CN112481412A - Nucleic acid molecule for detecting corn plant ND4403 and detection method thereof - Google Patents
Nucleic acid molecule for detecting corn plant ND4403 and detection method thereof Download PDFInfo
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Abstract
The invention relates to a nucleic acid molecule for detecting a corn plant ND4403 and a detection method thereof, wherein the nucleic acid molecule sequence of the corn plant comprises a sequence shown in SEQ ID NO. 1 or a complementary sequence thereof, or a sequence shown in SEQ ID NO. 2 or a complementary sequence thereof. The maize plant ND4403 of the present invention has high yield and/or nitrogen deficiency tolerance traits, and the detection method can accurately and rapidly identify whether a biological sample contains a DNA molecule of the transgenic maize event ND 4403.
Description
Technical Field
The invention relates to the technical field of plant biology. In particular to a nucleic acid molecule for detecting a corn plant ND4403 and a detection method thereof, in particular to a transgenic corn event ND4403 with high yield and/or nitrogen deficiency tolerance characters, a nucleic acid molecule for detecting whether a biological sample contains the specific transgenic corn event ND4403 and a detection method thereof.
Background
Nitrogen is one of the essential mineral nutrients of plants and an essential component for the synthesis of proteins, nucleic acids and many biologically active substances. Nitrogen deficiency usually leads to Plant growth retardation, green-yellow mature leaves with Accumulation of anthocyanins (Diaz C, Salibani V, Loudet O, et al, leaf Yellowing and Anthocynin Accumulation area Two genetic introduction Strategies in Response to Nitrogen Limitation in Arabidopsis thaliana [ J ]. Plant & Cell Physiology,2006,47(1): 74-83.). The large use of nitrogen fertilizer in agricultural production greatly improves crop yield. The current global annual application of about 8-9 million tons of nitrogen fertilizer is predicted to increase to 2.4 million tons in 2050 (Timman D. Global environmental impacts of agricultural expansion: the connected for stable and effective pathogens [ J ]. Proc Natl Acad Sci U S.1999, 96(11): 5995-. The increase of energy cost leads to the continuous increase of the price of the nitrogen fertilizer, thereby increasing the agricultural production cost, and simultaneously the loss of the nitrogen fertilizer gradually deteriorates the ecological environment. Based on double considerations of economic benefit and environmental protection, planting nitrogen-efficient crop varieties in modern agricultural production is an effective way for solving the problems of low utilization rate of nitrogen fertilizer, reduction of production cost and reduction of environmental pollution, and is also a basic requirement for agricultural sustainable development and enhancement of international competitiveness of products. The traditional breeding of new species is a very long-term process, and nowadays, the continuous maturation of genetic engineering makes it possible to rapidly obtain nitrogen-efficient species by means of genetic engineering. The maize ZmNRT1.1 gene encodes a nitrate transporter with low affinity, which can influence the absorption of nitrate nitrogen by the root system of a plant, thereby improving the utilization efficiency of nitrogen (Quaggiotti S, Ruperti B, Pizzeghello D, et al. Effect of low molecular size maize substructures on nitrate uptake and expression of genes involved in nitrate transport in mail (Zea Mays L.) [ J ]. Journal of Experimental Boy, 2004,55(398):816 and 823.), and can be used for improving the tolerance of maize under the condition of nitrogen deficiency and finally increasing the yield.
It is known that expression of foreign genes in plants is influenced by their chromosomal location, possibly 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 in order to be able to identify a commercializable event (i.e., an event in which the introduced gene of interest is optimally expressed). For example, it has been observed in plants and other organisms that the amount of expression of an introduced gene may 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, in that the actual expression pattern may not be consistent with the expression pattern expected from the transcriptional regulatory elements in the introduced gene construct, resulting in differences in the performance of the transformation event in the trait. Thus, it is often necessary to generate hundreds to thousands of different events and to screen those events for a single event with the amount and pattern of transgene expression expected for commercial purposes. Events with expected expression levels and patterns of transgenes can be used to introgress the transgenes into other genetic backgrounds by sexual outcrossing using conventional breeding methods. Progeny produced by this crossing pattern retain the transgene expression characteristics of the original transformation event. The use of this strategy ensures reliable gene expression in many varieties that are well adapted to local growth conditions. Therefore, there is a need for trait identification and screening of more transformation events to obtain superior transformation events with superior overall trait performance and commercial prospects.
It would be beneficial to be able to detect the presence of particular events to determine whether progeny of a sexual cross contain a gene of interest. Furthermore, methods of detecting specific events will also help to comply with relevant regulations, such as that foods derived from recombinant crops need to be officially approved and labeled before being placed on the market. It is possible to detect the presence of a transgene by any well-known polynucleotide detection method, 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, and the like. Thus, unless the sequence of chromosomal DNA adjacent to the inserted transgenic DNA ("flanking DNA") is known, this method described above cannot be used to distinguish between different events, particularly those produced with the same DNA construct. Therefore, it is now common to identify specific events of a transgene by PCR using a pair of primers spanning the junction of the inserted transgene and flanking DNA, specifically a first primer comprising the flanking sequence and a second primer comprising the inserted sequence.
Disclosure of Invention
The invention aims to provide a corn transformation event with excellent yield traits and/or nitrogen deficiency tolerance traits, a nucleic acid molecule for detecting a corn plant ND4403 and a detection method thereof. Transgenic corn event ND4403 exhibits high yield and/or nitrogen deficiency tolerance traits due to its high nitrogen use efficiency, and the detection method can accurately and rapidly identify whether a biological sample contains a DNA molecule of a particular transgenic corn event ND 4403.
In order to realize the purpose, 45 ZmNRTT 1.1A and bar gene-transferred maize transformation events are obtained by transforming the pCAMBIA 1301-ZmNRTT 1.1A expression vector into maize y822 immature embryos by utilizing an agrobacterium infection technology. Among them, ND4403 shows stable generational inheritance, nitrogen deficiency tolerance and outstanding yield traits, and has good application prospects.
In order to characterize the identity of ND4403, the invention provides a nucleic acid molecule comprising the sequence shown in SEQ ID NO. 1 and/or SEQ ID NO. 2, or the complement thereof.
Further, the nucleic acid molecule sequence comprises a sequence shown as SEQ ID NO. 3, or a complementary sequence thereof.
Further, the nucleic acid molecule sequence comprises a sequence shown as SEQ ID NO. 4, or a complementary sequence thereof.
Further, the nucleic acid molecule sequence comprises the sequence shown in SEQ ID NO. 5 or a complementary sequence thereof.
In another aspect, the invention provides a probe for detecting a maize transformation event, comprising the sequence shown in SEQ ID NO 1 or 2 or 3 or 4 or 5 or a fragment thereof or a variant or complement thereof.
The invention also provides a primer pair for detecting a corn transformation event, which is characterized by comprising:
a primer which specifically recognizes the nucleotide sequences 1 to 647 of the sequence shown by SEQ ID NO. 5 and a primer which specifically recognizes the nucleotide sequences 648-5737 of the sequence shown by SEQ ID NO. 5; and/or
A primer which specifically recognizes the nucleotide sequence at position 648-5737 of the sequence shown by SEQ ID NO. 5 and a primer which specifically recognizes the nucleotide sequence at position 5738-6093 of the sequence shown by SEQ ID NO. 5;
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 as SEQ ID NO 6 and SEQ ID NO 7 or the complement thereof; or the sequences shown in SEQ ID NO 8 and SEQ ID NO 9 or the complementary sequences thereof.
The invention also provides a kit or microarray for detecting a maize transformation event, characterized in that it comprises the probe described above and/or the primer pair described above.
The invention also provides a method for detecting corn transformation events, which is characterized by detecting whether the transformation events exist in a sample to be detected by using the probe or the primer pair or the probe and primer pair or the kit or microarray.
The invention also provides a method for breeding corn, which is characterized by comprising the following steps:
1) obtaining corn comprising the nucleic acid molecule;
2) subjecting the corn obtained in step 1) to pollen culture, unfertilized embryo culture, doubling culture, cell culture, tissue culture, selfing or crossing or a combination thereof to obtain a corn plant, seed, plant cell, progeny plant or plant part; and optionally also (c) a second set of one or more of,
3) subjecting the progeny plants obtained in step 2) to yield trait identification and/or nitrogen deficiency tolerance identification and testing for the presence of said transformation event therein using the methods described above.
Further, the invention also provides products made of the corn plants, seeds, plant cells, progeny plants or plant parts obtained by the method, including food, feed or industrial raw materials.
In the present invention, the nucleic acid molecule sequence may be at least 11 or more contiguous polynucleotides of any portion of the transgene insert sequence in said SEQ ID NO:1 or the complement thereof (first nucleic acid molecule) or at least 11 or more contiguous polynucleotides of any portion of the 5' left flank corn genomic DNA region in said SEQ ID NO:1 or the complement thereof (second nucleic acid molecule). The nucleic acid molecule may further be homologous or complementary to a portion of said SEQ ID NO. 3 comprising the entire said SEQ ID NO. 5. When the first nucleic acid molecule and the second nucleic acid molecule are used together, these nucleic acid molecules comprise a DNA primer pair in a DNA amplification method that produces an amplification product. The presence of transgenic maize event ND4403 or its progeny can be diagnosed when the amplification product produced in the DNA amplification method using the DNA primer pair is an amplification product comprising SEQ ID NO 1 or SEQ ID NO 3 or SEQ ID NO 5.
