CN112662799B - Nucleic acid molecule for detecting corn plant ND4401 and detection method thereof - Google Patents

Abstract

The invention relates to a nucleic acid molecule for detecting corn plant ND4401 and a detection method thereof, wherein the nucleic acid molecule sequence of the corn plant comprises a sequence shown as SEQ ID NO. 1 or a complementary sequence thereof, or a sequence shown as SEQ ID NO. 2 or a complementary sequence thereof. The corn plant ND4401 has high yield and/or nitrogen deficiency tolerance, and the detection method can accurately and quickly identify whether the biological sample contains the DNA molecule of the transgenic corn event ND 4401.

Description

A kind of nucleic acid molecule for detecting corn plant ND4401 and its detection method

Technical field

The present invention relates to the field of plant biotechnology. Specifically, it relates to a nucleic acid molecule for detecting corn plant ND4401 and a detection method thereof, in particular to a transgenic corn event ND4401 with high yield and/or nitrogen deficiency tolerance traits and for detecting whether biological samples contain Nucleic acid molecules of specific transgenic maize event ND4401 and their detection methods.

Background technique

Nitrogen is one of the essential mineral nutrients for plants and an essential component for the synthesis of proteins, nucleic acids and many biologically active substances. Nitrogen deficiency usually results in plant growth retardation and chlorosis of mature leaves accompanied by anthocyanin accumulation (DiazC, Saliba-Colombani V, Loudet O, et al. Leaf Yellowing and AnthocyaninAccumulation are Two Genetically Independent Strategies in Response to Nitrogen Limitation in Arabidopsis thaliana[J].Plant&Cell Physiology,2006,47(1):74-83.). The extensive use of nitrogen fertilizers in agricultural production has greatly increased crop yields. Currently, approximately 80-90 million tons of nitrogen fertilizer are applied globally every year, which is predicted to increase to 240 million tons by 2050 (Tilman D. Global environmental impacts of agricultural expansion: the need for sustainable and efficient practices[J]. Proc Natl Acad Sci U S A .1999,96(11):5995-6000.). The increase in energy costs has led to the continuous increase in the price of nitrogen fertilizer, thereby increasing the cost of agricultural production. At the same time, the loss of nitrogen fertilizer is also gradually deteriorating the ecological environment. Based on the dual considerations of economic benefits and environmental protection, planting nitrogen-efficient crop varieties in modern agricultural production is an effective way to solve the problem of low nitrogen fertilizer utilization, reduce production costs, and reduce environmental pollution. It is also an effective way to achieve sustainable agricultural development and enhance the international competitiveness of products. basic requirements. Traditionally, breeding new varieties is a very long-term process. Nowadays, the continuous maturity of genetic engineering makes it possible for us to quickly obtain nitrogen-efficient varieties through genetic engineering. The maize ZmNRT1.1 gene encodes a low-affinity nitrate transporter that can affect the absorption of nitrate nitrogen by plant roots, thereby improving nitrogen use efficiency (Quaggiotti S, Ruperti B, Pizzeghello D, et al. Effect of low molecularsize humic substances on nitrate uptake and expression of genes involved innitrate transport in maize(Zea mays L.)[J].Journal of Experimental Botany,2004,55(398):816-823.), using this gene can improve Tolerance of corn under nitrogen deficiency conditions and ultimately increased yield.

It is known that the expression of foreign genes in plants is affected by their chromosomal location, possibly due to the proximity of chromatin structure (e.g., heterochromatin) or transcriptional regulatory elements (e.g., enhancers) to the integration site. To this end, it is usually necessary to screen a large number of events before it is possible to identify events that can be commercialized (that is, 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 an introduced gene can vary significantly between events; there may also be differences in the spatial or temporal patterns of expression, such as the relative expression of the transgene between different plant tissues. Differences exist, and this difference is manifested in the fact that the actual expression pattern may be inconsistent with the expression pattern expected based on the transcriptional regulatory elements in the introduced gene construct, resulting in differences in trait expression during the transformation event. Therefore, it is often necessary to generate hundreds or thousands of different events and screen these events for a single event with the desired transgene expression amount and expression pattern for commercialization purposes. Events with expected transgene expression amounts and expression patterns can be used to introgress the transgene into other genetic backgrounds via sexual outcrossing using conventional breeding methods. The progeny produced by this cross maintain 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 growing conditions. Therefore, more transformation events need to be characterized and screened to obtain excellent transformation events with excellent comprehensive trait performance and commercial prospects.

It would be beneficial to be able to detect the presence of specific events to determine whether the progeny of a sexual cross contains the gene of interest. In addition, methods to detect specific events will help comply with regulations such as the need for formal approval and labeling of food derived from recombinant crops before being placed on the market. Detection of the presence of a transgene is possible by any of the well-known polynucleotide detection methods, such as polymerase chain reaction (PCR) or DNA hybridization using polynucleotide probes. 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 adjacent to the inserted transgenic DNA ("flanking DNA") is known, this method cannot be used to distinguish between different events, especially those produced with the same DNA construct. event. Therefore, it is now common to identify transgene-specific events by PCR using a pair of primers spanning the junction of the inserted transgene and flanking DNA, specifically a first primer containing the flanking sequence and a second primer containing the inserted sequence.

Contents of the invention

The purpose of the present invention is to provide a corn transformation event with excellent yield traits and/or nitrogen deficiency tolerance traits, as well as nucleic acid molecules for detecting corn plant ND4401 and detection methods thereof. The genetically modified corn event ND4401 exhibits high yield and/or nitrogen deficiency tolerance due to its high nitrogen utilization efficiency, and the detection method can accurately and quickly identify whether biological samples contain DNA molecules of the specific genetically modified corn event ND4401.

In order to achieve the above purpose, the present invention uses Agrobacterium infection technology to transform the pCAMBIA1301-ZmNRT1.1A expression vector into maize y822 immature embryos, and obtains 45 transformation events of ZmNRT1.1A and bar gene-transfected maize. Among them, ND4401 shows stable heritability from generation to generation, outstanding nitrogen deficiency tolerance and yield traits, and has good application prospects.

In order to characterize the identity characteristics of ND4401, the present invention provides a nucleic acid molecule comprising the sequence shown in SEQ ID NO: 1 and/or SEQ ID NO: 2, or its complementary sequence.

Further, the nucleic acid molecule sequence includes the sequence shown in SEQ ID NO: 3, or its complementary sequence.

Furthermore, the nucleic acid molecule sequence includes the sequence shown in SEQ ID NO: 4 or its complementary sequence.

On the other hand, the present invention provides a probe for detecting maize transformation events, which is characterized in that it 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 its sequence. Fragment or variant thereof or complementary sequence thereof.

The present invention also provides a primer pair for detecting maize transformation events, which is characterized in that the primer pair includes:

A primer that specifically recognizes the nucleotide sequence at positions 1-625 of the sequence shown in SEQ ID NO:4 and a primer that specifically recognizes the nucleotide sequence at positions 626-4920 of the sequence shown in SEQ ID NO:4; and/ or

A primer that specifically recognizes the nucleotide sequence at positions 626-4920 of the sequence shown in SEQ ID NO:4 and a primer that specifically recognizes the nucleotide sequence at positions 4921-5052 of the sequence shown in SEQ ID NO:4;

In some embodiments, the amplification product of the primer pair comprises the sequence of claim 2;

In some embodiments, the primer pair is the sequence shown in SEQ ID NO:5 and SEQ ID NO:6 or the complement thereof.

The present invention also provides a kit or microarray for detecting maize transformation events, which is characterized by comprising the above-mentioned probe and/or the above-mentioned primer pair.

The present invention also provides a method for detecting corn transformation events, which is characterized in that it includes 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 sample to be tested is The conversion event exists.

