A nucleic acid molecule for detecting maize plant nd7702 and a detection method thereof

Abstract

The present application relates to a kind of nucleic acid molecules for detecting corn ND7702, detection method and its application, the nucleic acid molecules of the corn ND7702 contain the sequence shown in the 1-7359 nucleotide of SEQ ID NO.3, the 584-7426 nucleotide of SEQ ID NO.5 and SEQ ID NO.4 or its reverse complement sequence.The present application corn ND7702 has high yield and / or nitrogen deficiency tolerance character, and detection method can accurately and quickly identify whether the DNA molecule of transgenic corn event ND7702 is contained in biological sample.

Description

Technical Field

[0001] This invention relates to the field of plant biotechnology. Specifically, it relates to a nucleic acid molecule for detecting ND7702 in maize plants and a method thereof, particularly to a transgenic maize event ND7702 with high yield and / or nitrogen deficiency tolerance traits, and a nucleic acid molecule for detecting whether a biological sample contains a specific transgenic maize event ND7702 and a method thereof. Background Technology

[0002] Nitrogen is one of the essential mineral nutrients for plants and is a necessary component for the synthesis of proteins, nucleic acids and many bioactive substances. Nitrogen deficiency usually leads to slow plant growth, chlorosis of mature leaves and anthocyanin accumulation (Diaz C, Saliba-Colombani V, Loudet O, et al. Leaf Yellowing and Anthocyanin Accumulation are Two Genetically Independent Strategies in Response to Nitrogen Limitation in Arabidopsis thaliana[J]. Plant & Cell Physiology, 2006, 47(1):74-83).

[0003] The extensive use of nitrogen fertilizer in agricultural production has greatly increased crop yields. Currently, approximately 80-90 million tons of nitrogen fertilizer are applied globally each year, and it is predicted that this will increase to 240 million tons by 2050 (Tilman D. Global environmental impacts of agricultural expansion: the need for sustainable and efficient practices[J]. Proc Natl Acad Sci US A. 1999, 96(11): 5995-6000). The increase in energy costs has led to a continuous rise in nitrogen fertilizer prices, increasing agricultural production costs while nitrogen fertilizer loss also damages the ecological environment. Based on the dual considerations of economic benefits and environmental protection, planting nitrogen-efficient crop varieties in modern agricultural production is an effective way to solve the problem of low nitrogen fertilizer utilization, reduce production costs, and reduce environmental pollution. It is also a basic requirement for sustainable agricultural development and enhancing the international competitiveness of products. Traditionally, breeding new varieties is a very long-term process. Nowadays, the continuous maturation of genetic engineering makes it possible for us to quickly obtain crop varieties with high nitrogen fertilizer utilization through genetic engineering.

[0004] Maize ZmNRT1.1A and ZmNRT2.1A encode two different nitrate transport proteins, both of which promote the uptake of nitrate nitrogen by maize roots, thereby improving the nitrogen fertilizer use efficiency of maize plants. (Quaggiotti S, Ruperti B, Pizzeghello D, et al. Effect of low molecular size humic substances on nitrate uptake and expression of genes involved in nitrate transport in maize (Zea mays L.)[J]. Journal of Experimental Botany, 2004, 55(398): 816-823; Quaggiotti S, Ruperti B, Borsa P, et al. Expression of a putative high-affinity NO3–transporter and of a H+-ATPase in relation to whole plant nitrate transport physiology in two maize genotypes differently responsive to low nitrogen availability ... Botany, 2003, 57, 1023–1031. Researchers overexpressed the ZmNRT1.1A gene in maize plants and disclosed three different transgenic maize transformation events, ND4401, ND4403, and ND6603 (patent application numbers 2020115519757, 2020115652840, and 2020115638646, respectively), to improve maize's tolerance to nitrogen deficiency under low-nitrogen conditions. However, ND4401, ND4403, and ND6603 are more suitable for planting under low-nitrogen conditions, and their effects on nitrogen use efficiency and yield improvement are not significant under medium- and high-nitrogen planting conditions in actual maize production.

