CN111394500A

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

Info

Publication number: CN111394500A
Application number: CN202010320879.3A
Authority: CN
Inventors: 王天宇; 张登峰; 曾廷儒; 李永祥; 李春辉; 宋燕春; 石云素; 黎裕
Original assignee: Institute of Crop Sciences of Chinese Academy of Agricultural Sciences
Current assignee: Institute of Crop Sciences of Chinese Academy of Agricultural Sciences
Priority date: 2020-04-22
Filing date: 2020-04-22
Publication date: 2020-07-10
Anticipated expiration: 2040-04-22
Also published as: CN111394500B

Abstract

The invention discloses a method for identifying whether a plant sample to be tested is derived from SbSNAC1-382 event or progeny thereof. The inventor of the invention transfers SbSNAC1 gene into the genome of Zheng 58 of maize inbred line by agrobacterium-mediated method to obtain a transgenic maize event SbSANC1-382, which is called SbSNAC1-382 for short. Drought resistance identification shows that the drought resistance of the SbSNAC1-382 event is obviously improved compared with that of Zheng 58 of a maize inbred line. Furthermore, detected, T₃generation-T₅The SbSNAC1-382 generation event is genetically stable and can be stably inherited from generation to generation. Thus, the SbSNAC1-382 event has the potential to enter commercial planting. The SbSNAC1-382 event has been preserved in China general microbiological culture Collection center (CGMCC, address No. 3 Xilu 1 Kyoto north Chen of the south facing Yang district in Beijing city) in 2019 at 04.04.9, and the preservation number is CGMCC No. 17493. The inventionThe method for identifying whether the plant sample is derived from the SbSNAC1-382 event or the progeny thereof can specifically detect the SbSNAC1-382 event and better supervise and manage the SbSNAC1-382 event. The invention has important application value.

Description

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

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a method for identifying whether a plant sample to be detected is from an SbSNAC1-382 event or a progeny thereof.

Background

The world grain production is seriously influenced by water resource shortage, the grain yield reduction caused by the water resource shortage accounts for more than 50 percent of the crop yield reduction every year, and the direct economic loss is difficult to estimate. The arid and semiarid regions of China occupy more than half of the soil area of China. According to statistics, the perennial drought area of China reaches about 20 percent of the total cultivated land area, and the grain yield reduction caused by drought reaches more than 1000 hundred million kilograms every year in nearly 40 years. Corn is one of the most important crops of grains, feeds and related industrial raw materials in the world. In China, corn has become the first large grain crop since 2009. The traditional corn genetic breeding plays a certain role in improving the drought resistance of the corn, but the genetic limitation among species is difficult to break through so as to obviously improve the drought resistance of the corn. The transgenic technology can break species boundaries, carry out directional modification and recombination transfer on genes, improve the drought resistance of the existing varieties and greatly improve the drought resistance of the corn. Practices of cultivating and popularizing transgenic corns in developed countries, particularly in the United states, have also proved that the drought resistance of corns can be remarkably improved by cultivating new varieties by the transgenic technology, and the method is the most realistic and effective way for greatly improving the yield and the quality and becomes the main direction of international corn breeding development. In 12 months 2011, the american ministry of agriculture animal and plant health quarantine (APHIS) officially approved monsanto transgenic drought resistant corn MON87460, which means that the first transgenic drought resistant corn in the world can be popularized and utilized in large scale in production. The drought-resistant corn transgene is a CspB gene (the CspB gene is from bacillus subtilis and codes an RNA chaperone protein), and under the condition of stress, the gene can enhance the function of plant cells and reduce the yield loss under the condition of water deficiency. Under the drought condition, the corn inbred line and the hybrid transformed by the gene can obviously improve the biomass and the yield of the corn, and the drought-resistant corn field test in the western drought region of the United states has already reached and even exceeds the yield-increasing target of 6 to 10 percent.

The SbSNAC1 gene is a NAC family gene cloned from Xinjiang a drought-tolerant local sorghum variety XG L-1 by Chinese academy of agricultural sciences crop science, and the overexpression of the gene in Arabidopsis can obviously improve the drought tolerance of transgenic Arabidopsis plants (L u et al, 2013).

Disclosure of Invention

The invention aims to identify whether a plant sample is derived from SbSNAC1-382 event or its progeny, wherein the SbSNAC1-382 event is maize Zea mays SbSNAC1-382CGMCC No. 17493. The plant sample may be a plant leaf, seed, or 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 comprise 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.

In the above method, the method for detecting whether the genomic DNA of the plant sample to be tested contains the DNA fragment a and/or the DNA fragment B may be S1), 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 primer pair Y is DNA molecule Y.

S3) carrying out Southern hybridization on the genomic DNA of the plant sample to be tested by using a probe A capable of specifically binding to the DNA molecule X and/or a 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.

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

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

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

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

The nucleotide sequence of the primer P1 can be shown as the sequence 5 in the sequence table.

The nucleotide sequence of the primer P2 can be shown as the sequence 6 in the sequence table.

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

The nucleotide sequence of the primer P4 can be shown as the sequence 8 in the sequence table.

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.

The primer pair Y1 may be composed of a primer S1 and a primer S3.

The primer pair Y2 may be composed of a primer S2 and a primer S3.

The primer pair Y3 may be composed of a primer S1 and a primer S4.

The primer pair Y4 may be composed of a primer S2 and a primer S4.

The nucleotide sequence of the primer S1 can be shown as the sequence 9 in the sequence table.

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

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

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

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

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

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

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

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

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

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

The 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 detected is from the SbSNAC1-382 event or the progeny thereof can be specifically a kit A; the kit A can comprise any one of the primer pair X and/or the primer pair Y; the SbSNAC1-382 event can be maize Zea mays SbSNAC1-382CGMCC No. 17493.

The kit A specifically comprises any one of the primer pair X and/or the primer pair Y.

The kit for identifying whether the plant sample to be detected is from the SbSNAC1-382 event or the progeny thereof can be specifically a kit B; the kit B can comprise any probe A capable of specifically binding to the DNA molecule X and/or any probe B capable of specifically binding to the DNA molecule Y; the SbSNAC1-382 event can be maize Zea mays SbSNAC1-382CGMCC No. 17493.

The kit B can specifically comprise any one of the probes A and/or any one of the probes B.

The application of any one of the primer pairs X and/or any one of the primer pairs Y in identifying whether a plant sample to be detected is from SbSNAC1-382 event or progeny thereof also belongs to the protection scope of the invention; the SbSNAC1-382 event can be maize Zea mays SbSNAC1-382CGMCC No. 17493.