The SEQ ID NO:1 or its complement is a 971 nucleotide long sequence located near the insertion junction at the 5' end of the insertion in transgenic maize event ND4403, the SEQ ID NO:1 or its complement consists of a 647 nucleotide maize left flank genomic DNA sequence (nucleotides 1-647 of SEQ ID NO: 1), a 125 nucleotide pCAMBIA 1301-ZmNRTT 1.1A construct left border DNA sequence (nucleotides 648-772 of SEQ ID NO: 1) and a 199 nucleotide first expression cassette DNA sequence of the glufosinate-resistant gene (nucleotides 773-971 of SEQ ID NO: 1), comprising the SEQ ID NO:1 or its complement is identifiable as the presence of transgenic maize event ND 4403.
The nucleic acid molecule sequence may be at least 11 or more contiguous polynucleotides of any portion of the transgene insert sequence in SEQ ID No. 2 or its complement (third nucleic acid molecule) or at least 11 or more contiguous polynucleotides of any portion of the 3' right flanking corn genomic DNA region in SEQ ID No. 2 or its complement (fourth nucleic acid molecule). When the third nucleic acid molecule and the fourth nucleic acid molecule are used together, these nucleic acid molecules comprise a set of DNA primers in a DNA amplification method that produces an amplification product. The presence of transgenic maize event ND4403 or its progeny can be diagnosed when the amplification product produced in the DNA amplification method using the DNA primer pair is an amplification product comprising SEQ ID NO 2 or SEQ ID NO 4 or SEQ ID NO 5.
The SEQ ID NO:2 or a complementary sequence thereof is a 1031 nucleotide sequence located near the insertion junction at the 3 'end of the insertion sequence in the transgenic maize event ND4403, the SEQ ID NO:2 or the complementary sequence thereof is composed of a 3' end DNA sequence of the second expression cassette of the 377-nucleotide nitrogen efficient utilization gene (nucleotides 1-377 of the SEQ ID NO: 2), a pCAMBIA 1301-ZmNRTT 1.1A construct right border DNA sequence of 298 nucleotides (nucleotides 378-675 of the SEQ ID NO: 2) and a maize integration site right flank genomic DNA sequence of 356 nucleotides (nucleotides 676-1031 of the SEQ ID NO: 2), and the inclusion of the SEQ ID NO:2 or the complementary sequence thereof is identified as the presence of the transgenic maize event 440ND 3.
5 or its complement is a 6093 nucleotide long sequence characterizing transgenic maize event ND4403, specifically comprising genomic and genetic elements as shown in table 1. Inclusion of said SEQ ID No. 5 or the complement thereof identifies the presence of transgenic maize event ND 4403.
TABLE 1 genomic and genetic elements encompassed by SEQ ID NO 5
1: the 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 of only DNA, but may also include RNA, a mixture of DNA and RNA, or a combination of DNA, RNA, or other nucleotides or analogs thereof that do not serve as templates for one or more polymerases. In addition, the probe or primer of the invention should be at least about 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22 contiguous nucleotides in length, which may be selected from the nucleotides set forth in SEQ ID NO 1, SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4 or SEQ ID NO 5. When selected from the nucleotides set forth in SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, or SEQ ID NO. 5, the probes and primers may be contiguous nucleotides of at least about 21 to about 50 or more in length.
The present invention also provides a method for increasing maize yield and/or nitrogen deficiency tolerance, comprising growing in soil at least one transgenic maize plant comprising in its genome the nucleic acid sequence of SEQ ID NO. 5, position 2243 6093 or comprising in its genome the nucleic acid sequence of SEQ ID NO. 5; the transgenic maize plants have high yield and/or nitrogen deficiency tolerance traits.
The invention also provides a method for improving the yield and/or the nitrogen deficiency tolerance of corn, which is characterized in that an expression cassette for expressing a nitrogen high-efficiency utilization gene with a sequence shown as the 2243-5439 nucleotide sequence of SEQ ID NO. 5 is introduced into the genome of the corn
In the nucleic acid molecules and methods for detecting maize plants of the present invention, the following definitions and methods may better define the invention and guide those of ordinary skill in the art in the practice of the invention, unless otherwise indicated, the terms are understood according to their conventional usage by those of ordinary skill in the art.
The "corn" refers to maize (Zea mays) and includes all plant species that can be mated with corn, including wild corn species.
The term "comprising" 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 calli, plant clumps (plant granules), and plant cells intact in plants or plant parts such as embryos, pollen, ovules, seeds, leaves, flowers, branches, fruits, stalks, roots, root tips, anthers, and the like. It is to be understood that parts of transgenic plants within the scope of the present invention, which are derived from transgenic plants or progeny thereof which have been previously transformed with a DNA molecule of the invention and thus consist at least in part of transgenic cells, include, but are not limited to, plant cells, protoplasts, tissue, callus, embryos, and flowers, stems, fruits, leaves, and roots.
The term "gene" refers to a nucleic acid fragment that expresses a particular protein, including regulatory sequences preceding the coding sequence (5 'non-coding sequences) and regulatory sequences following the coding sequence (3' non-coding sequences). "native gene" refers to a gene that is naturally found to have its own regulatory sequences. "chimeric gene" refers to any gene that is not a native gene, comprising regulatory and coding sequences not found in nature. "endogenous gene" refers to a native gene that is located in its natural location in the genome of an organism. "foreign gene" is a foreign gene that exists in the genome of an organism and does not originally exist, and also refers to a gene that has been introduced into a recipient cell through a transgenic step. The foreign gene may comprise a native gene or a chimeric gene inserted into a non-native organism. A "transgene" is a gene that has been introduced into the genome by a transformation procedure. The site in the plant genome at which the recombinant DNA has been inserted may be referred to as an "insertion site" or "target site".
"flanking DNA" may comprise the genome as it occurs naturally in an organism such as a plant or foreign (heterologous) DNA introduced by the transformation process, such as a fragment associated with the transformation event. Thus, flanking DNA may include a combination of native and foreign DNA. In the present invention, a "flanking region" or "flanking sequence" or "genomic boundary region" or "genomic boundary sequence" refers to a sequence of at least 3, 5, 10, 11, 15, 20, 50, 100, 200, 300, 400, 1000, 1500, 2000, 2500, or 5000 base pairs or more in length, which is located directly upstream or downstream of and adjacent to the originally exogenously inserted DNA molecule. When the flanking region is located upstream, it may also be referred to as "left border flanking" or "5 'genomic flanking region" or "genomic 5' flanking sequence" or the like. When the flanking region is located downstream, it may also be referred to as "right border flanking" or "3 'genomic flanking region" or "genomic 3' flanking sequence" or the like.
Transformation procedures that result in random integration of the exogenous DNA will result in transformation events that contain different flanking regions that are specific for each transformation event. When the recombinant DNA is introduced into a plant by conventional crossing, its flanking regions are not usually altered. Transformation events will also contain unique junctions between the heterologous insert DNA and segments of genomic DNA or between two segments of heterologous DNA. "junction" is the point at which two specific DNA fragments are joined. For example, the junction is present where the insert DNA joins the flanking DNA. A junction point is also present in a transformed organism where two DNA fragments are joined together in a manner that is modified in the way found in the native organism. "junction DNA" refers to DNA comprising a junction site.
The present invention provides a transgenic corn event designated ND4403, namely, corn plant ND4403, and progeny thereof, said transgenic corn event ND4403 being a corn plant ND4403, comprising plants and seeds of transgenic corn event ND4403 and plant cells thereof or regenerable parts thereof, said plant parts of transgenic corn event ND4403 including, but not limited to, cells, pollen, ovules, flowers, buds, roots, stems, silks, inflorescences, ears, leaves, and products from corn plant ND4403, such as corn meal, corn flour, corn oil, corn steep liquor, silks, corn starch, and biomass remaining in a corn crop field.
Transgenic maize event ND4403 of the invention comprises a DNA construct which when expressed in a plant cell, achieves yield enhancement and/or nitrogen deficiency tolerance traits in said transgenic maize event ND 4403. The DNA construct comprises an expression cassette comprising a suitable promoter for expression in plants operably linked to a nucleic acid molecule encoding a maize nitrate transporter gene ZmNAR1.1A, said ZmNAR1.1A being tolerant to nitrogen deficiency and a suitable polyadenylation signal sequence. The DNA construct comprises a further expression cassette comprising a suitable promoter for expression in plants operably linked to the bar gene encoding Phosphinothricin Acetyltransferase (PAT) protein whose nucleic acid molecule is tolerant to glufosinate herbicides and which can be used as a selection marker for the transformation stage, and a suitable polyadenylation signal sequence. Further, the promoter may be a suitable promoter isolated from a plant, including constitutive, inducible and/or tissue specific promoters, suitable promoters include, but are not limited to, the cauliflower mosaic virus (CaMV)35S promoter, the Figwort Mosaic Virus (FMV)35S promoter, the Ubiquitin protein (Ubiquitin) promoter, the Actin (Actin) promoter, the Agrobacterium tumefaciens (Agrobacterium tumefaciens) nopaline synthase (NOS) promoter, the octopine synthase (OCS) promoter, the nocturnal (Cestrum) yellow leaf curly virus promoter, the potato tuber storage protein (Patatin) promoter, the ribulose-1, 5-bisphosphate carboxylase/oxygenase (rusco) promoter, the glutathione thiotransferase (GST) promoter, the E9 promoter, the GOS promoter, the alcA/alcR promoter, the Agrobacterium rhizogenes (Agrobacterium rhizogenes) RolD promoter, and the Arabidopsis thaliana (Arabidopsis thaliana) Suc2 promoter. The polyadenylation signal sequence may be a suitable polyadenylation signal sequence that functions in plants, including, but not limited to, polyadenylation signal sequence derived from the Agrobacterium tumefaciens nopaline synthase (NOS) gene, cauliflower mosaic virus (CaMV)35S terminator, polyadenylation signal sequence derived from the protease inhibitor II (PIN II) gene, and polyadenylation signal sequence derived from the alpha-tubulin (alpha-tubulin) gene.