The present invention also provides a method for breeding corn, which is characterized in that the method includes the following steps:

1) Obtain corn containing the above nucleic acid molecules;

2) Using the corn obtained in step 1) through pollen culture, unfertilized embryo culture, double culture, cell culture, tissue culture, selfing or hybridization or a combination thereof to obtain corn plants, seeds, plant cells, progeny plants or plant parts ; and optionally,

3) Conduct yield trait identification and/or nitrogen deficiency tolerance identification on the progeny plants obtained in step 2), and use the above method to detect whether the transformation event exists therein.

Furthermore, the present invention also provides products made from corn plants, seeds, plant cells, progeny plants or plant parts obtained by the above method, including food, feed or industrial raw materials.

In the present invention, the nucleic acid molecule sequence may be at least 11 or more consecutive polynucleotides (first nucleic acid molecule) of any part of the transgene insertion sequence in SEQ ID NO: 1 or its complementary sequence, or At least 11 or more contiguous polynucleotides (second nucleic acid molecules) of any part of the 5' left flanking maize genomic DNA region in SEQ ID NO: 1 or its complementary sequence. The nucleic acid molecule may further be homologous to or complementary to a portion of said SEQ ID NO:3 comprising the entirety of said SEQ ID NO:5. When a first nucleic acid molecule and a second nucleic acid molecule are used together, these nucleic acid molecules include a pair of DNA primers in a DNA amplification method that produces an amplification product. When the amplification product generated in the DNA amplification method using a DNA primer pair is an amplification product including SEQ ID NO: 1 or SEQ ID NO: 3 or SEQ ID NO: 4, the transgenic maize event ND4401 or its progeny can be diagnosed. exist.

The SEQ ID NO: 1 or its complementary sequence is a sequence of 1363 nucleotides in length located near the insertion junction site at the 5' end of the inserted sequence in the transgenic maize event ND4401. The SEQ ID NO: 1 or its complementary sequence is The complementary sequence consists of the 625 nucleotide left flank genomic DNA sequence of maize (nucleotides 1-625 of SEQ ID NO: 1), the 69 nucleotide left border DNA sequence of the pCAMBIA1301-ZmNRT1.1A construct (SEQ It consists of nucleotides 626-694 of ID NO:1) and the first expression cassette DNA sequence of 669 nucleotides of the glufosinate-resistant gene (nucleotides 695-1363 of SEQ ID NO:1), comprising the SEQ ID NO: 1 or its complementary sequence can identify the presence of transgenic maize event ND4401.

The nucleic acid molecule sequence may be at least 11 or more contiguous polynucleotides (third nucleic acid molecule) of any part of the transgene insert sequence in SEQ ID NO:2 or its complementary sequence, or be the SEQ ID At least 11 or more contiguous polynucleotides (fourth nucleic acid molecule) of any part of the 3' right flanking maize genomic DNA region in NO:2 or its complementary sequence. When the third nucleic acid molecule and the fourth nucleic acid molecule are used together, these nucleic acid molecules include a set of DNA primers in a DNA amplification method that produces an amplification product. When the amplification product generated in a DNA amplification method using a DNA primer pair is an amplification product including SEQ ID NO:2 or SEQ ID NO:4, the presence of transgenic maize event ND4401 or its progeny can be diagnosed.

The SEQ ID NO: 2 or its complementary sequence is a sequence of 720 nucleotides in length located near the insertion junction site at the 3' end of the inserted sequence in the transgenic maize event ND4401. The SEQ ID NO: 2 or its complementary sequence is The complementary sequence consists of the 3' end DNA sequence of the second expression cassette of the nitrogen efficient utilization gene of 581 nucleotides (nucleotides 1-581 of SEQ ID NO: 2), and the 7-nucleotide pCAMBIA1301-ZmNRT1.1A The construct right border DNA sequence (nucleotides 582-588 of SEQ ID NO:2) and the 132 nucleotide right flanking genomic DNA sequence of the maize integration site (nucleotides 589-720 of SEQ ID NO:2 ) composition, the presence of the transgenic maize event ND4401 can be identified by including the SEQ ID NO:2 or its complementary sequence.

The SEQ ID NO: 4 or its complementary sequence is a sequence of 5052 nucleotides in length that characterizes the transgenic maize event ND4401. The specific genome and genetic elements it contains are shown in Table 1. The presence of the transgenic maize event ND4401 can be identified by including the SEQ ID NO:4 or its complementary sequence.

Table 1 Genome and genetic elements contained in SEQ ID NO:4

1: Unit bp.

It is well known to those skilled in the art that the first and second nucleic acid molecules or the third and fourth nucleic acid molecules need not consist solely of DNA, but may also include RNA, a mixture of DNA and RNA, or DNA, RNA or other molecules that are not one or Combinations of multiple polymerase template nucleotides or analogs thereof. In addition, the probe or primer described in the present invention should be at least about 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22 consecutive nucleotides in length, which may be selected from The nucleotide described in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:4. When selected from the nucleotides shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4, the probe and primer may be at least about 21 to about 50 nucleotides in length or more consecutive nucleotides.

The present invention also provides a method for improving corn yield and/or nitrogen deficiency tolerance, which is characterized by comprising planting at least one transgenic corn plant in soil, the transgenic corn plant comprising SEQ ID NO: 4. The nucleic acid sequence at positions 2165-5052, or the genome of the transgenic corn plant contains SEQ ID NO: 4; the transgenic corn plant has high yield and/or nitrogen deficiency tolerance traits.

The present invention also provides a method for improving corn yield and/or nitrogen deficiency tolerance, which is characterized by using an expression cassette expressing a nitrogen efficient utilization gene with a sequence shown in nucleotides 2165-4913 of SEQ ID NO:4. Imported into the maize genome.

In the nucleic acid molecules for detecting corn plants and their detection methods of 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 stated, according to ordinary skill in the art People understand the term in its conventional usage.

The "corn" refers to Zea mays and includes all plant species that can mate with corn, including wild corn species.

The "include" means "including but not limited to".

The term "plant" includes whole plants, plant cells, plant organs, plant protoplasts, plant cell tissue cultures from which plants can be regenerated, plant callus, plant clumps and plants or plant parts intact. Cells, the plant parts such as embryos, pollen, ovules, seeds, leaves, flowers, branches, fruits, stems, roots, root tips, anthers, etc. It should be understood that parts of transgenic plants within the scope of the present invention include, but are not limited to, plant cells, protoplasts, tissues, callus, embryos, as well as flowers, stems, fruits, leaves and roots, and the above plant parts are derived from plants previously treated with the present invention. A transgenic plant or its progeny that has been transformed with a DNA molecule and thus consists at least in part of transgenic cells.

The term "gene" refers to a nucleic acid fragment that expresses a specific protein, including regulatory sequences before the coding sequence (5' non-coding sequence) and regulatory sequences after the coding sequence (3' non-coding sequence). "Native gene" refers to a gene found naturally with its own regulatory sequences. A "chimeric gene" refers to any gene that is not native and contains regulatory and coding sequences not found in nature. An "endogenous gene" refers to a native gene located in its native location in the genome of an organism. "Exogenous genes" are foreign genes that are present in the genome of an organism and did not originally exist. They also refer to genes introduced into recipient cells through transgenic steps. Foreign genes may include native genes or chimeric genes inserted into a non-native organism. A "transgene" is a gene that has been introduced into the genome through a transformation procedure. The site in the plant genome where recombinant DNA has been inserted may be called an "insertion site" or a "target site."

"Flanking DNA" may comprise the genome naturally occurring in an organism such as a plant or foreign (heterologous) DNA introduced through a transformation process, such as fragments associated with a transformation event. Thus, flanking DNA may include a combination of native and exogenous DNA. In the present invention, "flanking region" or "flanking sequence" or "genome boundary region" or "genome boundary sequence" refers to at least 3, 5, 10, 11, 15, 20, 50, 100, 200, 300, 400 , 1000, 1500, 2000, 2500 or 5000 base pairs or longer sequences located directly upstream or downstream of and adjacent to the initial exogenously inserted DNA molecule. When the flanking region is located upstream, it may also be called the "left border flank" or "5' flanking" or "5' genome flanking region" or "genomic 5' flanking sequence", etc. When the flanking region is located downstream, it may also be called the "right border flanking" or "3' flanking" or "3' genome flanking region" or "genomic 3' flanking sequence", etc.

