CN111394500B - Method for identifying whether a test plant sample is derived from the SbSNAC1-382 event or a progeny thereof - Google Patents

Method for identifying whether a test plant sample is derived from the SbSNAC1-382 event or a progeny thereof Download PDF

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CN111394500B
CN111394500B CN202010320879.3A CN202010320879A CN111394500B CN 111394500 B CN111394500 B CN 111394500B CN 202010320879 A CN202010320879 A CN 202010320879A CN 111394500 B CN111394500 B CN 111394500B
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CN111394500A (en
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王天宇
张登峰
曾廷儒
李永祥
李春辉
宋燕春
石云素
黎裕
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Institute of Crop Sciences of Chinese Academy of Agricultural Sciences
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Abstract

The invention discloses a method for identifying whether a plant sample to be tested is derived from the SbSNAC1-382 event or a progeny thereof. The inventor of the invention transfers the SbSNAC1 gene into the genome of the maize inbred line Zheng 58 by an agrobacterium-mediated method, and obtains a transgenic maize event SbSANC-382, abbreviated as SbSNAC1-382 event. Drought resistance identification shows that the SbSNAC1-382 event is obviously improved in drought resistance compared with the maize inbred line Zheng 58. In addition, the T 3 generation-T 5 generation SbSNAC1-382 event has genetic stability and can be inherited stably in different generations through detection. Thus, the SbSNAC1-382 event is likely to enter commercial planting. The SbSNAC1-382 event is preserved in China general microbiological culture collection center (CGMCC) with the preservation number of CGMCC No.17493 in the 04 th month of 2019 in the Ganyang area North Star West-way No.1 of Beijing city. 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 monitor and manage the SbSNAC1-382 event. The invention has important application value.

Description

Method for identifying whether a test plant sample is derived from the SbSNAC1-382 event or a progeny thereof
Technical Field
The invention belongs to the field of biotechnology, and particularly relates to a method for identifying whether a plant sample to be tested is derived from an SbSNAC1-382 event or a progeny thereof.
Background
The shortage of water resources seriously affects the world grain production, and the grain yield reduction caused by the shortage of water resources accounts for more than 50% of the crop yield reduction every year, so that the direct economic loss is difficult to estimate. The arid and semiarid regions of China occupy more than half of the national land area. According to statistics, the perennial drought area of China reaches about 20% of the total area of cultivated land, and the yield reduction of grains caused by drought only reaches more than 1000 hundred million kilograms in recent 40 years. Corn is one of the most important crops in the world, both as food, feed and related industrial materials. In our country, corn has become the first large grain crop since 2009. Traditional corn genetic breeding plays a certain role in improving drought resistance of corn, but is difficult to break through genetic restriction among seeds to obviously improve drought resistance of corn. The transgenic technology can break species boundaries, carry out directional transformation and recombination transfer on genes, improve drought resistance of the existing variety and greatly improve drought resistance of corn. Practice of cultivating and popularizing transgenic corn in developed countries, particularly in the United states, has also proved that the transgenic technology for cultivating new varieties can remarkably improve drought resistance of corn, is the most realistic and effective way for greatly improving yield and quality, and has become the main direction of international corn breeding development. In 12 months 2011, the U.S. department of agriculture animal and plant health quarantine Agency (APHIS) formally approved the monsanto transgenic drought-resistant corn MON87460, which means that the first transgenic drought-resistant corn in the world can be popularized and utilized in production on a large scale. The drought-resistant corn is transgenic into a CspB gene (the CspB gene is from bacillus subtilis and codes for an RNA chaperone protein), and the gene can enhance the function of plant cells under adverse conditions and reduce the loss of yield under the condition of water deficiency. Under drought conditions, the corn inbred line and hybrid with the transgenic gene can remarkably improve the biomass and yield of corn, and the drought-resistant corn field test in the western United states drought area reaches or even exceeds the yield increase target of 6-10%.
