CN111394500A - Method for identifying whether plant sample to be detected is derived from SbSNAC1-382 event or progeny thereof - Google Patents

Abstract

The invention discloses a method for identifying whether a plant sample to be tested is derived from the SbSNAC1-382 event or its progeny. The inventors of the present invention transferred the SbSNAC1 gene into the genome of the maize inbred line Zheng 58 by the method mediated by Agrobacterium, and obtained a transgenic maize event SbSANC1-382, referred to as the SbSNAC1-382 event. The drought resistance identification showed that the drought resistance of the SbSNAC1‑382 event was significantly higher than that of the maize inbred line Zheng 58. In addition, after testing, the SbSNAC1-382 event in the T _3rd -T _5th generation has genetic stability and can be stably inherited between different generations. Therefore, the SbSNAC1‑382 event has the potential to enter commercial cultivation. The SbSNAC1‑382 incident has been deposited in the General Microbiology Center of the China Microorganism Culture Collection Management Committee (abbreviated as CGMCC, address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing) on April 4, 2019, and the deposit number is CGMCC No. .17493. The method for identifying whether a plant sample is derived from the SbSNAC1-382 event or its progeny provided by the present invention can specifically detect the SbSNAC1-382 event, and better supervise and manage the SbSNAC1-382 event. The invention has important application value.

Description

A method for identifying whether the plant sample to be tested is derived from the SbSNAC1-382 event or method of its descendants

technical field

The invention belongs to the field of biotechnology, and in particular relates to a method for identifying whether a plant sample to be tested is derived from the SbSNAC1-382 event or its progeny.

Background technique

Water shortage has seriously affected the world's food production. The annual reduction of food production due to water shortage accounts for more than 50% of crop production reduction, and the direct economic losses caused are difficult to estimate. my country's arid and semi-arid regions account for more than half of the country's land area. According to statistics, the perennial drought-affected area in my country accounts for about 20% of the total cultivated land area. In the past 40 years, the annual decrease in grain production due to drought alone has reached more than 100 billion kilograms. Corn is one of the most important food, feed and related industrial raw materials in the world. In my country, corn has become the largest food crop since 2009. The traditional maize genetics and breeding have played a certain role in improving the drought resistance of maize, but it is difficult to break through the genetic restriction between species and significantly improve the drought resistance of maize. Transgenic technology can break the boundaries of species, carry out directional transformation and recombination transfer of genes, and improve the drought resistance of existing varieties, which can greatly improve the drought resistance of maize. The practice of cultivating and promoting genetically modified corn in developed countries, especially in the United States, has also proved that the cultivation of new varieties with genetically modified technology can significantly improve the drought resistance of corn, and is the most realistic and effective way to greatly increase yield and improve quality. It has become an international corn breeding development. the main direction. In December 2011, the U.S. Department of Agriculture's Animal and Plant Health Inspection Service (APHIS) officially approved Monsanto's genetically modified drought-resistant corn MON87460, which means that the world's first genetically modified drought-resistant corn can be promoted and utilized on a large scale in production. The drought-resistant maize is transgenic for CspB gene (CspB gene is from Bacillus subtilis, encoding an RNA chaperone protein), which can enhance the function of plant cells under adverse conditions and reduce yield loss under water-deficient conditions. Under drought conditions, maize inbreds and hybrids transgenic for this gene can significantly increase maize biomass and yield, and field trials of drought-resistant maize in arid regions of the western United States have achieved or even exceeded 6% to 10% increase in yield Target.

The SbSNAC1 gene is a NAC family gene cloned from XGL-1, a drought-tolerant local sorghum cultivar in Xinjiang, by the Institute of Crop Science, Chinese Academy of Agricultural Sciences. Overexpression of this gene in Arabidopsis can significantly improve the drought tolerance of transgenic Arabidopsis plants (Lu et al., 2013).

SUMMARY OF THE INVENTION

The purpose of the present invention is to identify whether a plant sample is derived from the SbSNAC1-382 event or its progeny, the SbSNAC1-382 event being Zea mays SbSNAC1-382CGMCC No.17493. Plant samples can be plant leaves, seeds, and the like.

The present invention first protects a method for identifying whether a plant sample is derived from the SbSNAC1-382 event or its progeny, which may include the following steps: detecting whether the genomic DNA of the plant sample to be tested contains DNA fragment A and/or DNA fragment B; Judgment as follows: if the genomic DNA of the plant sample to be tested contains DNA fragment A and/or DNA fragment B, the plant sample to be tested is derived from the SbSNAC1-382 event or its descendants; if the genomic DNA of the plant sample to be tested does not contain DNA fragment A and/or DNA fragment B, the plant sample to be tested is not derived from the SbSNAC1-382 event or its progeny;

The nucleotide sequence of DNA fragment A is shown in sequence 3 in the sequence listing;

The nucleotide sequence of DNA fragment B is shown in sequence 4 in the sequence listing;

The SbSNAC1-382 event is maize Zea mays SbSNAC1-382 CGMCC No. 17493.

In the above method, the method of "detecting whether the genomic DNA of the plant sample to be tested contains DNA fragment A and/or DNA fragment B" may be S1) or S2) or S3).

S1) Direct sequencing.

S2) use primer pair X and/or primer pair Y to carry out PCR amplification of the genomic DNA of the plant sample to be tested, and then judge as follows: if the target amplification product is obtained, the plant sample to be tested is derived from the SbSNAC1-382 event or its progeny ; If the target amplification product is not obtained, the plant sample to be tested is not derived from the SbSNAC1-382 event or its progeny;

Primer pair X consists of an upstream primer FX and a downstream primer RX; the upstream primer FX is a part of the DNA molecule shown in the 1-451 position from the 5' end of the sequence 3 in the sequence table; the downstream primer RX is the sequence 3 from the sequence table. The reverse complementary sequence of a part of the DNA molecule shown at positions 452-933 from the 5' end; the target amplification product of primer pair X is DNA molecule X;

Primer pair Y consists of an upstream primer FY and a downstream primer RY; the upstream primer FY is a part of the DNA molecule shown in the 1-352 position from the 5' end of the sequence 4 in the sequence listing; the downstream primer RY is the sequence 4 in the sequence listing. The reverse complementary sequence of a part of the DNA molecule shown at positions 353-547 from the 5' end; the target amplification product of primer pair Y is DNA molecule Y.

S3) Perform Southern hybridization on the genomic DNA of the plant sample to be tested with probe A that can specifically bind to the DNA molecule X and/or probe B that can specifically bind to the DNA molecule Y, and then judge as follows: if the hybridization is obtained If the hybrid fragment cannot be obtained, the plant sample to be tested is not derived from the SbSNAC1-382 event or its progeny.

The primer pair X may be at least one of primer pair X1, primer pair X2, and primer pair X3.

Primer pair X1 may consist of primer P1 and primer P2.

Primer pair X2 may consist of primer P3 and primer P4.

Primer pair X3 may consist of primer P1 and primer P4.

The nucleotide sequence of primer P1 can be shown in SEQ ID NO: 5 in the sequence listing.

The nucleotide sequence of primer P2 can be shown as SEQ ID NO: 6 in the sequence listing.

The nucleotide sequence of primer P3 can be shown as sequence 7 in the sequence listing.

The nucleotide sequence of primer P4 can be shown as SEQ ID NO: 8 in the sequence listing.

The primer pair Y may be at least one of a primer pair Y1, a primer pair Y2, a primer pair Y3, and a primer pair Y4.