Transformation procedures that result in random integration of foreign DNA will result in transformation events containing different flanking regions that are specific to each transformation event. When recombinant DNA is introduced into a plant through traditional crossing, its flanking regions are usually not altered. Transformation events may also involve unique junctions between a heterologous insert DNA and a segment of genomic DNA, or between two segments of genomic DNA, or between two segments of heterologous DNA. A "junction" is the point where two specific DNA fragments join. For example, a junction exists where the insert DNA joins the flanking DNA. Junctions are also present in transformed organisms, where two DNA segments are joined together in a modified manner from that found in natural organisms. "Jog DNA" refers to DNA containing junction points.

The present invention provides a transgenic corn event called ND4401 and its progeny. The transgenic corn event ND4401 is a corn plant ND4401, which includes plants and seeds of the transgenic corn event ND4401 and its plant cells or regenerable parts thereof. The transgenic corn event ND4401 is a corn plant ND4401. Plant parts of the corn plant ND4401, including but not limited to cells, pollen, ovules, flowers, buds, roots, stems, silks, inflorescences, ears, leaves and products from the corn plant ND4401, such as corn meal, cornmeal, corn oil , corn steep liquor, corn silk, corn starch and biomass left in the corn crop field.

The transgenic maize event ND4401 of the present invention includes a DNA construct, and when expressed in plant cells, the transgenic maize event ND4401 obtains yield enhancement and/or nitrogen deficiency tolerance traits. The DNA construct comprises an expression cassette comprising a suitable promoter for expression in plants operably linked to a gene encoding a maize nitrate transporter and a suitable polyadenylation signal sequence. ZmNAR1.1A, the nucleic acid molecule of ZmNAR1.1A is tolerant to nitrogen deficiency. The DNA construct comprises another expression cassette comprising a suitable promoter for expression in plants and a suitable polyadenylation signal sequence operably linked to the promoter encoding phosphinothricin acetyl Transferase (PAT) gene bar. The nucleic acid molecule of the PAT protein is resistant to glufosinate herbicide and can be used as a screening marker in the transformation stage. Further, the promoter may be a suitable promoter isolated from a plant, including constitutive, inducible and/or tissue-specific promoters, and the suitable promoter includes, but is not limited to, Cauliflower Mosaic Virus (CaMV) 35S Promoter, FMV 35S promoter, Ubiquitin promoter, Actin promoter, Agrobacterium tumefaciens nopaline synthase (NOS) promoter, Octopine synthase (OCS) promoter, Cestrum yellow leaf curl virus promoter, potato tuber storage protein (Patatin) promoter, ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) promoter, glutathione sulfotransferase (GST) promoter, E9 promoter, GOS promoter, alcA/alcR promoter, Agrobacterium rhizogenes RolD promoter and Arabidopsis thaliana )Suc2 promoter. The polyadenylation signal sequence may be a suitable polyadenylation signal sequence that functions in plants, and the suitable polyadenylation signal sequence includes, but is not limited to, derived from Agrobacterium tumefaciens The polyadenylation signal sequence of the nopaline synthase (NOS) gene, the polyadenylation signal sequence derived from the cauliflower mosaic virus (CaMV) 35S terminator, and the polyadenylation signal sequence derived from the protease inhibitor II (PINII) gene and their sources The polyadenylation signal sequence of the α-tubulin gene.

In addition, the expression cassette may also include other genetic elements including, but not limited to, enhancers and signal peptides/transit peptides. The enhancer can enhance the expression level of the gene, and the enhancer includes, but is not limited to, tobacco etch virus (TEV) translation activator, CaMV35S enhancer and FMV35S enhancer. The signal peptide/transit peptide can guide the EPSPS protein to be transported to specific organelles or compartments outside the cell or within the cell, for example, targeting the chloroplast using a sequence encoding a chloroplast transit peptide, or targeting the endoplasmic reticulum using a 'KDEL' retention sequence.

The DNA construct is introduced into the plant using a transformation method, which includes, but is not limited to, Agrobacterium-mediated transformation, biolistic transformation, and pollen tube channel transformation.

The Agrobacterium-mediated transformation method is a common method for plant transformation. The exogenous DNA to be introduced into the plant is cloned into the vector between the left and right border consensus sequences, that is, the T-DNA region. The vector is transformed into Agrobacterium cells, which are subsequently used to infect plant tissue, and the T-DNA region of the vector containing foreign DNA is inserted into the plant genome.

After transformation, transgenic plants must be regenerated from the transformed plant tissue and the progeny with foreign DNA selected using appropriate markers.

A DNA construct is an assembly of DNA molecules interconnected to provide one or more expression cassettes. The DNA construct is preferably a plasmid that is capable of self-replication in bacterial cells and contains different restriction endonuclease sites for the introduction of a functional genetic element, that is, a promoter. , introns, leader sequences, coding sequences, 3' terminator regions and other sequences of DNA molecules. The expression cassette contained in the DNA construct includes the genetic elements necessary to provide for the transcription of the messenger RNA and can be designed for expression in prokaryotic or eukaryotic cells. The expression cassettes of the present invention are designed for expression most preferably within plant cells.

A transgenic "event" is obtained by transforming plant cells with a heterologous DNA construct, that is, including at least one nucleic acid expression cassette containing a target gene, which is inserted into the plant genome through a transgenic method to produce a plant population, and then regenerates the plant population. , and select specific plants with characteristics of insertion into specific genomic loci. The term "event" refers to the original transformation event including heterologous DNA and the progeny of that transformation event. The term "event" also refers to the offspring resulting from a sexual cross between a transformation event and an individual of another breed containing heterologous DNA, even after repeated backcrossing to the backcross parent, the inserted DNA and flanking DNA from the transformation event parent Genomic DNA is also present at the same chromosomal location in the hybrid offspring. The term "event" also refers to a DNA sequence from the original transformation event that contains the inserted DNA and flanking genomic sequences in close proximity to the inserted DNA, which DNA sequence is expected to be transferred into progeny consisting of progeny containing the inserted DNA A parent line (eg, the original transformation event and its progeny from selfing) is produced by sexual crossing with a parent line that does not contain the inserted DNA, and the progeny receives the inserted DNA containing the target gene.

"Recombinant" in the present invention refers to forms of DNA and/or proteins and/or organisms that are not normally found in nature and are therefore produced by artificial intervention. This artificial intervention can produce recombinant DNA molecules and/or recombinant plants. The "recombinant DNA molecule" is obtained by artificially combining two otherwise separate sequence segments, such as by chemical synthesis or by manipulating isolated nucleic acid segments through genetic engineering techniques. Techniques for performing nucleic acid manipulations are well known.

The term "transgene" includes any cell, cell line, callus, tissue, plant part or plant whose genotype has been altered by the presence of a heterologous nucleic acid, including a transgene originally so altered as well as by The offspring individuals produced by the original transgenic body through sexual hybridization or asexual reproduction. In the present invention, the term "transgene" does not include changes in the genome (chromosomal or extrachromosomal) by conventional plant breeding methods or naturally occurring events such as random allogeneic fertilization, non-recombinant viral infection, non-recombinant Bacterial transformation, non-recombinant transposition, or spontaneous mutation.

"Heterologous" as used herein means that the first molecule is not normally found in combination with the second molecule in nature. For example, a molecule can be derived from a first species and inserted into the genome of a second species. The molecule is therefore heterologous to the host and is artificially introduced into the genome of the host cell.