The application of any probe A capable of specifically binding to the DNA molecule X and/or any probe B capable of specifically binding to the DNA molecule Y in identifying whether the plant sample to be detected is from SbSNAC1-382 event or progeny thereof also belongs to the protection scope of the invention; the SbSNAC1-382 event can be maize Zea mays SbSNAC1-382CGMCC No. 17493.

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

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

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

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

The inventor of the invention transfers SbSNAC1 gene into the genome of Zheng 58 of maize inbred line by agrobacterium-mediated method to obtain a transgenic maize event SbSANC1-382 (SbSNAC 1-382 for short). Drought resistance identification shows that the drought resistance of the SbSNAC1-382 event is obviously improved compared with that of a control corn (namely Zheng 58 of a corn inbred line). Furthermore, detected, T₃generation-T₅The SbSNAC1-382 generation event is genetically stable and can be stably inherited from generation to generation. Therefore, the SbSNAC1-382 event is likely to enter into commercial planting, the SbSNAC1-382 event is preserved in China general microbiological culture Collection center (CGMCC, address: No. 3 Xilu 1 Beichen of the rising district in Beijing city) in 04.2019 at 04.M., and the preservation number is CGMCC No. 17493. The method for identifying whether the plant sample is derived from the SbSNAC1-382 event or the progeny thereof can specifically detect the SbSNAC1-382 event and better supervise and manage the SbSNAC1-382 event. The invention has important application value.

Drawings

FIG. 1 shows the result of agarose gel electrophoresis in step three of example 1.

FIG. 2 shows the results of step five 2 in example 1.

FIG. 3 shows the results of step five 3 in example 1.

FIG. 4 shows the result of step seven in example 1.

FIG. 5 is the observation result of the eighth grouting stage in example 1.

FIG. 6 shows the results of the eight steps in example 1 after harvesting.

FIG. 7 is a vector diagram of recombinant plasmid 35S: SbSNAC 1.

FIG. 8 shows the results of the first step experiment in example 2.

FIG. 9 shows the results of the experiment in step two of example 2.

FIG. 10 shows the results of the experiment in step three of example 2.

FIG. 11 shows the results of the experiment in step four of example 2.

FIG. 12 shows the results of the first step in example 3.

FIG. 13 shows the results of the experiment in step two of example 3.

Detailed Description

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

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

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

The quantitative tests in the following examples, all set up three replicates and the results averaged.

The plasmid pCAMBIA3301 is a product of Cambia corporation.

Example 1 acquisition and drought resistance identification of SbSNAC1 transgenic maize

Cloning of the SbSNAC1 Gene

1. Leaves and roots of a sorghum variety XG L-1 (provided by grain crop research institute of Sinkiang academy of agricultural sciences) are mixed and used as a material.

2. And (3) after the step 1 is finished, taking the material, extracting total RNA, and then carrying out reverse transcription to obtain the cDNA of the sorghum variety XG L-1.

3. After the step 3 is finished, the cDNA of the sorghum variety XG L-1 is taken as a template, and 5' -TTT is adoptedCCATGGGATTGCCGGTGAT-3 '(recognition site for restriction enzyme NcoI is underlined) and 5' -TTTGGTGACCAGCCTCAGAATGGCCCCAAC-3' (recognition sites of restriction enzyme BstE II are underlined) were subjected to PCR amplification to obtain a PCR amplification product of about 985 bp.

4. And (3) connecting the PCR amplification product obtained in the step (3) with a PMD18-T vector to obtain a recombinant plasmid PMD18-SbSNAC 1.

The recombinant plasmid PMD18-SbSNAC1 was sequenced. The sequencing result shows that the recombinant plasmid PMD18-SbSNAC1 contains DNA molecules shown in 1 st to 966 th positions from the 5' tail end of a sequence 1 in a sequence table.

Second, construction of recombinant plasmid 35S, SbSNAC1

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

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

3. The DNA fragment recovered in step 1 was ligated with the vector backbone recovered in step 2 to obtain recombinant plasmid 35S: SbSNAC 1.

Recombinant plasmid 35S:SbSNAC 1 was sequenced. According to the sequencing results, the structure of recombinant plasmid 35S: SbSNAC1 is described as follows: a small fragment between the recognition sequences for restriction enzymes NcoI and BstE II of plasmid pCAMBIA3301 was replaced with a DNA molecule shown in the 5 th to 970 th positions from the 5' end of sequence 1 in the sequence listing. Recombinant plasmid 35S, wherein the protein SbSNAC1 shown in a sequence 2 in a sequence table is expressed by SbSNAC 1.

Thirdly, obtaining of positive recombinant agrobacterium

1. The recombinant plasmid 35S is transformed into agrobacterium tumefaciens EH105 by adopting a freeze-thawing method, so as to obtain the recombinant agrobacterium tumefaciens.

2. After the step 1 is completed, the monoclonals of the recombinant agrobacterium are respectively inoculated to YEB liquid culture medium and cultured for 16h at 28 ℃ and 200rpm, so that agrobacterium liquid is obtained.

3. And (3) after the step 2 is finished, performing PCR amplification by respectively taking the agrobacterium liquid, water and recombinant plasmid 35S, wherein SbSNAC1 is used as a template, and 5'-TTTCCATGGGATTGCCGGTGAT-3' and 5'-TTTGGTGACCAGCCTCAGAATGGCCCCAAC-3' are used as primers to obtain PCR amplification products. Recombinant plasmid 35S: SbSNAC1 was used as a positive control. Water was used as a negative control.

4. After the step 3 is completed, carrying out 1% (m/v) agarose gel electrophoresis on the PCR amplification product, and judging according to an electrophoresis result as follows: if the PCR amplification product of a certain recombinant agrobacterium contains a DNA fragment of about 985bp, the recombinant agrobacterium is a positive recombinant agrobacterium.

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

Four, T₀Obtaining of maize with SbSNAC1 gene

1. Inoculating the monoclonal of the positive recombinant agrobacterium obtained in the step three to a YEB liquid culture medium, culturing at 28 ℃ and 200rpm to obtain OD_550nm0.3-0.4 of agrobacterium liquid.

2. After the step 1 is finished, taking agrobacterium liquid, centrifuging for 10min at 4 ℃ and 10000rpm, and collecting thalli.