In addition, the expression cassette may also include other genetic elements including, but not limited to, enhancers and signal/transit peptides. The enhancer may enhance the expression level of a gene, including, but not limited to, Tobacco Etch Virus (TEV) translational activator, CaMV35S enhancer, and FMV35S enhancer. The signal peptide/transit peptide may direct the transportation of the EPSPS protein to a particular organelle or compartment outside 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 a plant using transformation methods including, but not limited to, Agrobacterium-mediated transformation, biolistic transformation, and pollen tube channel transformation.
The Agrobacterium-mediated transformation method is a commonly used method for plant transformation. The foreign DNA to be introduced into the plant is cloned between the left and right border consensus sequences of the vector, i.e.the T-DNA region. Said vector is transformed into an agrobacterium cell, which is subsequently used to infect plant tissue, said T-DNA region of the vector comprising the foreign DNA being inserted into the plant genome.
After transformation, transgenic plants must be regenerated from the transformed plant tissue and progeny with exogenous DNA selected using appropriate markers.
A DNA construct is a combination of DNA molecules linked together to provide one or more expression cassettes. The DNA construct is preferably a plasmid capable of autonomous replication in bacterial cells and containing different restriction enzyme sites for the introduction of DNA molecules providing functional genetic elements, i.e. promoters, introns, leader sequences, coding sequences, 3' terminator regions and other sequences. The expression cassette contained in the DNA construct, which includes the genetic elements necessary to provide for transcription of messenger RNA, can be designed for expression in prokaryotic or eukaryotic cells. The expression cassette of the invention is designed to be most preferably expressed in plant cells.
A transgenic "event" is obtained by transforming a plant cell with a heterologous DNA construct, i.e., comprising at least one nucleic acid expression cassette containing a gene of interest, inserting into the plant genome by transgenic means to produce a population of plants, regenerating said population of plants, and selecting for a particular plant characterized by the insertion of a particular genomic locus. The term "event" refers to both the original transformation event comprising the heterologous DNA and the progeny of the transformation event. The term "event" also refers to progeny resulting from sexual crosses between a transformation event and individuals of other varieties containing heterologous DNA, where the inserted DNA and flanking genomic DNA from the parent of the transformation event are present in the same chromosomal location in the progeny of the cross, even after repeated backcrosses with the backcross parents. The term "event" also refers to a DNA sequence from an original transformation event that comprises an inserted DNA and flanking genomic sequences immediately adjacent to the inserted DNA, which DNA sequence is expected to be transferred to progeny that result from sexual crossing of a parental line containing the inserted DNA (e.g., progeny resulting from the original transformation event and its selfing) with a parental line that does not contain the inserted DNA, and which progeny received the inserted DNA comprising the gene of interest.
"recombinant" in the context of the present invention refers to a form of DNA and/or protein and/or organism that is not normally found in nature and is therefore produced by human intervention. Such manual intervention may result in recombinant DNA molecules and/or recombinant plants. Such "recombinant DNA molecules" are obtained by artificially combining two otherwise isolated sequence segments, for example, by chemical synthesis or by manipulating an isolated nucleic acid segment by 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, the genotype of which is altered by the presence of heterologous nucleic acid, including the transgene body that was originally so altered, as well as progeny individuals generated from the original transgene body by sexual crossing or asexual reproduction. In the present invention, the term "transgene" does not include changes (chromosomal or extra-chromosomal) in the genome by conventional plant breeding methods or naturally occurring events such as random allofertilization, non-recombinant viral infection, non-recombinant bacterial transformation, non-recombinant transposition, or spontaneous mutation.
By "heterologous" in the context of the present invention is meant that the first molecule is not normally found in combination with the second molecule in nature. For example, a molecule may be derived from a first species and inserted into the genome of a second species. Such molecules are therefore heterologous to the host and are artificially introduced into the genome of the host cell.
Culturing a transgenic maize event ND4403 that is highly productive and/or tolerant to nitrogen deficiency by: first sexually crossing a first parent corn plant consisting of a corn plant bred from transgenic corn event ND4403 and its progeny obtained by transformation with a nitrogen efficient use expression cassette of the present invention with a second parent corn plant lacking high yield and/or nitrogen deficiency tolerance, thereby producing a multiplicity of first generation progeny plants; progeny plants that are tolerant to nitrogen deficiency or have increased yield are then selected and maize plants that are tolerant to nitrogen deficiency or have increased yield can be grown. These steps can further include backcrossing the high-yielding and/or nitrogen-deficient tolerant progeny plants with the second or third parent corn plant, and then selecting the progeny through identification of a trait-related molecular marker (e.g., a DNA molecule comprising the junction site identified at the 5 'end and 3' end of the insert sequence in transgenic corn event ND 4403) to produce yield-improved and/or nitrogen-deficient tolerant corn plants.
It is also understood that two different transgenic plants may also be crossed to produce progeny containing two separate, separately added exogenous genes. Selfing of appropriate progeny can yield progeny plants that are homozygous for both added exogenous 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 having a conventional detectable label or reporter molecule, e.g., a radioisotope, ligand, chemiluminescent agent or enzyme, bound thereto. Such a probe is complementary to one strand of the target nucleic acid, and in the present invention, the probe is complementary to one DNA strand from the genome of transgenic corn event ND4403, whether the genomic DNA is from transgenic corn event ND4403 or seed or a plant or seed or extract derived from transgenic corn event ND 4403. Probes of the invention include not only deoxyribonucleic or ribonucleic acids, but also polyamides 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 by nucleic acid hybridization, annealing, forming a hybrid between the primer and the target DNA strand, and then extending along the target DNA strand under the action of a polymerase (e.g., a DNA polymerase). The primer pairs of the present invention are directed to their use in the amplification of a target nucleic acid molecule, for example, by Polymerase Chain Reaction (PCR) or other conventional nucleic acid amplification methods.
The length of the 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 or more. Such probes and primers hybridize specifically to the target sequence under highly stringent hybridization conditions. Although probes that are different from and maintain the ability to hybridize to the target DNA sequence can be designed by conventional methods, it is preferred that the probes and primers of the present invention have complete DNA sequence identity to the contiguous nucleic acid of the target sequence.
As used herein, "kit" or "microarray" refers to a set of reagents or chips for the purpose of identification and/or detection of corn transformation events in a biological sample. For the purpose of quality control (e.g. purity of seed lot), detection of events in or in a material comprising or derived from plant material, such as, but not limited to, food or feed products, kits or chips may be used, and components thereof may be specifically adjusted.
Primers and probes based on the flanking genomic DNA and insertion sequences of the present invention can be determined by conventional methods, for example, by isolating the corresponding DNA molecule from plant material derived from transgenic maize event ND4403 and determining the nucleic acid sequence of the DNA molecule. The DNA molecule comprises a transgene insert and a maize genomic flanking region, and fragments of the DNA molecule can be used as primers or probes.
The nucleic acid probes and primers of the invention hybridize to a target DNA sequence under stringent conditions. Any conventional nucleic acid hybridization or amplification method can be used to identify the presence of DNA derived from transgenic corn event ND4403 in a sample. Nucleic acid molecules or fragments thereof are capable of specifically hybridizing to other nucleic acid molecules under certain circumstances. As used herein, two nucleic acid molecules can be said to be capable of specifically hybridizing to each other if they are capable of forming an antiparallel, double-stranded nucleic acid structure. Two nucleic acid molecules are said to be "complements" of one another if they exhibit complete complementarity. As used herein, a nucleic acid molecule is said to exhibit "perfect complementarity" when each nucleotide of the nucleic acid molecule is complementary to a corresponding nucleotide of another nucleic acid molecule. Two nucleic acid molecules are said to be "minimally complementary" if they are capable of hybridizing to each other with sufficient stability to allow them to anneal and bind to each other under at least conventional "low stringency" conditions. Similarly, two nucleic acid molecules are said to have "complementarity" if they are capable of hybridizing to each other with sufficient stability to allow them to anneal and bind to each other under conventional "highly stringent" conditions. Deviations from perfect complementarity may be tolerated as long as such deviations do not completely prevent the formation of a double-stranded structure by the two molecules. In order to allow a nucleic acid molecule to act as a primer or probe, it is only necessary to ensure sufficient complementarity in sequence to allow the formation of a stable double-stranded structure in the particular solvent and salt concentrations employed.