Transgenic corn event ND4401, which is high-yielding and/or tolerant to nitrogen deficiency, is cultivated by first sexually crossing a first parent corn plant with a second parent corn plant, thereby producing a diverse first generation of progeny plants , the first parent corn plant consists of corn plants cultivated from transgenic corn event ND4401 and its progeny, which are obtained by transformation using the nitrogen efficient utilization expression cassette of the present invention, and the second Parent corn plants lack high yields and/or nitrogen deficiency tolerance; then selecting progeny plants that are tolerant to nitrogen deficiency or have increased yields can produce corn plants that are tolerant to nitrogen deficiencies or have increased yields. These steps may further include backcrossing high-yielding and/or nitrogen-deficiency-tolerant progeny plants to a second parent corn plant or a third parent corn plant, and then identifying the trait-associated molecular markers (e.g., containing transgenic corn events). DNA molecules of the junction sites identified at the 5' and 3' ends of the inserted sequence in ND4401) to select progeny to produce corn plants with increased yield and/or tolerance to nitrogen deficiency.

It will also be understood that two different transgenic plants can also be crossed to produce progeny containing two independent, discretely added foreign genes. Selfing of appropriate progeny can result in progeny plants that are homozygous for both added foreign genes. Backcrossing of parental plants and outcrossing with non-transgenic plants as described above is also contemplated, as is asexual propagation.

The term "probe" is an isolated nucleic acid molecule to which a conventional detectable label or reporter molecule is bound, such as a radioisotope, ligand, chemiluminescent agent, or enzyme. This probe is complementary to a strand of the target nucleic acid. In the present invention, the probe is complementary to a DNA strand from the genome of transgenic maize event ND4401, whether the genomic DNA is from transgenic maize event ND4401 or seeds or from transgenics. Plant or seed or extract of Zea mays event ND4401. The probes of the present invention include not only deoxyribonucleic acid or ribonucleic acid, but also polyamide and other probe materials that specifically bind to a target DNA sequence and can be used to detect the presence of the target DNA sequence.

The term "primer" is an isolated nucleic acid molecule that binds to a complementary target DNA strand through nucleic acid hybridization, annealing, and formation of a hybrid between the primer and target DNA strand, followed by the action of a polymerase (e.g., DNA polymerase) down, extending along the target DNA strand. The primer pairs of the present invention relate to their use in the amplification of target nucleic acid molecules, for example, by polymerase chain reaction (PCR) or other conventional nucleic acid amplification methods.

The length of probes and primers is generally 11 polynucleotides or more, preferably 18 polynucleotides or more, more preferably 24 polynucleotides or more, and most preferably 30 polynucleotides Sour or more. Such probes and primers hybridize specifically to target sequences under highly stringent hybridization conditions. Although probes that are different from the target DNA sequence and maintain hybridization ability to the target DNA sequence can be designed by conventional methods, preferably, the probes and primers in the present invention have a complete DNA sequence with the continuous nucleic acid of the target sequence. Identity.

As used herein, "kit" or "microarray" refers to a reagent set or chip used for the purpose of identification and/or detection of maize transformation events in biological samples. Kits or chips may be used for the purpose of quality control (e.g. purity of seed lots), detection of events in or containing or derived from plant material such as, but not limited to, food or feed products, and their The components can be adjusted specifically.

Primers and probes based on the flanking genomic DNA and insert sequences of the present invention can be determined by conventional methods, for example, by isolating the corresponding DNA molecules from plant material derived from transgenic maize event ND4401 and determining the nucleic acid sequence of the DNA molecules. The DNA molecule contains the transgene insert sequence and the maize genome flanking regions, and fragments of the DNA molecule can be used as primers or probes.

The nucleic acid probes and primers of the present invention hybridize to target DNA sequences under stringent conditions. Any conventional nucleic acid hybridization or amplification method can be used to identify the presence of DNA derived from transgenic maize event ND4401 in a sample. Nucleic acid molecules or fragments thereof can specifically hybridize with other nucleic acid molecules under certain circumstances. As used in the present invention, if two nucleic acid molecules can form an antiparallel double-stranded nucleic acid structure, it can be said that the two nucleic acid molecules can specifically hybridize with each other. If two nucleic acid molecules show complete complementarity, one of the nucleic acid molecules is said to be the "complement" of the other. As used herein, two nucleic acid molecules are said to exhibit "complete complementarity" when each nucleotide of one nucleic acid molecule is complementary to the corresponding nucleotide of the other nucleic acid molecule. Two nucleic acid molecules are said to be "minimally complementary" if they are able to hybridize to each other with sufficient stability such that they anneal and bind to each other under at least conventional "low stringency" conditions. Similarly, two nucleic acid molecules are said to be "complementary" if they can hybridize to each other with sufficient stability such that they anneal and bind to each other under conventional "highly stringent" conditions. Deviations from perfect complementarity are permissible as long as such departures do not completely prevent the two molecules from forming a double-stranded structure. In order for a nucleic acid molecule to serve as a primer or probe, it is only necessary to ensure that it has sufficient sequence complementarity to form a stable double-stranded structure under the specific solvent and salt concentration used.

As used in the present invention, a substantially homologous sequence is a nucleic acid molecule that is capable of specifically hybridizing to a matching complementary strand of another nucleic acid molecule under highly stringent conditions. Suitable stringent conditions to promote DNA hybridization, for example, treatment with 6.0× sodium chloride/sodium citrate (SSC) at approximately 45°C, followed by washing with 2.0× SSC at 50°C, will be apparent to those skilled in the art. is publicly known. For example, the salt concentration in the wash step can be selected from low stringency conditions of about 2.0×SSC, 50°C to highly stringent conditions of about 0.2×SSC, 50°C. In addition, the temperature conditions in the washing step can be increased from about 22°C at room temperature for low stringency conditions to about 65°C for highly stringent conditions. Temperature conditions and salt concentration can both change, or one variable can remain constant while the other variable changes. Preferably, a nucleic acid molecule of the invention can be combined with SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: under moderately stringent conditions, such as at about 2.0×SSC and about 65°C. 4. Specific hybridization occurs between one or more nucleic acid molecules in SEQ ID NO:4, SEQ ID NO:6 and SEQ ID NO:7 or their complementary sequences, or any fragment of the above sequences. More preferably, a nucleic acid molecule of the present invention is in contact with one or more nucleic acid molecules of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4 or their complementary sequences under highly stringent conditions. , or any fragment of the above sequence specifically hybridizes. In the present invention, the preferred marker nucleic acid molecule has SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4 or its complementary sequence, or any fragment of the above sequence.

Another preferred marker nucleic acid molecule of the present invention has 80% to 100% or 90% to 100% sequence identity. SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:4 can be used as markers in plant breeding methods to identify the progeny of genetic crosses. Hybridization between the probe and the target DNA molecule can be detected by any method known to those skilled in the art, including but not limited to fluorescent labels, radioactive labels, antibody labels, and chemiluminescent labels.

With respect to the amplification of a target nucleic acid molecule using specific amplification primers (e.g., by PCR), "stringent conditions" refer to conditions that only allow hybridization of the primer to the target nucleic acid molecule in a thermal DNA amplification reaction, with the The primer of the corresponding sequence of the target nucleic acid molecule (or its complementary sequence) can bind to the target nucleic acid molecule, and preferably generates a unique amplification product, which is an amplicon.

The term "specific binding (target sequence)" means that under stringent hybridization conditions the probe or primer hybridizes only to the target molecule in a sample containing the target molecule.

As used herein, "amplified DNA" or "amplicon" refers to the nucleic acid amplification product of a target nucleic acid molecule that is part of a nucleic acid template. For example, to determine whether a corn plant was produced by sexual crossing containing the transgenic corn event ND4401 of the present invention, or whether a corn sample collected from a field contains the transgenic corn event ND4401, or whether a corn extract, such as meal, meal or oil contains Genetically modified corn event ND4401, DNA extracted from corn plant tissue samples or extracts can be used by nucleic acid amplification methods using primer pairs to produce amplicons that are diagnostic for the presence of genetically modified corn event ND4401 DNA. The primer pair includes a first primer derived from a flanking sequence adjacent to the insertion site of the inserted exogenous DNA in the plant genome, and a second primer derived from the inserted exogenous DNA. The amplicons are of a length and sequence that are also diagnostic for the transgenic maize event ND4401.

