA1 expression vector to transform rice Nipponbare by the Agrobacterium-mediated method, and obtains a batch of positive transformation events. Through the identification of insect resistance and herbicide tolerance traits, it is found that the transformation event RN85N-e10 is a transformant with 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 characteristics of RN85N-e10, the present invention provides a nucleic acid molecule, which comprises the sequence shown in SEQ ID NO: 1 and / or SEQ ID NO: 2, or its reverse complementary sequence.

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

[0012] Even further, 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] Even further, the nucleic acid molecule comprises the sequence shown in SEQ ID NO: 5 or its reverse complementary sequence.

[0014] The present invention also provides a probe for detecting a rice transformation event, which is characterized by comprising 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 its fragment, or its variant, or its reverse complementary sequence.

[0015] The present invention also provides a primer pair for detecting a rice transformation event, which is characterized in that the amplification product of the primer pair 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, or its fragment, or its variant, or its reverse complementary sequence.

[0016] In some embodiments, the above 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 a rice transformation event, characterized by comprising the above-mentioned probe and / or primer pair.

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

[0019] The present invention also provides a method for breeding rice, characterized in that the method comprises the following steps: 1) Obtaining rice containing the above nucleic acid molecule; 2) Obtaining a rice plant, seed, plant cell, progeny plant or plant part from the rice obtained in step 1) through pollen culture, unfertilized embryo culture, doubling culture, cell culture, tissue culture, self-crossing or hybridization or a combination of the above; and optionally, 3) Identifying the insect-resistant trait and / or herbicide resistance of the progeny plant obtained in step 2), and using the above method to detect whether the transformation event exists therein.

[0020] Furthermore, the present invention also provides a product made from the rice plant, seed, plant cell, progeny plant or plant part obtained by the above method, including food, feed or industrial raw materials.

[0021] The 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 RN85N-e10. The SEQ ID NO: 1 spans the left flanking genomic DNA sequence of the rice insertion site and the 5' end DNA sequence of the left border of the inserted sequence. The presence of the transgenic rice event RN85N-e10 can be identified by including the SEQ ID NO: 1 or its reverse complementary sequence. The 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 RN85N-e10. The SEQ ID NO: 2 spans the 3' end DNA sequence of the right border of the inserted sequence and the right flanking genomic DNA sequence of the rice insertion site. The presence of the transgenic rice event RN85N-e10 can be identified by including the SEQ ID NO: 2 or its reverse complementary sequence.

[0022] In the present invention, the nucleic acid sequence may be at least 11 or more consecutive polynucleotides of any part of the transgene insertion sequence in SEQ ID NO: 3 or its reverse complementary sequence (the first nucleic acid sequence), or at least 11 or more consecutive polynucleotides of any part of the 5'-left flanking rice genomic DNA region in SEQ ID NO: 3 or its reverse complementary sequence (the second nucleic acid sequence). The nucleic acid sequence may further be a part of SEQ ID NO: 3 that is homologous to or reverse complementary to SEQ ID NO: 1 or SEQ ID NO: 6 in its entirety. When the first nucleic acid sequence and the second nucleic acid sequence are used together, these nucleic acid sequences include a DNA primer pair in a DNA amplification method for generating an amplification product. When the amplification product generated by using the DNA primer pair in the DNA amplification method is an amplification product including SEQ ID NO: 1 or SEQ ID NO: 3 or SEQ ID NO: 6 or its reverse complementary sequence, the presence of the transgenic rice event RN85N-e10 or its progeny can be diagnosed.

[0023] SEQ ID NO: 3 is a sequence with a length of 1420 nucleotides located near the insertion junction at the 5'-end of the insertion sequence in the transgenic rice event RN85N-e10. SEQ ID NO: 3 consists of a 538-nucleotide rice left flanking genomic DNA sequence (nucleotides 1-538 of SEQ ID NO: 3), a 91-nucleotide left border DNA sequence of the pBWA(V)HS-bar-LC85AcA1 construct (nucleotides 539-629 of SEQ ID NO: 3), and a 5'-end DNA sequence of the first expression cassette of the glufosinate-resistant gene with 791 nucleotides (nucleotides 630-1420 of SEQ ID NO: 3). The presence of the transgenic rice event RN85N-e10 can be identified by including SEQ ID NO: 3 or its reverse complementary sequence.