[0005] It is known that the expression of exogenous genes in plants is influenced by their chromosomal location, possibly due to the proximity of chromatin structures (such as heterochromatin) or transcriptional regulatory elements (such as enhancers) to the integration site. Therefore, screening a large number of events is often required to identify commercially viable events (i.e., events where the introduced target gene is optimally expressed). For example, significant differences in the expression levels of introduced genes have been observed between events in plants and other organisms; differences may also exist in spatial or temporal patterns of expression, such as the relative expression of transgenes in different plant tissues. This difference manifests as the actual expression pattern potentially differing from the expected expression pattern based on the transcriptional regulatory elements in the introduced gene construct, leading to variations in phenotypic expression of the transformation event. Therefore, it is often necessary to generate hundreds or thousands of different events and screen for a single event with the expected transgene expression levels and patterns for commercial purposes. Events with the expected transgene expression levels and patterns can be used to introduce transgenes into other genetic backgrounds through sexual crossbreeding using conventional breeding methods. Offspring produced through this crossbreeding retain the transgene expression characteristics of the original transformation event. Applying this strategy can ensure reliable gene expression in many varieties that are well adapted to local growing conditions. Therefore, more transformation events need to be identified and screened to obtain superior transformation events with excellent overall phenotypic performance and commercial potential.

[0006] Being able to detect the presence of specific events to determine whether the offspring of sexual hybridization contain the target gene would be beneficial. Furthermore, methods for detecting specific events would help comply with relevant regulations, such as the requirement for formal approval and labeling of foods derived from recombinant crops before they can be placed on the market. Detecting the presence of transgenes using any well-known polynucleotide detection method is possible, such as polymerase chain reaction (PCR) or DNA hybridization using polynucleotide probes. These methods typically focus on commonly used genetic elements, such as promoters, terminators, and marker genes. Therefore, unless the sequence of the chromosomal DNA adjacent to the inserted transgene DNA (“flanking DNA”) is known, the above methods cannot be used to distinguish different events, especially those produced using the same DNA construct. Therefore, it is currently common to use a pair of primers spanning the junction of the inserted transgene and the flanking DNA via PCR to identify transgene-specific events; specifically, a first primer containing the flanking sequence and a second primer containing the inserted sequence. Summary of the Invention

[0007] This invention overexpresses the ZmNRT2.1 gene in maize and measures the nitrogen deficiency tolerance of transgenic maize plants under different nitrogen conditions. The results are compared with those of the ZmNRT1.1A gene transgenic maize events ND4401, ND4403, and ND6603. The aim is to obtain a maize transformation event and its detection method that exhibits superior yield traits and / or nitrogen deficiency tolerance traits in actual production applications (medium to high nitrogen conditions).

[0008] To achieve the above objectives, this invention utilizes Agrobacterium-mediated transformation technology to convert the pCAMBIA1301-ZmNRT2.1A expression vector into the immature embryos of maize Y822, resulting in 29 transformation events involving the ZmNRT2.1A and bar genes. Among these, ND7702 exhibits stable generational inheritance, strong nitrogen deficiency tolerance under low-nitrogen conditions, and significant yield-increasing effects under medium-to-high nitrogen planting conditions, demonstrating promising application prospects.

[0009] To characterize ND7702, the present invention provides a nucleic acid molecule comprising the sequence shown in SEQ ID NO.1 and / or SEQ ID NO.2, or their complementary sequence.

[0010] Furthermore, the nucleic acid molecule sequence comprises the sequence shown in SEQ ID NO.3, or its complementary sequence.

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

[0012] On the other hand, the present invention provides a probe for detecting maize transformation events, characterized in that it comprises the sequence shown in SEQ ID NO.1 or SEQ ID NO.2 or SEQ ID NO.3 or SEQ ID NO.4 or SEQ ID NO.6, a fragment thereof, a variant thereof, or a complementary sequence thereof.

[0013] The present invention also provides primer pairs for detecting maize transformation events, characterized in that the amplification products of the primer pairs contain the sequence shown in SEQ ID NO.1 or SEQ ID NO.2 or SEQ ID NO.3 or SEQ ID NO.4 or SEQ ID NO.6, or a fragment thereof, or a variant thereof, or an inverse complementary sequence thereof.

