Insect-resistant and herbicide-resistant transgenic rice RN85N-e3 and detection method thereof

Abstract

The invention relates to a nucleic acid molecule of a rice transformation event RN85N-e3 and a detection method. The nucleic acid molecule of the rice transformation event RN85N-e3 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-e3 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-e3 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-e3, and in particular to a transgenic rice event RN85N-e3 that is resistant to insects and tolerant to the application of glufosinate-ammonium herbicides, and a nucleic acid molecule and a detection method thereof for detecting whether a biological sample contains a specific transgenic rice event RN85N-e3. Background Art

[0002] Rice is one of the most widely planted crops in my country. The entire growth period of rice is affected by a variety of pests, among which the Chilo suppressalis is one of the main pests of rice. It has a wide range of damage and poses a serious threat to a large number of rice fields worldwide every year. Especially in the key seasons of rice growth, such as summer and autumn, the rampage of Chilo suppressalis often leads to a significant drop in rice yields. Cultivating insect-resistant and herbicide-resistant transgenic rice varieties and promoting transgenic rice will be an effective means for us to increase yields and ensure food security.

[0003] The use of a single Bt protein control strategy can easily cause target pests to develop resistance, so it is necessary to develop new insecticidal proteins to improve insecticidal efficiency and prolong control effects. LC85-Ac protein is a new insecticidal protein that can be used to control lepidopteran pests including fall armyworm, striped stem borer, oriental armyworm, beet armyworm, armyworm, cutworm, and two-spotted armyworm (patent application number 202410332785.6).

[0004] Field weeds compete with crops for water, fertilizer, light energy and growth space, directly affecting crop yield and quality. At the same time, many weeds are intermediate hosts for crop pathogens and pests, and are one of the important biological limiting factors for crop yield. According to statistics from the Food and Agriculture Organization of the United Nations, the global food production loss caused by weeds is as high as US$95 billion per year, equivalent to a loss of 380 million tons of wheat, which is more than half of the global wheat production in 2009. Therefore, effective control of field weeds is one of the important measures to promote food production. In addition, the scale and mechanization of agricultural planting is a foreseeable trend, which makes traditional manual weeding methods unrealistic. The promotion and use of herbicides can greatly reduce the difficulty of field management and reduce labor intensity. The development of new high-efficiency, low-toxic, and residue-free herbicide products is expensive, time-consuming and difficult. This problem can be overcome by cultivating herbicide-resistant rice through genetic engineering technology. Spraying 1-2 times during the rice growth period can effectively solve the weed problem, reducing the amount of herbicides used and input costs. Therefore, herbicide-resistant transgenic rice has very broad application value and market potential.

[0005] The present invention connects the insect-resistant gene expression cassette and the herbicide-resistant gene expression cassette in series, so that they are efficiently expressed in transgenic rice, have both insect-resistant and herbicide-resistant traits, and further enhance 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 location, which may be due to chromatin structure (e.g., heterochromatin) or the proximity of transcriptional regulatory elements (e.g., enhancers) to the integration site. For this reason, it is often necessary to screen a large number of events before it is possible to identify events that are commercially viable (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 amount of expression of the introduced gene can vary greatly between events; there may also be differences in the spatial or temporal pattern of expression, such as differences in the relative expression of the transgene between different plant tissues, which can be manifested in the actual expression pattern being inconsistent with the expression pattern expected based on the transcriptional regulatory elements in the introduced gene construct, resulting in differences in the trait expression of the transformation events. Therefore, it is often necessary to generate hundreds or thousands of different events and screen these events to find a single event with the expected transgene expression amount and expression pattern for commercial purposes. Events with the expected transgene expression amount and expression pattern can be used to introgress the transgene into other genetic backgrounds using conventional breeding methods through sexual outcrossing. The progeny produced by this cross retain the transgene expression characteristics of the original transformation event. Applying this strategy model 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 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 contains the target gene. In addition, methods for detecting specific events will also help to comply with relevant regulations, such as the need to obtain formal approval and labeling of foods derived from recombinant crops before they are put on the market. It is possible to detect the presence of transgenics 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, the above method cannot be used to distinguish different events, especially those generated with the same DNA construct. Therefore, a pair of primers that span the junction of the inserted transgenic and flanking DNA are often used to identify transgenic specific events by PCR, specifically a first primer containing a flanking sequence and a second primer containing an inserted sequence. Summary of the invention

[0008] The purpose 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 rice RN85N-e3 and a detection method thereof. The transgenic rice event RN85N-e3 has excellent insect resistance and good tolerance to glufosinate herbicide, and the detection method can accurately and quickly identify whether a biological sample contains a DNA molecule of a specific transgenic rice event RN85N-e3.

