CN112574987A - Nucleic acid molecule for detecting corn plant ND6603 and detection method thereof - Google Patents
Nucleic acid molecule for detecting corn plant ND6603 and detection method thereof Download PDFInfo
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- CN112574987A CN112574987A CN202011563864.6A CN202011563864A CN112574987A CN 112574987 A CN112574987 A CN 112574987A CN 202011563864 A CN202011563864 A CN 202011563864A CN 112574987 A CN112574987 A CN 112574987A
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Abstract
The invention relates to a nucleic acid molecule for detecting a corn plant ND6603 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 ND6603 of the invention has a nitrogen deficiency tolerance trait, and the detection method can accurately and rapidly identify whether a biological sample contains a DNA molecule of the transgenic maize event ND 6603.
Description
Technical Field
The invention relates to the technical field of plant biology. In particular to a nucleic acid molecule for detecting a maize plant ND6603 and a detection method thereof, in particular to a transgenic maize event ND6603 with a nitrogen deficiency tolerance character, a nucleic acid molecule for detecting whether a biological sample contains a specific transgenic maize event ND6603 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 nitrogen deficiency tolerance character, a nucleic acid molecule for detecting a corn plant ND6603 and a detection method thereof. The transgenic maize event ND6603 exhibits nitrogen deficiency tolerance traits due to its higher nitrogen use efficiency, and the detection method can accurately and rapidly identify whether a biological sample contains the DNA molecule of a particular transgenic maize event ND 6603.
In order to realize the purpose, 56 ZmNRTT 1.1B and bar gene-transferred maize transformation events are obtained by transforming the pCAMBIA 1301-ZmNRTT 1.1B expression vector into maize y822 immature embryos by utilizing an agrobacterium infection technology. Among them, ND6603 shows stable generation inheritance, has outstanding nitrogen deficiency tolerance, and has good application prospect.
In order to characterize the identity of ND6603, the present 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 can specifically recognize 1 st to 316 th nucleotide sequences of the sequence shown in SEQ ID NO. 5 and a primer which can specifically recognize 317 th and 5420 th nucleotide sequences of the sequence shown in SEQ ID NO. 5; and/or
A primer for specifically recognizing the nucleotide sequence 317-5420 of the sequence shown by the SEQ ID NO. 5 and a primer for specifically recognizing the nucleotide sequence 5421-5950 of the sequence shown by the 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 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 ND6603 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 640 nucleotide sequence located near the insertion junction at the 5' end of the insertion sequence in the transgenic maize event ND6603, the SEQ ID NO 1 or its complement is composed of a 316 nucleotide maize left flank genomic DNA sequence (nucleotides 1-316 of SEQ ID NO 1), a 125 nucleotide pCAMBIA1301-ZmNRT1.1B construct left border DNA sequence (nucleotides 317-.
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 ND6603 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 its complement is a 1213 nucleotide sequence located near the insertion junction at the 3 'end of the insertion sequence in transgenic maize event ND6603, the SEQ ID NO:2 or its complement is composed of the DNA sequence at the 3' end of the second expression cassette for the 335 nucleotide nitrogen efficient use gene (nucleotides 1-335 of SEQ ID NO: 2), the pCAMBIA 1301-ZmNRTT 1.1B construct right border DNA sequence of 348 nucleotides (nucleotides 336-683 of SEQ ID NO: 2) and the genomic DNA sequence right flanking the maize integration site of 530 nucleotides (nucleotides 684-1213 of SEQ ID NO: 2), comprising the sequence identified as being the presence of the SEQ ID NO:2 or its complement as the instant transgenic maize event ND 6603.
5 or the complement thereof is a 5950 nucleotide long sequence characteristic of transgenic maize event ND6603, 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 6603.
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 nitrogen deficiency tolerance, comprising growing in soil at least one transgenic maize plant comprising in its genome the nucleic acid sequence at positions 1912-5950 of SEQ ID NO 5 or comprising in its genome SEQ ID NO 5; the transgenic corn plants have a nitrogen deficiency tolerance trait.