Primer pair Y1 may consist of primer S1 and primer S3.

Primer pair Y2 may consist of primer S2 and primer S3.

Primer pair Y3 may consist of primer S1 and primer S4.

Primer pair Y4 may consist of primer S2 and primer S4.

The nucleotide sequence of primer S1 can be shown as SEQ ID NO: 9 in the sequence listing.

The nucleotide sequence of primer S2 can be shown as sequence 10 in the sequence listing.

The nucleotide sequence of primer S3 can be shown as sequence 11 in the sequence listing.

The nucleotide sequence of primer S4 can be shown as sequence 12 in the sequence listing.

The nucleotide sequence of the target amplification product (that is, DNA molecule X) of the primer pair X1 can be shown as the 215-525th position from the 5' end of sequence 3 in the sequence listing.

The nucleotide sequence of the target amplification product (that is, DNA molecule X) of the primer pair X2 can be shown as the 323-564th position from the 5' end of sequence 3 in the sequence listing.

The nucleotide sequence of the target amplification product (that is, DNA molecule X) of the primer pair X3 can be shown as the 215-564th position from the 5' end of sequence 3 in the sequence listing.

The nucleotide sequence of the target amplification product of primer pair Y1 (that is, DNA molecule Y) can be shown as the 45-547th position from the 5' end of sequence 4 in the sequence listing.

The nucleotide sequence of the target amplification product of primer pair Y2 (that is, DNA molecule Y) can be shown as the 1-547th position from the 5' end of sequence 4 in the sequence listing.

The nucleotide sequence of the target amplification product of primer pair Y3 (that is, DNA molecule Y) can be shown as the 215-436th position from the 5' end of sequence 4 in the sequence listing.

The nucleotide sequence of the target amplification product of primer pair Y4 (that is, DNA molecule Y) can be shown as the 1-436th position from the 5' end of sequence 4 in the sequence listing.

The present invention also protects a kit for identifying whether a plant sample to be tested is derived from the SbSNAC1-382 event or its progeny.

The kit for identifying whether the plant sample to be tested is derived from the SbSNAC1-382 event or its progeny protected by the present invention may specifically be kit A; the kit A may include any one of the above-mentioned primer pairs X and/or Primer pair Y; the SbSNAC1-382 event may be Zea mays SbSNAC1-382 CGMCC No. 17493 of maize.

The kit A can specifically consist of any one of the primer pair X and/or primer pair Y described above.

The kit protected by the present invention for identifying whether the plant sample to be tested is derived from the SbSNAC1-382 event or its progeny can be specifically kit B; the kit B can include any of the above-mentioned DNA molecules X that can specifically bind Probe A and/or any of the above-mentioned probe B that can specifically bind to DNA molecule Y; the SbSNAC1-382 event can be Zea mays SbSNAC1-382 CGMCC No.17493.

The kit B can specifically be composed of any of the above-mentioned probes A and/or any of the above-mentioned probes B.

The application of any of the above-mentioned primer pairs X and/or any of the above-mentioned primer pairs Y in identifying whether the plant sample to be tested is derived from the SbSNAC1-382 event or its progeny also belongs to the protection scope of the present invention; the SbSNAC1-382 event It may be Zea mays SbSNAC1-382 CGMCC No. 17493 of corn.

Any of the above-mentioned probe A that can specifically bind to DNA molecule X and/or any of the above-mentioned probe B that can specifically bind to DNA molecule Y is used in identifying whether the plant sample to be tested is derived from the SbSNAC1-382 event or its progeny. The application of SbSNAC1-382 also falls within the protection scope of the present invention; the SbSNAC1-382 event can be Zea mays SbSNAC1-382CGMCC No.17493.

The application of DNA fragment A and/or DNA fragment B in identifying whether the plant sample to be tested is derived from the SbSNAC1-382 event or its progeny also belongs to the protection scope of the present invention; the SbSNAC1-382 event can be Zea mays SbSNAC1-382CGMCC No. 17493;

The nucleotide sequence of DNA fragment A can be shown as sequence 3 in the sequence listing;

The nucleotide sequence of DNA fragment B can be shown in SEQ ID NO: 4 in the Sequence Listing.

The present invention also protects DNA fragment A and/or DNA fragment B;

The nucleotide sequence of DNA fragment A can be shown as sequence 3 in the sequence listing;

The nucleotide sequence of DNA fragment B can be shown in SEQ ID NO: 4 in the Sequence Listing.

The inventors of the present invention transferred the SbSNAC1 gene into the genome of the maize inbred line Zheng 58 by the method mediated by Agrobacterium, and obtained a transgenic maize event SbSANC1-382 (SbSNAC1-382 event for short). The drought resistance identification showed that the drought resistance of the SbSNAC1-382 event was significantly improved compared with the control maize (ie, the maize inbred line Zheng 58). In addition, after testing, the T ₃ generation-T ₅ generation SbSNAC1-382 event has genetic stability and can be stably inherited between different generations. Therefore, the SbSNAC1-382 event is likely to enter commercial cultivation. The SbSNAC1-382 event has been deposited in the General Microbiology Center of the China Microorganism Culture Collection Management Committee (CGMCC for short) on April 4, 2019. The address is: Beichen West, Chaoyang District, Beijing Road No. 1 Courtyard No. 3), the preservation number is CGMCC No.17493. The method for identifying whether a plant sample is derived from the SbSNAC1-382 event or its progeny provided by the present invention can specifically detect the SbSNAC1-382 event and better supervise and manage the SbSNAC1-382 event. The invention has important application value.

Description of drawings

Fig. 1 is the agarose gel electrophoresis result of step 3 in Example 1.

Fig. 2 is the result of step five 2 in embodiment 1.

FIG. 3 is the result of step 53 in Example 1.

FIG. 4 is the result of Step 7 in Example 1.

Fig. 5 is the observation result of the eighth grain filling period in Example 1.

Fig. 6 is the result of measuring yield after the eighth step in Example 1 after harvesting.

Figure 7 is a schematic diagram of the vector of the recombinant plasmid 35S::SbSNAC1.

FIG. 8 is the experimental result of step 1 in Example 2. FIG.

FIG. 9 is the experimental result of step 2 in Example 2.

FIG. 10 is the experimental result of step 3 in Example 2.

FIG. 11 is the experimental result of step 4 in Example 2.

FIG. 12 is the experimental result of step 1 in Example 3. FIG.

13 is the experimental result of step 2 in Example 3.

Detailed ways

The following examples facilitate a better understanding of the present invention, but do not limit the present invention.

The experimental methods in the following examples are conventional methods unless otherwise specified.

The test materials used in the following examples were purchased from conventional biochemical reagent stores unless otherwise specified.

The quantitative tests in the following examples are all set to repeat the experiments three times, and the results are averaged.

Plasmid pCAMBIA3301 is a product of Cambia Company.

Example 1. Acquisition and drought resistance identification of transgenic SbSNAC1 maize

1. Cloning of SbSNAC1 gene

1. Take the leaves and roots of sorghum variety XGL-1 (provided by the Institute of Food Crops, Xinjiang Academy of Agricultural Sciences), mix them, and use them as materials.

2. After completing step 1, take the material, first extract total RNA, and then reverse transcribed to obtain the cDNA of sorghum variety XGL-1.