[0014] In some embodiments, the primer pairs described above are the sequences shown in SEQ ID NO.7 and SEQ ID NO.8 or their complementary sequences.

[0015] The present invention also provides a kit or microarray for detecting maize transformation events, characterized in that it comprises the probes and / or primer pairs described above.

[0016] The present invention also provides a method for detecting maize transformation events, characterized in that it includes using the above-described probe or primer pair or probe and primer pair or kit or microarray to detect whether the transformation event exists in the sample to be tested.

[0017] The present invention also provides a method for breeding maize, characterized in that the method includes the following steps:

[0018] 1) Obtain corn containing the above-mentioned nucleic acid molecules;

[0019] 2) Obtain maize plants, seeds, plant cells, progeny plants, or plant parts by means of pollen culture, unfertilized embryo culture, doubling culture, cell culture, tissue culture, self-pollination, or hybridization, or a combination thereof, using the maize obtained in step 1). Optionally,

[0020] 3) The progeny plants obtained in step 2) are subjected to yield trait identification and / or nitrogen deficiency tolerance identification, and the above methods are used to detect whether the transformation event exists therein.

[0021] Furthermore, the present invention also provides articles made from corn plants, seeds, plant cells, progeny plants or plant parts obtained by the above method, including food, feed or industrial raw materials.

[0022] The SEQ ID NO.1 is a 22-nucleotide sequence located near the insertion junction at the 5' end of the inserted sequence in transgenic maize event ND7702. The SEQ ID NO.1 spans the left wing genomic DNA sequence of the maize insertion site and the DNA sequence at the 5' end of the left boundary of the inserted sequence. The presence of the SEQ ID NO.1 or its reverse complementary sequence can be used to identify the existence of transgenic maize event ND7702.

[0023] The SEQ ID NO.2 is a 22-nucleotide sequence located near the insertion junction at the 3' end of the inserted sequence in transgenic maize event ND7702. The SEQ ID NO.2 spans the DNA sequence at the 3' end of the right boundary of the inserted sequence and the right wing genomic DNA sequence of the maize insertion site. The presence of the SEQ ID NO.2 or its reverse complementary sequence can be used to identify the transgenic maize event ND7702.

[0024] In this invention, the nucleic acid sequence can be at least 11 or more consecutive polynucleotides (first nucleic acid sequence) of any portion of the transgenic insertion sequence in SEQ ID NO.3 or its reverse complementary sequence, or at least 11 or more consecutive polynucleotides (second nucleic acid sequence) of any portion of the 5' left-wing maize genomic DNA region in SEQ ID NO.3 or its reverse complementary sequence. The nucleic acid sequence can further be a portion homologous to or reverse complementary to a portion of SEQ ID NO.3 containing the complete SEQ ID NO.1. When the first and second nucleic acid sequences are used together, these nucleic acid sequences include a DNA primer pair in the DNA amplification method for generating the amplification product. When the amplification product generated in the DNA amplification method using the DNA primer pair is an amplification product including SEQ ID NO.1 or SEQ ID NO.3 or its reverse complementary sequence, the presence of transgenic maize event ND7702 or its progeny can be diagnosed.

[0025] SEQ ID NO.3 is a 7922-nucleotide sequence located near the insertion junction at the 5' end of the inserted sequence in transgenic maize event ND7702. SEQ ID NO.3 consists of an 842-nucleotide left-wing genomic DNA sequence of maize (nucleotides 1-842 of SEQ ID NO.3), a 6517-nucleotide vector backbone sequence (nucleotides 843-7359 of SEQ ID NO.3), and a 563-nucleotide partial DNA sequence of the first expression cassette of the bar gene (nucleotides 7360-7922 of SEQ ID NO.3). The presence of SEQ ID NO.3 or its reverse complementary sequence is sufficient to identify the transgenic maize event ND7702.