3. Adding the thallus collected in the step 2 into an infection culture medium (MS culture medium containing 100 mu M acetosyringone) for resuspension to obtain OD_550nm0.3-0.4 of the staining solution. Collecting young embryo of Zheng 58 of corn inbred line pollinated for 11-12 days and with size of 1.0-1.5mm, infecting (soaking) with infection solution in dark condition for 5min, placing on co-culture medium, sealing with air permeable adhesive tape, and culturing at 20 deg.C in dark for 3 days.

4. After the step 3 is completed, the young embryo of Zheng 58 of the maize inbred line is taken and placed in a resting culture medium for culture for 7 days at 28 ℃.

5. After the step 4 is finished, placing the young embryo of Zheng 58 of the maize inbred line on a selective medium 1, and culturing for 14 days at 28 ℃; then placing on a selection culture medium 2, and culturing for 14 days at 28 ℃ to obtain the resistant callus.

6. After step 5, the resistant callus was taken and placed in regeneration medium 1, and dark culture was carried out at 25 ℃ for 14 days to obtain mature somatic embryos.

7. After the step 6 is completed, transferring the mature somatic embryos to a regeneration culture medium 2, and alternately culturing for 7-10 days at 25 ℃ in light and dark to obtain resistant seedlings. Culturing alternately in 16h and 8h under illumination at illumination intensity of 80-100 μ E/m 2/s.

8. After the step 7 is finished, the resistant seedlings are transplanted into soil to obtain T₀Substitution of the SbSNAC1 baseDue to the corn.

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

Five, T₀Identification of maize transgenic for the SbSNAC1 gene

1. Spraying Basta

Respectively to T₀The leaf of the plant which is used for replacing the SbSNAC1 gene transferred corn is sprayed with Basta (the concentration is 2 per mill), and the leaf is observed after 5 to 7 days. If a certain T₀When no withering occurs in leaves of the maize with the SbSNAC1 gene, the T is₀Preliminary identification of transgenic SbSNAC1 gene in maize as T₀Transgenic SbSNAC1 gene maize.

Preliminary identification of T by spraying Basta₀The 7 lines of maize transgenic for the sbSNAC1 gene were designated as SbSNC1-382, SbSNAC1-383, SbSNAC1-389, SbSNAC1-466, SbSNAC1-467, SbSNAC1-471 and SbSNAC1-474, respectively.

2. PCR detection of SbSNAC1 Gene

(1) Separately extracting T₀The genomic DNA of leaves of maize (SbSNC1-382, SbSNAC1-383, SbSNAC1-389, SbSNAC1-466, SbSNAC1-467, SbSNAC1-471 or SbSNAC1-474) transgenic for SbSNAC1 gene was used as a template for PCR amplification using a specific primer pair (consisting of SbSNAC 1-F: 5'-GACCGCAAGTACCCAAACGG-3' and SbSNAC 1-R: 5'-CACCCAGTCATCCAGCCTGAG-3', wherein SbSNAC1-F spans two exons) for amplifying SbSNAC1 gene to obtain PCR amplification products.

According to the steps, the template is replaced by the genome DNA of Zheng 58 leaves of the maize inbred line, and other steps are not changed and are used as negative control.

According to the steps, the template is replaced by water, and other steps are unchanged and used as water contrast.

The template was replaced with recombinant plasmid 35S, SbSNAC1, as a positive control, following the above procedure, with no other steps.

Reaction conditions are as follows: 95 ℃ for 5 min; 30s at 95 ℃, 30s at 60 ℃, 30s at 72 ℃ and 34 cycles; 5min at 72 ℃; storing at 15 ℃.

(2) Subjecting each PCR amplification product to 1% (m/v) agarose gel electrophoresis, and judging according to the electrophoresis result as follows: if the PCR amplification product of a certain strain contains a DNA fragment of about 249bp, the strain is identified as T again₀Transgenic SbSNAC1 gene maize.

The results of the agarose gel electrophoresis are shown in FIG. 2(Marker is DNA Marker, 1 to 7 are SbSNC1-382, SbSNAC1-383, SbSNAC1-389, SbSNAC1-466, SbSNAC1-467, SbSNAC1-471 and SbSNAC1-474 in this order).

3. PCR detection of Bar Gene

(1) Extraction of T₀The PCR amplification product was obtained by transferring the genomic DNA of leaves of maize (SbSNC1-382, SbSNAC1-383, SbSNAC1-389, SbSNAC1-466, SbSNAC1-467, SbSNAC1-471 or SbSNAC1-474) which is an SbSNAC1 gene into a genome and using the genomic DNA as a template, and performing PCR amplification using a primer pair (consisting of 5'-GAAGTCCAGCTGCCAGAAAC-3' and 5'-GTCTGCACCATCGTCAACC-3') specific to the Bar gene.

According to the steps, the template is replaced by water, and other steps are unchanged and used as blank control.

(2) Subjecting each PCR amplification product to 1% (m/v) agarose gel electrophoresis, and judging according to the electrophoresis result as follows: if the PCR amplification product of a certain strain contains a DNA fragment of about 444bp, the strain is identified as T again₀Transgenic SbSNAC1 gene maize.

The results of the partial agarose gel electrophoresis are shown in FIG. 3(Marker is DNA Marker, lanes 1 to 7 are SbSNC1-382, SbSNAC1-383, SbSNAC1-389, SbSNAC1-466, SbSNAC1-467, SbSNAC1-471 and SbSNAC1-474 in this order, lane 11 is a positive control, and lane 12 is a negative control).

The above results indicate that SbSNC1-382, SbSNAC1-383, SbSNAC1-389, SbSNAC1-466, SbSNAC1-467, SbSNAC1-471 and SbSNAC1-474 are all T₀Transgenic SbSNAC1 gene maize.

Sixth, T₁generation-T₅Generation of SbSNAC1-382 Generation

Get T₀Transforming SbSNAC1 gene corn seeds, selfing to obtain T₁The SbSNAC1 gene is transferred to the corn seeds. T is₁Continuous selfing of the seeds of the corn with the SbSNAC1 gene to obtain T₂Seed and T of corn with SbSNAC1 gene₃Seed and T of corn with SbSNAC1 gene₄Seed and T of SbSNAC1 gene transferred corn₅The SbSNAC1 gene is transferred to the corn seeds.

Selection of T₁Transgenic SbSNAC1 gene maize-T₅Subsequent experiments were performed with SbSNAC1 gene transgenic maize.

Seven, T₁generation-T₅Greenhouse drought resistance identification of transgenic SbSNAC1 gene corn

The corn to be tested is T₁generation-T₅Maize transgenic for SbSNAC1 gene (SbSNC1-382, SbSNAC1-383, SbSNAC1-389, SbSNAC1-466, SbSNAC1-467, SbSNAC1-471 or SbSNAC1-474) or Zea mays inbred line Zheng 58. Zheng 58 of maize inbred line was used as control.