The SbSNAC1 gene is a NAC family gene cloned from a drought-enduring local sorghum variety XGL-1 in Xinjiang by the national academy of agricultural science crop research. The gene can be over-expressed in arabidopsis thaliana, so that drought tolerance of transgenic arabidopsis thaliana plants can be obviously improved (Lu et al, 2013).
Disclosure of Invention
The object 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. 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 a SbSNAC1-382 event or progeny thereof, which may include the steps of: detecting whether the genome 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 DNA fragment A and/or DNA fragment B, the plant sample to be tested is derived from SbSNAC1-382 event or its progeny; 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 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 was maize 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) or S2) or S3).
S1) direct sequencing.
S2) carrying out PCR amplification on genomic DNA of the plant sample to be detected by using the primer pair X and/or the primer pair Y, and then judging as follows: if the target amplification product is obtained, the plant sample to be detected is derived from SbSNAC1-382 event or the progeny thereof; if the target amplification product is not obtained, the plant sample to be tested is not derived from the SbSNAC1-382 event or the progeny thereof;
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 the 1 st to 451 st positions from the 5' end of a sequence 3 in a sequence table; the downstream primer RX is the reverse complementary sequence of a part of DNA molecules shown in 452-933 from the 5' end of the sequence 3 in the sequence table; the target amplification product of the 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 DNA molecules shown in the 1 st to 352 th positions from the 5' end of the sequence 4 in the sequence table; the downstream primer RY is the reverse complementary sequence of a part of DNA molecules shown in 353-547 from the 5' end of the sequence 4 in the sequence table; the target amplified product of the primer pair Y is a DNA molecule Y.
S3) carrying out Southern hybridization on genomic DNA of the plant sample to be detected by using a probe A capable of specifically binding to the DNA molecule X and/or using a probe B capable of specifically binding to the DNA molecule Y, and then judging as follows: if the hybrid fragment is obtained, the plant sample to be tested is derived from the SbSNAC1-382 event or a progeny thereof; if no hybrid fragment is available, 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 the primer P1 can be shown as a sequence 5 in a sequence table.
The nucleotide sequence of the primer P2 can be shown as a sequence 6 in a sequence table.
The nucleotide sequence of the primer P3 can be shown as a sequence 7 in a sequence table.
The nucleotide sequence of the primer P4 can be shown as a sequence 8 in a sequence table.
The primer pair Y may be at least one of primer pair Y1, primer pair Y2, primer pair Y3, and 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 the primer S1 can be shown as a sequence 9 in a sequence table.
The nucleotide sequence of the primer S2 can be shown as a sequence 10 in a sequence table.
The nucleotide sequence of the primer S3 can be shown as a sequence 11 in a sequence table.
The nucleotide sequence of the primer S4 can be shown as a sequence 12 in a sequence table.
The nucleotide sequence of the target amplification product (namely DNA molecule X) of the primer pair X1 can be shown as 215-525 from the 5' end of the sequence 3 in the sequence table.
The nucleotide sequence of the target amplification product (namely DNA molecule X) of the primer pair X2 can be shown as 323-564 th from the 5' end of the sequence 3 in the sequence table.
The nucleotide sequence of the target amplification product (namely 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 (namely DNA molecule Y) of the primer pair Y1 can be shown as 45-547 positions from the 5' end of the sequence 4 in the sequence table.
The nucleotide sequence of the target amplification product (namely DNA molecule Y) of the primer pair Y2 can be shown in the 1 st to 547 th positions from the 5' tail end of the sequence 4 in the sequence table.
The nucleotide sequence of the target amplification product (namely DNA molecule Y) of the primer pair Y3 can be shown as 215-436 from the 5' end of the sequence 4 in the sequence table.
The nucleotide sequence of the target amplification product (namely DNA molecule Y) of the primer pair Y4 can be shown as 1 st to 436 st from the 5' end of the sequence 4 in the sequence table.
The invention also protects a kit for identifying whether a plant sample to be tested originates from the SbSNAC1-382 event or its progeny.