[0026] The nucleic acid sequence may be at least 11 or more consecutive polynucleotides (third nucleic acid sequence) of any portion of the transgenic insertion sequence in SEQ ID NO.4 or its reverse complementary sequence, or at least 11 or more consecutive polynucleotides (fourth nucleic acid sequence) of any portion of the 3' right-wing maize genomic DNA region in SEQ ID NO.4 or its reverse complementary sequence. The nucleic acid sequence may further be homologous to or reverse complementary to a portion of SEQ ID NO.4 containing the complete SEQ ID NO.2 or SEQ ID NO.7. When the third and fourth nucleic acid sequences are used together, these nucleic acid sequences include a DNA primer set in the DNA amplification method that generates the amplification product. When the amplification product generated in the DNA amplification method using the DNA primer pair is an amplification product including SEQ ID NO.2, SEQ ID NO.4, or SEQ ID NO.7 or its reverse complementary sequence, the presence of transgenic maize event ND7702 or its progeny can be diagnosed.

[0027] SEQ ID NO.4 is a 7426-nucleotide sequence located near the insertion junction at the 3' end of the inserted sequence in transgenic maize event ND7702. SEQ ID NO.4 consists of a partial DNA sequence of the second expression cassette of the ZmNRT2.1A gene (nucleotides 1-289 of SEQ ID NO.4), a DNA sequence of the right boundary of the pCAMBIA1301-ZmNRT2.1A construct (nucleotides 290-583 of SEQ ID NO.4), a vector backbone sequence of 6557 nucleotides (nucleotides 584-7140 of SEQ ID NO.3), and a genomic DNA sequence of the right wing of the maize integration site (nucleotides 7141-1426 of SEQ ID NO.4). The presence of SEQ ID NO.4 or its reverse complementary sequence is sufficient to identify the transgenic maize event ND7702.

[0028] The sequences shown in SEQ ID NO. 3 (nucleotides 1-7359) and SEQ ID NO. 5 and SEQ ID NO. 4 (nucleotides 584-7426) are full-length insertion sequences (including exogenous insertion sequences and upstream and downstream flanking genomic sequences) of 19055 nucleotides characterizing transgenic maize event ND7702. The specific genomic and genetic elements contained therein are shown in Table 1. The presence of the above full-length insertion sequence or its reverse complementary sequence is sufficient to identify the transgenic maize event ND7702.

[0029] Table 1. Genome and genetic elements contained in the full-length insert sequence.

[0030]

[0031] 1: Unit: bp.

[0032] As is well known to those skilled in the art, the first and second nucleic acid molecules, or the third and fourth nucleic acid molecules, need not consist solely of DNA; they may also include RNA, a mixture of DNA and RNA, or a combination of DNA, RNA, or other nucleotides or analogues that do not serve as templates for one or more polymerases. Furthermore, the probes or primers described in this invention should be at least approximately 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22 consecutive nucleotides in length, which may be selected from the nucleotides described in SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, or SEQ ID NO. 4. When selected from the nucleotides shown in SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, or SEQ ID NO. 4, the probes and primers may be at least approximately 21 to approximately 50 or more consecutive nucleotides in length.

[0033] The present invention also provides a method for improving maize yield and / or nitrogen deficiency tolerance, characterized in that it includes planting at least one transgenic maize plant in soil, wherein the transgenic maize plant contains the nucleic acid sequence of SEQ ID NO. 5 from position 1571 to 4853 in its genome, or the genome of the transgenic maize plant contains nucleotides 1-7359 of SEQ ID NO. 3 and nucleotides 584-7426 of SEQ ID NO. 5 and SEQ ID NO. 4; the transgenic maize plant has high yield and / or nitrogen deficiency tolerance traits.

[0034] The present invention also provides a method for improving maize yield and / or nitrogen deficiency tolerance, characterized in that an expression cassette of a nitrogen-efficient utilization gene, as shown in the sequence of nucleotides 1571-4559 of SEQ ID NO.5, is introduced into the maize genome.

[0035] In the present invention for detecting nucleic acid molecules in maize plants and the detection method thereof, the following definitions and methods are intended to better define the present invention and guide those skilled in the art to implement it. Unless otherwise stated, the terms shall be understood according to their conventional usage by those skilled in the art.

[0036] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0037] Figure 1 A schematic diagram of the binding site between the transgenic insertion sequence and the maize genome.