(1) Planting 3 corns to be tested in a flowerpot, and planting each strain for 5 times; then placing in a greenhouse for conventional management.

(2) And (3) after the step (1) is finished, stopping watering when the corn to be detected grows until the visible leaves of the corn reach 4 leaves and 1 heart, and continuously drying for 21 days. And observing the growth state of the corn to be detected.

The growth state of part of the maize to be tested is shown in figure 4(SbSNC1-382 is T₅And the SbSNC1-382, Zheng 58 is a maize inbred line Zheng 58). The results show that the leaves of Zheng 58 of the maize inbred line are curled and wilting phenomenon appears, and T₁generation-T₅The leaves of the corn transformed with the SbSNAC1 gene were extended and remained green. Thus, it can be seen that T is compared with Zheng 58 of maize inbred line₁generation-T₅Transgenic SbSNAC 1-gene maizeThe drought resistance is obviously improved.

Eight, T₃generation-T₅Identification of field drought resistance of SbSNC1-382 generation

In 2015, T₃And (3) carrying out field drought resistance identification on the SbSNC1-382 and Zheng 58 of the maize inbred line.

2016, get T₄And (3) carrying out field drought resistance identification on the SbSNC1-382 and Zheng 58 of the maize inbred line.

In 2017, get T₅And (3) carrying out field drought resistance identification on the SbSNC1-382 and Zheng 58 of the maize inbred line.

The drought resistance identification is carried out by water treatment and dry treatment, wherein each material is planted in 6 rows, the row length is 5m, the process is repeated for 3 times, the water treatment and drip irrigation are carried out for 7 times, the water amount is 350 square/mu, and the dry treatment and the watering are 150 square/mu.

The corns for drought resistance identification are all planted in the Xinjiang Wulu wood-Qianning ditch test field. The growth state and yield during the grouting period were observed.

The test results are as follows:

1. the observation result in the grouting period shows (FIG. 5, SbSNC1-382 is T₃Substituted SbSNC 1-382): in the arid region, with T₃Substituted SbSNC1-382, T₄Substituted SbSNC1-382 or T₅Compared with SbSNC1-382, the plant height of Zheng 58 of corn inbred line is obviously reduced, and the leaf color is obviously green and yellow;

2. post harvest measurements (Table 1 and FIG. 6), in the drought region, T₃Substituted SbSNC1-382, T₄Substituted SbSNC1-382 or T₅The yield of the generation SbSNC1-382 is obviously increased compared with the Zheng 58 of the maize inbred line.

TABLE 1 average yield per plant of SbSNAC1-382 in the Water and Dry regions

Note: the data in the table are mean values ± standard error; the average yield per plant was the yield of the seeds (14% moisture).

The test result shows that compared with Zheng 58 of a corn inbred line, T₃Substituted SbSNC1-382, T₄Substituted SbSNC1-382 or T₅The drought resistance of the SbSNC1-382 generation is obviously improved.

Example 2, T₃generation-T₅Genetic stability test of the SbSNAC1-382 Generation

Recombinant plasmid 35S: A schematic of the vector for SbSNAC1 is shown in FIG. 7. Recombinant plasmid 35S, SbSNAC1, the size of which is 10.24kb, has only one restriction enzyme HindIII and one EcoRI site on the vector, so that a linear fragment of 10.24kb can be obtained by single digestion with the two enzymes. In addition, the vector has no restriction sites for BglII and DraI.

The genomic DNA of SbSNC1-382 event or Zea mays inbred line Zheng 58 was single digested with restriction enzymes HindIII and EcoRI, respectively, and hybridization was performed using probes specific to SbSNAC1 gene and Bar gene, respectively. The results showed that the SbSNAC1-382 event contained 2 copies of the SbSNAC1 gene and the Bar gene, and that these two copies were in the transgene T₃、T₄And T₅Generations can be stably inherited. Therefore, these 2 copies are likely to be inserted into the same position of the maize genome. To verify this hypothesis, the genomic DNA of SbSNAC1-382 event and Zea mays inbred line Zheng 58 was digested with restriction enzymes BglII and DraI, respectively, which did not have a cleavage site in the insertion sequence (T-DNA), thereby determining the number of insertion sites (the number of integration sites of T-DNA in Zea mays genome). Southern hybridization revealed the SbSNAC1-382 event (i.e., T)₃generation-T₅Generations SbSNAC1-382) is a single site insertion of 2 copies of the SbSNAC1 and Bar genes into the maize genome and is stably inherited from different generations. The specific results are as follows:

first, the Southern hybridization was carried out using SbSNAC1 gene probe, restriction enzymes HindIII and EcoRI

1. PCR amplification was carried out using recombinant plasmid 35S as template and SbSNAC1 as primers 5'-CGCGTGGGGTCAAGACGGACTG-3' and 5'-GGGAACGAGTCCAGCTCCGGGAAC-3' to obtain a DNA fragment of about 386 bp. This fragment is the SbSNAC1 gene probe.

2. T was treated with restriction enzymes HindIII and EcoRI respectively₃SbSNAC1-382, T₄SbSNAC1-382, T₅For SbSNAC1-382 or Zheng 58 of maize inbred lineThe genomic DNA was cleaved with an enzyme and hybridized with the SbSNAC1 gene probe obtained in step 1.

The recombinant plasmid 35S was digested with the restriction enzyme HindIII, SbSNAC1 and hybridized with the SbSNAC1 gene probe obtained in step 1. As a positive control.