The length of the amplicon may range from the combined length of the primer pair plus one nucleotide base pair, preferably plus about fifty nucleotide base pairs, more preferably plus about two hundred fifty nucleotides base pairs, most preferably plus about four hundred and fifty nucleotide base pairs or more.

Alternatively, primer pairs can be derived from flanking genomic sequences on both sides of the inserted DNA to generate amplicons that include the entire inserted nucleotide sequence. One of the primer pairs derived from the plant genomic sequence can be located at a distance from the inserted DNA sequence, which distance can range from one nucleotide base pair to about twenty thousand nucleotide base pairs. The use of the term "amplicon" specifically excludes primer dimers formed in thermal amplification reactions of DNA.

Nucleic acid amplification reaction can be achieved by any nucleic acid amplification reaction method known in the art, including polymerase chain reaction (PCR). Various nucleic acid amplification methods are well known to those skilled in the art. PCR amplification methods have been developed to amplify 22 kb of genomic DNA and 42 kb of phage DNA. These methods, as well as other DNA amplification methods known in the art, can be used in the present invention. The inserted foreign DNA sequence and the flanking DNA sequence from the transgenic maize event ND4401 can be amplified by using the provided primer sequences to amplify the genome of the transgenic maize event ND4401. After amplification, the PCR amplicon or cloned DNA is subjected to standard DNA sequencing.

DNA detection kits based on DNA amplification methods contain DNA primer molecules that specifically hybridize to target DNA under appropriate reaction conditions and amplify diagnostic amplicons. Kits may provide agarose gel-based detection methods or any of the many methods known in the art for detecting diagnostic amplicons. A kit containing DNA primers that are homologous or complementary to any part of the maize genomic region of SEQ ID NO: 1 or SEQ ID NO: 2 and to any part of the transgene insert region of SEQ ID NO: 4 is provided by the present invention. A primer pair specifically identified as useful in DNA amplification methods is SEQ ID NO: 5 and SEQ ID NO: 6, which amplify diagnostic amplification of homology to a portion of the 5' transgene/genomic region of transgenic maize event ND4401 amplicon, wherein the amplicon includes SEQ ID NO:3.

Amplicons generated by these methods can be detected by a variety of techniques. One such method is GeneticBit Analysis, which designs a DNA oligonucleotide strand that spans both the inserted DNA sequence and adjacent flanking genomic DNA sequences. The oligonucleotide chain is fixed in the microwell of a microplate, and after PCR amplification of the target region (using one primer each within the insert sequence and the adjacent flanking genomic sequence), the single-stranded PCR product Hybridizes to an immobilized oligonucleotide strand and serves as a template for a single-base extension reaction using DNA polymerase and ddNTPs specifically labeled for the next expected base. Results can be obtained by fluorescence or ELISA-type methods. The signal represents the presence of insert/flanking sequences, indicating that the amplification, hybridization, and single-base extension reactions were successful.

Another method is Pyrosequencing (pyrosequencing) technology. This method designs an oligonucleotide strand that spans the inserted DNA sequence and the adjacent genomic DNA binding site. The oligonucleotide strand is hybridized to a single-stranded PCR product of the target region (using one primer each within the insert sequence and in the adjacent flanking genomic sequence), and then mixed with DNA polymerase, ATP, sulfonylase, and fluorescein. enzyme, apyrase, adenosine-5'-phosphosulfate and luciferin were incubated together. Add dNTPs respectively and measure the resulting optical signal. The optical signal represents the presence of insert/flanking sequences, indicating that amplification, hybridization, and single- or multi-base extension reactions were successful.

Fluorescence polarization phenomenon is also a method that can be used to detect the amplicon of the present invention (Chen X, Levine L, and Kwok P Y. Fluorescence polarization in homogeneous nucleic acid analysis [J]. Genome Res, 1999, 9 (5): 492-8.). Using this approach requires designing an oligonucleotide strand that spans the inserted DNA sequence and the adjacent genomic DNA binding site. The oligonucleotide strand is hybridized to a single-stranded PCR product of the target region (using one primer within the insert and one primer in the adjacent flanking genomic sequence) and then combined with DNA polymerase and a fluorescently labeled ddNTP Incubate. Single base extension results in insertion of ddNTPs. This insertion allows a change in polarization to be measured using a fluorometer. Changes in polarization represent the presence of insert/flanking sequences, indicating that amplification, hybridization, and single-base extension reactions were successful.

Taqman is described as a method for detecting and quantifying the presence of DNA sequences and is detailed in the instructions for use provided by the manufacturer. A brief example is given below to design a FRET oligonucleotide probe that spans the inserted DNA sequence and adjacent genomic flanking binding sites. The FRET probe and PCR primers (one primer each within the insert sequence and adjacent flanking genomic sequences) are cycled in the presence of a thermostable polymerase and dNTPs. Hybridization of the FRET probe results in the splitting of the fluorescent and quenching moieties on the FRET probe and the release of the fluorescent moiety. The generation of a fluorescent signal represents the presence of insert/flanking sequences, indicating that amplification and hybridization were successful.

Based on the principle of hybridization, suitable techniques for detecting plant material derived from transgenic maize event ND4401 may also include Southern blot hybridization, Northern blot hybridization and in situ hybridization. In particular, such suitable techniques include incubating the probe and sample, washing to remove unbound probe and detecting whether the probe has hybridized. The detection method depends on the type of label attached to the probe. For example, radioactively labeled probes can be detected by exposure and development of X-ray films, or enzyme-labeled probes can be detected by achieving color changes through substrate conversion.

Molecular markers can also be used to detect sequences (Tyagi S and Kramer F R. Molecular beacons: probes that fluoresce upon hybridization [J]. Nat Biotechnol, 1996, 14(3): 303-8.). Design a FRET oligonucleotide probe that spans the inserted DNA sequence and adjacent genomic flanking binding sites. The unique structure of this FRET probe results in it containing a secondary structure that is able to maintain fluorescent and quenching moieties in close proximity. The FRET probe and PCR primers (one primer each within the insert sequence and adjacent flanking genomic sequences) are cycled in the presence of a thermostable polymerase and dNTPs. After successful PCR amplification, the hybridization of the FRET probe and the target sequence results in the loss of the secondary structure of the probe, resulting in the spatial separation of the fluorescent part and the quenching part, generating a fluorescent signal. The generation of a fluorescent signal represents the presence of insert/flanking sequences, indicating that amplification and hybridization were successful.

Other methods described, such as microfluidics, provide methods and devices for isolating and amplifying DNA samples. Photodyes are used to detect and measure specific DNA molecules. Nanotube devices containing electronic sensors for detecting DNA molecules or nanobeads that bind specific DNA molecules and thus can be detected are useful for detecting the DNA molecules of the present invention.

DNA detection kits can be developed using the compositions of the present invention and methods described or known in the field of DNA detection. The kit is useful for identifying whether DNA of transgenic maize event ND4401 exists in a sample, and can also be used to cultivate corn plants containing DNA of transgenic maize event ND4401. The kit may contain DNA primers or probes that are homologous to or complementary to at least a portion of SEQ ID NO: 1, 2, 3 or 4, or other DNA primers or probes that are homologous to or complementary to DNA contained in transgenic genetic elements of DNA. These DNA sequences can be used in DNA amplification reactions or as probes in DNA hybridization methods. The DNA structure of the site where the transgene insert sequence is combined with the maize genome contained in the maize genome and illustrated in Table 1 includes: the left flanking genomic region of maize ND4401 located at the 5' end of the transgene insert sequence, the left border region from the vector Part of the inserted sequence, the first expression cassette consists of the 35S promoter of cauliflower mosaic virus (CaMV 35S promoter), operably linked to the glufosinate (glufosinate ammonium) resistance gene sequence (bar), and operably It is composed of the 35S terminator of cauliflower mosaic virus (CaMV 35S polyA); the second expression cassette consists of the maize ubiquitin gene promoter (ubiquitin promoter), operably linked to the maize nitrate transporter gene ZmNAR1.1A , and is operably linked to the nopaline synthase gene terminator (noterminator), consisting of a portion of the insert sequence from the right border region of the vector, and the right flank genomic region of the corn plant ND4401 located at the 3' end of the transgene insert sequence (SEQ ID NO:4). In the DNA amplification method, the DNA molecules used as primers can be any part of the transgene insertion sequence derived from the transgenic maize event ND4401, or any part of the DNA region derived from the flanking maize genome in the transgenic maize event ND4401.