[0038] Figure 2 This is a physical spectrum of the plant expression vector pCAMBIA1301-ZmNRT2.1. The English names and abbreviations of each element are listed below:

[0039]

[0040] Figure 3 Results of ZmNRT2;1 gene amplification in transgenic maize ND7702 and negative control maize Y822.

[0041] M: DNA marker, band size is indicated next to it; 1: blank control; 2: Y822; 3: positive control plasmid; 4-9: ND7702.

[0042] Figure 4 Results of bar gene amplification in transgenic maize ND7702 and non-transgenic maize Y822.

[0043] M: DNA marker, band size is indicated next to it; 1: blank control; 2: Y822; 3: positive control plasmid; 4-9: ND7702.

[0044] Figure 5 PCR results of the right flanking sequence specificity of transgenic maize ND7702.

[0045] M: DNA marker, band size is indicated next to it; T: ND7702; CK: Y822. Detailed Implementation

[0046] The transformation event ND7702 involved in this application refers to maize plants obtained through genetic transformation using the maize inbred line Y822 as the recipient, resulting in the insertion of a foreign gene insert (T-DNA insert) between specific genomic sequences. In a specific embodiment, the expression vector used for the transgenic process has... Figure 2 The physical map shown indicates that the obtained T-DNA insert has the sequence shown in SEQ ID NO. 3 (nucleotides 843-7359) and SEQ ID NO. 5 and SEQ ID NO. 4 (nucleotides 584-7140). Transformation event ND7702 can refer to this transgenic process, the combination of the T-DNA insert and flanking sequences within the genome obtained by this process, or the maize plant obtained by this transgenic process. In specific instances, this event also applies to plants obtained by transforming other recipient varieties with the same expression vector, thereby inserting the T-DNA insert into the same genomic location. Transformation event ND7702 can also refer to offspring plants obtained by asexual reproduction, sexual reproduction, doubling or multiplication, or a combination thereof, from the aforementioned plants.

[0047] Example 1: Acquisition of Transformation Events and Identification of Traits

[0048] The cDNA sequence of the ZmNRT2.1A gene was cloned from maize, digested with enzymes, and inserted into the intermediate vector pCAMBIA1301-Ubi. This intermediate vector already contains the bar gene, thus constructing the pCAMBIA1301-ZmNRT2.1A expression vector. The physical map of the vector is shown below. Figure 2 Recombinant plasmids were efficiently transformed into the immature embryos of the self-selected maize line Y822 at the Institute of Agricultural Biotechnology, Jilin Academy of Agricultural Sciences, using Agrobacterium-mediated transformation. Twenty-nine transformation events yielded maize transgenic ZmNRT2.1A and bar genes. Among them, the transgenic maize event ND7702, containing the exogenous genes ZmNRT2.1A and bar, exhibited stable generational heritability and outstanding yield traits and nitrogen deficiency tolerance, showing promising application prospects.

[0049] The detection methods for exogenous ZmNRT2.1A and bar genes were as follows: using maize genomic DNA as a template, PCR amplification was performed using specific primer pairs for ZmNRT2.1A and bar (Table 2). Materials yielding bands of 834 bp (ZmNRT2.1A gene) and 441 bp (bar gene), respectively, were considered plants containing the ZmNRT2.1A and bar genes. The electrophoresis results of the PCR amplification products are shown below. Figure 3 and Figure 4 As shown.

[0050] Table 2 Primer sequences for PCR detection of ZmNRT2.1A and bar genes.

[0051]

[0052] Example 2: Identification of nitrogen-efficient utilization and yield traits in conversion event ND7702

[0053] The maize ZmNRT2.1A protein promotes nitrate uptake. This invention uses a constitutive expression approach to express the ZmNRT2.1A protein in maize, thereby enhancing the maize's nitrate uptake capacity, improving nitrogen use efficiency, and ultimately increasing yield. This invention also identified the field growth morphology and physiological traits of ND7702 plants under different nitrogen fertilizer gradients.

[0054] The experiment was conducted at the transgenic experimental base of the Jilin Academy of Agricultural Sciences in Gongzhuling City, Siping City, Jilin Province. The soil was sandy loam with flat terrain. The topsoil (20cm) contained 125.94 mg / kg of available nitrogen, 23.96 mg / kg of available phosphorus, 149.24 mg / kg of available potassium, and 25.22 g / kg of organic matter. The plots were 5m long, 0.60m apart, and 0.25m apart, with 6 rows per plot.