The invention also provides a method for improving the nitrogen deficiency tolerance of corn, which is characterized in that an expression cassette of a nitrogen-expressing efficient utilization gene with a sequence shown as 1912-5072 th nucleotide of SEQ ID NO. 5 is introduced into a corn genome
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 ND6603, and its progeny, said transgenic corn event ND6603 being a corn plant ND6603, comprising plants and seeds of transgenic corn event ND6603 and plant cells thereof or regenerable parts thereof, said plant parts of transgenic corn event ND6603 including, but not limited to, cells, pollen, ovules, flowers, shoots, roots, stems, silks, inflorescences, ears, leaves, and products from corn plant ND6603 such as corn flour, corn oil, corn steep liquor, corn silks, corn starch and biomass left in the field of corn crops.
The transgenic maize event ND6603 of the present invention comprises a DNA construct which when expressed in a plant cell, acquires the nitrogen deficiency tolerance trait of said transgenic maize event ND 6603. The DNA construct comprises an expression cassette comprising a suitable promoter for expression in plants operably linked to a nucleic acid molecule encoding the maize nitrate transporter gene ZmNRTT 1.1B, which ZmNRTT 1.1B is 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 "xenogenic" 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 ND6603 tolerant to nitrogen deficiency by: first sexually crossing a first parent corn plant consisting of a corn plant bred from a transgenic corn event ND6603 and its progeny obtained by transformation using the nitrogen high efficiency utilization expression cassette of the present invention with a second parent corn plant lacking nitrogen deficiency tolerance, thereby producing diverse first generation progeny plants; progeny plants tolerant to nitrogen deficiency are then selected and maize plants tolerant to nitrogen deficiency can be grown. These steps can further include backcrossing the nitrogen-deficient tolerant progeny plant 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 in transgenic corn event ND 6603) to produce a nitrogen-deficient tolerant corn plant.
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 strand of DNA from the genome of transgenic corn event ND6603, whether the genomic DNA is from transgenic corn event ND6603 or seed or a plant or seed or extract derived from transgenic corn event ND 6603. 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 ND6603 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 ND6603 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 with a transgenic corn event ND6603 of the present invention, or whether a corn sample collected from a field comprised a transgenic corn event ND6603, or whether a corn extract, such as meal, flour, or oil, comprised a transgenic corn event ND6603, 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 6603. 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 maize event ND 6603.
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 ND6603 can be amplified by the use of the provided primer sequences to the genome of transgenic maize event ND6603 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 ND6603, 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 ND6603, 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 ND6603 based on hybridization principles may also include Southern blot hybridization, Northern blot hybridization and in situ hybridization. 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 the transgenic corn event ND6603 in a sample and can also be used to cultivate corn plants containing DNA of the transgenic corn event ND 6603. 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 ND6603 left flanking genomic region located 5' to the transgenic insert, a portion of the insert from the left border region of the vector, a first expression cassette consisting of the 35S promoter of cauliflower mosaic virus (CaMV 35S promoter), operably linked to the glufosinate (glufosinate) resistance gene sequence (bar), and operably linked to 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 ZmNRTT 1.1B, and operably linked to the nopaline synthase gene terminator (NOs terminator), a portion of the insertion sequence from the right border region of the vector, and the maize plant ND6603 right wing genomic region located 3' of the transgene insertion sequence (SEQ ID NO: 5). In the DNA amplification method, the DNA molecule that serves as a primer can be any portion derived from the transgene insertion sequence in transgenic maize event ND6603, or can be any portion derived from the DNA region flanking the maize genome in transgenic maize event ND 6603.
Transgenic corn event ND6603 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 maize event ND6603 of the present invention, can provide improved hybrid transgenic maize varieties with a variety of 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 ND6603 has a nitrogen deficiency tolerance character. The corn plant with the character expresses a nitrate transport protein ZmNRT1.1B protein, which endows the plant with a nitrogen efficient utilization character so as to increase a nitrogen deficiency tolerance character (a herbicide resistance gene bar is used as a screening marker of a transformation stage). Meanwhile, in the detection method, the nucleic acid molecules 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 DNA primers or probes to generate an amplification product diagnosed as the transgenic corn event ND6603 or the progeny thereof, and the presence of plant material derived from the transgenic corn event ND6603 can be identified quickly, 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-ZmNRT1.1B. 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.
ZmNRT1.1B maize nitrate transporter gene.