[0024] The nucleic acid sequence may be at least 11 or more consecutive polynucleotides (third nucleic acid sequence) of any part 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 part of the 3'-right flanking rice genomic DNA region in SEQ ID NO: 4 or its reverse complementary sequence. The nucleic acid sequence may further be a part of SEQ ID NO: 4 that is homologous to or reverse complementary to SEQ ID NO: 2 or SEQ ID NO: 7 in its entirety. When the third nucleic acid sequence and the fourth nucleic acid sequence are used together, these nucleic acid sequences include a DNA primer set in a DNA amplification method for generating an amplification product. When the amplification product generated by using a DNA primer pair in a DNA amplification method is an amplification product including SEQ ID NO: 2 or SEQ ID NO: 4 or SEQ ID NO: 7 or its reverse complementary sequence, the presence of the transgenic rice event RN85N-e10 or its progeny can be diagnosed.

[0025] SEQ ID NO: 4 is a sequence 1340 nucleotides in length located near the insertion junction at the 3'-end of the insertion sequence in the transgenic rice event RN85N-e10. SEQ ID NO: 4 consists of the 3'-end DNA sequence (nucleotides 1-389) of the second expression cassette of the insect-resistant gene of 389 nucleotides, the right border DNA sequence of the pBWA(V)HS-bar-LC85AcA1 construct of 460 nucleotides (nucleotides 390-849) of SEQ ID NO: 4, and the genomic DNA sequence of 491 nucleotides of the right flanking region of the rice integration site (nucleotides 850-1340) of SEQ ID NO: 4. The presence of the transgenic rice event RN85N-e10 can be identified by including SEQ ID NO: 4 or its reverse complementary sequence.

[0026] SEQ ID NO: 5 is a sequence 7210 nucleotides in length characterizing the transgenic rice event RN85N-e10. The presence of the transgenic rice event RN85N-e10 can be identified by including SEQ ID NO: 5 or its reverse complementary sequence.

[0027] The present invention also provides a method for protecting rice plants from damage caused by herbicides, which is characterized by including applying an effective dose of glufosinate herbicide to a field planted with at least one transgenic rice plant, wherein the transgenic rice plant sequentially contains SEQ ID NO: 1, the nucleic acid sequence at positions 539-6719 of SEQ ID NO: 5, 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 has tolerance to glufosinate herbicide.

[0028] The present invention also provides a method for protecting rice plants from insect infestation, which is characterized by including providing at least one transgenic rice plant cell in the diet of a target insect, wherein the transgenic rice plant cell sequentially contains SEQ ID NO: 1, the nucleic acid sequence at positions 539-6719 of SEQ ID NO: 5, and SEQ ID NO: 2 in its genome, or the genome of the transgenic rice plant cell contains SEQ ID NO: 5; the target insect feeding on the transgenic rice plant cell is inhibited from further feeding on the rice plant.

[0029] In the nucleic acid molecule and its detection method for detecting rice plants according to the present invention, the following definitions and methods can better define the present invention and guide those of ordinary skill in the art to implement the present invention. Unless otherwise specified, the terms are understood according to the conventional usage of those of ordinary skill in the art.

[0030] The present invention provides a nucleic acid molecule for detecting rice plants and its detection method. The transgenic rice event RN85N-e10 has insect-resistant traits and the effect of tolerating glufosinate herbicide. Rice plants with such traits express LC85-Ac protein and phosphinothricin acetyltransferase (PAT) protein, which confer insect resistance and tolerance to glufosinate on the plants. At the same time, in the detection method of the present invention, 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 can be used as DNA primers or probes to generate amplification products diagnostic of the transgenic rice event RN85N-e10 or its offspring, and can quickly, accurately, and stably identify the presence of plant materials derived from the transgenic rice event RN85N-e10.

[0031] The transgenic rice event RN85N-e10 has strong tolerance to glufosinate and prominent insect-resistant traits. These characteristics enable the RN85N-e10 transformant to be used to improve the glufosinate herbicide tolerance and insect-resistant traits of rice, thereby cultivating new rice varieties with insect resistance and herbicide tolerance.

[0032] The technical solutions of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Description of the Drawings

[0033] Figure 1 Physical map of the recombinant expression vector pBWA(V)HS-bar-LC85AcA1.

[0034] Figure 2 Indoor bioassay effect of Chilo suppressalis.