As used herein, a substantially homologous sequence is a nucleic acid molecule that specifically hybridizes under highly stringent conditions to the complementary strand of a matched nucleic acid molecule. Suitable stringency conditions for promoting DNA hybridization include, for example, treatment with 6.0 XSSC/sodium citrate (SSC) at about 45 ℃ followed by a wash with 2.0 XSSC at 50 ℃, as is well known to those skilled in the art. For example, the salt concentration in the washing step can be selected from the group consisting of about 2.0 XSSC for low stringency conditions, 50 ℃ to about 0.2 XSSC for high stringency conditions, 50 ℃. In addition, the temperature conditions in the washing step can be raised from about 22 ℃ at room temperature for low stringency conditions to about 65 ℃ for high stringency conditions. Both the temperature conditions and the salt concentration may be varied, or one may be held constant while the other is varied. Preferably, a nucleic acid molecule of the invention can specifically hybridize to one or more of SEQ ID NO 1, SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 5, SEQ ID NO 6 and SEQ ID NO 7, or complements thereof, or any fragment thereof, under moderately stringent conditions, such as at about 2.0 XSSC and about 65 ℃. More preferably, a nucleic acid molecule of the invention specifically hybridizes under highly stringent conditions to one or more of SEQ ID NO 1, SEQ ID NO 2, SEQ ID NO 3 and SEQ ID NO 5 or to a complementary sequence thereof, or to a fragment of any of the foregoing. In the context of the present invention, preferred marker nucleic acid molecules have SEQ ID NO 1, SEQ ID NO 2, SEQ ID NO 3 or SEQ ID NO 5 or their complements or any fragment of the aforementioned sequences.
Another preferred marker nucleic acid molecule of the invention has 80% to 100% or 90% to 100% sequence identity with SEQ ID NO 1, 2, 3, 4 or 5 or the complement thereof or any fragment thereof. SEQ ID NO 1, SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4 or SEQ ID NO 5 can be used as markers in plant breeding methods to identify progeny of a genetic cross. Hybridization of the probe to the target DNA molecule can be detected by any method known to those skilled in the art, including, but not limited to, fluorescent, radioactive, antibody-based, and chemiluminescent labels.
With respect to amplification of a target nucleic acid molecule using a particular amplification primer (e.g., by PCR), "stringent conditions" refer to conditions that allow only hybridization of the primer to the target nucleic acid molecule in a DNA thermal amplification reaction, a primer having a sequence corresponding to the target nucleic acid molecule (or its complement) that is capable of binding to the target nucleic acid molecule and preferably producing a unique amplification product, i.e., an amplicon.
The term "specifically binds (target sequence)" means that the probe or primer hybridizes only to the target molecule in a sample containing the target molecule under stringent hybridization conditions.
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 hybridization comprising the transgenic corn event ND4403 of the present invention, or whether a corn sample collected from a field comprises the transgenic corn event ND4403, or whether a corn extract, such as meal, flour, or oil, comprises the transgenic corn event ND4403, DNA extracted from a corn plant tissue sample or extract can be subjected to a nucleic acid amplification method using a primer pair to produce an amplicon diagnostic for the presence of DNA of the transgenic corn event ND 4403. The primer pair includes a first primer derived from a flanking sequence adjacent to the insertion site of the inserted foreign DNA in the plant genome, and a second primer derived from the inserted foreign DNA. The amplicon has a length and sequence that is also diagnostic for the transgenic corn event ND 4403.
The amplicon can range in length from the bound length of the primer pair plus one nucleotide base pair, preferably plus about fifty nucleotide base pairs, more preferably plus about two hundred fifty nucleotide base pairs, and most preferably plus about four hundred fifty nucleotide base pairs or more.
Alternatively, primer pairs may be derived from flanking genomic sequences flanking the inserted DNA to produce amplicons that include the entire inserted nucleotide sequence. One of the primer pairs derived from a plant genomic sequence may be located at a distance from the inserted DNA sequence that may 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 the DNA thermal amplification reaction.
The nucleic acid amplification reaction may be carried out by any of the nucleic acid amplification reaction methods known in the art, including the Polymerase Chain Reaction (PCR). Various nucleic acid amplification methods are well known to those skilled in the art. The PCR amplification method has been developed to amplify 22kb of genomic DNA and 42kb 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 exogenous DNA sequence and flanking DNA sequences from transgenic maize event ND4403 can be obtained by amplifying the genome of transgenic maize event ND4403 using the provided primer sequences, followed by standard DNA sequencing of the PCR amplicons or cloned DNA.
DNA detection kits based on DNA amplification methods contain DNA primer molecules that specifically hybridize to the target DNA and amplify the diagnostic amplicon under appropriate reaction conditions. The kit may provide an agarose gel based detection method or a number of methods known in the art for detecting diagnostic amplicons. Kits containing DNA primers homologous or complementary to any portion of the maize genomic region of SEQ ID NO 1 or SEQ ID NO 2 and to any portion of the transgene insert region of SEQ ID NO 5 are provided by the present invention. In particular, the primer pairs identified as useful in the DNA amplification method are SEQ ID NO 6 and SEQ ID NO 7, which amplify a diagnostic amplicon homologous to a portion of the 5' transgene/genomic region of transgenic maize event ND4403, wherein the amplicon comprises SEQ ID NO 3. The primer pairs identified as useful in the DNA amplification method can also be SEQ ID NO 8 and SEQ ID NO 9, which amplify a diagnostic amplicon homologous to a portion of the 3' transgene/genomic region of transgenic corn event ND4403, wherein the amplicon comprises SEQ ID NO 4.
Amplicons produced by these methods can be detected by a variety of techniques. One such method is Genetic Bit Analysis, which designs a DNA oligonucleotide strand spanning an intervening DNA sequence and adjacent flanking genomic DNA sequences. The oligonucleotide strand is immobilized within a microwell of a microwell plate, and after PCR amplification of the target region (using one primer in each of the intervening sequence and adjacent flanking genomic sequence), a single-stranded PCR product can hybridize to the immobilized oligonucleotide strand and serve as a template for a single-base extension reaction using DNA polymerase and ddNTPs specifically labeled for the next desired base. The results can be obtained by fluorescence or ELISA-like methods. The signal represents the presence of the inserted/flanking sequence, indicating that the amplification, hybridization and single base extension reactions were successful.
Another method is Pyrosequencing (Pyrosequencing) technology. The method designs an oligonucleotide strand that spans the junction of the inserted DNA sequence and the adjacent genomic DNA. The oligonucleotide strand is hybridized to the single-stranded PCR product of the target region (using one primer in each of the intervening and adjacent flanking genomic sequences), and then incubated with DNA polymerase, ATP, sulfurylase, luciferase, apyrase, adenosine-5' -phosphothioate, and luciferin. dNTPs were added separately and the resulting optical signals were measured. The light signal represents the presence of the inserted/flanking sequence, indicating that the amplification, hybridization, and single or multiple base extension reactions were successful.
Fluorescence polarization is also a method that can be used to detect the amplicons of the invention (Chen X, Levine L, and Kwok P Y. fluorescence polarization in genetic nucleic acid analysis [ J ]. Genome Res,1999,9(5): 492-8.). Using this method requires the design of an oligonucleotide strand that spans the intervening DNA sequence and adjacent genomic DNA binding sites. The oligonucleotide strand is hybridized to the single stranded PCR product of the target region (using one primer each within the insert sequence and adjacent flanking genomic sequence) and then incubated with DNA polymerase and a fluorescently labeled ddNTP. Single base extension will result in insertion of ddNTPs. This insertion can be measured for changes in its polarization using a fluorometer. The change in polarization represents the presence of the inserted/flanking sequence, indicating that the amplification, hybridization and single base extension reactions were successful.
Taqman is described as a method for detecting and quantifying the presence of DNA sequences, which is described in detail in the instructions provided by the manufacturer. Briefly, as illustrated below, a FRET oligonucleotide probe is designed to span the inserted DNA sequence and adjacent genomic flanking binding sites. The FRET probe and PCR primers (one primer for each of the flanking genomic sequences within the insert and adjacent) are cycled in the presence of a thermostable polymerase and dNTPs. Hybridization of the FRET probe results in cleavage of the fluorescent and quenching moieties on the FRET probe and release of the fluorescent moiety. The generation of a fluorescent signal represents the presence of the inserted/flanking sequence, indicating that amplification and hybridization were successful.
Suitable techniques for detecting plant material derived from transgenic maize event ND4403 can also include Southern blot hybridization, Northern blot hybridization and in situ hybridization based on hybridization principles. In particular, the suitable techniques include incubating the probe and sample, washing to remove unbound probe and detecting whether the probe has hybridised. The detection method depends on the type of label attached to the probe, for example, a radiolabeled probe can be detected by X-ray exposure and development, or an enzymatically labeled probe can be detected by a color change achieved by substrate conversion.
Sequences can also be detected using molecular markers (Tyagi S and Kramer F R. molecular beacons: probes that are fluorescent upon hybridization [ J ]. Nat Biotechnol,1996,14(3): 303-8.). A FRET oligonucleotide probe is designed that spans the inserted DNA sequence and the adjacent genomic flanking binding sites. The unique structure of the FRET probe results in it containing a secondary structure that is capable of retaining both a fluorescent moiety and a quenching moiety in close proximity. The FRET probe and PCR primers (one primer for each of the flanking genomic sequences within the insert and adjacent) are cycled in the presence of a thermostable polymerase and dNTPs. Upon successful PCR amplification, hybridization of the FRET probe to the target sequence results in loss of the secondary structure of the probe, thereby spatially separating the fluorescent moiety from the quencher moiety to produce a fluorescent signal. The generation of a fluorescent signal represents the presence of the inserted/flanking sequence, indicating that amplification and hybridization were successful.
Other described methods, such as microfluidics (microfluidics), provide methods and apparatus for isolating and amplifying DNA samples. The fluorochromes are used to detect and measure specific DNA molecules. A nano-tube (nanotube) device comprising an electronic sensor for detecting DNA molecules or nano-beads binding to specific DNA molecules and thus being detectable is useful for detecting the DNA molecules of the present invention.