A promoter of a ubiquitin promoter maize ubiquitin 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: as a result of the amplification of the 5' end, the expected size of the fragment is 593bp (SEQ ID NO: 3); b: as a result of amplification of the 3' end, the expected size of the fragment was 1142bp (SEQ ID NO: 4). M: DL2000 DNA marker; t: ND6603 corn material; CK: non-transgenic maize.
Detailed Description
The transformation event ND6603 referred to herein refers to a maize plant that has been genetically transformed with maize inbred line Y822 as recipient to obtain an exogenous gene insert (T-DNA insert) inserted between specific genomic 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 in nucleotide 317 and 5420 of SEQ ID NO. 5. Transformation event ND6603 may refer to this transgenic process, may also refer to the combination of the T-DNA insert and flanking sequences within the genome resulting from this process, or may refer to the 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 ND6603 can also refer to progeny plants resulting from vegetative propagation, sexual propagation, doubling or doubling of the above plants, or a combination thereof.
Example 1 acquisition and screening of transformation events
The cDNA sequence of ZmNRT1.1B gene is cloned from corn, and is inserted into an intermediate vector pCAMBIA1301-Ubi through enzyme digestion connection, the intermediate vector contains bar gene, and finally the intermediate vector is constructed into a pCAMBIA1301-ZmNRT1.1B 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 56 corn transformation events of ZmNRTT 1.1B and bar genes are obtained. The transgenic corn event ND6603 contains exogenous genes ZmNRT1.1B and bar, shows stable generation inheritance and outstanding nitrogen deficiency tolerance, and has a good application prospect.
The detection method of the exogenous ZmNRT1.1B 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 ZmNRTT 1.1B and bar, so as to respectively obtain the materials of 470bp (ZmNRTT 1.1B gene) and 441bp (bar gene) bands as plants containing the ZmNRTT 1.1B gene and the bar gene.
TABLE 2 PCR detection primer sequences for the ZmNRT1.1B and bar genes
Example 2 nitrogen-efficient utilization and nitrogen deficiency tolerance characterization of transformation event ND6603
The corn ZmNRT1.1B protein has the function of promoting nitrate absorption. According to the invention, a constitutive expression mode is adopted to express ZmNRT1.1B protein in corn, and the nitrogen utilization efficiency of corn is improved by improving the absorption capacity of corn nitrate, so that the tolerance under the condition of nitrogen deficiency is finally improved. The invention identifies the field growth form and physiological characters of ND6603 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, ear length and hundred grain weight average of the positive plant of the corn ND6603 are obviously higher than that of the negative plant under the condition of low medium nitrogen supply, and the yield increasing effect is obvious. Specifically, the weight of the spike is increased by 19.3 percent under the condition of low nitrogen, the length of the spike is increased by 18.1 percent, the row number of the spike is increased by about 1 row, and the weight of the hundred grains is increased by 7.3 percent. The ear weight and ear length increase 5.6% and 5.6% under the condition of medium nitrogen. There were no significant differences between positive and negative plants under high nitrogen conditions (see table 3 for details). The yield of ND6603 negative plants under low nitrogen (nitrogen deficiency) conditions was only 46% (104.13/225.78) under high nitrogen (nitrogen sufficient) conditions, whereas the yield of ND6603 positive plants under low nitrogen (nitrogen deficiency) conditions reached 62% (144.26/231.06) under high nitrogen (nitrogen sufficient) conditions, indicating that ND6603 positive plants were better tolerant to nitrogen deficiency conditions.
TABLE 3 comparison of agronomic trait identification of ND6603 and controls
From the results of the total nitrogen content of the plant stalks under different nitrogen conditions, the ND6603 has the total nitrogen content lower than that of a receptor control material under different nitrogen conditions. Especially under high nitrogen conditions, the control stalk total nitrogen content was 3.00g/kg, while ND6603 total nitrogen content was only 1.90g/kg (Table 4). This indicates that the nitrogen utilization efficiency of ND6603 is higher than that of the wild-type control, which is why ND6603 is produced in a higher amount.
TABLE 4 measurement of total nitrogen content of plant stalks by ND6603 and acceptor control
Example 3 flanking sequences and maize genomic insertion sites of the exogenous sequence of transformation event ND6603
To clarify the identity of transformation event ND6603, the present invention further identifies the insertion site of the ND6603 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 6603. The third generation sequencing technology re-sequenced the transformant ND6603 by using the Nanopore sequencing technology of Oxford Nanopore Technologies, ONT for short, in the UK, with a sequencing depth of 10 times, an average read length of 20kb, a maximum read length of 60kb, and an accuracy of 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 33kb contained the target sequence, indicating that the foreign sequence in ND6603 was a single copy insertion. The flanking sequences of the genome of the exogenous sequence of 316bp at the 5 'end and 530bp at the 3' end obtained by sequencing are respectively 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 8, 935586-.