[0035] A, D: Nipponbare (leaves); B, C, E, F: RN85N-e10 (leaves).

[0036] Figure 3 Specific PCR verification results of the RN85N-e10 transformation event.

[0037] M: DNA Marker, size marked beside (unit: bp); N: Blank control water; C: Genomic DNA of the receptor control Nipponbare; 1, 2: Genomic DNA of the transformant RN85N-e10; 3: Sample containing RN85N-e10.

[0038] A: Expected size of the left border PCR fragment is 1014 bp; B: Expected size of the right border PCR fragment is 737 bp. Specific Embodiments

[0039] The transformation event RN85N-e10 involved in this application refers to a rice plant obtained by genetic transformation using Nipponbare as the receptor, with an exogenous gene insert (T-DNA insert) inserted between specific genomic sequences. In a specific embodiment, the expression vector used for transgenic transformation has Figure 1For the physical map shown, the resulting T-DNA insert has the sequence shown by nucleotides 539 - 6719 of SEQ ID NO: 5. The transformation event RN85N-e10 can refer to this transgenic process, or to the T-DNA insert in the genome obtained from this process, or to the combination of the T-DNA insert and the flanking sequences, or can refer to the rice plants obtained from this transgenic process. In a specific example, this event is also applicable to other recipient varieties transformed with the same expression vector, so as to obtain plants with the T-DNA insert inserted into the same genomic position. The transformation event RN85N-e10 can also refer to the progeny plants obtained by asexual reproduction, sexual reproduction, haploidization or doubling reproduction or a combination of the above of the above plants.

[0040] Example 1 Obtaining and Character Identification of Transformation Events LC85-AcA1 The gene encodes the protein LC85-Ac, which has a significant control effect on Lepidoptera pests such as Chilo suppressalis; bar The gene encodes phosphinothricin acetyltransferase, which can improve the tolerance of plants to glufosinate herbicides. The present invention uses the pBWA(V)HS-bar-LC85AcA1 expression vector (see the physical map of the vector in Figure 1 , containing LC85-AcA1 gene expression cassette and bar gene expression cassette), and transforms the recipient Nipponbare by the Agrobacterium-mediated method to obtain 44 positive transformation events, and the herbicide tolerance and insect resistance of the T1 generation plants of these transformed seedlings are identified and screened.

[0041] 1. Herbicide Resistance Screening Using Nipponbare as a reference, by spraying glufosinate at twice the medium recommended concentration in the field at the seedling stage, the transformants with better herbicide tolerance were screened. The results showed that the tolerance of 11 transformation events to glufosinate herbicide was significantly higher than that of the control (Table 1), and they were named RN85N-e1~RN85N-e11.

[0042] Table 1 Herbicide Tolerance Performance

[0043] The values are the mean ± standard deviation from 3 biological replicates. Statistical analysis was performed using LSD for multiple comparisons (α = 0.05), and different letters indicate significant differences in the data in the same column at the same herbicide concentration.

[0044] 2. Insect Resistance Identification Using Nipponbare as a reference, the transformants with better insect resistance were screened from the above 11 transformation events by the method of in-vitro leaf bioassay. The newly hatched larvae of Chilo suppressalis were fed with detached rice leaves to evaluate the insect resistance of the materials.

[0045] The indoor bioassay results are shown in Table 2. Among these 11 transformants, the resistance levels of RN85N-e3 and RN85N-e10 to Chilo suppressalis were high resistance, and the others were resistant, moderately resistant, low resistant or susceptible.

[0046] Figure 2 The indoor bioassay effect of the leaves of transformant RN85N-e10 on Chilo suppressalis was shown.

[0047] Table 2 Indoor bioassay

[0048] The values were expressed as the mean ± standard deviation of 4 biological replicates, and the significant differences of the data in the same column were analyzed by the LSD method (α = 0.05).

[0049] Generally speaking, RN85N-e3 and RN85N-e10 are transformants with excellent performance in herbicide tolerance and insect resistance, which can be used to improve the glufosinate herbicide tolerance and insect resistance traits of rice, so as to cultivate new rice varieties with insect resistance and herbicide tolerance.

[0050] Example 2 Molecular characterization of transformation event RN85N-e10 In order to further clarify the identity characteristics of transformation event RN85N-e10, the present invention analyzed the insertion site of the exogenous sequence of RN85N-e10 on the rice genome and the insertion structure of the exogenous sequence.