3. After completing step 3, using the cDNA of sorghum variety XGL-1 as a template, 5'-TTT CCATGG GATTGCCGGTGAT-3' (underlined is the recognition site of restriction endonuclease NcoI) and 5'-TTT GGTGACC AGCCTCAGAATGGCCCCAAC- The primer pair consisting of 3' (underlined is the recognition site of the restriction endonuclease BstE II) was PCR amplified to obtain a PCR amplification product of about 985 bp.

4. Connect the PCR amplification product obtained in step 3 with the PMD18-T vector to obtain a recombinant plasmid PMD18-SbSNAC1.

The recombinant plasmid PMD18-SbSNAC1 was sequenced. Sequencing results showed that the recombinant plasmid PMD18-SbSNAC1 contained the DNA molecules shown in the sequence 1 from the 5' end of the 1st to 966th positions in the sequence listing.

2. Construction of recombinant plasmid 35S::SbSNAC1

1. The recombinant plasmid PMD18-SbSNAC1 was digested with restriction enzymes NcoI and BstE II, and a DNA fragment of about 980 bp was recovered.

2. The plasmid pCAMBIA3301 was digested with the restriction enzymes NcoI and BstE II, and the vector backbone of about 9250bp was recovered.

3. Connect the DNA fragment recovered in step 1 and the vector backbone recovered in step 2 to obtain a recombinant plasmid 35S::SbSNAC1.

The recombinant plasmid 35S::SbSNAC1 was sequenced. According to the sequencing results, the structure of the recombinant plasmid 35S::SbSNAC1 is described as follows: The small fragment between the restriction endonucleases NcoI and BstE II recognition sequences of plasmid pCAMBIA3301 is replaced by the sequence 1 in the sequence table from the 5' end of the 5th to the The DNA molecule shown at position 970. The recombinant plasmid 35S::SbSNAC1 expresses the protein SbSNAC1 shown in sequence 2 in the sequence listing.

Third, the acquisition of positive recombinant Agrobacterium

1. The recombinant plasmid 35S::SbSNAC1 was transformed into Agrobacterium tumefaciens EH105 by freeze-thaw method to obtain recombinant Agrobacterium.

2. After completing step 1, inoculate the single clones of each recombinant Agrobacterium into YEB liquid medium respectively, and cultivate at 28° C. and 200 rpm for 16 hours to obtain an Agrobacterium liquid.

3. After completing step 2, use each Agrobacterium liquid, water and recombinant plasmid 35S::SbSNAC1 as templates, and 5'-TTTCCATGGGATTGCCGGTGAT-3' and 5'-TTTGGTGACCAGCCTCAGAATGGCCCCAAC-3' as primers for PCR amplification to obtain PCR amplification product. The recombinant plasmid 35S::SbSNAC1 served as a positive control. Water served as a negative control.

4. After completing step 3, the PCR amplification product is subjected to 1% (m/v) agarose gel electrophoresis, and the following judgment is made according to the electrophoresis results: if the PCR amplification product of a recombinant Agrobacterium contains about 985bp of DNA fragment, the recombinant Agrobacterium is a positive recombinant Agrobacterium.

Agarose gel electrophoresis is shown in Figure 1 ("+" is a positive control, "-" is a negative control, and lanes 1 to 7 are all recombinant Agrobacterium).

4. The acquisition of SbSNAC1 gene transgenic maize in T ₀ generation

1. Inoculate the single clone of the positive recombinant Agrobacterium obtained in step 3 into YEB liquid medium, cultivate at 28° C. and 200 rpm, and obtain an Agrobacterium liquid with an OD _550nm of 0.3-0.4.

2. After completing step 1, take the Agrobacterium bacteria solution, centrifuge at 4°C and 10,000 rpm for 10 minutes, and collect the bacteria.

3. Take the bacterial cells collected in step 2, add invasion medium (MS medium containing 100 μM acetosyringone) to resuspend, and obtain an infection solution with an OD _550nm of 0.3-0.4. Take the immature embryo of the corn inbred line Zheng 58 with a size of 1.0-1.5 mm after pollination for 11-12 days, infect (i.e. soak) with an infection solution for 5 min under dark conditions, and then place it on a co-cultivation medium with Seal with breathable tape and incubate in the dark at 20°C for 3 days.

4. After step 3 is completed, the immature embryos of the maize inbred line Zheng 58 are taken, placed in a resting medium, and cultured at 28° C. for 7 days.

5. After completing step 4, the immature embryos of the maize inbred line Zheng 58 were taken, first placed on selective medium 1, and cultured at 28°C for 14 days; Sexual healing.

6. After completing step 5, the resistant callus was taken, placed in regeneration medium 1, and cultured in the dark at 25°C for 14 days to obtain mature somatic embryos.

7. After step 6 is completed, the mature somatic embryos are transferred to regeneration medium 2, and cultured at 25° C. and light and dark alternately for 7-10 days to obtain resistant seedlings. The light and dark alternate culture is 16h light culture and 8h dark culture, and the light intensity during light culture is 80-100μE/m2/s.

8. After completing step 7, transplant the resistant seedlings into the soil to obtain the T ₀ generation maize to be transformed with the SbSNAC1 gene.

Co-cultivation medium, resting medium, selective medium 1 (selective medium containing 1.5 mg/L bialaphos), selective medium 2 (selective medium containing 3 mg/L bialaphos), regeneration medium Both 1 and regeneration medium 2 are described in Frame et al., 2011.

5. Identification of the SbSNAC1 gene-transformed maize in T ₀ generation

1. Spray Basta

Basta (2‰) was sprayed on the leaves of the plants of the T ₀ generation to be transfected with SbSNAC1 gene, and the leaves were observed after 5-7 days. If the leaves of a certain T ₀ generation of SbSNAC1 transgenic maize did not wither, the T ₀ generation of SbSNAC1 transgenic maize was preliminarily identified as a T ₀ generation of SbSNAC1 transgenic maize.

Seven lines of maize that were preliminarily identified as SbSNAC1 gene transgenic T ₀ generation by spraying Basta were named as SbSNC1-382, SbSNAC1-383, SbSNAC1-389, SbSNAC1-466, SbSNAC1-467, SbSNAC1-471 and SbSNAC1-474, respectively. .

2. PCR detection of SbSNAC1 gene

(1) The genomic DNA of the leaves of the SbSNAC1 gene transgenic maize (SbSNC1-382, SbSNAC1-383, SbSNAC1-389, SbSNAC1-466, SbSNAC1-467, SbSNAC1-471 or SbSNAC1-474) of the T ₀ generation were extracted and used as a template , using a specific primer pair for amplifying the SbSNAC1 gene (composed of SbSNAC1-F: 5'-GACCGCAAGTACCCAAACGG-3' and SbSNAC1-R: 5'-CACCCAGTCATCCAGCCTGAG-3', where SbSNAC1-F spans two exons) to carry out PCR Amplification to obtain PCR amplification products.

According to the above steps, the template was replaced with the genomic DNA of the leaves of the maize inbred line Zheng 58, and other steps remained unchanged, which was used as a negative control.

According to the above steps, the template was replaced with water, and other steps remained unchanged, which was used as a water control.

According to the above steps, the template was replaced with the recombinant plasmid 35S::SbSNAC1, and other steps remained unchanged, which was used as a positive control.

Reaction conditions: 95°C, 5 min; 95°C for 30s, 60°C for 30s, 72°C for 30s, 34 cycles; 72°C for 5 min; storage at 15°C.

(2) Carry out 1% (m/v) agarose gel electrophoresis on each PCR amplification product, and judge according to the electrophoresis results as follows: if the PCR amplification product of a certain strain contains a DNA fragment of about 249 bp, then the strain The line was re-identified as T ₀ generation transgenic maize with SbSNAC1 gene.