And at the left boundary of the insertion site, 316bp 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 siteDesigning a primer pair by taking 530bp and an exogenous insertion sequence on a genome, so that an F primer can be combined with the exogenous insertion sequence, an R primer can be combined with the genome sequence, and an amplification product is fused with a part of the exogenous insertion sequence and a part of a corn genome sequence. And performing PCR detection on ND6603 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 ND6603 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 sequence and the insertion position to verify the foreign fragment insertion position. The results are shown in FIG. 2. The results demonstrated that the ND6603 foreign fragment was stably inserted into chromosome 8 at position 935586-935694.
Example 4 detection method of transformation event ND6603
Breeding can be performed from transgenic maize event ND6603 and a new variety developed to produce, for example, an agricultural or commercial product. The agricultural or commodity product is expected to contain a nucleotide sequence capable of diagnosing the presence of transgenic maize event ND6603 material in the agricultural or commodity product if a sufficient amount is detected 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 ND6603 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 6603.
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 to be transgenic ND6603 and non-transgenic corn y 822. Detecting the specific boundary sequence in the ND6603 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 ND6603 transformation event. Moreover, the PCR method can track the existence of transformation events, thereby being applied to breeding work.
In summary, the transgenic maize event ND6603 of the present invention has a higher tolerance to nitrogen deficiency, and the detection method can accurately and rapidly identify whether a biological sample contains the DNA molecule of the transgenic maize event ND 6603.
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
<110> Jilin province academy of agricultural sciences
<120> nucleic acid molecule for detecting corn plant ND6603 and detection method thereof
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acaaacacgg atctaacaca aacatgaaca gaagtagaac taccgggccc taaccatgga 240
ccggaacgcc gatctagaga aggtagagag gggggggggg ggaggacgag cggcgtacct 300
tgaagcggag gtgccgacgg gtggatttgg gggagttgtg ggaccactcg atttccccga 360
tcgttcaaac atttggcaat aaagtttctt aagattgaat cctgttgccg gtcttgcgat 420
gattatcata taatttctgt tgaattacgt taagcatgta ataattaaca tgtaatgcat 480
gacgttattt atgagatggg tttttatgat tagagtcccg caattataca tttaatacgc 540
gatagaaaac aaaatatagc gcgcaaacta ggataaatta tcgcgcgcgg tgtcatctat 600
gttactagat cgggaattaa actatcagtg tttagcagct cggtgaagtc gctgctgtcg 660
gacctgctag gcagcgctgt tagcaagcaa gaacccggag acaaggttga gcttgaatga 720
gaaggaaccg ctcacatggt ggcgggggtc ggagctcgca gctgggagag gacagaggac 780
acacgcttgg cttgatgaag gtggatgagc aaagcactgg gaactctcgc ggctgctgct 840
tggctttgtg tagactgtgg agtgctacag gagggaaggt cgtcgggaga acggccaggc 900