The genetically modified corn event ND4401 can be combined with other genetically modified corn varieties, such as corns that are tolerant to herbicides (such as glyphosate, dicamba, etc.), or genetically modified corn varieties that carry insect-resistant genes. Various combinations of all these different transgenic events, bred together with the transgenic corn event ND4401 of the present invention, can provide improved hybrid transgenic corn varieties with a variety of traits. These varieties can show superior characteristics such as increased yield compared to non-GM varieties and single-trait GM varieties.

The invention provides a nucleic acid molecule for detecting corn plants and a detection method thereof. The transgenic corn event ND4401 has high yield and/or nitrogen deficiency tolerance traits. The corn plants with this trait express the nitrate transporter ZmNAR1.1A protein, which imparts efficient nitrogen utilization to the plant, thereby increasing yield and/or nitrogen deficiency tolerance (the herbicide resistance gene bar is used as a screening marker in the transformation stage). At the same time, in the detection method of the present invention, the nucleic acid molecules of SEQ ID NO: 1 or its complementary sequence, SEQ ID NO: 2 or its complementary sequence, SEQ ID NO: 3 or its complementary sequence, SEQ ID NO: 4 or its complementary sequence can be used as DNA. The primers or probes are used to produce amplification products diagnosed as transgenic maize event ND4401 or its progeny, and the presence of plant materials derived from transgenic maize event ND4401 can be quickly, accurately and stably identified.

The technical solution of the present invention will be further described in detail below through the accompanying drawings and examples.

Description of the drawings

Figure 1 Physical map of the recombinant expression vector pCAMBIA1301-ZmNRT1.1A. The English and abbreviation meanings of each component are listed below:

T-Border(left) T-DNA left border sequence.

CaMV 35S polyA 35S terminator of Cauliflower Mosaic Virus (CaMV).

bar encodes PAT protein, which relieves glufosinate-ammonium toxicity.

CaMV 35S promoter 35S promoter of cauliflower mosaic virus (CaMV).

nos terminator The terminator of the nopaline synthase gene.

ZmNAR1.1A AnVP1 gene CDS.

ubiquitin promoter promoter of the maize ubiquitin gene.

nos terminator The terminator of the nopaline synthase gene.

T-Border(right) T-DNA right border sequence.

PVS1 sta Plasmid stabilizing site for pVS1 plasmid.

PVS1 rep Replication origin site of pVS1 plasmid.

PBR322 bom bom site of pBR322 plasmid.

PBR322 ori Replication origin site of pBR322 plasmid.

kanamycin(R) encodes an aminoglycoside phosphotransferase protein that confers kanamycin resistance to bacteria.

Figure 2 Transformation event-specific PCR verification results. M: DL2000 DNA marker; T: ND4401 corn material; CK: non-GMO corn. The expected size of the PCR fragment is 1069bp.

Detailed ways

The transformation event ND4401 involved in this application refers to a corn plant in which a foreign gene insert (T-DNA insert) is inserted between specific genome sequences after genetic transformation using the corn inbred line Y822 as the recipient. In a specific embodiment, the expression vector used for transgene has the physical map shown in Figure 1, and the resulting T-DNA insert has the sequence shown in nucleotides 695-4913 of SEQ ID NO: 4. Transformation event ND4401 may refer to this transgenic process, or may refer to the combination of a T-DNA insert and flanking sequences in the genome resulting from this process, or may refer to the maize plant resulting from 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 position. Transformation event ND4401 may also refer to progeny plants obtained from the above-mentioned plants through asexual reproduction, sexual reproduction, redundant or doubled propagation, or a combination thereof.

Example 1 Obtaining and screening of transformation events

The cDNA sequence of the ZmNRT1.1A gene was cloned from maize and inserted into the intermediate vector pCAMBIA1301-Ubi through enzyme digestion. The intermediate vector already contains the bar gene and was finally constructed into the pCAMBIA1301-ZmNRT1.1A expression vector. The physical map of the vector is shown in Figure 1 . Agrobacterium infection technology was used to efficiently transform the recombinant plasmid into young embryos of maize y822, a self-selected line of the Institute of Agricultural Biotechnology, Jilin Academy of Agricultural Sciences, and 45 transformation events of maize transfected with ZmNRT1.1A and bar genes were obtained. Among them, the transgenic corn event ND4401 contains exogenous genes ZmNRT1.1A and bar, which shows stable generation heritability and outstanding yield traits and nitrogen deficiency tolerance, and has good application prospects.

The detection method of exogenous ZmNRT1.1A and bar genes is as follows: using corn genomic DNA as a template, using the specific primer pairs of ZmNRT1.1A and bar for PCR amplification, and obtaining 452bp size (ZmNRT1.1A gene) and 441bp respectively. The materials for the large and small (bar gene) bands are plants containing ZmNRT1.1A gene and bar gene.

Table 2 Primer sequences for PCR detection of ZmNRT1.1A gene and bar gene

Example 2 Efficient nitrogen utilization and identification of yield traits of transformation event ND4401

Corn ZmNRT1.1A protein has the function of promoting nitrate absorption. The present invention uses a constitutive expression method to express the ZmNRT1.1A protein in corn, thereby improving the corn's nitrate absorption capacity, thereby improving the nitrogen utilization efficiency of the corn, and ultimately increasing the yield. The present invention identifies the field growth morphology and physiological traits of ND4401 plants under different nitrogen fertilizer gradients.

The test location is the genetically modified test base of the Jilin Academy of Agricultural Sciences in Gongzhuling City, Siping City, Jilin Province. The test soil is sandy loam with flat terrain. The soil alkali-hydrolyzable nitrogen content in the cultivated layer (20cm) is 125.94mg/kg, the available phosphorus content is 23.96mg/kg, the available potassium content is 149.24mg/kg, and the organic matter content is 25.22g/kg. The row length of the plot is 5m, the row spacing is 0.60m, the plant spacing is 0.25m, and each plot has 6 rows.

Test conditions: Referring to the "Regional Formula and Fertilization Recommendations for the Three Major Food Crops of Wheat, Corn, and Rice (2013)" issued by the Ministry of Agriculture and Rural Affairs, the high nitrogen treatment (HN) applied 38kg/acre of formula fertilizer according to the amount of fertilizer used in normal farming; Medium nitrogen treatment (MN): apply 70% formula fertilizer, supplement phosphorus fertilizer and potash fertilizer and the same as the high nitrogen treatment; low nitrogen treatment (LN) do not apply formula fertilizer, supplement phosphorus fertilizer and potash fertilizer and the same as the high nitrogen treatment.

Experimental design: The experiment adopts a random block design. 10 corn plants with roughly the same growth status are selected from each plot to measure relevant morphological and physiological indicators. Specific indicators include: ear weight, ear length, ear row, 100-kernel weight, and total nitrogen above the ground of the plant. content.