The results of agarose gel electrophoresis are shown in Figure 2 (Marker is DNA Marker, 1 to 7 are SbSNAC1-382, SbSNAC1-383, SbSNAC1-389, SbSNAC1-466, SbSNAC1-467, SbSNAC1-471 and SbSNAC1-474 in sequence).

3. PCR detection of Bar gene

(1) Extract the genomic DNA of the leaves of the T ₀ generation transgenic SbSNAC1 gene maize (SbSNC1-382, SbSNAC1-383, SbSNAC1-389, SbSNAC1-466, SbSNAC1-467, SbSNAC1-471 or SbSNAC1-474) and use it as a template, A specific primer pair for amplifying the Bar gene (consisting of 5'-GAAGTCCAGCTGCCAGAAAC-3' and 5'-GTCTGCACCATCGTCAACC-3') was used for PCR amplification to obtain PCR amplification products.

According to the above steps, the template was replaced with the genomic DNA of the leaves of the maize inbred line Zheng 58, and other steps remained unchanged, which was used as a negative control.

According to the above steps, the template was replaced with water, and other steps remained unchanged, which was used as a blank control.

According to the above steps, the template was replaced with the recombinant plasmid 35S::SbSNAC1, and other steps remained unchanged, which was used as a positive control.

Reaction conditions: 95°C, 5 min; 95°C for 30s, 60°C for 30s, 72°C for 30s, 34 cycles; 72°C for 5 min; storage at 15°C.

(2) Perform 1% (m/v) agarose gel electrophoresis on each PCR amplification product, and judge according to the electrophoresis results as follows: if the PCR amplification product of a certain strain contains a DNA fragment of about 444 bp, then the strain The line was re-identified as T ₀ generation transgenic maize with SbSNAC1 gene.

Part of the agarose gel electrophoresis results are shown in Figure 3 (Marker is DNA Marker, lanes 1 to 7 are SbSNAC1-382, SbSNAC1-383, SbSNAC1-389, SbSNAC1-466, SbSNAC1-467, SbSNAC1-471 and SbSNAC1-474, Lane 11 is the positive control and lane 12 is the negative control).

The above results showed that SbSNC1-382, SbSNAC1-383, SbSNAC1-389, SbSNAC1-466, SbSNAC1-467, SbSNAC1-471 and SbSNAC1-474 were all T ₀ generation transgenic SbSNAC1 maize.

6. Acquisition of T _1st generation-T _5th generation SbSNAC1-382

The seeds of the T ₀ generation transgenic maize with SbSNAC1 gene were taken and self-crossed to obtain the seeds of the T ₁ generation transgenic maize with SbSNAC1 gene. The seeds of the T ₁ -generation SbSNAC1 transgenic maize were continuously selfed to obtain the T ₂ -generation SbSNAC1-transgenic maize seeds, the T ₃ -generation SbSNAC1-transgenic maize seeds, the T ₄ -generation SbSNAC1-transgenic maize seeds and the T ₅ -generation SbSNAC1-transgenic maize seeds. Genetic corn seeds.

The T ₁ generation transgenic maize with SbSNAC1 gene and the T ₅ generation transgenic maize with SbSNAC1 gene were selected for follow-up experiments.

7. Identification of drought resistance in greenhouse of SbSNAC1 transgenic maize from T ₁ generation to T ₅ generation

The maize to be tested is T ₁ generation-T ₅ generation transgenic maize with SbSNAC1 gene (SbSNC1-382, SbSNAC1-383, SbSNAC1-389, SbSNAC1-466, SbSNAC1-467, SbSNAC1-471 or SbSNAC1-474) or maize inbred line Zheng 58. The maize inbred line Zheng 58 was used as a control.

(1) Plant 3 corns to be tested in flowerpots, and each line is planted with 5 replicates; then placed in a greenhouse for routine management.

(2) After completing step (1), stop watering when the corn to be tested grows until the visible leaves of the corn reach 4 leaves and 1 heart, and it is continuously dry for 21 days. Observe the growth state of the corn to be tested.

The growth state of some corn to be tested is shown in Figure 4 (SbSNC1-382 is the T ₅ generation SbSNC1-382, and Zheng 58 is a corn inbred line Zheng 58). The results showed that the leaves of the maize inbred line Zheng 58 were curled and wilted, while the leaves of the T ₁ -T ₅ -generation SbSNAC1 transgenic maize were stretched and remained green. It can be seen that compared with the maize inbred line Zheng 58, the drought resistance of SbSNAC1 gene transgenic maize from T ₁ to T ₅ generations was significantly improved.

8. Field drought resistance identification of T ₃ generation-T ₅ generation SbSNC1-382

In 2015, the T ₃ generation SbSNC1-382 and the maize inbred line Zheng 58 were identified for drought resistance in the field.

In 2016, the T ₄ generation SbSNC1-382 and the maize inbred line Zheng 58 were identified for drought resistance in the field.

In 2017, the T ₅ generation SbSNC1-382 and the maize inbred line Zheng 58 were identified for drought resistance in the field.

Two treatments of water and drought were set for drought resistance identification. Each material was planted in 6 rows with a row length of 5 meters, repeated 3 times.

The maize for drought resistance identification was planted in the Anningqu test field in Urumqi, Xinjiang. The growth status and yield at the grain filling stage were observed.

The test results are as follows:

1. The observation results at the grain filling stage (Fig. 5, SbSNC1-382 is T ₃ generation SbSNC1-382): in arid regions, compared with T ₃ generation SbSNC1-382, T ₄ generation SbSNC1-382 or T ₅ generation SbSNC1-382 , the plant height of the maize inbred line Zheng 58 decreased significantly, and the leaf color was obviously turned green and yellow;

2. The yield test after harvest showed (Table 1 and Figure 6) that in arid regions, the yield of T ₃ generation SbSNC1-382, T ₄ generation SbSNC1-382 or T ₅ generation SbSNC1-382 was significantly higher than that of the maize inbred line Zheng 58 .

Table 1. Average yield per plant of SbSNAC1-382 in water and arid regions

Note: The data in the table is the mean ± standard error; the average yield per plant is the grain yield (water 14%).

The test results showed that compared with the maize inbred line Zheng 58, the drought resistance of T ₃ generation SbSNC1-382, T ₄ generation SbSNC1-382 or T ₅ generation SbSNC1-382 was significantly improved.

Embodiment 2, the genetic stability detection of T ₃ generation-T ₅ generation SbSNAC1-382

The vector diagram of the recombinant plasmid 35S::SbSNAC1 is shown in Figure 7. The size of the recombinant plasmid 35S::SbSNAC1 is 10.24kb, and there is only one restriction endonuclease HindIII and one EcoRI digestion site on this vector, so using these two enzymes for single digestion, you can get a 10.24kb linear segment. In addition, there are no restriction endonucleases BglII and DraI cleavage sites on this vector.