ggtggtgcac gcgcggacga ggaaacaccc gccccagcag gccgccttcc cgcccttcac 960
cttcacctgc agcagcagag ctcaagcaca acaaaggcag cagccggtgc cgccgctgcc 1020
caagacggga ggaggcggcc tctttgccga cttctcctcg gccgccgccg gcctagacgc 1080
ccctcgtgac aacccgacct cgacgacacc gtgccgtgga tccactaccc catccccatc 1140
gt 1142
<210> 5
<211> 5950
<212> DNA
<213> Artificial Synthesis (unknown)
<400> 5
gtaacaagct tgtggttgtg ggatcagcca actgcgcaac gaaaaataat tgccactgta 60
tgcccaactc aactcctagc taggagagga agaagagtac aacgaagaga ctgtggcaag 120
cacagcacag tggatacaca cctagcttgg ccggtacaga gcaccgtgct tgcttgtcca 180
tttccgtcac agctagcgct tgcccaggtc ccccacctta tcaacaacaa ctgtgcccct 240
cctgcccccg ctcgccgcga gagctcccgt cccgacgacc ttccctcctg tagcactcta 300
cagtctacac caagcactgg gaacagagga cacacgcttg gcttgatgag gacacacgct 360
tggcttgatg acgcttagac aacttaataa cacattgcgg acgtttttaa tgtactgaat 420
taacgccgaa ttaattcggg ggatctggat tttagtactg gattttggtt ttaggaatta 480
gaaattttat tgatagaagt attttacaaa tacaaataca tactaagggt ttcttatatg 540
ctcaacacat gagcgaaacc ctataggaac cctaattccc ttatctggga actactcaca 600
cattattatg gagaaactcg agtcaaatct cggtgacggg caggaccgga cggggcggta 660
ccggcaggct gaagtccagc tgccagaaac ccacgtcatg ccagttcccg tgcttgaagc 720
cggccgcccg cagcatgccg cggggggcat atccgagcgc ctcgtgcatg cgcacgctcg 780
ggtcgttggg cagcccgatg acagcgacca cgctcttgaa gccctgtgcc tccagggact 840
tcagcaggtg ggtgtagagc gtggagccca gtcccgtccg ctggtggcgg ggggagacgt 900
acacggtcga ctcggccgtc cagtcgtagg cgttgcgtgc cttccagggg cccgcgtagg 960
cgatgccggc gacctcgccg tccacctcgg cgacgagcca gggatagcgc tcccgcagac 1020
ggacgaggtc gtccgtccac tcctgcggtt cctgcggctc ggtacggaag ttgaccgtgc 1080
ttgtctcgat gtagtggttg acgatggtgc agaccgccgg catgtccgcc tcggtggcac 1140
ggcggatgtc ggccgggcgt cgttctgggc tcatggtaga ctcgagagag atagatttgt 1200
agagagagac tggtgatttc agcgtgtcct ctccaaatga aatgaacttc cttatataga 1260
ggaaggtctt gcgaaggata gtgggattgt gcgtcatccc ttacgtcagt ggagatatca 1320
catcaatcca cttgctttga agacgtggtt ggaacgtctt ctttttccac gatgctcctc 1380
gtgggtgggg gtccatcttt gggaccactg tcggcagagg catcttgaac gatagccttt 1440
cctttatcgc aatgatggca tttgtaggtg ccaccttcct tttctactgt ccttttgatg 1500
aagtgacaga tagctgggca atggaatccg aggaggtttc ccgatattac cctttgttga 1560
aaagtctcaa tagccctttg gtcttctgag actgtatctt tgatattctt ggagtagacg 1620
agagtgtcgt gctccaccat gttggcaagc tgctctagcc aatacgcaaa ccgcctctcc 1680
ccgcgcgttg gccgattcat taatgcagct ggcacgacag gtttcccgac tggaaagcgg 1740
gcagtgagcg caacgcaatt aatgtgagtt agctcactca ttaggcaccc caggctttac 1800
actttatgct tccggctcgt atgttgtgtg gaattgtgag cggataacaa tttcacacag 1860
gaaacagcta tgacatgatt acgaattctc atgtttgaca gcttatcatc ggatctagta 1920
acatagatga caccgcgcgc gataatttat cctagtttgc gcgctatatt ttgttttcta 1980
tcgcgtatta aatgtataat tgcgggactc taatcataaa aacccatctc ataaataacg 2040
tcatgcatta catgttaatt attacatgct taacgtaatt caacagaaat tatatgataa 2100
tcatcgcaag accggcaaca ggattcaatc ttaagaaact ttattgccaa atgtttgaac 2160
gatcgatcca ctagtcaatc actagtgaat tcgagctcga gctcggtacc cggggatcct 2220
cagtggccga cggcaataga ctcctcgtct gcgagctcga tgccggcgtc ggccaggcgc 2280
ttctccttat agacgtagcc cctggcggcg aacgtgaaga ggacgaggtt gatggcgctg 2340
atgacggcga gcagccagta gaagtagtcg agcctcccgt cgtcgaggtt gtcggcgagc 2400