The kit for identifying whether the plant sample to be tested is derived from SbSNAC1-382 event or the progeny thereof, which is protected by the invention, can be specifically a kit A; the kit A may comprise any one of the primer pairs X and/or primer pairs Y described above; the SbSNAC1-382 event may be maize Zea mays SbSNAC1-382CGMCC No.17493.
The kit A specifically can be composed of any one of the primer pairs X and/or the primer pairs Y.
The kit for identifying whether the plant sample to be tested is derived from SbSNAC1-382 event or the progeny thereof, which is protected by the invention, can be specifically a kit B; the kit B can comprise any one of the probes A capable of specifically binding to the DNA molecule X and/or any one of the probes B capable of specifically binding to the DNA molecule Y; the SbSNAC1-382 event may be maize Zea mays SbSNAC1-382CGMCC No.17493.
The kit B specifically comprises any one of the probes A and/or any one of the probes B.
The use of any of the above-described primer pairs X and/or any of the above-described primer pairs Y in the identification of whether a plant sample to be tested is derived from the SbSNAC1-382 event or progeny thereof is also within the scope of the present invention; the SbSNAC1-382 event may be maize Zea mays SbSNAC1-382CGMCC No.17493.
The use of any one of the above-described probes A capable of specifically binding to DNA molecule X and/or any one of the above-described probes B capable of specifically binding to DNA molecule Y for identifying whether a plant sample to be tested is derived from the SbSNAC1-382 event or a progeny thereof also falls within the scope of the present invention; the SbSNAC1-382 event may be maize Zea mays SbSNAC1-382CGMCC No.17493.
The use of DNA fragment a and/or DNA fragment B in the identification of whether a plant sample to be tested is derived from the SbSNAC1-382 event or its progeny is also within the scope of the present invention; the SbSNAC1-382 event may be maize 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 DNA fragment A and/or DNA fragment B;
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 inventor of the present invention transfers the SbSNAC1 gene into the genome of the maize inbred line Zheng 58 by an agrobacterium-mediated method, and obtains a transgenic maize event SbSANC-382 (SbSNAC 1-382 event for short). Drought resistance identification shows that the SbSNAC1-382 event has obviously improved drought resistance compared with that of control corn (namely corn inbred line Zheng 58). In addition, the T 3 generation-T 5 generation SbSNAC1-382 event has genetic stability and can be inherited stably in different generations through detection. Therefore, the SbSNAC1-382 event is likely to enter commercial planting, and the SbSNAC1-382 event is preserved in the China general microbiological culture Collection center (CGMCC, address is 1 st West Song No. 3 of the Games-induced cation area North Star of Beijing) in 04 th year 2019, 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 monitor 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 result of step five 2 in example 1.
FIG. 3 shows the result of step five 3 in example 1.
Fig. 4 shows the result of step seven in example 1.
FIG. 5 is the result of the eight grouting period observation in the step of example 1.
FIG. 6 shows the results of the post-harvest measurements of step eight of example 1.
FIG. 7 is a schematic diagram of a vector for recombinant plasmid 35 S:SbSNAC 1.
FIG. 8 shows the experimental results of step one in example 2.
FIG. 9 shows the experimental results of step two in example 2.
FIG. 10 shows the experimental results of step three in example 2.
FIG. 11 shows the experimental results of step four in example 2.
FIG. 12 shows the experimental results of step one in example 3.
FIG. 13 shows the experimental results of step two in example 3.
Detailed Description
The following examples facilitate a better understanding of the present invention, but are not intended to limit the same.
The experimental methods in the following examples are conventional methods unless otherwise specified.
The test materials used in the examples described below, unless otherwise specified, were purchased from conventional biochemical reagent stores.
The quantitative tests in the following examples were all set up in triplicate and the results averaged.
Plasmid pCAMBIA3301 is a product of Camcia company.
Example 1 obtaining and drought resistance identification of SbSNAC1 Gene-transferred maize
1. Cloning of the SbSNAC1 Gene
1. Leaves and root systems of sorghum variety XGL-1 (supplied by the institute of food crops, xinjiang academy of agricultural sciences) were taken and mixed as materials.