The results are shown in FIG. 8 (lane 1, restriction enzyme HindIII digested T₃Genomic DNA from SbSNAC1-382, lane 2 is T digested with the restriction enzyme HindIII₄Genomic DNA from SbSNAC1-382, lane 3 is T digested with the restriction enzyme HindIII₅The genomic DNA of the inbred line Zheng 58 of maize was digested with restriction enzyme HindIII in lane 4 and restriction enzyme EcoRI in lane 5 instead of SbSNAC1-382₃Genomic DNA from SbSNAC1-382, lane 6 restriction enzyme EcoRI digested T₄Genomic DNA from SbSNAC1-382, lane 7 restriction enzyme EcoRI digested T₅The genomic DNA of the inbred line Zheng 58 of maize was digested with restriction enzyme EcoRI in place of SbSNAC1-382 in lane 8, and restriction enzyme HindIII in lane 9, and recombinant plasmid 35S digested with restriction enzyme HindIII in lane 9, SbSNAC1 and Takara D L15 kb DNA marker in lane 10, it was shown that 10.24kb fragment was hybridized from recombinant plasmid 35S digested with restriction enzyme HindIII in lane SbSNAC1, and that T was digested with restriction enzyme HindIII₃generation-T₅The sbSNAC1-382 has 2 specific hybridization bands between 2.5-5kb relative to Zheng 58, and endogenous background hybridization bands are present in Zheng 58, which are signals generated by nonspecific hybridization of SbSNAC1 gene probe with homologous sequences in corn genome, and the same hybridization band is observed in the event of SbSNAC1-382 regardless of insertion, thus being endogenous background hybridization band; after cleavage with the restriction enzyme EcoRI, T₃generation-T₅The sbSNAC1-382 has 2 specific hybridization bands between 2.5-5kb relative to Zheng 58, and endogenous background hybridization bands are present in Zheng 58, which are signals generated by nonspecific hybridization of SbSNAC1 gene probe with homologous sequences in maize genome, and the same hybridization bands are observed in the SbSNAC1-382 event regardless of insertion,thus, it is an endogenous background hybridization band. As the recombinant plasmid 35S has only one restriction enzyme HindIII and one EcoRI cutting site in SbSNAC1, the specific number of the inserted copies represented by the specific number of the bands obtained by cutting and hybridizing the transgenic corn genome DNA with the two restriction enzymes respectively. The hybridization results showed that the insertion of the SbSNAC1-382 event had 2 copies of the SbSNAC1 gene.

Second, the results of Southern hybridization using Bar Gene Probe, restriction enzymes HindIII and EcoRI

1. PCR amplification was carried out using recombinant plasmid 35S as template and SbSNAC1 as primers 5'-ATGAGCCCAGAACGACGCCCG-3' and 5'-TCAAATCTCGGTGACGGGCAGGAC-3' to obtain a DNA fragment of about 552 bp. The fragment is the Bar gene probe.

2. T was treated with restriction enzymes HindIII and EcoRI respectively₃SbSNAC1-382, T₄SbSNAC1-382, T₅Cutting the genome DNA of the generation SbSNAC1-382 or Zheng 58 of the maize inbred line by enzyme, and hybridizing the Bar gene probe obtained in the step 1.

The recombinant plasmid 35S:SbSNAC 1 was digested with the restriction enzyme HindIII and hybridized with the Bar gene probe obtained in step 1. As a positive control.

The results are shown in FIG. 9 (lane 1, restriction enzyme HindIII digested T₃Genomic DNA from SbSNAC1-382, lane 2 is T digested with the restriction enzyme HindIII₄Genomic DNA from SbSNAC1-382, lane 3 is T digested with the restriction enzyme HindIII₅The genomic DNA of the inbred line Zheng 58 of maize was digested with restriction enzyme HindIII in lane 4 and restriction enzyme EcoRI in lane 5 instead of SbSNAC1-382₃Genomic DNA from SbSNAC1-382, lane 6 restriction enzyme EcoRI digested T₄Genomic DNA from SbSNAC1-382, lane 7 restriction enzyme EcoRI digested T₅The genomic DNA of the inbred line Zheng 58 of maize was digested with restriction enzyme EcoRI in lane 8, and HindIII in lane 9, and SbSNAC1, Takara D L15 kb DNA marker in lane 10 in lane 9 instead of SbSNAC1-382The recombinant plasmid 35S cut by the endonuclease HindIII, wherein a 10.24kb fragment is hybridized by SbSNAC1 (a hybridization band is weaker due to the lower loading amount of the plasmid); after cleavage with the restriction enzyme HindIII, T₃generation-T₅The generation SbSNAC1-382 has a band near 2.5kb and 4kb respectively, and no band appears in Zheng 58 of the maize inbred line; after cleavage with the restriction enzyme EcoRI, T₃generation-T₅The sbSNAC1-382 has a band near 4kb and 5kb, and no band appears in Zheng 58 of maize inbred line; as the recombinant plasmid 35S has only one restriction enzyme HindIII and one EcoRI cutting site in SbSNAC1, the specific number of the inserted copies represented by the specific number of the bands obtained by cutting and hybridizing the transgenic corn genome DNA with the two restriction enzymes respectively. The hybridization results showed that the insertion of the SbSNAC1-382 event had 2 copies of the Bar gene.

Third, the result of Southern hybridization using SbSNAC1 gene probe, restriction enzymes BglII and DraI

1. The same as step 1.

2. For T with restriction enzymes BglII and DraI respectively₃SbSNAC1-382, T₄SbSNAC1-382, T₅Cutting the genome DNA of the generation SbSNAC1-382 or Zheng 58 of the maize inbred line by enzyme, and hybridizing the gene probe SbSNAC1 obtained in the step 1.

The recombinant plasmid 35S:SbSNAC 1 was digested with restriction enzyme DraI and hybridized with the SbSNAC1 gene probe obtained in step 1. As a positive control.

The results are shown in FIG. 10 (lane 1 shows the restriction enzyme BglII digested T₃Genomic DNA from SbSNAC1-382, lane 2, restriction endonuclease BglII cut T₄Genomic DNA from SbSNAC1-382, lane 3 is T cut with the restriction enzyme BglII₅The genomic DNA of the inbred line Zheng 58 of maize was digested with the restriction enzyme BglII in lane 4 and the restriction enzyme DraI in lane 5 instead of SbSNAC1-382₃Genomic DNA from SbSNAC1-382, lane 6, restriction endonuclease DraI cut T₄Genomic DNA from SbSNAC1-382, lane 7, restriction endonuclease DraI cut T₅Sb substituteGenomic DNA of SNAC1-382, lane 8 is genomic DNA of Zheng 58 from the inbred line of maize digested with restriction enzyme DraI, lane 9 is recombinant plasmid 35S digested with restriction enzyme HindIII, SbSNAC1, lane 10 is Takara D L15 kb DNA marker.) the results show that 10.24kb fragment was hybridized from recombinant plasmid 35S digested with restriction enzyme HindIII, SbSNAC1, and T after digestion with restriction enzyme BglII₃generation-T₅The generation of SbSNAC1-382 has 1 specific hybridization band at the position of more than 15kb relative to Zheng 58 of maize inbred line, endogenous background hybridization bands exist in Zheng 58 of maize inbred line, these signals are generated by nonspecific hybridization of SbSNAC1 gene probe and homologous sequences in maize genome, and the same hybridization band can be observed in the event of SbSNAC1-382 regardless of insertion, thus being endogenous background hybridization band; after cleavage with restriction enzyme DraI, T₃generation-T₅The sbSNAC1-382 has 1 specific hybridization band between 10-15kb relative to Zheng 58, and endogenous background hybridization band in Zheng 58, which are signals generated by nonspecific hybridization of SbSNAC1 gene probe with homologous sequence in corn genome, and independent of insertion, the same hybridization band can be observed in the event of SbSNAC1-382, thus being endogenous background hybridization band. The hybridization results showed that the 2 copies of the SbSNAC1 gene from SbSNAC1-382 event were single site insertions in the genome.