[0055] Test conditions: Referring to the "Regional Formula and Fertilization Recommendations for the Three Major Grain Crops (Wheat, Corn and Rice) (2013)" issued by the Ministry of Agriculture and Rural Affairs, the high nitrogen treatment (HN) was applied with 38 kg / mu of formula fertilizer according to the normal tillage fertilization rate; the medium nitrogen treatment (MN) was applied with 70% formula fertilizer, and the supplementation of phosphorus and potassium fertilizer was the same as the high nitrogen treatment; the low nitrogen treatment (LN) was not applied with formula fertilizer, and the supplementation of phosphorus and potassium fertilizer was the same as the high nitrogen treatment.

[0056] Experimental design: The experiment adopted a randomized block design. Ten maize plants with roughly the same growth were selected from each plot to measure relevant morphological and physiological indicators, including ear weight, ear length, ear row, 100-grain weight, and total nitrogen content in the aboveground parts of the plant.

[0057] 1. Identification of yield traits such as ear weight, ear length, ear row size, and 100-kernel weight in maize ND7702.

[0058] The results of the ear weight, ear length, ear row length, and 100-kernel weight measurements are shown in Table 3. The results show that the ear weight, ear length, and 100-kernel weight of the positive maize ND7702 plants were significantly higher than those of the negative control Y822 plants under low and medium nitrogen supply conditions, indicating a significant yield increase. Specifically, under low nitrogen conditions, ear weight increased by 15.3%, ear length by 11.6%, and 100-kernel weight by 6.7%. Under medium nitrogen conditions, ear weight increased by 5.7%, ear length by 5.8%, and 100-kernel weight by 7.4%. Under high nitrogen conditions, there was no significant difference compared to the control. The control plants, under nitrogen-deficient low-nitrogen and medium-nitrogen conditions, yielded 149.96 g and 277.36 g per ear, respectively, representing 50.11% and 92.68% of the yields under nitrogen-sufficient high-nitrogen conditions. In contrast, the ND7702 positive plants, under low-nitrogen and medium-nitrogen conditions, yielded 172.92 g and 293.20 g per ear, respectively, representing 57.74% and 97.90% of the yields under high-nitrogen conditions. These results indicate that the ND7702 positive plants exhibit significantly higher tolerance to nitrogen deficiency than the control.

[0059] Table 3 Comparison of agronomic traits between ND7702 and the control

[0060]

[0061]

[0062] 2. Identification of total nitrogen content in the stems of maize ND7702 plants

[0063] The results of total nitrogen content in plant stems under different nitrogen conditions (Table 4) show that the total nitrogen content in the stems of the ND7702 transformant was lower than that of the control material Y822 under all nitrogen conditions. Especially under high nitrogen conditions, the total nitrogen content in the stems of the control was 3.0 g / kg, while that of the ND7702 transformant was only 1.94 g / kg, significantly higher than the control. This indicates that ND7702 maize can still ensure sufficient nitrogen absorption for normal plant growth even under low nitrogen soil conditions, with more nitrogen being transported to the grains to increase yield.

[0064] Table 4. Determination of total nitrogen content in the stems of ND7702 and the receptor control Y822.

[0065]

[0066] Example 2: Flanking sequences of the exogenous sequence in transformation event ND7702 and the insertion site in the maize genome.

[0067] To clarify the identity of the transformation event ND7702, this invention further identified the insertion site of the exogenous sequence of ND7702 in the maize genome.

[0068] The invention employs third-generation sequencing to locate the T-DNA insertion site in transgenic maize ND7702. The third-generation sequencing technology utilizes Oxford Nanopore Technologies (ONT) nanopore sequencing technology to re-sequencing the ND7702 transformant, achieving a 10-fold sequencing depth, an average read length of 20kb, a maximum read length of 60kb, and an accuracy of 85%.