The genomic DNA of the SbSNC1-382 event or the maize inbred line Zheng 58 was digested with restriction endonucleases HindIII and EcoRI, respectively, and the specific probes for the SbSNAC1 gene and the Bar gene were used for hybridization, respectively. The results showed that the SbSNAC1-382 event contained two copies of the SbSNAC1 gene and the Bar gene, and these two copies were stably inherited in the transgenic T ₃ , T ₄ and T ₅ generations. Therefore, it is likely that the two copies were inserted into the same location in the maize genome. In order to test this hypothesis, the SbSNAC1-382 event and the genomic DNA of the maize inbred line Zheng 58 were digested with restriction enzymes BglII and DraI without restriction sites in the inserted sequence (T-DNA), respectively. The number of insertion sites (the number of T-DNA integration sites within the maize genome) was determined. The results of Southern hybridization showed that the T-DNA in the SbSNAC1-382 event (ie T ₃ generation-T ₅ generation SbSNAC1-382) was inserted into two copies of SbSNAC1 and Bar genes at a single site in the maize genome, and could be transferred between different generations stable inheritance. The specific results are as follows:

1. The results of Southern hybridization using SbSNAC1 gene probe, restriction enzymes HindIII and EcoRI

1. Using the recombinant plasmid 35S::SbSNAC1 as the template and 5'-CGCGTGGGGTCAAGACGGACTG-3' and 5'-GGGAACGAGTCCAGCTCCGGGAAC-3' as the primers for PCR amplification, a DNA fragment of about 386bp was obtained. This fragment is the SbSNAC1 gene probe.

2. Use restriction endonucleases HindIII and EcoRI to digest the genomic DNA of T ₃ generation SbSNAC1-382, T ₄ generation SbSNAC1-382, T ₅ generation SbSNAC1-382 or the inbred line Zheng 58 of maize, respectively. Step 1 The obtained SbSNAC1 gene probe was hybridized.

The recombinant plasmid 35S::SbSNAC1 was digested with restriction endonuclease HindIII, and the SbSNAC1 gene probe obtained in step 1 was used for hybridization. as a positive control.

The experimental results are shown in Figure 8 (swimming lane 1 is the genomic DNA of T ₃ generation SbSNAC1-382 cut by restriction endonuclease HindIII, and swimming lane 2 is the genomic DNA of T ₄ generation SbSNAC1-382 cut by restriction endonuclease HindIII, Swimming lane 3 is the genomic DNA of T ₅ generation SbSNAC1-382 digested by restriction endonuclease HindIII, swimming lane 4 is the genomic DNA of the maize inbred line Zheng 58 digested by restriction endonuclease HindIII, and swimming lane 5 is the restriction endonuclease. Genomic DNA of T ₃ generation SbSNAC1-382 digested by Dicer EcoRI, lane 6 is the genomic DNA of T ₄ generation SbSNAC1-382 digested by restriction endonuclease EcoRI, and lane 7 is the genomic DNA of T 4 generation SbSNAC1-382 digested by restriction endonuclease EcoRI T _5th generation SbSNAC1-382 genomic DNA, lane 8 is the genomic DNA of the maize inbred line Zheng 58 digested by restriction endonuclease EcoRI, lane 9 is the recombinant plasmid 35S::SbSNAC1 digested by restriction endonuclease HindIII , lane 10 is Takara DL15kb DNA marker). The results showed that the recombinant plasmid 35S::SbSNAC1 digested with the restriction endonuclease HindIII hybridized a fragment of 10.24 kb; after the restriction endonuclease HindIII, the SbSNAC1-382 of the T _3rd generation to the _5th generation of the maize The inbred line Zheng 58 has 2 specific hybridization bands between 2.5-5kb, and the maize inbred line Zheng 58 has endogenous background hybridization bands. These signals are due to the homologous sequence between the SbSNAC1 gene probe and the maize genome. Generated by non-specific hybridization, irrespective of insertion, the same hybridization band was also observed in the SbSNAC1-382 event, thus endogenous background hybridization band; after digestion with restriction enzyme EcoRI, T ₃ generation- Compared with the maize inbred line Zheng 58, T _5th generation SbSNAC1-382 has 2 specific hybrid bands between 2.5 and 5 kb, and the maize inbred line Zheng 58 has endogenous background hybrid bands. These signals are due to the SbSNAC1 gene probe. Non-specific hybridization of the needle to homologous sequences within the maize genome resulted, regardless of the insertion, the same hybridized band was also observed in the SbSNAC1-382 event, and therefore an endogenous background hybridized band. Since there is only one restriction endonuclease HindIII and one EcoRI restriction site in the recombinant plasmid 35S::SbSNAC1, the specific passage numbers obtained by the restriction enzyme digestion and hybridization of the transgenic maize genomic DNA with these two enzymes respectively represent the inserted copy number. The hybridization results showed that the insert sequence of the SbSNAC1-382 event had 2 copies of the SbSNAC1 gene.

2. The results of Southern hybridization using Bar gene probe, restriction enzymes HindIII and EcoRI

1. The recombinant plasmid 35S::SbSNAC1 was used as the template, and 5'-ATGAGCCCAGAACGACGCCCG-3' and 5'-TCAAATCTCGGTGACGGGCAGGAC-3' were used as primers for PCR amplification to obtain a DNA fragment of about 552 bp. This fragment is the Bar gene probe.

2. Use restriction endonucleases HindIII and EcoRI to digest the genomic DNA of T ₃ generation SbSNAC1-382, T ₄ generation SbSNAC1-382, T ₅ generation SbSNAC1-382 or the inbred line Zheng 58 of maize, respectively. Step 1 The obtained Bar gene probe was hybridized.

The recombinant plasmid 35S::SbSNAC1 was digested with restriction endonuclease HindIII, and the Bar gene probe obtained in step 1 was used for hybridization. as a positive control.

The experimental results are shown in Figure 9 (swimming lane 1 is the genomic DNA of T ₃ generation SbSNAC1-382 cut by restriction endonuclease HindIII, and swimming lane 2 is the genomic DNA of T ₄ generation SbSNAC1-382 cut by restriction endonuclease HindIII, Swimming lane 3 is the genomic DNA of T ₅ generation SbSNAC1-382 digested by restriction endonuclease HindIII, swimming lane 4 is the genomic DNA of the maize inbred line Zheng 58 digested by restriction endonuclease HindIII, and swimming lane 5 is the restriction endonuclease. Genomic DNA of T ₃ generation SbSNAC1-382 digested by Dicer EcoRI, lane 6 is the genomic DNA of T ₄ generation SbSNAC1-382 digested by restriction endonuclease EcoRI, and lane 7 is the genomic DNA of T 4 generation SbSNAC1-382 digested by restriction endonuclease EcoRI T _5th generation SbSNAC1-382 genomic DNA, lane 8 is the genomic DNA of the maize inbred line Zheng 58 digested by restriction endonuclease EcoRI, lane 9 is the recombinant plasmid 35S::SbSNAC1 digested by restriction endonuclease HindIII , lane 10 is Takara DL15kb DNA marker). The results showed that the recombinant plasmid 35S::SbSNAC1 digested with the restriction endonuclease HindIII hybridized a fragment of 10.24kb (due to the low loading of the plasmid, resulting in a weaker hybridization band); use the restriction endonuclease HindIII enzyme After cutting, T ₃ generation-T ₅ generation SbSNAC1-382 had a band near 2.5kb and 4kb respectively, but no band appeared in the maize inbred line Zheng 58 _; after digestion with restriction endonuclease EcoRI, T3 Generation-T _5th generation SbSNAC1-382 has a band near 4kb and 5kb, but no band appears in the maize inbred line Zheng 58; because the recombinant plasmid 35S::SbSNAC1 has only one restriction enzyme HindIII and one EcoRI Therefore, the number of specific strips obtained by the enzyme digestion and hybridization of transgenic maize genomic DNA with these two enzymes represents the number of inserted copies. The hybridization results showed that the insert sequence of the SbSNAC1-382 event had 2 copies of the Bar gene.