The DNA detection kit can be developed using the compositions described herein and methods described or known in the DNA detection art. The kit is useful for identifying the presence of DNA of transgenic corn event ND4403 in a sample, and can also be used for breeding corn plants containing DNA of transgenic corn event ND 4403. The kit may contain DNA primers or probes homologous or complementary to at least a portion of SEQ ID NO 1, 2, 3, 4 or 5, or other DNA primers or probes homologous or complementary to DNA contained in the transgenic genetic element of DNA, which DNA sequences may be used in DNA amplification reactions or as probes in DNA hybridization methods. The DNA structure of the site of the maize genome containing the transgene insert and illustrated in table 1 comprises: a maize ND4403 left flanking genomic region 5' of the transgene insert, a portion of the insert from the left border region of the vector, a first expression cassette consisting of a cauliflower mosaic virus 35S promoter (CaMV 35S promoter), operably linked to a glufosinate (glufosinate) resistance gene sequence (bar), and operably linked to a cauliflower mosaic virus 35S terminator (CaMV 35S polyA); the second expression cassette consists of a maize ubiquitin gene promoter (ubiquitin promoter), operably linked to the maize nitrate transporter gene ZmNAR1.1A, and operably linked to a nopaline synthase gene terminator (NOs terminator), a portion of the insertion sequence from the right border region of the vector, and the maize plant ND4403 right wing genomic region located 3' of the transgene insertion sequence (SEQ ID NO: 5). In a DNA amplification method, the DNA molecule that serves as a primer can be any portion of a DNA sequence derived from the transgene insertion sequence in transgenic corn event ND4403, or any portion of a DNA region flanking the corn genome in transgenic corn event ND 4403.
Transgenic corn event ND4403 can be combined with other transgenic corn varieties, such as herbicide (e.g., glyphosate, dicamba, etc.) tolerant corn, or transgenic corn varieties carrying insect-resistant genes. All of these various combinations of different transgenic events, when bred with transgenic corn event ND4403 of the present invention, can provide improved hybrid transgenic corn varieties with multiple traits. These varieties can exhibit more excellent characteristics such as an increase in yield as compared with non-transgenic varieties and single-trait transgenic varieties.
The invention provides a nucleic acid molecule for detecting corn plants and a detection method thereof, wherein a transgenic corn event ND4403 has high-yield and/or nitrogen-deficiency tolerance traits. The maize plants of this trait express the nitrate transporter zmnar1.1a protein, which confers to plants a nitrogen efficient use trait to increase yield and/or nitrogen deficiency tolerance traits (herbicide resistance gene bar as a selection marker at the transformation stage). Meanwhile, in the detection method of the invention, the nucleic acid molecule of SEQ ID NO. 1 or a complementary sequence thereof, SEQ ID NO. 2 or a complementary sequence thereof, SEQ ID NO. 3 or a complementary sequence thereof, SEQ ID NO. 4 or a complementary sequence thereof, and SEQ ID NO. 5 or a complementary sequence thereof can be used as a DNA primer or probe to generate an amplification product diagnosed as transgenic corn event ND4403 or its progeny, and the presence of plant material derived from transgenic corn event ND4403 can be identified rapidly, accurately and stably.
The technical solution of the present invention is further described in detail by the accompanying drawings and embodiments.
Drawings
FIG. 1 physical map of recombinant expression vector pCAMBIA 1301-ZmNRT1.1A. The English and abbreviated meanings of each element are listed as follows:
T-Border (left) T-DNA left border sequence.
35S terminator of CaMV35S polyA cauliflower mosaic virus (CaMV).
bar codes PAT protein and relieves glufosinate toxicity.
The 35S promoter of CaMV35S promoter cauliflower mosaic virus (CaMV).
The terminator of the nos terminator nopaline synthase gene.
ZmNAR1.1A AnVP1 gene CDS.
A promoter of a ubiquitin promoter maize ubiquitin gene.
The terminator of the nos terminator nopaline synthase gene.
T-Border (right) T-DNA right border sequence.
Plasmid stabilization site of PVS1 sta pVS1 plasmid.
The replication initiation site of the PVS1 rep pVS1 plasmid.
The bom site of the PBR322 bom pBR322 plasmid.
Origin of replication of the PBR322 ori pBR322 plasmid.
kanamycins (R) encode aminoglycoside phosphotransferase proteins that confer kanamycin resistance to bacteria.
FIG. 2 transformation event specific PCR validation results. A: 5' end amplification result, the expected size of the fragment is 559bp (SEQ ID NO: 3); b: as a result of amplification of the 3' end, the expected size of the fragment was 728bp (SEQ ID NO: 4). M: DL2000 DNAmarker; t: ND4403 corn material; CK: non-transgenic maize.
Detailed Description
The transformation event ND4403 refers to a maize plant which is obtained by taking a maize inbred line Y822 as a receptor and performing genetic transformation to insert an exogenous gene insert (T-DNA insert) between specific genome sequences. In a specific example, the expression vector used for the transgene has the physical map shown in FIG. 1, and the resulting T-DNA insert has the sequence shown as nucleotide 648-5737 of SEQ ID NO: 5. Transformation event ND4403 may refer to this transgenic process, may also refer to the combination of a T-DNA insert and flanking sequences within the genome resulting from this process, or may refer to a maize plant resulting from 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 and inserting the T-DNA insert into the same genomic position. Transformation event ND4403 may also refer to progeny plants derived from the above plants by vegetative propagation, sexual propagation, doubling or doubling, or a combination thereof.
Example 1 acquisition and screening of transformation events
The cDNA sequence of ZmNRT1.1A gene is cloned from corn, and is inserted into an intermediate vector pCAMBIA1301-Ubi through enzyme digestion connection, the intermediate vector already contains bar gene, and finally the intermediate vector is constructed into a pCAMBIA1301-ZmNRT1.1A expression vector, and the physical map of the vector is shown in figure 1. The recombinant plasmid is efficiently transformed into the young embryo of the corn y822, a selected line of agricultural biotechnology research institute of agricultural academy of sciences of Jilin province by utilizing an agrobacterium infection technology, and 45 transformation events of the corn with the ZmNRT1.1A and bar genes are obtained. The transgenic corn event ND4403 contains exogenous genes ZmNRT1.1A and bar, shows stable generation inheritance and outstanding yield traits and nitrogen deficiency tolerance, and has a good application prospect.
The detection method of the exogenous ZmNRT1.1A and bar genes comprises the following steps: the genome DNA of corn is used as a template, and PCR amplification is carried out by using specific primer pairs of ZmNRT1.1A and bar to respectively obtain the materials of strips with 452bp size (ZmNRT1.1A gene) and 441bp size (bar gene) as plants containing the ZmNRT1.1A gene and the bar gene.
TABLE 2 PCR detection primer sequences for the ZmNRT1.1A and bar genes
Example 2 nitrogen efficient utilization and yield trait identification of transformation event ND4403
The corn ZmNRT1.1A protein has the function of promoting nitrate absorption. The ZmNRT1.1A protein is expressed in the corn in a constitutive expression mode, and the nitrogen utilization efficiency of the corn is improved by improving the absorption capacity of the corn nitrate, so that the yield is finally improved. The invention identifies the field growth form and physiological characters of the ND4403 plants under different nitrogen fertilizer gradients.
The test site is a transgenic test base of agricultural scientific institute of Jilin province, four Ping city, principals, and four. The test soil is sandy loam with flat terrain, the content of alkaline hydrolysis nitrogen in a plough layer (20cm) soil is 125.94mg/kg, the content of quick-acting phosphorus is 23.96mg/kg, the content of quick-acting potassium is 149.24mg/kg, and the content of organic matters is 25.22 g/kg. The row length of each cell is 5m, the row spacing is 0.60m, the planting spacing is 0.25m, and each cell has 6 rows.
And (3) testing conditions are as follows: according to large formula and fertilization suggestions (2013) of three large grain crop regions, namely wheat, corn and rice, issued by rural parts in agriculture, the high nitrogen treatment (HN) is applied with 38 kg/mu of formula fertilizer according to the fertilization amount of normal farming; medium nitrogen treatment (MN): applying 70% of formula fertilizer, and supplementing phosphate fertilizer and potash fertilizer as same as high-nitrogen treatment; low nitrogen treatment (LN) no formulated fertilizer is applied, and phosphate and potash supplementation is the same as high nitrogen treatment.
Experiment design: the test adopts a random block design, 10 corns with approximately same growth vigor are taken in each cell, and relevant forms and physiological indexes are determined, wherein the specific indexes comprise: ear weight, ear length, ear row, hundred grain weight and total nitrogen content of the overground part of the plant.
The result shows that the ear weight and ear length of the corn ND4403 positive plant are obviously higher than those of the negative plant under various nitrogen supply conditions, and the yield increasing effect is obvious. Specifically, the weight of the panicle under the low nitrogen condition is increased by 3.1%. Under the condition of medium nitrogen, the weight of the spike is increased by 17.2%, the length of the spike is increased by 5.0%, and the row number of the spike is increased by about 1 row. The ear weight increased by 28.8% and the ear length increased by 4.2% under high nitrogen conditions (see table 3 for details). This indicates that ND4403 positive plants are well tolerant to nitrogen deficiency conditions and have yield increasing properties.