The results showed that under various nitrogen supply conditions, the ear weight, ear length, and 100-grain weight of corn ND4401-positive plants were significantly higher than those of negative plants, and the yield increase effect was significant. Specifically, under low nitrogen conditions, ear weight increased by 22.8%, ear length increased by 23.3%, and 100-grain weight increased by 15.4%. Under medium nitrogen conditions, ear weight increased by 36.9%, ear length increased by 26.3%, ear row number increased by about 1 row, and 100-grain weight increased by 8.5%. Under high nitrogen conditions, ear weight increased by 37.1%, ear length increased by 24.9%, and 100-grain weight increased by 31.4% (see Table 3 for details). The yield of ND4401-negative plants under low-nitrogen (nitrogen deficiency) conditions is only 76% (132.62/173.92) of that under high-nitrogen (nitrogen-sufficient) conditions, while the yield of ND4401-positive plants under low-nitrogen (nitrogen deficiency) conditions reaches the level of high-nitrogen 85% (202.70/238.48) under (nitrogen sufficient) conditions, which indicates that ND4401 positive plants have better tolerance to nitrogen deficiency conditions.

Table 3 Comparison of agronomic traits identification between ND4401 and control

From the results of the total nitrogen content of plant stems under different nitrogen conditions, the total nitrogen content of ND4401 plants under different nitrogen conditions was lower than that of the recipient control material. Especially under high nitrogen conditions, the total nitrogen content of the control stems was 3.00g/kg, while the total nitrogen content of ND4401 was only 2.17g/kg (Table 4). This indicates that the nitrogen utilization efficiency of ND4401 is higher than that of the wild-type control, which is also the reason for the higher yield of ND4401.

Table 4 Determination of total nitrogen content in plant stems of ND4401 and recipient control

Example 3 Transformation event ND4401 flanking sequence of exogenous sequence and maize genome insertion position

In order to clarify the identity of the transformation event ND4401, the present invention further identified the insertion site of the ND4401 exogenous sequence on the maize genome.

The present invention uses the third generation sequencing method to locate the T-DNA insertion position of transgenic maize ND4401. The third generation sequencing technology uses the nanopore sequencing technology of Oxford Nanopore Technologies (ONT) in the UK to resequence the transformant ND4401. The sequencing depth is 10 times, the average read length of the data is 20kb, and the longest read length is 60kb. The accuracy is 85%.

Using T-DNA exogenous sequences to conduct BLAST search and comparison of the third-generation sequencing results, it was found that a contg with a read length of approximately 14 kb contained the target sequence, indicating that the exogenous sequence in ND4401 was a single copy insertion. The 5'-end 625bp and 3'-end 132bp genomic flanking sequences of the exogenous sequences obtained by sequencing were subjected to BLAST nucleic acid- Nucleic acid comparison showed that the foreign sequence was integrated at positions 38327236-38327270 on chromosome 5 of maize.

At the left border of the insertion site, select 625 bp on the genome and the exogenous insertion sequence to design a primer pair, so that the F primer can combine with the genome sequence, the R primer can combine with the exogenous insertion sequence, and the amplification product fuses a part of the maize genome sequence and a part of the foreign insertion sequence. Source insertion sequence. Use the designed primers to perform PCR detection on ND4401. PCR amplification was performed using the genomic DNA of transgenic corn lines as template. The PCR reaction was performed in a 20 μL system. The amplification cycle program is: pre-denaturation at 94°C for 3 min; denaturation at 94°C for 30 s, T _m annealing for 30 s, extension at 72°C for a certain time (set according to the size of the product fragment), 35 cycles; extension at 72°C for 5 min.

Based on the results of flanking sequences and insertion positions, PCR amplification of the ND4401 transformation event was performed using the genome upstream primer (SEQ ID NO: 5) and the vector left border primer (SEQ ID NO: 6) to verify the insertion position of the foreign fragment. The results are shown in Figure 2. The results showed that the ND4401 exogenous fragment was stably inserted into chromosome 5 at positions 38327236-38327270.

Example 4 Detection method of transformation event ND4401

Genetically modified corn event ND4401 can be used for breeding, and the new varieties can be used to produce agricultural products or commodities. If detected in sufficient amounts in the agricultural product or commodity, the agricultural product or commodity is expected to contain a nucleotide sequence capable of diagnosing the presence of transgenic corn event ND4401 material in the agricultural product or commodity. Such agricultural products or commodities include, but are not limited to, corn oil, corn meal, cornmeal, corn gluten, corn tortillas, cornstarch, and any other food product that is to be used as a food source for animal consumption, or otherwise as a bulking agent or in cosmetic compositions The ingredients are used for cosmetic purposes etc. Nucleic acid detection methods and/or kits based on probes or primer pairs can be developed to detect whether biological samples contain nucleic acid analysis of genetically modified maize event ND4401, wherein the probe sequence or primer amplification sequence is selected from the group consisting of SEQ ID NO: 1, The sequence shown in SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:4 to diagnose the presence of transgenic maize event ND4401.

One of the detection methods is:

The common CTAB method was used to extract genomic DNA from corn samples. The specific operation process was implemented in accordance with the Ministry of Agriculture Announcement No. 1485-4-2010, DNA extraction and purification for detection of genetically modified plants and product components. Wherein, the corn samples include the corn to be transgenic ND4401 and the non-transgenic corn y822. The PCR method was used to detect the specific border sequences in ND4401 plants. The PCR primer pairs used were SEQ ID NO:5 and SEQ ID NO:6 respectively. The PCR reaction system was:

The reaction procedure is:

Steps 2 to 4: Loop 35 times

The PCR product was electrophoresed in 1% (w/v) 1×TAE agarose gel and the results are shown in Figure 2. The expected target band (SEQ ID NO: 3) can be amplified in the ND4401 transformation event. Moreover, this PCR method can track the presence of transformation events and thus be applied to breeding efforts.

In summary, the transgenic corn event ND4401 of the present invention has higher yield and/or tolerance to nitrogen deficiency, and the detection method can accurately and quickly identify whether the biological sample contains DNA molecules of the transgenic corn event ND4401.

Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not limiting. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art will understand that the technical solutions of the present invention can be modified. The solution may be modified or equivalently substituted without departing from the spirit and scope of the technical solution of the present invention.

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gcctcggtgg cacggcggat gtcggccggg cgtcgttctg ggctcatggt agactcgaga 1440