Fourth, the Southern hybridization results using Bar Gene Probe, restriction enzymes BglII and DraI

1. The same as step 1 in the second step.

2. For T with restriction enzymes BglII and DraI respectively₃SbSNAC1-382, T₄SbSNAC1-382, T₅Cutting the genome DNA of the generation SbSNAC1-382 or Zheng 58 of the maize inbred line by enzyme, and hybridizing the Bar gene probe obtained in the step 1.

The results are shown in FIG. 11 (lane 1 shows the restriction enzyme BglII digested T₃Substituted for SbSNAC1-382Genomic DNA, lane 2 restriction endonuclease BglII digested T₄Genomic DNA from SbSNAC1-382, lane 3 is T cut with the restriction enzyme BglII₅The genomic DNA of the inbred line Zheng 58 of maize was digested with the restriction enzyme BglII in lane 4 and the restriction enzyme DraI in lane 5 instead of SbSNAC1-382₃Genomic DNA from SbSNAC1-382, lane 6, restriction endonuclease DraI cut T₄Genomic DNA from SbSNAC1-382, lane 7, restriction endonuclease DraI cut T₅The genomic DNA of the inbred line Zheng 58 of maize was digested with restriction enzyme DraI in lane 8 instead of SbSNAC1-382, and recombinant plasmid 35S digested with restriction enzyme HindIII in lane 9: SbSNAC1 in lane 10, Takara D L15 kb DNA marker).

The result shows that the recombinant plasmid 35S cut by the restriction enzyme HindIII hybridizes to a 10.24kb fragment from SbSNAC 1; after cleavage with the restriction enzyme BglII, T₃passage-T5 SbSNAC1-382 has 1 specific hybridization band at the position larger than 15kb relative to Zea mays inbred line Zheng 58, which is consistent with the result of hybridization using SbSNAC1 gene probe (the size of the specific band hybridized using SbSNAC1 gene probe and Bar gene probe is consistent); after cleavage with restriction enzyme DraI, T₃passage-T5 SbSNAC1-382 has 1 specific hybridization band between 10-15kb relative to Zea mays inbred line Zheng 58, which is consistent with the results of hybridization using SbSNAC1 gene probe (the size of the specific band hybridized using SbSNAC1 gene probe and Bar gene probe is consistent). Hybridization results showed that the 2 copies of the Bar gene at SbSNAC1-382 event were single site insertions in the genome.

The Southern hybridization results indicated that the SbSNAC1-382 event T-DNA fragment was a single site insertion in the maize genome with 2 copies of the SbSNAC1 gene and the Bar gene and was stably inherited from different generations.

The SbSNAC1-382 event has been preserved in China general microbiological culture Collection center (CGMCC, address No. 3 Xilu 1 Kyoto north Chen of the south-facing-Yang district in Beijing) in 04.2019 and the preservation number is CGMCC No. 17493. The whole sequence of the SbSNAC1-382 event is called Zea mays SbSNAC1-382CGMCC No.17493, which is abbreviated as SbSNAC1-382 event.

Example 3 determination of the 5 'and 3' flanking sequences of the foreign insert at the SbSNAC1-382 event insertion site

The flanking sequences of a particular transgenic event are specific. Thus, the use of flanking sequences allows the specific detection of transgenic events. Such as hybridization with a probe comprising at least part of the flanking sequence and at least part of the foreign insert, or design of primers for specific amplification comprising at least part of the flanking sequence and at least part of the foreign insert, PCR amplification, detection of specific bands, etc. Can design upstream specific primers according to the flanking sequence at the 5' part, design downstream specific primers according to exogenous insert fragments, and amplify specific fragments; or designing an upstream specific primer according to the exogenous insert fragment, designing a downstream specific primer according to the 3' end flanking sequence, and amplifying the specific fragment.

Obtaining and verifying flanking sequence of one and 5' ends

1. Extraction of T₅The Genome DNA of SbSNAC1-382 leaf is substituted and used as a template, a specific primer GSP1: 5'-TATCCCTGGCTCGTCGCCGA-3', a specific primer GSP 2: 5'-AGGGCTTCAAGAGCGTGGTCGCT-3', a specific primer GSP 3: 5'-CCGTCACCGAGATTTGACTCGAGTTTC-3' and a random primer (a component in a Genome walking Kit; the Genome walking Kit is a product of TaKaRa company and has the product number of 6108) are used for carrying out TAI L-PCR reaction, and the sequence of the left boundary of the integration site of the corn Genome of the exogenous gene is obtained, the sequence length is 933bp, and is specifically shown as a sequence 3 in a sequence table, wherein the 1 st to 451 th positions of the sequence 3 in the sequence table from the 5' end are the corn Genome sequence, and the 452 nd to 933 position are a vector sequence.

Designing and synthesizing a specific upstream primer P1 according to the DNA molecule shown in the 1 st to 451 th sites from the 5' end of the sequence 3 in the sequence table: 5'-AGAATCATACACCAGTAACAAGCC-3' and P3: 5'-GGAATGAACCTCATCCCAATGA-3' are provided. Designing and synthesizing a downstream identification primer P2 according to the DNA molecule shown in the No. 452-933 site from the 5' end of the sequence 3 in the sequence table: 5'-CAGTACATTAAAAACGTCCGCA-3' and P4: 5'-ACTAAAATCCAGATCCCCCGAA-3' are provided.

2. Taking the genome DNA of SbSNAC1-382 leaf blade, water, the genome DNA of Zheng 58 leaf blade of maize inbred line or the genome DNA of SbSNAC1-383 leaf blade as a template, and carrying out PCR amplification by adopting a primer pair A (consisting of P1 and P2), a primer pair B (consisting of P3 and P4) or a primer pair C (consisting of P1 and P4) to obtain a PCR amplification product.