[0069] BLAST search and alignment of the third-generation sequencing results with the exogenous T-DNA sequence revealed a 44kb contg containing the target sequence, indicating that the exogenous T-DNA sequence in ND7702 was a single-copy insertion. The 5' end 842bp and 3' end 286bp flanking sequences of the obtained exogenous sequence were retrieved from the public maize genome database (…). (http: / / ensembl.gramene.org / Zea_mays / Info / Annotation / #assembly) with Zm-B73- REFERENCE-NAM-5.0 BLAST nucleic acid-nucleic acid alignment was performed using the reference genome, and the foreign sequence was found to be integrated at position 84393783 in maize Chr05.

[0070] Primer pairs were designed at the right boundary of the insertion site, using a 286 bp section of the genome and the exogenous insertion sequence. Primer F was designed to bind to the genome sequence, and primer R to bind to the exogenous insertion sequence. The amplification product fused a portion of the maize genome sequence and a portion of the exogenous insertion sequence. PCR detection of ND7702 was performed using the designed primers. PCR amplification was performed using genomic DNA from transgenic maize lines as a template. The PCR reaction was carried out in a 20 μL system. The amplification cycle program was: 95℃ pre-denaturation for 5 min; 98℃ denaturation for 10 s, 62℃ annealing for 30 s, 72℃ extension for 60 s, 35 cycles; 72℃ extension for 5 min.

[0071] Based on the flanking sequence and insertion site results, PCR amplification of the ND7702 transformation event was performed using the downstream primer of the genome (SEQ ID NO.8) and the upstream primer of the vector's right boundary (SEQ ID NO.7) to verify the insertion site of the exogenous fragment. Results are shown below. Figure 5 The results showed that the exogenous fragment ND7702 was stably inserted into the maize genome at position Chr05:84393783.

[0072] Further BLAST search and analysis were performed on the third-generation sequencing results based on the exogenous vector sequence, and the exogenous insertion sequence of maize ND7702 (nucleotides 843-7359 of SEQ ID NO. 3, and nucleotides 584-7140 of SEQ ID NO. 5 and SEQ ID NO. 4) was obtained. The insertion structure is shown below. Figure 1 As shown, it contains one copy of the T-DNA sequence and two copies of the vector backbone sequence. Generally speaking, the insertion of long vector backbone sequences can have a certain impact on the agronomic traits of the transformant. Therefore, this invention further measured the agronomic traits of maize ND7702, such as ear weight and yield.

[0073] Example 3: Identification of agronomic traits of transformation event ND7702

[0074] This invention used three maize transformation events ND4401, ND4403, and ND6603, which overexpress the ZmNRT1.1A gene, as control materials to identify agronomic traits such as ear weight and yield in maize ND7702. The results are shown in Table 5.

[0075] The results showed that maize plants with positive ND7702 conversion under low nitrogen supply conditions had higher yields than ND6603 but lower yields than ND4401 and ND4403. However, under medium and high nitrogen supply conditions, their yields were significantly higher than ND4401, ND4403, and ND6603. In actual production, fertilization standards for medium and high nitrogen are usually applied, so the ND7702 conversion event is more suitable for widespread application in actual maize production.

[0076] Table 5 Comparison of agronomic traits of ND7702 with ND4401, ND4403 and ND6603

[0077]

[0078] Example 4: Detection method for conversion event ND7702

[0079] Genetically modified maize event ND7702 can be bred, and the resulting new varieties can be used to produce agricultural products or commodities. If a sufficient amount is detected in said agricultural product or commodity, it is expected to contain a nucleotide sequence capable of diagnosing the presence of genetically modified maize event ND7702 material in said agricultural product or commodity. The agricultural product or commodity includes, but is not limited to, corn oil, cornmeal, corn gluten, corn tortillas, corn starch, and any other food intended for animal consumption as a food source, or additionally as a growth promoter or ingredient in cosmetic compositions for cosmetic purposes. Nucleic acid detection methods and / or kits based on probe or primer pairs can be developed to detect the presence of genetically modified maize event ND7702 in biological samples, wherein the probe sequence or primer amplification sequence is selected from sequences shown in SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, or SEQ ID NO. 4, to diagnose the presence of genetically modified maize event ND7702.