TABLE 3 comparison of agronomic traits for ND4403 and controls
From the results of the total nitrogen content of the plant stalks under different nitrogen conditions, the total nitrogen content of the ND4403 plants under different nitrogen conditions is lower than that of the receptor control material. Especially under high nitrogen conditions, the total nitrogen content of the control stalk is 3.00g/kg, while the total nitrogen content of ND4403 is only 2.00g/kg (Table 4). This indicates that the nitrogen utilization efficiency of ND4403 was higher than that of the wild-type control, and this is also the reason why ND4403 was produced in a higher amount.
TABLE 4 measurement of total nitrogen content of plant stalks by ND4403 and acceptor control
Example 3 flanking sequences and maize genomic insertion position of transformation event ND4403 exogenous sequence
To clarify the identity of transformation event ND4403, the present invention further identifies the insertion site of the ND4403 exogenous sequence on the maize genome.
The invention adopts a third generation sequencing method to position the T-DNA insertion position of the transgenic corn ND 4403. The third generation sequencing technology utilizes Nanopore sequencing technology of Oxford Nanopore Technologies, ONT for short, in England to perform re-sequencing on the transformant ND4403, the sequencing depth is 10 times, the average read length of data is 20kb, the longest read length is 60kb, and the accuracy is 85%.
BLAST search alignment of the third generation sequencing results using the T-DNA foreign sequence revealed that a contig with a read length of approximately 10kb contained the target sequence, indicating that the foreign sequence in ND4403 was a single copy insertion. The 5 '-terminal 647bp and 3' -terminal 356bp genome flanking sequences of the exogenous sequence obtained by sequencing are subjected to BLAST nucleic acid-nucleic acid alignment in a maize public genome database (http:// ensemble. gram. org/Zea _ mays/Info/association/# assembly), and the exogenous sequence is integrated at the position of the maize chromosome 3 7866623-.
And at the left boundary of the insertion site, 647bp and an exogenous insertion sequence on the genome are selected to design a primer pair, so that the F primer can be combined with the genome sequence, the R primer can be combined with the exogenous insertion sequence, and an amplification product is fused with a part of corn genome sequence and a part of exogenous insertion sequence. Or at the right border of the insertion site, 356bp and the exogenous insertion sequence on the genome are selected to design a primer pair, so that the F primer can be combined with the exogenous insertion sequence, the R primer can be combined with the genome sequence, and the amplification product is fused with a part of the exogenous insertion sequence and a part of the maize genome sequence. And carrying out PCR detection on ND4403 by using the designed primer. And carrying out PCR amplification by using the genome DNA of the transgenic corn strain as a template. The PCR reaction was carried out in a 20. mu.L system. The amplification cycle program was: pre-denaturation at 94 ℃ for 3 min; denaturation at 94 ℃ for 30s, TmAnnealing for 30s, extending for a certain time (according to the size of a product fragment) at 72 ℃, and performing 35 cycles; extension at 72 ℃ for 5 min.
The ND4403 transformation event was PCR amplified using the genome upstream primer (SEQ ID NO:6) and the vector left border primer (SEQ ID NO:7) and the vector right border primer (SEQ ID NO:8) and the genome downstream primer (SEQ ID NO:9), respectively, based on the results of the flanking sequences and the insertion positions, to verify the foreign fragment insertion positions. The results are shown in FIG. 2. The results demonstrated that the ND4403 foreign fragment was stably inserted into the position of 7866623 and 7866705 No. 3 chromosome.
Example 4 detection method of transformation event ND4403
Breeding can be performed from transgenic maize event ND4403 and producing, for example, an agricultural or commodity product from the bred new variety. If a sufficient amount is detected in the agricultural or commodity product, the agricultural or commodity product is expected to contain a nucleotide sequence capable of diagnosing the presence of transgenic corn event ND4403 material in the agricultural or commodity product. Such agricultural or commercial products include, but are not limited to, corn oil, corn meal, corn flour, corn gluten, corn tortillas, corn starch, and any other food product intended for consumption by an animal as a food source, or otherwise for cosmetic use as an ingredient in a bulking or cosmetic composition, and the like. A probe or primer pair based nucleic acid detection method and/or kit can be developed to detect a nucleic acid analysis of whether a transgenic corn event ND4403 is contained in a biological sample, wherein the probe sequence or primer amplification sequence is selected from the group consisting of the sequences as set forth in SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4 or SEQ ID No. 5, to diagnose the presence of the transgenic corn event ND 4403.
One detection method comprises the following steps:
the genome DNA of a corn sample is extracted by adopting a common CTAB method, and the specific operation process is implemented according to No. 1485 bulletin-4-2010 of Ministry of agriculture, and the DNA extraction and purification of the transgenic plants and the product components thereof. Wherein the corn sample comprises corn ND4403 to be transgenic and non-transgenic corn y 822. Detecting the specific boundary sequence in the ND4403 plant by using a PCR method, wherein the used PCR primer pairs are respectively SEQ ID NO. 6 and SEQ ID NO. 7, and SEQ ID NO. 8 and SEQ ID NO. 9, and the PCR reaction system comprises:
the reaction procedure is as follows:
and 2, step 4: circulating for 35 times
The PCR product was electrophoretically detected on a 1% (w/v)1 XTAE agarose gel, and the results are shown in FIG. 2. The expected target bands (SEQ ID NO:3 and SEQ ID NO:4) were amplified in the ND4403 transformation event. Moreover, the PCR method can track the existence of transformation events, thereby being applied to breeding work.
In summary, the transgenic corn event ND4403 of the invention has higher yield and/or tolerance to nitrogen deficiency, and the detection method can accurately and rapidly identify whether the biological sample contains the DNA molecule of the transgenic corn event ND 4403.
Finally, it should be noted that the above embodiments are only for illustrating the technical solutions of the present invention and not for limiting, and although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions may be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.
Sequence listing
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<213> Artificial Synthesis (unknown)
<400> 1
tctttatcag ctataagttt cgctgaaggc ttgagtccgt tactactatc aaaacagcag 60
cagcagtaac aacaacaaga tgtttagatc tttcaggaag ggagtaatgc catttctagg 120
agacaaaagt aaaaggcgat ctatggcgct gtgttgtcgt ctgttacagg gaagacgtag 180
ccttttcccc tatatctgct gcagttttcc ataacggatt cgtacaacgc tacccggccg 240
ttagctacga attgtgacga tgcccctgct ttagcaaaag ctagcggaat tattgagttc 300
tggcaccgtc ggtttgtcgg tcgatctctc aacgtaattg ctgttcctcg cggaattgcg 360
cgatgatatg aagtgaagct agctgtcacg aatagcttga gctcagacct gcagcacgcc 420
tgcatctgtt cctcgcagat agattcagct cgcctgccgg ccggtgacaa caattagtat 480
gtgttgcaat ttgcaagtga ggtgacgatc gacgtgcagt gcagagagag atgcatggaa 540
ttaatgggtg atgcatcatc gctagtcaaa gtgtggctgg cccaatgtgg ggagggctcg 600
aattttgttt ctttccgccg cgcgccactc gatcgtcttg tcatgtctgt acctagacta 660