3. The results of Southern hybridization using SbSNAC1 gene probe, restriction enzymes BglII and DraI

1. Same as 1 in step 1.

2. The genomic DNA of T ₃ generation SbSNAC1-382, T ₄ generation SbSNAC1-382, T ₅ generation SbSNAC1-382 or maize inbred line Zheng 58 was digested with restriction enzymes BglII and DraI, respectively, with step 1. The obtained SbSNAC1 gene probe was hybridized.

The recombinant plasmid 35S::SbSNAC1 was digested with restriction endonuclease DraI, and the SbSNAC1 gene probe obtained in step 1 was used for hybridization. as a positive control.

The experimental results are shown in Figure 10 (swimming lane 1 is the genomic DNA of T ₃ generation SbSNAC1-382 cut by restriction endonuclease BglII, and swimming lane 2 is the genomic DNA of T ₄ generation SbSNAC1-382 cut by restriction endonuclease BglII, Swimming lane 3 is the genomic DNA of T ₅ generation SbSNAC1-382 digested by restriction endonuclease BglII, swimming lane 4 is the genomic DNA of the maize inbred line Zheng 58 digested by restriction endonuclease BglII, and swimming lane 5 is the restriction endonuclease. Genomic DNA of T ₃ generation SbSNAC1-382 digested by nuclease DraI, lane 6 is the genomic DNA of T ₄ generation SbSNAC1-382 digested by restriction endonuclease DraI, and lane 7 is the genomic DNA of T 4 generation SbSNAC1-382 digested by restriction endonuclease DraI T _5th generation SbSNAC1-382 genomic DNA, lane 8 is the genomic DNA of the maize inbred line Zheng 58 digested by restriction endonuclease DraI, lane 9 is the recombinant plasmid 35S::SbSNAC1 digested by restriction endonuclease HindIII , lane 10 is Takara DL 15kb DNA marker). The results showed that the recombinant plasmid 35S::SbSNAC1 digested with the restriction endonuclease HindIII hybridized a fragment of 10.24 kb; after the restriction endonuclease BglII, the T ₃ generation-T ₅ generation SbSNAC1-382 was compared with the maize The inbred line Zheng 58 has a specific hybridization band at a position greater than 15kb, and the maize inbred line Zheng 58 has an endogenous background hybridization band. These signals are due to the non-specificity of the SbSNAC1 gene probe and the homologous sequence in the maize genome. Hybridization produced, regardless of insertion, the same hybridized band was also observed in the SbSNAC1-382 event, so it was an endogenous background hybridized band; after digestion with restriction enzyme DraI, T ₃ generation-T ₅ Compared with the maize inbred line Zheng 58, the generation SbSNAC1-382 has a specific hybridization band between 10 and 15 kb, and the maize inbred line Zheng 58 has an endogenous background hybridization band. These signals are due to the SbSNAC1 gene probe and the Non-specific hybridization of homologous sequences within the maize genome resulted, regardless of insertion, the same hybridized band was also observed in the SbSNAC1-382 event, thus endogenous background hybridized band. The hybridization results showed that the two copies of the SbSNAC1 gene in the SbSNAC1-382 event were inserted at a single site in the genome.

4. The results of Southern hybridization using Bar gene probe, restriction enzymes BglII and DraI

1. Same as 1 in step 2.

2. The genomic DNA of T ₃ generation SbSNAC1-382, T ₄ generation SbSNAC1-382, T ₅ generation SbSNAC1-382 or maize inbred line Zheng 58 was digested with restriction enzymes BglII and DraI, respectively, with step 1. The obtained Bar gene probe was hybridized.

The recombinant plasmid 35S::SbSNAC1 was digested with restriction endonuclease HindIII, and the Bar gene probe obtained in step 1 was used for hybridization. as a positive control.

The experimental results are shown in Figure 11 (lane 1 is the genomic DNA of T ₃ generation SbSNAC1-382 digested by restriction endonuclease BglII, and swimming lane 2 is the genomic DNA of T ₄ generation SbSNAC1-382 digested by restriction endonuclease BglII, Swimming lane 3 is the genomic DNA of T ₅ generation SbSNAC1-382 digested by restriction endonuclease BglII, swimming lane 4 is the genomic DNA of the maize inbred line Zheng 58 digested by restriction endonuclease BglII, and swimming lane 5 is the restriction endonuclease. Genomic DNA of T ₃ generation SbSNAC1-382 digested by nuclease DraI, lane 6 is the genomic DNA of T ₄ generation SbSNAC1-382 digested by restriction endonuclease DraI, and lane 7 is the genomic DNA of T 4 generation SbSNAC1-382 digested by restriction endonuclease DraI T _5th generation SbSNAC1-382 genomic DNA, lane 8 is the genomic DNA of the maize inbred line Zheng 58 digested by restriction endonuclease DraI, lane 9 is the recombinant plasmid 35S::SbSNAC1 digested by restriction endonuclease HindIII , lane 10 is Takara DL15kb DNA marker).

The results showed that the recombinant plasmid 35S::SbSNAC1 digested with the restriction endonuclease HindIII hybridized a fragment of 10.24 kb; after the restriction endonuclease BglII, the T3 _- T5 generation SbSNAC1-382 was compared with the maize The inbred line Zheng 58 has a specific hybridization band at a position greater than 15kb, which is consistent with the result of hybridization using the SbSNAC1 gene probe (the size of the specific band hybridized using the SbSNAC1 gene probe and the Bar gene probe is consistent); After digestion with endonuclease DraI, SbSNAC1-382 of T3 _- T5 generation had a specific hybridization band between 10-15kb compared with the maize inbred line Zheng 58, which was consistent with the result of hybridization using the SbSNAC1 gene probe (The specific bands hybridized with the SbSNAC1 gene probe and the Bar gene probe have the same size). The hybridization results showed that the two copies of the Bar gene in the SbSNAC1-382 event were inserted at a single site in the genome.

The above Southern hybridization results showed that the T-DNA fragment of the SbSNAC1-382 event was inserted at a single site in the maize genome, and there were two copies of the SbSNAC1 gene and the Bar gene at the insertion site, which could be stably inherited between different generations.

The SbSNAC1-382 incident has been deposited in the General Microbiology Center of the China Microorganism Culture Collection Management Committee (abbreviated as CGMCC, address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing) on April 4, 2019, and the deposit number is CGMCCNo. 17493. The full name of the SbSNAC1-382 event is Zea mays SbSNAC1-382 CGMCC No. 17493, and the abbreviated name is the SbSNAC1-382 event.

Example 3. Determination of the 5'-end flanking sequences and 3'-end flanking sequences of the exogenous insert at the SbSNAC1-382 event insertion site

The flanking sequences are specific for a particular transgenic event. Therefore, the use of flanking sequences can specifically detect transgenic events. PCR amplification, such as by hybridization with a probe comprising at least a portion of the flanking sequence and at least a portion of the exogenous insert, or a primer designed to specifically amplify a primer comprising at least a portion of the flanking sequence and at least a portion of the foreign insert, Detection of specific bands, etc. The upstream specific primer can be designed according to the sequence flanking the 5', the downstream specific primer can be designed according to the exogenous insert, and the specific fragment can be amplified; Sequence design of downstream specific primers to amplify specific fragments.