[0009] To achieve the above purpose, the present invention uses the pBWA(V)HS-bar-LC85AcA1 expression vector to transform rice Nipponbare by Agrobacterium-mediated method, and obtains a batch of positive transformation events. Through the identification of insect resistance and herbicide resistance traits, it was found that the transformation event RN85N-e3 is a transformant with excellent herbicide tolerance and insect resistance, which can be used to improve the insect resistance and herbicide resistance traits of rice.

[0010] In order to characterize the identity characteristics of RN85N-e3, 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] 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 comprises 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 a 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 or variant thereof or a reverse complementary sequence thereof.

[0015] The present invention also provides a primer pair for detecting rice transformation events, characterized in that the amplification product of the primer pair comprises a 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 a reverse complementary sequence thereof.

[0016] In some embodiments, 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.

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

[0018] The present invention also provides a method for detecting rice transformation events, characterized in that it comprises 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 the 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 rice plants, seeds, plant cells, offspring plants or plant parts by pollen culture, unfertilized embryo culture, doubling culture, cell culture, tissue culture, selfing or hybridization or a combination thereof from the rice obtained in step 1); and optionally, 3) Identifying the insect resistance and / or herbicide resistance of the offspring plants obtained in step 2), and using the above-mentioned method to detect whether the transformation event exists therein.

[0020] Furthermore, the present invention also provides products made from the rice plants, seeds, plant cells, progeny plants or plant parts 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 insertion sequence in the transgenic rice event RN85N-e3, and the SEQ ID NO: 1 spans the DNA sequence at the left flank of the rice insertion site and the DNA sequence at the 5' end of the left border of the insertion sequence. The presence of the transgenic rice event RN85N-e3 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 insertion sequence in the transgenic rice event RN85N-e3, and the SEQ ID NO: 2 spans the DNA sequence at the 3' end of the right border of the insertion sequence and the DNA sequence at the right flank of the rice insertion site. The presence of the transgenic rice event RN85N-e3 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 continuous polynucleotides (first nucleic acid sequence) of any part of the transgenic insertion sequence in the SEQ ID NO: 3 or its reverse complementary sequence, or at least 11 or more continuous polynucleotides (second nucleic acid sequence) of any part of the 5' left wing rice genomic DNA region in the SEQ ID NO: 3 or its reverse complementary sequence. The nucleic acid sequence may further be a part homologous to or reverse complementary to the SEQ ID NO: 3, which part of the sequence must contain the complete SEQ ID NO: 1 or SEQ ID NO: 6 sequence. 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 producing an amplification product. When the amplification product produced in the DNA amplification method using the DNA primer pair 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-e3 or its progeny can be diagnosed.

[0023] The SEQ ID NO: 3 is a sequence with a length of 1444 nucleotides located near the insertion junction at the 5' end of the insertion sequence in the transgenic rice event RN85N-e3, and the SEQ ID NO: 3 consists of a 750-nucleotide rice left flank genomic DNA sequence (nucleotides 1-750 of SEQ ID NO: 3), an 83-nucleotide pBWA(V)HS-bar-LC85AcA1 construct left border DNA sequence (nucleotides 751-833 of SEQ ID NO: 3), and a 611-nucleotide 5' end DNA sequence of the first expression cassette of the glufosinate-tolerant gene (nucleotides 834-1444 of SEQ ID NO: 3). The presence of the transgenic rice event RN85N-e3 can be identified by containing the SEQ ID NO: 3 or its reverse complementary sequence.

[0024] The nucleic acid sequence may be at least 11 or more continuous polynucleotides of any part of the transgenic insertion sequence in the SEQ ID NO: 4 or its reverse complementary sequence (third nucleic acid sequence), or at least 11 or more continuous polynucleotides of any part of the 3' right wing rice genomic DNA region in the SEQ ID NO: 4 or its reverse complementary sequence (fourth nucleic acid sequence). The nucleic acid sequence may further be homologous to or reverse complementary to a part of the SEQ ID NO: 4 that includes the entire SEQ ID NO: 2 or SEQ ID NO: 7. 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 producing an amplification product. When the amplification product produced in the DNA amplification method using the DNA primer pair 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-e3 or its progeny can be diagnosed.