[0051] 1. Analysis of the flanking sequences of the insertion site of the exogenous sequence on the rice genome Take 100 mg of plant leaves, quickly grind them in liquid nitrogen, and extract total DNA using the CTAB method. After measuring the concentration of genomic DNA, ensure that the total amount of DNA > 1 mg. Using genomic resequencing, with each read length of 150 bp, obtain at least 10 Gb of data, and ensure that the data quality index Q30 ≥ 80% (i.e., the proportion of bases with a sequencing error rate greater than 0.1% is less than 20%). According to the genomic resequencing results, use the BWA software with the transgenic vector T-DNA sequence as a template to perform sequence homology comparison and screening with all the sequenced sequences (BWA, http: / / bio-bwa.sourceforge.net / , default settings). Further assemble and screen the selected sequences, remove the reads with all vector sequences, and finally obtain a class of read sequences, which are characterized by half being genomic sequences and the other half being vector sequences. Align the obtained genomic sequences with the Nipponbare MSU7.0 version reference genome (http: / / rice.uga.edu / pub / data / Eukaryotic_Projects / o_sativa / annotation_dbs / pseudomolecules / version_7.0 / all.dir / ) for homologous alignment analysis to obtain the specific position of the genomic sequence on the genome, which is the possible insertion site.

[0052] Align the sequencing results with the reference genome and the exogenous T-DNA sequence respectively to obtain the insertion position information of the exogenous insertion fragment, which is at the position of Chr6: 24872663 bp in the rice genome. Subsequently, use the Primerblast software on the NCBI website (https: / / blast.ncbi.nlm.nih.gov / Blast) to design forward and reverse primers respectively on the genomic flanking sequences and exogenous insertion sequences at the left and right boundaries of the insertion site (as shown in Table 3). Using the genomic DNA of the transgenic rice line as a template, verify the insertion site by PCR amplification. The amplified product combines a part of the rice genomic sequence and a part of the T-DNA sequence, and sequence analysis is performed on the PCR product.

[0053] Table 3 Primer information table

[0054] The specific PCR reaction system contains 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 the volume is made up to 20 μL with water.

[0055] The PCR amplification program was as follows: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 30 s, annealing for 30 s, extension at 72°C for a certain time (set at 60 s / kb), 30 cycles; extension at 72°C for 7 min.