caaccgtcac ggccggcgtg ggccgtgacc ttgtgcacga tggtgacgag cagggtgctg 2460
aagaagaacc cgagcgcgca ggtgctgagg aacaggcccg tgctcatggt cttcatgccc 2520
ttggggcact cgcgcaggaa gaaggcgagc tggcccatgt acgtgaatgc ctcgcccgcg 2580
ccgacgagca cgaactgcgg catgagcagg aacaccgtga gcgtggcccc gtggccggag 2640
gccacctggc ggtggcgctc gacgagcgcg gccacggcca tgccggcgac ggagaggagg 2700
aggccgacga agacgcgctg cagcggggtg aggccgtgcg ggttgcccgt gaggcggcgg 2760
gcgaggggcg ccaggaggcg gtcgtagacg ggcacggtga gcaggatgga gccgatgagg 2820
aagacggtga gggagcccgc ggggatgagg aagcccgagc cgccgccgag ggcccggtcc 2880
atgacctcgg cctgcgccac cgagaaggtg gtcatctgcg cgtggatggt ccagaacatg 2940
atggtggtgg cccagatggg cagcatccgc accacctgct tcacctcctc cacgtccgtc 3000
cgcgtgcaca gcgcccactt gctcgccgcc ggtgacgact cgccgccgcc gtcgacgacg 3060
acgatggccg cgtggtccag gaacctgcat tccttgctgt ggggcagttt ctccttcccc 3120
ttgacgtcgg cgccggcggc ggccgcgtcg tccacgtcgt agagcatgtc cgggtcggac 3180
ggcagcggca gcgcgcgctt gctccaggcg gcggccgtca cggcggccac ctgggtgagc 3240
gggctcccca ccagcttcct gaaccggtac ctccgggtgc ccagcaggaa gacgcccagc 3300
ccgcacagga tgccgacggc gcagatgccg tagccccagc ggcggcccac gttgtcctgc 3360
acgtacacca gcaccgtgac ggccagcagc gcgccgatgc tgacgaagaa gtagaaccag 3420
ttgaagaagc gcagcatcct cttgcgctcg ccgccgtgcg cctcgtcgaa ctggtcggag 3480
ccgaagcccg acacgctgga cttgagcccg cccgtgccca gcgccgtcag gtacagaccc 3540
aggtatagca ccccgagctg cgtcccgttc gccggcacgc agtcggggct cgcccccttg 3600
gcgtccgcac acgccggcgg acgcagcccg ggagcggccg ttgagatcgt caggatcatc 3660
acccccgtgg cctggacggc ggtgaagatg gcgatggtga ggtagcggcc gaggtaggtg 3720
tcggcgacga acccgccgag gaggcagagc atgaaggaag ccccgatgaa gttggtgacg 3780
gtgttggcgg cggaggcatt gccgaggtgc atggtgccgg tcatgtacgg caccaggttc 3840
accgcgatgc ccagcgtcgt catccgctcg aacagctccg cgcctagtat catggcggcg 3900
cacgcccagc cgccggtggt ggcgcggcta gcggggcggc ccttgtagtc ccaggcgtcc 3960
gtcaaggcct tgccatccga cgcagtatcc ggcaggacgg aggccatgga tcctctagag 4020
tcgacctgca gaagtaacac caaacaacag ggtgagcatc gacaaaagaa acagtaccaa 4080
gcaaataaat agcgtatgaa ggcagggcta aaaaaatcca catatagctg ctgcatatgc 4140
catcatccaa gtatatcaag atcaaaataa ttataaaaca tacttgttta ttataataga 4200
taggtactca aggttagagc atatgaatag atgctgcata tgccatcatg tatatgcatc 4260
agtaaaaccc acatcaacat gtatacctat cctagatcga tatttccatc catcttaaac 4320
tcgtaactat gaagatgtat gacacacaca tacagttcca aaattaataa atacaccagg 4380
tagtttgaaa cagtattcta ctccgatcta gaacgaatga acgaccgccc aaccacacca 4440
catcatcaca accaagcgaa caaaaagcat ctctgtatat gcatcagtaa aacccgcatc 4500
aacatgtata cctatcctag atcgatattt ccatccatca tcttcaattc gtaactatga 4560
atatgtatgg cacacacata cagatccaaa attaataaat ccaccaggta gtttgaaaca 4620
gaattctact ccgatctaga acgaccgccc aaccagacca catcatcaca accaagacaa 4680
aaaaaagcat gaaaagatga cccgacaaac aagtgcacgg catatattga aataaaggaa 4740
aagggcaaac caaaccctat gcaacgaaac aaaaaaaatc atgaaatcga tcccgtctgc 4800
ggaacggcta gagccatccc aggattcccc aaagagaaac actggcaagt tagcaatcag 4860
aacgtgtctg acgtacaggt cgcatccgtg tacgaacgct agcagcacgg atctaacaca 4920
aacacggatc taacacaaac atgaacagaa gtagaactac cgggccctaa ccatggaccg 4980
gaacgccgat ctagagaagg tagagagggg ggggggggga ggacgagcgg cgtaccttga 5040