gtagggctgg ttgctagaga gtgcttggtg taaacaaatt gacgcttaga caacttaata 720
acacattgcg gacgttttta atgtactgaa ttaacgccga attaattcgg gggatctgga 780
ttttagtact ggattttggt tttaggaatt agaaatttta ttgatagaag tattttacaa 840
atacaaatac atactaaggg tttcttatat gctcaacaca tgagcgaaac cctataggaa 900
ccctaattcc cttatctggg aactactcac acattattat ggagaaactc gagtcaaatc 960
tcggtgacgg g 971
<210> 2
<211> 1031
<212> DNA
<213> Artificial Synthesis (unknown)
<400> 2
gacccgacaa acaagtgcac ggcatatatt gaaataaagg aaaagggcaa accaaaccct 60
atgcaacgaa acaaaaaaaa tcatgaaatc gatcccgtct gcggaacggc tagagccatc 120
ccaggattcc ccaaagagaa acactggcaa gttagcaatc agaacgtgtc tgacgtacag 180
gtcgcatccg tgtacgaacg ctagcagcac ggatctaaca caaacacgga tctaacacaa 240
acatgaacag aagtagaact accgggccct aaccatggac cggaacgccg atctagagaa 300
ggtagagagg gggggggggg gaggacgagc ggcgtacctt gaagcggagg tgccgacggg 360
tggatttggg ggagatcgtg accagctcga atttccccga tcgttcaaac atttggcaat 420
aaagtttctt aagattgaat cctgttgccg gtcttgcgat gattatcata taatttctgt 480
tgaattacgt taagcatgta ataattaaca tgtaatgcat gacgttattt atgagatggg 540
tttttatgat tagagtcccg caattataca tttaatacgc gatagaaaac aaaatatagc 600
gcgcaaacta ggataaatta tcgcgcgcgg tgtcatctat gttactagat cgggaattaa 660
actatcagtg ttgatggcat gaggcgatgg atctggatca aaccgaaata ggaacaggat 720
ggacggcatc cccaatcgcc acgcaacggt tgactttatt tggctaaaga caaaagaaac 780
tagaatagca tattgatctc tgtcaaatat ttaaaatgaa gctttcgtta cttcaaaaat 840
catgttttag ttgttaaaat tagttgtagg catctaaaca gtttagctaa tagttcaact 900
attacctact tttagcaaaa cggctaatag ttagccaact atttgttagc tacctaatta 960
cattagtaac tttttagcca acttagagca actctaaaag aagccctaaa ctatcccaaa 1020
attaatatta g 1031
<210> 3
<211> 559
<212> DNA
<213> Artificial Synthesis (unknown)
<400> 3
agcacgcctg catctgttcc tcgcagatag attcagctcg cctgccggcc ggtgacaaca 60
attagtatgt gttgcaattt gcaagtgagg tgacgatcga cgtgcagtgc agagagagat 120
gcatggaatt aatgggtgat gcatcatcgc tagtcaaagt gtggctggcc caatgtgggg 180
agggctcgaa ttttgtttct ttccgccgcg cgccactcga tcgtcttgtc atgtctgtac 240
ctagactagt agggctggtt gctagagagt gcttggtgta aacaaattga cgcttagaca 300
acttaataac acattgcgga cgtttttaat gtactgaatt aacgccgaat taattcgggg 360
gatctggatt ttagtactgg attttggttt taggaattag aaattttatt gatagaagta 420
ttttacaaat acaaatacat actaagggtt tcttatatgc tcaacacatg agcgaaaccc 480
tataggaacc ctaattccct tatctgggaa ctactcacac attattatgg agaaactcga 540
gtcaaatctc ggtgacggg 559
<210> 4
<211> 728
<212> DNA
<213> Artificial Synthesis (unknown)
<400> 4
gacccgacaa acaagtgcac ggcatatatt gaaataaagg aaaagggcaa accaaaccct 60
atgcaacgaa acaaaaaaaa tcatgaaatc gatcccgtct gcggaacggc tagagccatc 120
ccaggattcc ccaaagagaa acactggcaa gttagcaatc agaacgtgtc tgacgtacag 180
gtcgcatccg tgtacgaacg ctagcagcac ggatctaaca caaacacgga tctaacacaa 240
acatgaacag aagtagaact accgggccct aaccatggac cggaacgccg atctagagaa 300
ggtagagagg gggggggggg gaggacgagc ggcgtacctt gaagcggagg tgccgacggg 360
tggatttggg ggagatcgtg accagctcga atttccccga tcgttcaaac atttggcaat 420
aaagtttctt aagattgaat cctgttgccg gtcttgcgat gattatcata taatttctgt 480
tgaattacgt taagcatgta ataattaaca tgtaatgcat gacgttattt atgagatggg 540
tttttatgat tagagtcccg caattataca tttaatacgc gatagaaaac aaaatatagc 600
gcgcaaacta ggataaatta tcgcgcgcgg tgtcatctat gttactagat cgggaattaa 660
actatcagtg ttgatggcat gaggcgatgg atctggatca aaccgaaata ggaacaggat 720
ggacggca 728
<210> 5
<211> 6093
<212> DNA
<213> Artificial Synthesis (unknown)
<400> 5
tctttatcag ctataagttt cgctgaaggc ttgagtccgt tactactatc aaaacagcag 60
cagcagtaac aacaacaaga tgtttagatc tttcaggaag ggagtaatgc catttctagg 120
agacaaaagt aaaaggcgat ctatggcgct gtgttgtcgt ctgttacagg gaagacgtag 180
ccttttcccc tatatctgct gcagttttcc ataacggatt cgtacaacgc tacccggccg 240
ttagctacga attgtgacga tgcccctgct ttagcaaaag ctagcggaat tattgagttc 300
tggcaccgtc ggtttgtcgg tcgatctctc aacgtaattg ctgttcctcg cggaattgcg 360
cgatgatatg aagtgaagct agctgtcacg aatagcttga gctcagacct gcagcacgcc 420
tgcatctgtt cctcgcagat agattcagct cgcctgccgg ccggtgacaa caattagtat 480
gtgttgcaat ttgcaagtga ggtgacgatc gacgtgcagt gcagagagag atgcatggaa 540
ttaatgggtg atgcatcatc gctagtcaaa gtgtggctgg cccaatgtgg ggagggctcg 600
aattttgttt ctttccgccg cgcgccactc gatcgtcttg tcatgtctgt acctagacta 660
gtagggctgg ttgctagaga gtgcttggtg taaacaaatt gacgcttaga caacttaata 720
acacattgcg gacgttttta atgtactgaa ttaacgccga attaattcgg gggatctgga 780
ttttagtact ggattttggt tttaggaatt agaaatttta ttgatagaag tattttacaa 840
atacaaatac atactaaggg tttcttatat gctcaacaca tgagcgaaac cctataggaa 900
ccctaattcc cttatctggg aactactcac acattattat ggagaaactc gagtcaaatc 960
tcggtgacgg gcaggaccgg acggggcggt accggcaggc tgaagtccag ctgccagaaa 1020
cccacgtcat gccagttccc gtgcttgaag ccggccgccc gcagcatgcc gcggggggca 1080
tatccgagcg cctcgtgcat gcgcacgctc gggtcgttgg gcagcccgat gacagcgacc 1140
acgctcttga agccctgtgc ctccagggac ttcagcaggt gggtgtagag cgtggagccc 1200
agtcccgtcc gctggtggcg gggggagacg tacacggtcg actcggccgt ccagtcgtag 1260
gcgttgcgtg ccttccaggg gcccgcgtag gcgatgccgg cgacctcgcc gtccacctcg 1320
gcgacgagcc agggatagcg ctcccgcaga cggacgaggt cgtccgtcca ctcctgcggt 1380
tcctgcggct cggtacggaa gttgaccgtg cttgtctcga tgtagtggtt gacgatggtg 1440
cagaccgccg gcatgtccgc ctcggtggca cggcggatgt cggccgggcg tcgttctggg 1500
ctcatggtag actcgagaga gatagatttg tagagagaga ctggtgattt cagcgtgtcc 1560
tctccaaatg aaatgaactt ccttatatag aggaaggtct tgcgaaggat agtgggattg 1620
tgcgtcatcc cttacgtcag tggagatatc acatcaatcc acttgctttg aagacgtggt 1680
tggaacgtct tctttttcca cgatgctcct cgtgggtggg ggtccatctt tgggaccact 1740
gtcggcagag gcatcttgaa cgatagcctt tcctttatcg caatgatggc atttgtaggt 1800
gccaccttcc ttttctactg tccttttgat gaagtgacag atagctgggc aatggaatcc 1860
gaggaggttt cccgatatta ccctttgttg aaaagtctca atagcccttt ggtcttctga 1920
gactgtatct ttgatattct tggagtagac gagagtgtcg tgctccacca tgttggcaag 1980
ctgctctagc caatacgcaa accgcctctc cccgcgcgtt ggccgattca ttaatgcagc 2040
tggcacgaca ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat taatgtgagt 2100
tagctcactc attaggcacc ccaggcttta cactttatgc ttccggctcg tatgttgtgt 2160
ggaattgtga gcggataaca atttcacaca ggaaacagct atgacatgat tacgaattct 2220
catgtttgac agcttatcat cggatctagt aacatagatg acaccgcgcg cgataattta 2280
tcctagtttg cgcgctatat tttgttttct atcgcgtatt aaatgtataa ttgcgggact 2340
ctaatcataa aaacccatct cataaataac gtcatgcatt acatgttaat tattacatgc 2400
ttaacgtaat tcaacagaaa ttatatgata atcatcgcaa gaccggcaac aggattcaat 2460
cttaagaaac tttattgcca aatgtttgaa cgatcgatcc actagtcaat cactagtgaa 2520
ttcgagctct cagtggagcg tgggctcgtc ggccatctcg acgccgttga cgctgccgtc 2580
ggcgccgggg cggcccgcct tgtacttgta ccagcgggcg gcgacgaggt agactagtag 2640
gttggcgagg cagacggcgg cgagcagcca gtagaagttg tcgaggcggc ccttgttgag 2700
gtcgttggcg atccaggggt ggcggtcgcc cgtgaccctg tgcacggcgg cgacgagcgc 2760
ggagctgacg aagaatccca gcgacagggt gctgaggaac agccccgtgc tcatggtctt 2820
catccccttg gggcactcgc gcaggaagaa gtcgagctgg ccgatgtacg tgaacgcctc 2880
gcccgccccc acgaggaaga actgcgggat gagccagaac acggacatgg gcacgacgcc 2940
tccggaggcg gactcggagg aatcgcgcgc gacgcggagg cggcggacct ccgtgagcgc 3000
ggcgcccgcc atggcgacga cggagagcgc gaggccgacg gcgatccgct gcagcggggt 3060
gaggccgtgc gggttgccgc tgacgcggcg cgcgacgggc accaccaggc ggtcgtagac 3120
gggcacggtg agcaggatgg agccgacgaa gaagacggtg agggagcccg cggggatctg 3180
gaacgagccc ccgacgcggc ggtccatggt ggtggcctgc gacaccgaga aggtggtcat 3240
ctgcgcgtac accgtccaga acatgatcgt ggtcgcccag atcggcagca tccgcgccac 3300
cgtcttcacc tcctccacgt ccgtcagcgt cgccagccgc cacttgctgc tgctgctcgc 3360
ccccgccgcc ggatcctcgt tgatcgccgc gtggtccagg aagcggaact ggtcggtgtg 3420
ggggaggcgc tccttgcgct tgctcttctt gctggaggac ccatcctcga cggcggcggc 3480
cttgccgacg tcgacgtcgt agagcatggc ggggtcggcg gggagaggga ggcggcgctt 3540
gcgccaggcg gcgacgacga cggcggcgat ctgcgtgagg gggctgccgg ccagcttctt 3600
gaagcggtac ctgcgtgtgc cggccaggaa gacgaggagg cccgcggcga tggcgcaggc 3660
gcaggcgccg tagccccagc gcctgcccag gttgtcctgg acgtacacca gcacggtgac 3720