[0080] One of the detection methods is:

[0081] Genomic DNA was extracted from maize samples using the standard CTAB method, following the procedures outlined in Ministry of Agriculture and Rural Affairs Announcement No. 1485-4-2010, "DNA Extraction and Purification for Detection of Components in Transgenic Plants and Their Products." The maize samples included the proposed transgenic maize ND7702 and non-transgenic maize Y822. Specific boundary sequences in ND7702 plants were detected using PCR, with the primer pairs SEQ ID NO. 7 and SEQ ID NO. 8 used.

[0082] PCR reaction system:

[0083]

[0084] The reaction procedure is as follows:

[0085]

[0086] Steps 2-4: Repeat 35 times.

[0087] The PCR products were electrophoresed on a 1% (w / v) 1×TAE agarose gel. The results are shown in the figure. Figure 5The ND7702 transformation event can amplify the expected target band (SEQ ID NO.6). Furthermore, this PCR method can track the existence of transformation events, thus enabling its application in breeding work.

[0088] In summary, the transgenic maize event ND7702 of this invention has high yield and / or tolerance to nitrogen deficiency, and the detection method can accurately and rapidly identify whether biological samples contain DNA molecules of transgenic maize event ND7702.

[0089] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.

Claims

1. A nucleic acid molecule for detecting the maize transformation event ND7702, characterized in that, The sequence of the nucleic acid molecule is any one of the following i)-iv): i) The sequences shown in SEQ ID NO.1 and / or SEQ ID NO.2; ii) The sequences shown in SEQ ID NO.3 and / or SEQ ID NO.4; iii) The sequence shown in SEQ ID NO.6; iv) The sequences shown in SEQ ID NO. 3, nucleotides 1-7359, and SEQ ID NO. 5 and SEQ ID NO. 4, nucleotides 584-7426.

2. A probe for detecting maize transformation event ND7702, characterized in that, This includes the sequence shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, or SEQ ID NO.

6.

3. A primer pair for detecting the maize transformation event ND7702, characterized in that, The primer pair is the sequence shown in SEQ ID NO.7 and SEQ ID NO.

8.

4. A kit or microarray for detecting maize transformation event ND7702, characterized in that, It includes the probe of claim 2 and / or the primer pair of claim 3.

5. A method for detecting maize transformation event ND7702, characterized in that, This includes using any of the following to detect the presence of the transformation event in the sample to be tested: i) The probe according to claim 2; ii) The primer pair as described in claim 3; iii) The probe of claim 2 and the primer pair of claim 3; iv) The kit or microarray according to claim 4.

6. A method for breeding maize, characterized in that, The method includes the following steps: 1) Obtaining corn containing the nucleic acid molecule of claim 1; 2) The corn obtained in step 1) is subjected to pollen culture, unfertilized embryo culture, doubling culture, cell culture, tissue culture, self-pollination or hybridization or a combination thereof to obtain corn plants, seeds, plant cells, progeny plants or plant parts.

7. The method according to claim 6, characterized in that, It also includes step 3): identifying nitrogen deficiency tolerance and / or yield traits in the progeny plants obtained in step 2), and detecting the presence of the transformation event therein using the method of claim 5.

8. The use of maize plants, seeds, plant cells, progeny plants, or plant parts obtained by the method of claim 6 and / or claim 7 in the preparation of articles, characterized in that, The products include food, feed, or industrial raw materials.

9. A method for improving maize yield and / or nitrogen deficiency tolerance, characterized in that, The invention includes planting at least one transgenic maize plant in soil, wherein the genome of the transgenic maize plant contains nucleotides 1571-4853 of SEQ ID NO. 5, or wherein the genome of the transgenic maize plant contains nucleotides 1-7359 of SEQ ID NO. 3, and nucleotides 584-7426 of SEQ ID NO. 5 and SEQ ID NO. 4; wherein the transgenic maize plant has high yield and / or nitrogen deficiency tolerance traits.

10. A method for improving maize yield and / or nitrogen deficiency tolerance, characterized in that, An expression cassette for a nitrogen-efficient utilization gene, as shown in the sequence of nucleotides 1571-4559 of SEQ ID NO.5, was introduced into the maize genome.

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