agcggaggtg ccgacgggtg gatttggggg agttgtggga ccactcgatt tccccgatcg 5100
ttcaaacatt tggcaataaa gtttcttaag attgaatcct gttgccggtc ttgcgatgat 5160
tatcatataa tttctgttga attacgttaa gcatgtaata attaacatgt aatgcatgac 5220
gttatttatg agatgggttt ttatgattag agtcccgcaa ttatacattt aatacgcgat 5280
agaaaacaaa atatagcgcg caaactagga taaattatcg cgcgcggtgt catctatgtt 5340
actagatcgg gaattaaact atcagtgttt agcagctcgg tgaagtcgct gctgtcggac 5400
ctgctaggca gcgctgttag caagcaagaa cccggagaca aggttgagct tgaatgagaa 5460
ggaaccgctc acatggtggc gggggtcgga gctcgcagct gggagaggac agaggacaca 5520
cgcttggctt gatgaaggtg gatgagcaaa gcactgggaa ctctcgcggc tgctgcttgg 5580
ctttgtgtag actgtggagt gctacaggag ggaaggtcgt cgggagaacg gccaggcggt 5640
ggtgcacgcg cggacgagga aacacccgcc ccagcaggcc gccttcccgc ccttcacctt 5700
cacctgcagc agcagagctc aagcacaaca aaggcagcag ccggtgccgc cgctgcccaa 5760
gacgggagga ggcggcctct ttgccgactt ctcctcggcc gccgccggcc tagacgcccc 5820
tcgtgacaac ccgacctcga cgacaccgtg ccgtggatcc actaccccat ccccatcgtc 5880
gacgaagcca gtcccgccgc gcctgccctg gcagatagct tcatcccaga tttcttctcg 5940
gagctgcatg 5950
<210> 6
<211> 18
<212> DNA
<213> Zea mays
<400> 6
aattgccact gtatgccc 18
<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
gaaaagggca aaccaaac 18
<210> 9
<211> 17
<212> DNA
<213> Zea mays
<400> 9
acgatgggga tggggta 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 can specifically recognize 1 st to 316 th nucleotide sequences of the sequence shown in SEQ ID NO. 5 and a primer which can specifically recognize 317 th and 5420 th nucleotide sequences of the sequence shown in SEQ ID NO. 5; and/or
A primer for specifically recognizing the nucleotide sequence 317-5420 of the sequence shown by the SEQ ID NO. 5 and a primer for specifically recognizing the nucleotide sequence 5421-5950 of the sequence shown by the 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) performing nitrogen deficiency tolerance identification on the progeny plant obtained in step 2) 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 nitrogen deficiency tolerance, comprising growing in soil at least one transgenic maize plant comprising in its genome the nucleic acid sequence at position 1912-5950 of SEQ ID NO 5 or comprising in its genome SEQ ID NO 5; the transgenic corn plants have a nitrogen deficiency tolerance trait.
9. A method for improving nitrogen deficiency tolerance of corn, which is characterized in that an expression cassette of a nitrogen high efficiency utilization gene with a sequence shown as 1912-5072 nucleotides of SEQ ID NO. 5 is introduced into a corn 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 |
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2020
- 2020-12-25 CN CN202011563864.6A patent/CN112574987B/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 |
Non-Patent Citations (2)
| Title |
|---|
| "PREDICTED: Zea mays protein NRT1/ PTR FAMILY 6.3 (LOC103637664), mRNA", NCBI REFERENCE SEQUENCE: XM_008660202.3 * |
| ZHENGYU WEN ET AL.: "Maize NPF6 Proteins Are Homologs of Arabidopsis CHL1 That Are Selective for Both Nitrate and Chloride", THE PLANT CELL * |
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