The reaction system is 20 mu L, and consists of 2 mu L10 × PCR buffer, 0.5 mu L0 dNTP (concentration is 10 mmol/L1), 0.5 mu L2 Taq enzyme (concentration is 5U/mu L3), 1.0 mu L template (concentration is 50 ng/mu L if the template is the genome DNA of a corn leaf), 0.5 mu L upstream primer (concentration is 10 mu mol/L), 0.5 mu L downstream primer (concentration is 10 mu mol/L) and 15 mu L ddH₂O。

The reaction procedure is as follows: 5min at 95 ℃; at 95 ℃ for 30s, 60 ℃ for 30s, and 72 ℃ for 1min, and 35 cycles; 5min at 72 ℃; storing at 15 ℃.

3. The PCR amplification products were subjected to 1% (m/v) agarose gel electrophoresis.

The results of the agarose gel electrophoresis are shown in FIG. 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,

lanes

1, 5 and 9 are genomic DNAs of SbSNAC1-382 leaves,

lanes

2, 6 and 10 are water,

lanes

3, 7 and 11 are genomic DNAs of Zheng 58 leaves of maize inbred line, and

lanes

4, 8 and 12 are genomic DNAs of SbSNAC1-383 leaves). The results showed that using the genomic DNA of SbSNAC1-382 leaf as template, a DNA fragment of about 311bp could be obtained using primer pair A, a DNA fragment of about 242bp could be obtained using primer pair B, and a DNA fragment of about 350bp could be obtained using primer pair C.

Obtaining and verifying flanking sequence of second and 3' end

1. Extraction of T₅Construction of Fosmid library (Takara Biotechnology (Dalian) Co., L td) from genomic DNA of SbSNAC1-382 leaf, PCR amplification with SbSNAC1 Gene-specific primers 5'-GACCGCAAGTACCCAAACGG-3' and 5'-CACCCAGTCATCCAGCCTGAG-3' (reaction conditions: 95 ℃, 5 min; denaturation at 95 ℃ for 30s, annealing at 60 ℃ for 30s, elongation at 72 ℃ for 30s, 34 cycles; elongation at 72 ℃ for 5 min; storage at 15 ℃), screening of positive single clones, and sequencing with PacBio RSII (Han future group Biotech Co., Ltd.) according to sequencing protocolAnd obtaining the sequence of the exogenous gene at the right border of the corn genome integration site. The sequence is 547bp in length and is specifically shown as a sequence 4 in a sequence table. Wherein, the 1 st-352 th site from the 5' end of the sequence 4 in the sequence table is a corn genome sequence, and the 353 rd-547 th site is a vector sequence.

Designing and synthesizing a specific primer S1 according to the DNA molecule shown in the 1 st-352 th site from the 5' end of the sequence 4 in the sequence table: 5'-AGTGCACATTGCAATCCTACAAGC-3' and S2: 5'-CCTAAGTTCATGCAACTAGAGGTTTCA-3' are provided. Designing and synthesizing S3 according to the DNA molecule shown in 353-547 site from the 5' end of the sequence 4 in the sequence table: 5'-GGTTTCGCTCATGTGTTGAGC-3' and S4: 5'-TCCAGATCCCCCGAATTAATTCG-3' are provided.

2. PCR amplification was carried out using genomic DNA of SbSNAC1-382 leaf as a template, and using primer set 1 (consisting of S1 and S3), primer set 2 (consisting of S2 and S3), primer set 3 (consisting of S1 and S4), primer set 4 (consisting of S2 and S4), or primer set 5 (consisting of 5'-GACCGCAAGTACCCAAACGG-3' and 5'-CACCCAGTCATCCAGCCTGAG-3') to obtain a PCR amplification product.

And (3) performing PCR amplification by using water as a template and adopting a primer pair 5 to obtain a PCR amplification product. As a negative control.

The results of the agarose gel electrophoresis are shown in FIG. 13 (lanes 1 to 5 are genomic DNAs of SbSNAC1-382 leaves, lane 6 is water,

lanes

1 and 6 are primer set 1, lane 2 is primer set 2, lane 3 is primer set 3, lane 4 is primer set 4, and lane 5 is primer set 5). As a result, it was found that a DNA fragment of about 503bp could be obtained by using the genomic DNA of SbSNAC1-382 leaf as a template, a DNA fragment of about 547bp could be obtained by using the primer set 2, a DNA fragment of about 392bp could be obtained by using the primer set 3, a DNA fragment of about 436bp could be obtained by using the primer set 4, and a DNA fragment of about 249bp could be obtained by using the primer set 5.

<110> institute of crop science of Chinese academy of agricultural sciences

<120> a method for identifying whether a test plant sample is derived from the sbSNAC1-382 event or its progeny