[0056] The results of PCR amplification are shown in Figure 3 . The PCR amplification products at the left and right boundaries were sequenced to obtain the T-DNA insertion boundary sequence and the flanking sequences on both sides of the RN85N-e10 transformant, and assembled as follows (capital letters are rice genome sequences, lowercase letters are T-DNA sequences, and the positions of the transformant-specific PCR primers are marked in bold): Left boundary: CGGATTTGCT ATTTCACTTT CGATTTTTTT TGTCCGTAGA GTCTTACATT AAGATTTGTG 60 TTCCTGTAGA TCCAACTTAT ACTTATACTC AGTTGCATAC CACTTAAATT TAGTTACAGT 120 GCGCCAAAAT TAGATTTTAC AACTGCTTTT GTCTCGATAT TTTTGGCATG TTTGTCCCTT 180 TTTGTTTATT TCGTTCAAAA CAGGCTGTAT CCGCTGGTTA AAAATTTCTT TAAGATTAAA 240 ATCTCAAAAg tggtgtaaac aaattgacgc ttagacaact taataacaca ttgcggacgt 300 ttttaatgtt agactgaatt aacgccgaat taattcgggg gatctggatt ttagtactgg 360 attttggttt taggaattag aaattttatt gatagaagta ttttacaaat acaaatacat 420 actaagggtt tcttatatgc tcaacacatg agcgaaaccc tataggaacc ctaattccct 480 tatctgggaa ctactcacac attattatgg agaaagcgat cgctcaaatc tcggtgacgg 540 gcaggaccgg acggggcggt accggcaggc tgaagtccag ctgccagaaa cccacgtcat 600 gccagttccc gtgcttgaag ccggccgccc gcagcatgcc gcggggggca tatccgagcg 660 cctcgtgcat gcgcacgctc gggtcgttgg gcagcccgat gacagcgacc acgctcttga 720 agccctgtgc ctccagggac ttcagcaggt gggtgtagag cgtggagccc agtcccgtcc 780 gctggtggcg gggggagacg tacacggtcg actcggccgt ccagtcgtag gcgttgcgtg 840 ccttccaggg gcccgcgtag gcgatgccgg cgacctcgcc gtccacctcg gcgacgagcc 900 agggatagcg ctcccgcaga cggacgaggt cgtccgtcca ctcctgcggt tcctgcggct 960 cggtacggaa gttgaccgtg cttgtctcga tgtagtggtt gacgatggtg caga 1014 Right boundary: cccgcaatta tacatttaat acgcgataga aaacaaaata tagcgcgcaa actaggataa 60 attatcgcgc gcggtgtcat ctatgttact agatcgggcc atccgcactg tagcggatgg 120 cctaaaaaaa aaactagaag agacgagtct gagactcagc gtctcggtcg cagtcataac 180 ttcgtatagc atacattata cgaagttatg ggccgcatta ccctgttatc cctaggccgc 240 ataacttcgt atagcctaca ttataggatg gagggatatc ctctcttaag gtagcgagca 300 agctctaaga ggagtgtcga caagcttggc actggccgtc gttttacaac gtcgtgactg 360 ggaaaaccct ggcgttaccc aacttaatcg ccttgcagca catccccctt tcgccagctg 420 gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa cagttgcgca gcctgaatgg 480 cgaatgctag agcagcttga gcttggatca gattgtcgtt tcccgccttc agtttaaact 540 atcagtgttt gacagGAAAT TAAAGCTGAA AAGAAACTGA AAAAGAAGGG AACAACAAGA 600 AAACCCGACA GCCTCGCATG TGTTTTATTG GGACATTCTC GCAATATCTT TGGTGCCGCA 660 CATAATCCAC ACCTTGATCT CATATGTGAT TTTGGTCACT AGTTGAGGCA ATTCAGTCTC 720 CTGATTGCGG AACAGTC 737 Sequencing analysis results showed that the exogenous fragment of RN85N-e10 was stably inserted at the position of Chr6:24872663 bp in the rice genome.

[0057] The receptor rice genome sequence at the insertion position of the transformant is shown (the genomic deletion sequence caused by the insertion of the transformant T-DNA is marked with an underline): tggaggggga attcggggtc ggatttgcta tttcactttc gatttttttt gtccgtagag 60 tcttacatta agatttgtgt tcctgtagat ccaacttata cttatactca gttgcatacc 120 acttaaattt agttacagtg cgccaaaatt agattttaca actgcttttg tctcgatatt 180 tttggcatgt ttgtcccttt ttgtttattt cgttcaaaac aggctgtatc cgctggttaa 240 aaatttcttt aagattaaaa tctcaaaa tc tgccaatctt tttaaaccga agccagcatt 300 tgctagtgtt atattaagta ggata gaaat tatttacaaa ctcaccaaag gtgaaagaga 360 taaagctgaa aagaaactga aaaagaaggg aacaacaaga aaacccgaca gcctcgcatg 420 tgttttattg ggacattctc gcaatatctt tggtgccgca cataatccac accttgatct 480 catatgtgat tttggtcact agttgaggca attcagtctc ctgattgcgg aacagtcatt 540 tgtttcgttc acaccagatt ttccaagcca ccatcatatg aattgatttc attgctttca 600 ttttgtgttt ttccccttgg gatg 624 Analysis of the left and right boundary sequences revealed that the insertion of the foreign sequence caused an 87-bp deletion mutation in the recipient rice genome; meanwhile, there was a 14-bp deletion at the LB end and a 19-bp deletion at the RB end of the T-DNA region. Analysis of the genomic sequences upstream and downstream of the insertion site showed that the insertion site of the foreign sequence was located in the gene intergenic region of the rice genome and there were no known functional genes.

[0058] 2. Analysis of the full-length inserted foreign sequence in the transformant Using SEQ ID NO: 8 and SEQ ID NO: 11 as primers and the genomic DNA of the transformant RN85N-e10 as the template, PCR amplification was carried out, and the obtained PCR product was the full-length inserted foreign sequence. The PCR product was subjected to second-generation sequencing analysis and sequence splicing to obtain the actual inserted sequence of the RN85N-e10 transformant and the left and right flanking rice genomic sequences, as shown in SEQ ID NO:5.