1. Acquisition and verification of the flanking sequence at the 5' end

1. Extract the genomic DNA of T _5th generation SbSNAC1-382 leaves and use it as a template, with specific primer GSP1: 5'-TATCCCTGGCTCGTCGCCGA-3', specific primer GSP2: 5'-AGGGCTTCAAGAGCGTGGTCGCT-3', specific primer GSP3 : 5'-CCGTCACCGAGATTTGACTCGAGTTTC-3' and random primers (components in the Genome walking Kit; Genome walking Kit is a product of TaKaRa company, the item number is 6108) to carry out TAIL-PCR reaction to obtain the left side of the integration site of the foreign gene in the maize genome. sequence of boundaries. The sequence is 933 bp long, as shown in sequence 3 in the sequence listing. Wherein, the 1-451st position of sequence 3 from the 5' end in the sequence listing is the maize genome sequence, and the 452nd-933th position is the vector sequence.

Specific upstream primers P1: 5'-AGAATCATACACCAGTAACAAGCC-3' and P3: 5'-GGAATGAACCTCATCCCAATGA-3' were designed and synthesized according to the DNA molecules shown at positions 1-451 from the 5' end of sequence 3 in the sequence listing. According to the DNA molecule shown at positions 452-933 from the 5' end of sequence 3 in the sequence listing, downstream identification primers P2: 5'-CAGTACATTAAAAACGTCCGCA-3' and P4: 5'-ACTAAAATCCAGATCCCCCGAA-3' were designed and synthesized.

2. Using the genomic DNA of the leaves of SbSNAC1-382, water, the genomic DNA of the leaves of the maize inbred line Zheng 58 or the genomic DNA of the leaves of SbSNAC1-383 as templates, primer pair A (composed of P1 and P2), primer pair B ( Composed of P3 and P4) or primer pair C (composed of P1 and P4) for PCR amplification to obtain PCR amplification products.

The reaction system is 20 μL, consisting of 2 μL 10× PCR buffer, 0.5 μL dNTP (concentration of 10 mmol/L), 0.5 μL Taq enzyme (concentration of 5 U/μL), 1.0 μL template (if it is the genomic DNA of corn leaves, the concentration is 50 ng/μL), 0.5 μL upstream primer (10 μmol/L concentration), 0.5 μL downstream primer (10 μmol/L concentration), and 15 μL ddH ₂ O.

The reaction program was: 95°C for 5 min; 95°C for 30 s, 60°C for 30 s, 72°C for 1 min, 35 cycles; 72°C for 5 min; storage at 15°C.

3. Perform 1% (m/v) agarose gel electrophoresis on the PCR amplification products.

The results of agarose gel electrophoresis are shown in Figure 12 (lanes 5 to 8 are primer pair A, lanes 1 to 4 are primer pair B, lanes 9 to 12 are primer pair C, and lanes 1, 5, and 9 are the genomes of SbSNAC1-382 leaves. DNA, lanes 2, 6, and 10 are water, lanes 3, 7, and 11 are genomic DNA from leaves of the maize inbred line Zheng 58, and lanes 4, 8, and 12 are genomic DNA from leaves of SbSNAC1-383). The results showed that using the genomic DNA of SbSNAC1-382 leaves as the template, a DNA fragment of about 311 bp could be obtained by using primer pair A, a DNA fragment of about 242 bp could be obtained by using primer pair B, and a DNA fragment of about 350 bp could be obtained by using primer pair C.

2. Acquisition and verification of the flanking sequence at the 3' end

1. The genomic DNA of T ₅ generation SbSNAC1-382 leaves was extracted to construct a Fosmid library (Takara Biotechnology (Dalian) Co., Ltd), and then the SbSNAC1 gene-specific primers 5'-GACCGCAAGTACCCAAACGG-3' and 5'-CACCCAGTCATCCAGCCTGAG-3' were used. PCR amplification (reaction conditions: 95°C, 5 min; denaturation at 95°C for 30s, annealing at 60°C for 30s, extension at 72°C for 30s, 34 cycles; extension at 72°C for 5 min; storage at 15°C), positive single clones were screened, and PacBio was used RSII was sequenced (Wuhan Future Group Biotechnology Co., Ltd.). According to the sequencing results, the sequence of the exogenous gene at the right border of the integration site of the maize genome was obtained. The sequence is 547 bp long, as shown in sequence 4 in the sequence listing. Wherein, the 1-352th position of sequence 4 from the 5' end in the sequence listing is the maize genome sequence, and the 353rd-547th position is the vector sequence.

Specific primers S1: 5'-AGTGCACATTGCAATCCTACAAGC-3' and S2: 5'-CCTAAGTTCATGCAACTAGAGGTTTCA-3' were designed and synthesized according to the DNA molecules shown at positions 1-352 from the 5' end of sequence 4 in the sequence listing. According to the DNA molecules shown at positions 353-547 from the 5' end of sequence 4 in the sequence listing, S3: 5'-GGTTTCGCTCATGTGTTGAGC-3' and S4: 5'-TCCAGATCCCCCGAATTAATTCG-3' were designed and synthesized.

2. Using the genomic DNA of SbSNAC1-382 leaves as the template, primer pair 1 (composed of S1 and S3), primer pair 2 (composed of S2 and S3), primer pair 3 (composed of S1 and S4), primer pair 4 were used (consisting of S2 and S4) or primer pair 5 (consisting of 5'-GACCGCAAGTACCCAAACGG-3' and 5'-CACCCAGTCATCCAGCCTGAG-3') for PCR amplification to obtain PCR amplification products.

Using water as a template, primer pair 5 was used for PCR amplification to obtain PCR amplification products. as a negative control.

The reaction system is 20 μL, consisting of 2 μL 10× PCR buffer, 0.5 μL dNTP (concentration of 10 mmol/L), 0.5 μL Taq enzyme (concentration of 5 U/μL), 1.0 μL template (if it is the genomic DNA of corn leaves, the concentration is 50 ng/μL), 0.5 μL upstream primer (10 μmol/L concentration), 0.5 μL downstream primer (10 μmol/L concentration), and 15 μL ddH ₂ O.

The reaction program was: 95°C for 5 min; 95°C for 30 s, 60°C for 30 s, 72°C for 1 min, 35 cycles; 72°C for 5 min; storage at 15°C.

3. Perform 1% (m/v) agarose gel electrophoresis on the PCR amplification products.

The results of agarose gel electrophoresis are shown in Figure 13 (lanes 1 to 5 are the genomic DNA of SbSNAC1-382 leaves, lane 6 is water, lanes 1 and 6 are primer pair 1, lane 2 is primer pair 2, and lane 3 is primer pair 3 , lane 4 is primer pair 4, and lane 5 is primer pair 5). The results showed that using the genomic DNA of SbSNAC1-382 leaves as a template, a DNA fragment of about 503 bp could be obtained with primer pair 1, a DNA fragment of about 547 bp could be obtained with primer pair 2, and a DNA fragment of about 392 bp could be obtained with primer pair 3. Using primer pair 4, a DNA fragment of about 436 bp can be obtained, and using primer pair 5, a DNA fragment of about 249 bp can be obtained.