ggccagcagc gaccccagcg agatgaagaa gaagaaccag ttgaagaagc gcatcatctg 3780
ccgcttctcc ccgccgtccg actcgtcgaa ctggtccgac ccgaaccccg acacgctcga 3840
ctttagccca cccgtgccca gcgccgtcag gtacagcgcc aggtacagca cccccagctg 3900
cgcccccgac gcccgcgcgc actccccgac gacgcctccg ccggtcgcgg agcaggacgc 3960
cggccgtagc cccggcgccg ccgtcgagat cgtcaggatc gtcacgcccg aggcctggac 4020
ggcggtgaag atggcgatgg tgaggtagcg gccgaggaag gagtcggcga cgaagccgcc 4080
gaggaggcag agcatgaagg aggtgcccat gaagttggtg acgacgttgg cggactcggc 4140
gttgcccagg tgcatggtgc ccgtcaggta cgtcaccagg ttcacggcga tgcccagcgt 4200
cgtcagccgc tcgttcagct ccgccactag gatcatggcg gcggcgcccc agcggccggt 4260
ggtggcgcgc ggggccggcc ggcccttgaa gtcccaggcg tcgaggagga cgtccgtctc 4320
cgccgcggca ttggtctcgg ggaggagtcc gaccatgagc tcggtacccg gggatcctct 4380
agagtcgacc tgcagaagta acaccaaaca acagggtgag catcgacaaa agaaacagta 4440
ccaagcaaat aaatagcgta tgaaggcagg gctaaaaaaa tccacatata gctgctgcat 4500
atgccatcat ccaagtatat caagatcaaa ataattataa aacatacttg tttattataa 4560
tagataggta ctcaaggtta gagcatatga atagatgctg catatgccat catgtatatg 4620
catcagtaaa acccacatca acatgtatac ctatcctaga tcgatatttc catccatctt 4680
aaactcgtaa ctatgaagat gtatgacaca cacatacagt tccaaaatta ataaatacac 4740
caggtagttt gaaacagtat tctactccga tctagaacga atgaacgacc gcccaaccac 4800
accacatcat cacaaccaag cgaacaaaaa gcatctctgt atatgcatca gtaaaacccg 4860
catcaacatg tatacctatc ctagatcgat atttccatcc atcatcttca attcgtaact 4920
atgaatatgt atggcacaca catacagatc caaaattaat aaatccacca ggtagtttga 4980
aacagaattc tactccgatc tagaacgacc gcccaaccag accacatcat cacaaccaag 5040
acaaaaaaaa gcatgaaaag atgacccgac aaacaagtgc acggcatata ttgaaataaa 5100
ggaaaagggc aaaccaaacc ctatgcaacg aaacaaaaaa aatcatgaaa tcgatcccgt 5160
ctgcggaacg gctagagcca tcccaggatt ccccaaagag aaacactggc aagttagcaa 5220
tcagaacgtg tctgacgtac aggtcgcatc cgtgtacgaa cgctagcagc acggatctaa 5280
cacaaacacg gatctaacac aaacatgaac agaagtagaa ctaccgggcc ctaaccatgg 5340
accggaacgc cgatctagag aaggtagaga gggggggggg gggaggacga gcggcgtacc 5400
ttgaagcgga ggtgccgacg ggtggatttg ggggagatcg tgaccagctc gaatttcccc 5460
gatcgttcaa acatttggca ataaagtttc ttaagattga atcctgttgc cggtcttgcg 5520
atgattatca tataatttct gttgaattac gttaagcatg taataattaa catgtaatgc 5580
atgacgttat ttatgagatg ggtttttatg attagagtcc cgcaattata catttaatac 5640
gcgatagaaa acaaaatata gcgcgcaaac taggataaat tatcgcgcgc ggtgtcatct 5700
atgttactag atcgggaatt aaactatcag tgttgatggc atgaggcgat ggatctggat 5760
caaaccgaaa taggaacagg atggacggca tccccaatcg ccacgcaacg gttgacttta 5820
tttggctaaa gacaaaagaa actagaatag catattgatc tctgtcaaat atttaaaatg 5880
aagctttcgt tacttcaaaa atcatgtttt agttgttaaa attagttgta ggcatctaaa 5940
cagtttagct aatagttcaa ctattaccta cttttagcaa aacggctaat agttagccaa 6000
ctatttgtta gctacctaat tacattagta actttttagc caacttagag caactctaaa 6060
agaagcccta aactatccca aaattaatat tag 6093
<210> 6
<211> 17
<212> DNA
<213> Zea mays
<400> 6
agcacgcctg catctgt 17
<210> 7
<211> 17
<212> DNA
<213> Artificial Synthesis (unknown)
<400> 7
cccgtcaccg agatttg 17
<210> 8
<211> 18
<212> DNA
<213> Artificial Synthesis (unknown)
<400> 8
gacccgacaa acaagtgc 18
<210> 9
<211> 17
<212> DNA
<213> Zea mays
<400> 9
tgccgtccat cctgttc 17
Claims (9)
1. A nucleic acid molecule, wherein the sequence comprises any one of:
i) comprises the sequence shown in SEQ ID NO. 1 and/or SEQ ID NO. 2, or the complementary sequence thereof;
ii) comprises the sequence shown in SEQ ID NO. 3, or a complementary sequence thereof;
ii) comprises the sequence shown in SEQ ID NO. 4, or a complementary sequence thereof;
iii) comprises the sequence shown in SEQ ID NO. 5, or the complement thereof.
2. A probe for detecting a maize transformation event comprising the sequence shown as SEQ ID NO 1 or 2 or 3 or 4 or 5 or a fragment or variant or complement thereof.
3. A primer pair for detecting a corn transformation event, wherein the primer pair comprises:
a primer which specifically recognizes the nucleotide sequences 1 to 647 of the sequence shown by SEQ ID NO. 5 and a primer which specifically recognizes the nucleotide sequences 648-5737 of the sequence shown by SEQ ID NO. 5; and/or
A primer which specifically recognizes the nucleotide sequence at position 648-5737 of the sequence shown by SEQ ID NO. 5 and a primer which specifically recognizes the nucleotide sequence at position 5738-6093 of the sequence shown by SEQ ID NO. 5;
optionally, the amplification product of the primer pair comprises the sequence of claim 2;
optionally, the primer pair is a sequence shown in SEQ ID NO. 6 and SEQ ID NO. 7 or a complementary sequence thereof; or the sequences shown in SEQ ID NO 8 and SEQ ID NO 9 or the complementary sequences thereof.
4. A kit or microarray for detecting a corn transformation event comprising the probe of claim 2 and/or the primer pair of claim 3.
5. A method for detecting a corn transformation event, comprising detecting the presence of said transformation event in a test sample using:
i) the probe of claim 2;
ii) the primer pair of claim 3;
iii) the probe of claim 2 and the primer pair of claim 3; or
iv) the kit or microarray of claim 4.
6. A method of breeding maize, comprising the steps of:
1) obtaining maize comprising the nucleic acid molecule of claim 1;
2) subjecting the corn obtained in step 1) to pollen culture, unfertilized embryo culture, doubling culture, cell culture, tissue culture, selfing or crossing or a combination thereof to obtain a corn plant, seed, plant cell, progeny plant or plant part; and optionally also (c) a second set of one or more of,
3) subjecting the progeny plant obtained in step 2) to nitrogen deficiency tolerance identification and/or yield trait identification and detecting the presence or absence of the transformation event therein using the method of claim 5.
7. A product made from the corn plant, seed, plant cell, progeny plant or plant part obtained by the method of claim 6, comprising a food, feed or industrial material.
8. A method for increasing maize yield and/or nitrogen deficiency tolerance comprising growing in soil at least one transgenic maize plant comprising in its genome the nucleic acid sequence of SEQ ID NO 5-2243-6093 or comprising in its genome the nucleic acid sequence of SEQ ID NO 5; the transgenic maize plants have high yield and/or nitrogen deficiency tolerance traits.
9. A method for increasing the yield and/or nitrogen deficiency tolerance of maize, characterized in that an expression cassette for a nitrogen-efficient gene expressing the sequence shown as nucleotides 2243-5439 of SEQ ID NO 5 is introduced into the maize genome.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2014164074A1 (en) * | 2013-03-13 | 2014-10-09 | Pioneer Hi-Bred International, Inc. | Enhanced nitrate uptake and nitrate translocation by over-expressing maize functional low-affinity nitrate transporters in transgenic maize |
| CN111206031A (en) * | 2020-03-11 | 2020-05-29 | 中国农业科学院生物技术研究所 | Nucleic acid sequence for detecting corn plant NAZ-4 and detection method thereof |
| CN111518941A (en) * | 2020-05-13 | 2020-08-11 | 四川农业大学 | Nucleic acid sequence for detecting corn plant AN1 and detection method thereof |
-
2020
- 2020-12-25 CN CN202011565284.0A patent/CN112481412B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2014164074A1 (en) * | 2013-03-13 | 2014-10-09 | Pioneer Hi-Bred International, Inc. | Enhanced nitrate uptake and nitrate translocation by over-expressing maize functional low-affinity nitrate transporters in transgenic maize |
| CN111206031A (en) * | 2020-03-11 | 2020-05-29 | 中国农业科学院生物技术研究所 | Nucleic acid sequence for detecting corn plant NAZ-4 and detection method thereof |
| CN111518941A (en) * | 2020-05-13 | 2020-08-11 | 四川农业大学 | Nucleic acid sequence for detecting corn plant AN1 and detection method thereof |
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