[0059] Detection Method for Transformation Event RN85N-e10 in Example 3 New varieties can be developed from the transgenic rice event RN85N-e10 to produce agricultural products or commodities. If a sufficient amount is detected in the agricultural product or commodity, the agricultural product or commodity is expected to contain nucleotide sequences capable of diagnosing the presence of transgenic rice event RN85N-e10 material in the agricultural product or commodity. The agricultural products or commodities include, but are not limited to, rice flour, rice oil, rice bran, rice germ, rice protein, rice starch, rice bran nutritional oil or rice bran polysaccharide, and any other food to be used as a food source for animal consumption, or cosmetics, industrial products, etc. Nucleic acid detection methods and / or kits based on probes or primer pairs can be developed to detect the transgenic rice event RN85N-e10 nucleotide sequences such as those shown in SEQ ID NO: 1 or SEQ ID NO: 2 in biological samples, where the probe sequences or primer amplification sequences are selected from the 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 transgenic rice event RN85N-e10.

[0060] One of the detection methods is: using the PCR method to detect samples containing RN85N-e10, 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).

[0061] PCR reaction system: Reaction program: 95°C, 5 min; (95°C, 30 sec; 55°C, 30 sec; 72°C, 60 sec) × 30 cycles; 72°C, 7 min.

[0062] Take the PCR product and electrophorese it in a 1% (w / v) 1×TAE agarose gel for detection, and the results are as Figure 3 shown.

[0063] In the RN85N-e10 transformation event, the expected target bands (SEQ ID NO: 6 and SEQ ID NO: 7 respectively) can be amplified. Moreover, this PCR method can trace the presence of the transformation event and thus be applied to breeding work. In summary, the transgenic rice event RN85N-e10 of the present invention can improve plant insect resistance and has high tolerance to glufosinate herbicide, and can be used to improve other rice germplasms and create new rice hybrid combinations. Its detection method can accurately and rapidly identify whether the DNA molecule of the transgenic rice event RN85N-e10 is contained in a biological sample.

[0064] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the spirit and scope of the technical solutions of the present invention.

Claims

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

2. Probe for detecting rice transformation events, characterized in that, Comprising 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 its fragment, or its variant, or its reverse complementary sequence.

3. Primer pairs for detecting rice transformation events, characterized in that, The amplification product of the primer pair comprises the sequence recited in claim 2.

4. The primer pair according to claim 3, wherein 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 kit or microarray for detecting rice transformation events, characterized in that, Comprising the probe recited in claim 2 and / or the primer pair recited in any one of claims 3-4.

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

7. A method for breeding rice, characterized in that, The method comprises the following steps: 1) Obtaining rice comprising the nucleic acid molecule recited in claim 1; 2) Obtaining rice plants, seeds, plant cells, progeny plants or plant parts from the rice obtained in step 1) by pollen culture, unfertilized embryo culture, doubling culture, cell culture, tissue culture, self-crossing or hybridization or a combination of the above.

8. The method according to claim 7, wherein The method comprises: evaluating the insect-resistant trait and / or identifying the herbicide resistance of the progeny plants obtained in step 2) of claim 7, and detecting whether the transformation event exists therein by the method recited in claim 6.

9. An article obtained by the method according to any one of claims 7-8, characterized in that, The article is made of rice plants, seeds, plant cells, progeny plants or plant parts, including food, feed or industrial raw materials.

10. A method for protecting rice plants from damage caused by herbicides, characterized in that, Comprising applying an effective dose of glufosinate herbicide to a field planted with at least one transgenic rice plant, wherein the transgenic rice plant sequentially comprises the nucleic acid sequences at positions 539-6719 of SEQ ID NO: 1, SEQ ID NO: 5 and SEQ ID NO: 2 in its genome, or the genome of the transgenic rice plant comprises SEQ ID NO: 5; the transgenic rice plant has tolerance to glufosinate herbicide.

11. A method for protecting rice plants from insect infestation, characterized in that, Comprising providing at least one transgenic rice plant cell in the diet of a target insect, the transgenic rice plant cell sequentially comprising SEQ ID NO: 1, the nucleic acid sequence at positions 539-6719 of SEQ ID NO: 5, and SEQ ID NO: 2 in its genome, or the genome of the transgenic rice plant cell comprising SEQ ID NO: 5; the target insect feeding on the transgenic rice plant cell is inhibited from further feeding on the rice plant.

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