<110> Institute of Crop Science, Chinese Academy of Agricultural Sciences

<120> A method for identifying whether a plant sample to be tested is derived from the SbSNAC1-382 event or its progeny

<160> 12

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<210> 10

<211> 27

<212> DNA

<213> Artificial sequence

<400> 10

cctaagttca tgcaactaga ggtttca 27

<210> 11

<211> 21

<212> DNA

<213> Artificial sequence

<400> 11

ggtttcgctc atgtgttgag c 21

<210> 12

<211> 23

<212> DNA

<213> Artificial sequence

<400> 12

tccagatccc ccgaattaat tcg 23

Claims (10)

1. A method for identifying whether a test plant sample is derived from the SbSNAC1-382 event or its progeny, comprising the steps of: detecting whether the genomic DNA of the plant sample to be detected contains a DNA fragment A and/or a DNA fragment B; then, the following judgment is made: if the genomic DNA of the plant sample to be tested contains the DNA fragment A and/or the DNA fragment B, the plant sample to be tested is derived from the SbSNAC1-382 event or the progeny thereof; if the genomic DNA of the plant sample to be tested does not contain the DNA fragment A and/or the DNA fragment B, the plant sample to be tested does not originate from the SbSNAC1-382 event or its progeny;

the nucleotide sequence of the DNA fragment A is shown as a sequence 3 in a sequence table;

the nucleotide sequence of the DNA fragment B is shown as a sequence 4 in a sequence table;

the SbSNAC1-382 event is corn Zea mays SbSNAC1-382CGMCC No. 17493.

2. The method of claim 1, wherein: the method for detecting whether the genomic DNA of the plant sample to be detected contains the DNA fragment A and/or the DNA fragment B is S1) or S2) or S3):

s1) direct sequencing;

s2) carrying out PCR amplification on the genome DNA of the plant sample to be detected by using the primer pair X and/or the primer pair Y, and then carrying out judgment as follows: if the target amplification product is obtained, the plant sample to be detected is derived from SbSNAC1-382 event or progeny thereof; if the target amplification product is not obtained, the plant sample to be detected does not originate from the SbSNAC1-382 event or the progeny thereof;

the primer pair X consists of an upstream primer FX and a downstream primer RX; the upstream primer FX is a part of a DNA molecule shown in 1 st to 451 th positions from the 5' end of a sequence 3 in a sequence table; the downstream primer RX is a reverse complementary sequence of a part of the DNA molecule shown in the 452-933 bit from the 5' end of the sequence 3 in the sequence table; the target amplification product of the primer pair X is a DNA molecule X;

the primer pair Y consists of an upstream primer FY and a downstream primer RY; the upstream primer FY is a part of a DNA molecule shown in 1 st to 352 th positions from the 5' end of a sequence 4 in a sequence table; the downstream primer RY is a reverse complementary sequence of a part of the DNA molecule shown in 353-547 th site from the 5' end of the sequence 4 in the sequence table; the target amplification product of the primer pair Y is a DNA molecule Y;

s3) carrying out Southern hybridization on the genomic DNA of the plant sample to be tested by using the probe A capable of specifically binding to the DNA molecule X and/or the probe B capable of specifically binding to the DNA molecule Y, and then carrying out the following judgment: if a hybrid fragment is obtained, the plant sample to be detected is derived from SbSNAC1-382 event or progeny thereof; if no hybrid fragments are available, the plant sample to be tested does not originate from the SbSNAC1-382 event or its progeny.

3. The method of claim 2, wherein:

the primer pair X is at least one of a primer pair X1, a primer pair X2 and a primer pair X3;

the primer pair X1 consists of a primer P1 and a primer P2;

the primer pair X2 consists of a primer P3 and a primer P4;

the primer pair X3 consists of a primer P1 and a primer P4;

the nucleotide sequence of the primer P1 is shown as a sequence 5 in the sequence table;

the nucleotide sequence of the primer P2 is shown as a sequence 6 in the sequence table;

the nucleotide sequence of the primer P3 is shown as a sequence 7 in the sequence table;

the nucleotide sequence of the primer P4 is shown as a sequence 8 in the sequence table;

the primer pair Y is at least one of a primer pair Y1, a primer pair Y2, a primer pair Y3 and a primer pair Y4;

the primer pair Y1 consists of a primer S1 and a primer S3;

the primer pair Y2 consists of a primer S2 and a primer S3;

the primer pair Y3 consists of a primer S1 and a primer S4;

the primer pair Y4 consists of a primer S2 and a primer S4;

the nucleotide sequence of the primer S1 is shown as a sequence 9 in the sequence table;

the nucleotide sequence of the primer S2 is shown as a sequence 10 in a sequence table;

the nucleotide sequence of the primer S3 is shown as a sequence 11 in the sequence table;

the nucleotide sequence of the primer S4 is shown as the sequence 12 in the sequence table.

4. A method according to claim 2 or 3, characterized by:

the nucleotide sequence of the target amplification product of the primer pair X1 is shown as the 215 th and 525 th positions from the 5' end of the sequence 3 in the sequence table;

the nucleotide sequence of the target amplification product of the primer pair X2 is shown as 323-564 th bits from the 5' end of the sequence 3 in the sequence table;

the nucleotide sequence of the target amplification product of the primer pair X3 is shown as 215-564 th site from the 5' end of the sequence 3 in the sequence table;

the nucleotide sequence of the target amplification product of the primer pair Y1 is shown as 45 th to 547 th sites from the 5' end of a sequence 4 in a sequence table;

the nucleotide sequence of the target amplification product of the primer pair Y2 is shown as 1 st-547 th site from the 5' end of a sequence 4 in a sequence table;

the nucleotide sequence of the target amplification product of the primer pair Y3 is shown as 215-436 th site from the 5' end of the sequence 4 in the sequence table;

the nucleotide sequence of the target amplification product of the primer pair Y4 is shown as 1 st to 436 th sites from the 5' end of the sequence 4 in the sequence table.

5. A kit for identifying whether a plant sample to be tested is derived from the SbSNAC1-382 event or its progeny, comprising primer pair X and/or primer pair Y according to any one of claims 2 to 4; the SbSNAC1-382 event is corn Zea mays SbSNAC1-382CGMCC No. 17493.

6. A kit for identifying whether a plant sample to be tested is derived from the SbSNAC1-382 event or its progeny, comprising a probe a capable of specifically binding to DNA molecule X and/or a probe b capable of specifically binding to DNA molecule Y according to any one of claims 2 to 4; the SbSNAC1-382 event is corn Zea mays SbSNAC1-382CGMCC No. 17493.

7. Use of primer pair X and/or primer pair Y according to any one of claims 2 to 4 for identifying whether a test plant sample is derived from the SbSNAC1-382 event or progeny thereof; the SbSNAC1-382 event is corn Zea mays SbSNAC1-382CGMCC No. 17493.

8. Use of a probe A capable of specifically binding to a DNA molecule X and/or a probe B capable of specifically binding to a DNA molecule Y according to any one of claims 2 to 4 for identifying whether a test plant sample originates from the SbSNAC1-382 event or from its progeny; the SbSNAC1-382 event is corn Zea mays SbSNAC1-382CGMCC No. 17493.

Use of DNA fragment a and/or DNA fragment B for identifying whether a plant sample to be tested is derived from SbSNAC1-382 event or progeny thereof; the SbSNAC1-382 event is corn Zea mays SbSNAC1-382CGMCC No. 17493;

the nucleotide sequence of the DNA fragment A is shown as a sequence 3 in a sequence table;

the nucleotide sequence of the DNA fragment B is shown as a sequence 4 in a sequence table.

DNA fragment A and/or DNA fragment B;

the nucleotide sequence of the DNA fragment A is shown as a sequence 3 in a sequence table;

the nucleotide sequence of the DNA fragment B is shown as a sequence 4 in a sequence table.

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