[0025] The SEQ ID NO: 4 is a 977-nucleotide sequence located near the insertion junction at the 3' end of the insertion sequence in the transgenic rice event RN85N-e3. The SEQ ID NO: 4 consists of a 507-nucleotide 3' end DNA sequence of the second expression cassette of the insect-resistant gene (nucleotides 1-507 of SEQ ID NO: 4), a 197-nucleotide pBWA(V)HS-bar-LC85AcA1 construct right border DNA sequence (nucleotides 508-704 of SEQ ID NO: 4), and a 273-nucleotide rice integration site right flank genomic DNA sequence (nucleotides 705-977 of SEQ ID NO: 4). The presence of the transgenic rice event RN85N-e3 can be identified by including the SEQ ID NO: 4 or its reverse complementary sequence.

[0026] The SEQ ID NO: 5 is a sequence of 6933 nucleotides in length that characterizes the transgenic rice event RN85N-e3. The presence of the transgenic rice event RN85N-e3 can be identified by including the 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, characterized in that it includes applying an effective dose of glufosinate herbicide to a field planted with at least one transgenic rice plant, wherein the transgenic rice plant comprises SEQ ID NO: 1, the nucleic acid sequences at positions 751-6660 of SEQ ID NO: 5 and SEQ ID NO: 2 in sequence in its genome, or the genome of the transgenic rice plant comprises SEQ ID NO: 5; and the transgenic rice plant has tolerance to glufosinate herbicide.

[0028] The present invention also provides a method for protecting rice plants from insect attack, characterized in that it includes providing at least one transgenic rice plant cell in the diet of the target insect, wherein the transgenic rice plant cell comprises SEQ ID NO: 1, the nucleic acid sequence at positions 751-6660 of SEQ ID NO: 5 and SEQ ID NO: 2 in sequence in its genome, or the genome of the transgenic rice plant cell comprises SEQ ID NO: 5; the target insect that feeds on the transgenic rice plant cell is inhibited from further feeding on the rice plant.

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

[0030] The invention provides a nucleic acid molecule for detecting rice plants and a detection method thereof. The transgenic rice event RN85N-e3 has insect resistance and tolerance to glufosinate herbicides. The rice plant with this trait expresses LC85-Ac protein and phosphinothricin acetyltransferase (PAT) protein, which endows the plant with insect resistance and tolerance to glufosinate. At the same time, in the detection method of the 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 a DNA primer or probe to produce an amplification product diagnosed as the transgenic rice event RN85N-e3 or its progeny, and the presence of plant materials derived from the transgenic rice event RN85N-e3 can be identified quickly, accurately and stably.

[0031] The transgenic rice event RN85N-e3 has strong tolerance to glufosinate ammonium and outstanding insect resistance. These characteristics make the RN85N-e3 transformant useful for improving the tolerance and insect resistance of rice to glufosinate ammonium herbicides, thereby breeding new rice varieties that are resistant to insects and herbicides.

[0032] The technical solution of the present invention is further described in detail below through the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0034] Figure 2 The effect of indoor biological testing on Chilo suppressalis.

[0035] A, D: Nipponbare (blades); B, C, E, F: RN85N-e3 (blades).

[0036] Figure 3 RN85N-e3 transformation event-specific PCR validation results.

[0037] M: DNA Marker, with size marked next to it (unit: bp); N: blank control water; C: receptor control Nipponbare genomic DNA; 1, 2: genomic DNA of transformant RN85N-e3; 3: Sample containing RN85N-e3 genomic DNA.

[0038] LB: The expected size of the left border PCR fragment is 717 bp; RB: The expected size of the right border PCR fragment is 694 bp. DETAILED DESCRIPTION

[0039] The transformation event RN85N-e3 involved in this application refers to a rice plant obtained by genetic transformation with rice Nipponbare as the recipient, in which a foreign gene insert (T-DNA insert) is inserted between specific genomic sequences. In a specific embodiment, the expression vector used for transgenic has Figure 1The physical map shown in FIG. 5 , and the obtained T-DNA insert has the sequence shown in nucleotides 751-6660 of SEQ ID NO: 5. Transformation event RN85N-e3 can refer to this transgenic process, or to the T-DNA insert in the genome obtained by this process, or to the combination of the T-DNA insert and the flanking sequence, or to the rice plant obtained by this transgenic process. In a specific example, the event is also applicable to plants obtained by transforming other recipient varieties with the same expression vector, thereby inserting the T-DNA insert into the same genomic position. Transformation event RN85N-e3 can also refer to offspring plants obtained by asexual reproduction, sexual reproduction, doubling or doubling reproduction of the above-mentioned plants, or a combination of the above.