<160>12

<170>PatentIn version 3.5

<210>1

<211>970

<212>DNA

<213>Sorghum bicolor（L.）Moench

<400>1

atgggattgc cggtgatgag gagggagagg gacgcggagg cggagctgaa cctgccgccg 60

gggttccggt tccaccccac agacgacgag ctggtggagc actacctgtg ccggaaagcg 120

gcggggcagc gcctcccggt gcccatcatc gcggaggtgg acctatacaa gttcgacccc 180

tgggacctgc cggagcgcgc gctgttcggg gtcagggagt ggtacttctt cacgcccagg 240

gaccgcaagt acccaaacgg gtcccgcccc aaccgcgccg ccggcaacgg gtactggaag 300

gccaccggcg ccgacaagcc cgtcgcgccg cggggccgca cgctcgggat caagaaggcg 360

ctcgtcttct acgccgggaa ggcgccgcgt ggggtcaaga cggactggat catgcacgag 420

tacaggctcg cggacgccgg ccgcgcagcc gcctccaaga agggatcgct caggctggat 480

gactgggtgc tgtgccgcct gtacaataag aagaacgagt gggagaagat gcagctgggg 540

aaggagtccg ccgccggcgt cggcaccgcc aaggaggagg cgatggacat gaccacctcg 600

cactcgcact cccactcgca gtcgcactcg cactcgcact cgtggggcga gacgcgcacg 660

ccggagtcgg agatcgtgga caacgacccg ttcccggagc tggactcgtt cccggcgttc 720

caggacccgg cggcggcgat gatgatggtg cccaagaagg agcaggtgga cgacggcagc 780

gccgccgcca acgccgccaa gagcagcgac ctgttcgtgg accttagcta cgacgacatc 840

cagggcatgt acagcggcct cgacatgctg cccccgccag gggaggactt cttctcctcg 900

ctcttcgcgt cgcccagggt caaggggaac cagcccgccg gagccgccgg gttggggcca 960

ttctgaggct 970

<210>2

<211>321

<212>PRT

<213>Sorghum bicolor（L.）Moench

<400>2

Met Gly Leu Pro Val Met Arg Arg Glu Arg Asp Ala Glu Ala Glu Leu

1 5 10 15

Asn Leu Pro Pro Gly Phe Arg Phe His Pro Thr Asp Asp Glu Leu Val

20 25 30

Glu His Tyr Leu Cys Arg Lys Ala Ala Gly Gln Arg Leu Pro Val Pro

35 40 45

Ile Ile Ala Glu Val Asp Leu Tyr Lys Phe Asp Pro Trp Asp Leu Pro

50 55 60

Glu Arg Ala Leu Phe Gly Val Arg Glu Trp Tyr Phe Phe Thr Pro Arg

65 70 75 80

Asp Arg Lys Tyr Pro Asn Gly Ser Arg Pro Asn Arg Ala Ala Gly Asn

85 90 95

Gly Tyr Trp Lys Ala Thr Gly Ala Asp Lys Pro Val Ala Pro Arg Gly

100105 110

Arg Thr Leu Gly Ile Lys Lys Ala Leu Val Phe Tyr Ala Gly Lys Ala

115 120 125

Pro Arg Gly Val Lys Thr Asp Trp Ile Met His Glu Tyr Arg Leu Ala

130 135 140

Asp Ala Gly Arg Ala Ala Ala Ser Lys Lys Gly Ser Leu Arg Leu Asp

145 150 155 160

Asp Trp Val Leu Cys Arg Leu Tyr Asn Lys Lys Asn Glu Trp Glu Lys

165 170 175

Met Gln Leu Gly Lys Glu Ser Ala Ala Gly Val Gly Thr Ala Lys Glu

180 185 190

Glu Ala Met Asp Met Thr Thr Ser His Ser His Ser His Ser Gln Ser

195 200 205

His Ser His Ser His Ser Trp Gly Glu Thr Arg Thr Pro Glu Ser Glu

210 215 220

Ile Val Asp Asn Asp Pro Phe Pro Glu Leu Asp Ser Phe Pro Ala Phe

225 230 235 240

Gln Asp Pro Ala Ala Ala Met Met Met Val Pro Lys Lys Glu Gln Val

245 250 255

Asp Asp Gly Ser Ala Ala Ala Asn Ala Ala Lys Ser Ser Asp Leu Phe

260265 270

Val Asp Leu Ser Tyr Asp Asp Ile Gln Gly Met Tyr Ser Gly Leu Asp

275 280 285

Met Leu Pro Pro Pro Gly Glu Asp Phe Phe Ser Ser Leu Phe Ala Ser

290 295 300

Pro Arg Val Lys Gly Asn Gln Pro Ala Gly Ala Ala Gly Leu Gly Pro

305 310 315 320

Phe

<210>3

<211>933

<212>DNA

<213>Artificial sequence

<400>3

agtgtagtat cataggaaaa gaattaaaag gtattaatga ctagaaattt gtatcaagtc 60

atgttataac acctaaaagc cagcaaaaat gagttttaga gaattaccca ctgttaaata 120

atagctgtag ttcaaagtac cccttctgcc ctaaaatttg gtaattttgt ccagagaaaa 180

ccattcactt tctgaccccc aaattttgag gcagagaatc atacaccagt aacaagccac 240

tgtaattttt ggaattttat aaaagcaact tgtagttcaa acctactcca aaacattaaa 300

agaataaaag aaaaggaaag aaggaatgaa cctcatccca atgagtctaa cttgagaact 360

tatcaattct ccctaagact taaaaataat tcagtagaaa cccaaaaata aacctaccac 420

ttaccttagc taagtttaac ccaatttacc aaggatatat tgtggtgtaa acaaattgac 480

gcttagacaa cttaataaca cattgcggac gtttttaatg tactgaatta acgccgaatt 540

aattcggggg atctggattt tagtactgga ttttggtttt aggaattaga aattttattg 600

atagaagtat tttacaaata caaatacata ctaagggttt cttatatgcc caacacatga 660

gcgaaaccct ataggaaccc taattccctt atctgggaac tactcacaca ttattatgga 720

gaaactcgag tcaaatctcg gtgacgggca ggaccggacg gggcggtacc ggcaggctga 780

agtccagctg ccagaaaccc acgtcatgcc agttcccgtg cttgaagccg gccgcccgca 840

gcatgccgcg gggggcatat ccgagcgcct cgtgcatgcg cacgctcggg tcgttgggca 900

gcccgatgac agcgaccacg ctcttgaagc cct 933

<210>4

<211>547

<212>DNA

<213>Artificial sequence

<400>4

cctaagttca tgcaactaga ggtttcaagc aactcctaca cttaagtgca cattgcaatc 60

ctacaagcat taagtgtagt aaagtagcat ataataatac ggttatgcat aaaaccgggg 120

cttgccttca attgctgggg ctgcggggag atcctcaata gcagcctctg aagcctgctc 180

ctggtcctcc tcttggacag gtccttgctc ggggatgagc acgtactctc cgtcggcaag 240

attacaatct aatgaaggca atgcgtaaga tatatgcatg atatgatatg tgcttttaga 300

aattacaact ttaaaggggt atgatctttt gagtttaaac aagttaacgc cgaattgacg 360

cttagacaac ttaataacac attgcggacg tttttaatgt actgaattaa cgccgaatta 420

attcggggga tctggatttt agtactggat tttggtttta ggaattagaa attttattga 480

tagaagtatt ttacaaatac aaatacatac taagggtttc ttatatgctc aacacatgag 540

cgaaacc 547

<210>5

<211>24

<212>DNA

<213>Artificial sequence

<400>5

agaatcatac accagtaaca agcc 24

<210>6

<211>22

<212>DNA

<213>Artificial sequence

<400>6

cagtacatta aaaacgtccg ca 22

<210>7

<211>22

<212>DNA

<213>Artificial sequence

<400>7

ggaatgaacc tcatcccaat ga 22

<210>8

<211>22

<212>DNA

<213>Artificial sequence

<400>8

actaaaatcc agatcccccg aa 22

<210>9

<211>24

<212>DNA

<213>Artificial sequence

<400>9

agtgcacatt gcaatcctac aagc 24

<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

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 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;

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 B is shown as a sequence 4 in a sequence table.

DNA fragment A and/or DNA fragment B;