gagatagatt tgtagagaga gactggtgat ttcagcgtgt cctctccaaa tgaaatgaac 1500

ttccttatat agaggaaggt cttgcgaagg atagtggggat tgtgcgtcat cccttacgtc 1560

agtggagata tcacatcaat ccacttgctt tgaagacgtg gttggaacgt cttctttttc 1620

cacgatgctc ctcgtgggtg ggggtccatc tttgggacca ctgtcggcag aggcatcttg 1680

aacgatagcc tttcctttat cgcaatgatg gcatttgtag gtgccacctt ccttttctac 1740

tgtccttttg atgaagtgac agatagctgg gcaatggaat ccgaggaggt ttcccgatat 1800

taccctttgt tgaaaagtct caatagccct ttggtcttct gagactgtat ctttgatatt 1860

cttggagtag acgagagtgt cgtgctccac catgttggca agctgctcta gccaatacgc 1920

aaaccgcctc tccccgcgcg ttggccgatt cattaatgca gctggcacga caggtttccc 1980

gactggaaag cgggcagtga gcgcaacgca attaatgtga gttagctcac tcattaggca 2040

ccccaggctt tacactttat gcttccggct cgtatgttgt gtggaattgt gagcggataa 2100

caatttcaca caggaaacag ctatgacatg attacgaatt ctcatgtttg acagctttc 2160

atcggatcta gtaacataga tgacaccgcg cgcgataatt tatcctagtt tgcgcgctat 2220

attttgtttt ctatcgcgta ttaaatgtat aattgcggga ctctaatcat aaaaacccat 2280

ctcataaata acgtcatgca ttacatgtta attattacat gcttaacgta attcaacaga 2340

aattatatga taatcatcgc aagaccggca acaggattca atcttaagaa actttattgc 2400

caaatgtttg aacgatcgat ccactagtca atcactagtg aattcgagct cgagctcggt 2460

acccggggat cctcagtgga gcgtgggctc gtcggccatc tcgacgccgt tgacgctgcc 2520

gtcggcgccg gggcggcccg ccttgtactt gtaccagcgg gcggcgacga ggtagactag 2580

taggttggcg aggcagacgg cggcgagcag ccagtagaag ttgtcgaggc ggcccttgtt 2640

gaggtcgttg gcgatccagg ggtggcggtc gcccgtgacc ctgtgcacgg cggcgacgag 2700

cgcggagctg acgaagaatc ccagcgacag ggtgctgagg aacagccccg tgctcatggt 2760

cttcatcccc ttggggcact cgcgcaggaa gaagtcgagc tggccgatgt acgtgaacgc 2820

ctcgcccgcc cccacgagga agaactgcgg gatgagccag aacacggaca tgggcacgac 2880

gcctccggag gcggactcgg aggaatcgcg cgcgacgcgg aggcggcgga cctccgtgag 2940

cgcggcgccc gccatggcga cgacggagag cgcgaggccg acggcgatcc gctgcagcgg 3000

ggtgaggccg tgcgggttgc cgctgacgcg gcgcgcgacg ggcaccacca ggcggtcgta 3060

gacgggcacg gtgagcagga tggagccgac gaagaagacg gtgagggagc ccgcggggat 3120

ctggaacgag cccccgacgc ggcggtccat ggtggtggcc tgcgacaccg agaaggtggt 3180

catctgcgcg tacaccgtcc agaacatgat cgtggtcgcc cagatcggca gcatccgcgc 3240

caccgtcttc acctcctcca cgtccgtcag cgtcgccagc cgccacttgc tgctgctgct 3300

cgcccccgcc gccggatcct cgttgatcgc cgcgtggtcc aggaagcgga actggtcggt 3360

gtgggggagg cgctccttgc gcttgctctt cttgctggag gacccatcct cgacggcggc 3420

ggccttgccg acgtcgacgt cgtagagcat ggcggggtcg gcggggagag ggaggcggcg 3480

cttgcgccag gcggcgacga cgacggcggc gatctgcgtg aggggggctgc cggccagctt 3540

cttgaagcgg tacctgcgtg tgccggccag gaagacgagg aggcccgcgg cgatggcgca 3600

ggcgcaggcg ccgtagcccc agcgcctgcc caggttgtcc tggacgtaca ccagcacggt 3660

gacggccagc agcgacccca gcgagatgaa gaagaagaac cagttgaaga agcgcatcat 3720

ctgccgcttc tccccgccgt ccgactcgtc gaactggtcc gacccgaacc ccgacacgct 3780

cgactttagc ccacccgtgc ccagcgccgt caggtacagc gccaggtaca gcacccccag 3840

ctgcgccccc gacgcccgcg cgcactcccc gacgacgcct ccgccggtcg cggagcagga 3900

cgccggccgt agccccggcg ccgccgtcga gatcgtcagg atcgtcacgc ccgaggcctg 3960

gacggcggtg aagatggcga tggtgaggta gcggccgagg aaggagtcgg cgacgaagcc 4020

gccgaggagg cagagcatga aggaggtgcc catgaagttg gtgacgacgt tggcggactc 4080

ggcgttgccc aggtgcatgg tgcccgtcag gtacgtcacc aggttcacgg cgatgcccag 4140

cgtcgtcagc cgctcgttca gctccgccac taggatcatg gcggcggcgc cccagcggcc 4200

ggtggtggcg cgcggggccg gccggccctt gaagtcccag gcgtcgagga ggacgtccgt 4260

ctccgccgcg gcattggtct cggggaggag tccgaccatg agctcggtac ccggggatcc 4320

tctagagtcg acctgcagaa gtaacaccaa acaacagggt gagcatcgac aaaagaaaca 4380

gtaccaagca aataaatagc gtatgaaggc agggctaaaa aaatccacat atagctgctg 4440

catatgccat catccaagta tatcaagatc aaaataatta taaaacatac ttgtttatta 4500

taatagatag gtactcaagg ttagagcata tgaatagatg ctgcatatgc catcatgtat 4560

atgcatcagt aaaacccaca tcaacatgta tacctatcct agatcgatat ttccatccat 4620

cttaaactcg taactatgaa gatgtatgac acacacatac agttccaaaa ttaataaata 4680

caccaggtag tttgaaacag tattctactc cgatctagaa cgaatgaacg accgcccaac 4740

cacaccacat catcacaacc aagcgaacaa aaagcatctc tgtatatgca tcagtaaaac 4800

ccgcatcaac atgtatacct atcctagatc gatatttcca tccatcatct tcaattcgta 4860

actatgaata tgtatggcac acacatacag atccaaaatt aataaatcca ccacccctcc 4920

cccctggtgc aggataatac aaggcttgtc ccacctcttg tgttccatcc cgcgccaacc 4980

catctgggca ggggcacgta gcgacaatct cactcgtcgg tccagggacc cccgggtccg 5040

aaacgccgac aa 5052

<210> 5

<211> 18

<212> DNA

<213> Zea mays

<400> 5

tctcagcccc ttacggca 18

<210> 6

<211> 18

<212> DNA

<213> Artificial synthesis (unknown)

<400> 6

gcaccatcgt caaccact 18

Claims (10)

1. A nucleic acid molecule, characterized in that the sequence comprises any one of the following:

i) Comprises the sequence shown in SEQ ID NO. 1 and/or SEQ ID NO. 2;

ii) comprises the sequence shown in SEQ ID NO. 3;

ii) comprises the sequence shown in SEQ ID No. 4.

2. A probe for detecting a maize transformation event comprising the sequence of SEQ ID NO. 1 or SEQ ID NO. 2 or SEQ ID NO. 3 or SEQ ID NO. 4.

3. A primer pair for detecting a maize transformation event, said primer pair comprising:

a primer specifically recognizing the 1 st to 625 th nucleotide sequence of the sequence shown in SEQ ID NO. 4 and a primer specifically recognizing the 626 th to 4920 th nucleotide sequence of the sequence shown in SEQ ID NO. 4; and/or

A primer specifically recognizing the 626 th to 4920 th nucleotide sequence of the sequence shown in SEQ ID NO. 4 and a primer specifically recognizing the 4921 th to 5052 th nucleotide sequence of the sequence shown in SEQ ID NO. 4.

4. A primer pair according to claim 3, wherein the amplification product of the primer pair comprises the sequence of claim 2.

5. The primer pair of claim 4, wherein the primer pair is a sequence shown in SEQ ID NO. 5 and SEQ ID NO. 6 or a complementary sequence thereof.

6. Kit or microarray for detecting maize transformation events, characterized in that it comprises a probe according to claim 2 and/or a primer pair according to any of claims 3-5.

7. A method for detecting a maize transformation event comprising detecting the presence or absence of said transformation event in a test sample using:

i) The probe of claim 2;

ii) the primer pair of any one of claims 3-5;

iii) The probe of claim 2 and the primer pair of any one of claims 3-5; or alternatively

iv) the kit or microarray of claim 6.

8. A method of breeding maize, the method comprising the steps of:

1) Obtaining corn comprising the nucleic acid molecule of claim 1;

2) Obtaining a maize plant, seed, plant cell, progeny plant or plant part from the maize obtained in step 1) by pollen culture, unfertilized embryo culture, doubling culture, cell culture, tissue culture, selfing or crossing or a combination thereof;

3) Identifying nitrogen deficiency tolerance in the progeny plant obtained in step 2) and detecting the presence or absence of said transformation event using the method of claim 7.

9. Use of a maize plant, seed, plant cell, progeny plant or plant part obtained by the method of claim 8 for the preparation of a product comprising a food, feed or industrial feedstock.

10. A method for increasing corn yield and/or nitrogen deficiency tolerance comprising planting at least one transgenic corn plant comprising SEQ ID NO 4 in the genome of said transgenic corn plant in soil; the transgenic maize plants have high yield and/or nitrogen deficiency tolerance traits.