[0040] Example 1 Acquisition of transformation events and character identification LC85-AcA1 The gene encodes a protein, LC85-Ac, which has a significant control effect on lepidopteran pests such as the 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 (the physical map of the vector is shown in Figure 1 ,Include LC85-AcA1 Gene expression cassette and bar The receptor Nipponbare was transformed by Agrobacterium-mediated method, and 44 positive transformation events were obtained. The herbicide tolerance and insect resistance of the T1 generation plants of these transformed seedlings were identified and screened.

[0041] 1. Herbicide resistance screening Using Nipponbare as a reference, we screened transformants with better herbicide tolerance by spraying glufosinate at twice the recommended concentration in the field at the seedling stage. 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 of three biological replicates. LSD was used for multiple comparisons in statistical analysis (α = 0.05), and different letters indicate significant differences in the same column of data at the same herbicide concentration.

[0044] 2. Identification of insect resistance Using Nipponbare as a reference, the transformants with better insect resistance were screened from the above 11 transformation events by leaf bioassay. The insect resistance of the materials was evaluated by feeding the newly hatched larvae of Chilo suppressalis with detached rice leaves.

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

[0046] Figure 2 The indoor bioassay effect of leaves of transformant RN85N-e3 on Chilo suppressalis was demonstrated.

[0047] Table 2 Indoor bioassay

[0048] The values ​​are expressed as the mean ± SD of 4 biological replicates, and the significance of the differences between the data in the same column was analyzed using the LSD method (α = 0.05).

[0049] In summary, RN85N-e3 and RN85N-e10 are transformants with excellent herbicide resistance and insect resistance, which can be used to improve the glufosinate herbicide tolerance and insect resistance of rice, thereby cultivating new rice varieties that are resistant to insects and herbicides.

[0050] Example 2 Molecular Characterization of Transformation Event RN85N-e3 In order to further clarify the identity characteristics of the transformation event RN85N-e3, the present invention analyzed the insertion site of the RN85N-e3 exogenous sequence on the rice genome and the insertion structure of the exogenous sequence.

[0051] 1. Analysis of flanking sequences of the insertion site of exogenous sequences in the rice genome 100 mg of plant leaves were taken, quickly ground with liquid nitrogen, and total DNA was extracted using the CTAB method. After the concentration of genomic DNA was measured, the total amount of DNA was guaranteed to be >1 mg. Using genome resequencing, each Read was 150 bp long, at least 10 Gb of data was obtained, and the data quality indicator Q30 was guaranteed to be ≥80% (i.e., the proportion of bases with a sequencing error rate greater than 0.1% was less than 20%). According to the genome resequencing results, the BWA software was used to compare and screen the sequence homology with all the sequences obtained by sequencing using the transgenic vector T-DNA sequence as a template (BWA, http: / / bio-bwa.sourceforge.net / , default settings). The screened sequences were further assembled and screened, and the Reads whose sequences were all vector sequences were removed. Finally, a type of Read sequence was obtained, which was characterized by half of the genome sequence and the other half of the vector sequence. The obtained genome sequence was compared with the Nipponbare MSU7.0 reference genome (http: / / rice.uga.edu / pub / data / Eukaryotic_Projects / o_sativa / annotation_dbs / pseudomolecules / version_7.0 / all.dir / ) to obtain the specific location of the genome sequence on the genome, which is the possible insertion site.

[0052] The sequencing results were compared with the reference genome and exogenous T-DNA sequences to obtain the insertion position information of the exogenous insertion fragment, which was located at the rice genome Chr9: 17256833-17256871 bp position. Subsequently, the forward and reverse primers were designed on the genome flanking sequences and exogenous insertion sequences at the left and right borders of the insertion site using the Primerblast software on the NCBI website (https: / / blast.ncbi.nlm.nih.gov / Blast) (as shown in Table 3). The insertion site was verified by PCR amplification using the genomic DNA of the transgenic rice line as a template. The amplified product fused a part of the rice genome sequence and a part of the T-DNA sequence, and the PCR product was sequenced and analyzed.

[0053] Table 3 Primer information

[0054] The PCR reaction system specifically includes 1.0 μL genomic DNA, 10.0 μL 2×PCR Mixture, 0.5 μL forward primer (10 μM) and 0.5 μL reverse primer (10 μM), and water is added to make up to 20 μL.

[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; and extension at 72°C for 7 min.

[0056] PCR amplification results are shown in Figure 3 The PCR amplification products of the left and right borders were sequenced to obtain the T-DNA insertion border sequence and flanking sequences of the RN85N-e3 transformant, which were assembled as follows (capital letters represent rice genome sequences, lowercase letters represent T-DNA sequences, and bold marks the positions of transformant-specific PCR primers): Left border: ACCCTGTTGA TTTGCTACCC TGTGCTGATT AGTTTCAATC GTGTATTGGC CAATCTGGTC 60 GTAAGAATAG CATTGCATTG CATTTATTAT GTTCTTATCA TGTGTTTTTT GGGGATGAAA 120 CGTACATGCA GCAAGATAAG TTCAACTTAT CCAGTTTTAT CGTCGTTCTG TACATATTAA 180 AATGTAGTTT AATACAGTAT AGTTTACTCC TACTTATACT TTAAGTATGT GCCACATCAT 240 CTAAAAGTAT ACATAGAGAT AGGACATACT AGGACTACTA GAATACAGTG GCGGAGCCAG 300 GATAAAAATA TAGCGGGGCT AAACCAAAAT GACCAACAGC AACTAAGAGA GATTTAGGAT 360 TAGGTTAGGA ATTTTTGGGC CTTCTTAACG TTTTGGGCCT TCTAACAACC TCAAACAAAG 420 TTTATATTAa acaaattgac gcttagacaa cttaataaca cattgcggac gtttttaatg 480 ttagactgaa ttaacgccga attaattcgg gggatctgga ttttagtact ggattttggt 540 tttaggaatt agaaatttta ttgatagaag tattttacaa atacaaatac atactaaggg 600 tttcttatat gctcaacaca tgagcgaaac cctataggaa ccctaattcc cttatctggg 660 aactactcac acattattat ggagaaagcg atcgctcaaa tctcggtgac gggcagg 717 Right boundary: gattgagatt ggagaaacgg aaggaacatt cattgttgat agcgtggagt tacttctgat 60 ggaggaatga gagctcgggc ccgaagactg accagctcga atttccccga tcgttcaaac 120 atttggcaat aaagtttctt aagattgaat cctgttgccg gtcttgcgat gattatcata 180 taatttctgt tgaattacgt taagcatgta ataattaaca tgtaatgcat gacgttattt 240 atgagatggg tttttatgat tagagtcccg caattataca tttaatacgc gatagaaaac 300 aaaatatagc gcgcaaacta ggataaatta tcgcgcgcgg tgtcatctat gttactagat 360 cgggccatcc gcactgtagc ggatggccta aaaaaaaaac tagaagagac gagtctgaga 420 ctcagcgtct cggtcgcagt cataacttcg tatagcatac attatacgaa gttatgggcc 480 gcattaccct gttatcccta ggccgcataa cttcgtatag cctacattat aggatggagg 540 gatatcctct cttaaggtGG GGGTATTATG AGGGCTCTAA TGGTCTATTA GAGGGTACGG 600 GGGTCTAAAA ACACCTCCACCCCACGCTG TCTCCGCCTC TGCTAGAATA CAATAATACA 660 ATAGTAGTTA CATTCTTCCA TTCCCTCAGC CATG 694 The results of sequencing analysis showed that the RN85N-e3 exogenous fragment was stably inserted into the rice genome Chr9: 17256833-17256871bp position.

[0057] The recipient rice genome sequence at the position of the transformant insertion (the underline marks the genome deletion sequence caused by the insertion of the transformant T-DNA): tatccagttt tatcgtcgtt ctgtacatat taaaatgtag tttaatacag tatagtttac 60 tcctacttat actttaagta tgtgccacat catctaaaag tatacataga gataggacat 120 actaggacta ctagaataca gtggcggagc caggataaaa atatagcggg gctaaaccaa 180 aatgaccaac agcaactaag agagatttag gattaggtta ggaatttttg ggccttctta 240 acgttttggg ccttctaaca acctcaaaca aagtttatat ta gcccctca tttataaata 300 tatggattag aattttagg g ggggtattat gagggctcta atggtctatt agagggtacg 360 ggggtctaaa aacacctcca cccccacgct gtctccgcct ctgctagaat acaataatac 420 aatagtagtt acattcttcc attccctcag ccatggacga agctacaagt atccagggat 480 gcctagggaa ccgatacata attttttttt agttatagtt tacctatttt catcatatat 540 acgtctcctc aattcaaact tagacatccg tatcaagcta aaatcgattc aaagtagagt 600 aaaatatgtg agtggaaccc ctctattttg ttttagcttt gtcaccgttt tcagtgtacg 660 taggtaacca ccgtatctaa tgctttgtcc cataatcaca tt 702 Analysis of the left and right border sequences showed that the insertion of the exogenous sequence caused a 37 bp deletion mutation in the recipient rice genome; at the same time, the LB end of the T-DNA region deleted 22 bp, and the RB end deleted 282 bp. Analysis of the upstream and downstream genomic sequences of the insertion site showed that the insertion site of the exogenous sequence was located in the intergenic region of the rice genome, and there was no known functional gene.

[0058] 2. Analysis of full-length insert sequences in vitro Using SEQ ID NO: 8 and SEQ ID NO: 11 as primers and the genomic DNA of the transformant RN85N-e3 as a template, PCR amplification was performed, and the obtained PCR product was the exogenous full-length insertion sequence. The PCR product was subjected to second-generation sequencing analysis and sequence splicing to obtain the actual insertion sequence of the RN85N-e3 transformant and the left and right flanking rice genome sequences, as shown in SEQ ID NO: 5.

[0059] Example 3 Detection method of transformation event RN85N-e3 New varieties can be bred from transgenic rice event RN85N-e3 and produce agricultural products or commodities. If sufficient amounts are detected in the agricultural products or commodities, the agricultural products or commodities are expected to contain nucleotide sequences that can diagnose the presence of transgenic rice event RN85N-e3 materials in the agricultural products or commodities. 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 polysaccharides, and any other food, cosmetics, industrial products, etc. that will be consumed as a food source for animals. Nucleic acid detection methods and / or kits based on probe or primer pairs can be developed to detect transgenic rice event RN85N-e3 nucleotide sequences such as those shown in SEQ ID NO: 1 or SEQ ID NO: 2 in biological samples, wherein the probe sequence or primer amplification sequence is 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-e3.

[0060] One of the detection methods is: using the PCR method to detect samples containing RN85N-e3, 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: The reaction procedure is: 95℃, 5 min; (95℃, 30 sec; 56℃, 30 sec; 72℃, 45 sec) × 30 cycles; 72℃, 7 min.

[0062] The PCR product was electrophoresed in 1% (w / v) 1×TAE agarose gel. Figure 3 shown.

[0063] The expected target bands (SEQ ID NO: 6 and SEQ ID NO: 7, respectively) can be amplified in the RN85N-e3 transformation event. Moreover, the PCR method can track the existence of the transformation event, so as to be applied to breeding work. In summary, the transgenic rice event RN85N-e3 of the present invention can improve plant insect resistance and has a 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 quickly identify whether the biological sample contains the DNA molecule of the transgenic rice event RN85N-e3.

[0064] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the spirit and scope of the technical solution of the present invention.

Claims

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

2. A probe for detecting rice transformation events, characterized in that: It includes the sequence shown by 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 variant or its reverse complementary sequence.

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

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

7. A method for breeding rice, characterized in that: The method comprises the following steps: 1) obtaining rice containing the nucleic acid molecule according to 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, offspring plants or plant parts.

8. The method according to claim 7, characterized in that The method comprises: evaluating the insect resistance traits and / or identifying the herbicide resistance of the offspring plants obtained in step 2) of claim 7, and using the method of claim 6 to detect whether there is a transformation event therein.

9. The product obtained by the method according to any one of claims 7 to 8, characterized in that The product is made from 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 The method comprises applying an effective dose of a glufosinate herbicide to a field planted with at least one transgenic rice plant, wherein the transgenic rice plant sequentially comprises the nucleic acid sequences of SEQ ID NO: 1, SEQ ID NO: 5 at positions 751-6660 and SEQ ID NO: 2 in its genome, or the genome of the transgenic rice plant comprises SEQ ID NO: 5; and the transgenic rice plant has tolerance to the glufosinate herbicide.

11. A method for protecting rice plants from insect attack, characterized in that The method comprises providing at least one transgenic rice plant cell in the diet of a target insect, wherein the transgenic rice plant cell comprises SEQ ID NO: 1, the nucleic acid sequence at positions 751-6660 of SEQ ID NO: 5 and SEQ ID NO: 2 in sequence in its genome, or the genome of the transgenic rice plant cell comprises SEQ ID NO: 5; and the target insect that ingests the transgenic rice plant cell is inhibited from further ingesting the rice plant.

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