2. After the step 1 is completed, the material is taken, total RNA is firstly extracted, and then reverse transcription is carried out, so that the cDNA of the sorghum variety XGL-1 is obtained.
3. After the step 3 is completed, PCR amplification is carried out by using cDNA of sorghum variety XGL-1 as a template and adopting a primer pair consisting of 5'-TTTCCATGGGATTGCCGGTGAT-3' (underlined as a recognition site of restriction enzyme NcoI) and 5'-TTTGGTGACCAGCCTCAGAATGGCCCCAAC-3' (underlined as a recognition site of restriction enzyme BstE II) 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-SbSNAC1.
The recombinant plasmid PMD18-SbSNAC1 was sequenced. Sequencing results show that the recombinant plasmid PMD18-SbSNAC1 contains DNA molecules shown in the 1 st to 966 th positions from the 5' end of the sequence 1 in the sequence table.
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 980bp was recovered.
2. Plasmid pCAMBIA3301 was digested with restriction enzymes NcoI and BstE II, and the vector backbone of about 9250bp was recovered.
3. And (3) connecting the DNA fragment recovered in the step (1) with the vector skeleton recovered in the step (2) to obtain a recombinant plasmid 35S (SbSNAC 1).
The recombinant plasmid 35S: sbSNAC1 was sequenced. Based on the sequencing results, the structure of recombinant plasmid 35S: sbSNAC1 was described as follows: the small fragment between the recognition sequences of restriction enzymes NcoI and BstE II of plasmid pCAMBIA3301 is replaced by a DNA molecule shown in the 5 th to 970 th positions from the 5' end of the sequence 1 in the sequence table. Recombinant plasmid 35S is SbSNAC1 expressed by a protein SbSNAC1 shown in a sequence 2 in a sequence table.
3. Acquisition of positive recombinant Agrobacterium
1. And (3) transforming the recombinant plasmid 35S by adopting a freeze thawing method, namely SbSNAC1 into agrobacterium tumefaciens EH105 to obtain recombinant agrobacterium.
2. After the step 1 is completed, the monoclone of each recombinant agrobacterium is respectively inoculated to a YEB liquid culture medium for 16 hours at 28 ℃ and 200rpm, and the agrobacterium liquid is obtained.
3. After the step 2 is completed, PCR amplification is carried out by taking each agrobacterium liquid, water and recombinant plasmid 35S, wherein SbSNAC1 is taken as a template, and 5'-TTTCCATGGGATTGCCGGTGAT-3' and 5'-TTTGGTGACCAGCCTCAGAATGGCCCCAAC-3' are taken as primers, so that a PCR amplification product is obtained. The recombinant plasmid 35S is SbSNAC1 as a positive control. Water was used as a negative control.
4. After the step 3 is completed, the PCR amplification product is subjected to 1% (m/v) agarose gel electrophoresis, and the following judgment is carried out according to the electrophoresis result: if the PCR amplification product of a 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 ("+" for positive control, "-" for negative control, lanes 1 to 7 were all recombinant Agrobacterium).
4. Obtaining of T 0 generation of quasi-transgenic SbSNAC1 gene corn
1. And (3) inoculating the monoclonal of the positive recombinant agrobacterium obtained in the step (III) into a YEB liquid culture medium, and culturing at 28 ℃ and 200rpm to obtain an agrobacterium liquid with OD 550nm of 0.3-0.4.
2. After the step 1 is completed, the agrobacterium liquid is taken, centrifuged at 10000rpm for 10min at 4 ℃ and the thalli are collected.
3. And (3) adding the thalli collected in the step (2) into an infection culture medium (MS culture medium containing 100 mu M acetosyringone) for resuspension to obtain an infection solution with OD 550nm of 0.3-0.4. Taking young embryo of the maize inbred line Zheng 58 with the pollination time of 11-12 days and the size of 1.0-1.5mm, infecting (i.e. soaking) the young embryo with an infecting liquid for 5min under a dark condition, then placing the young embryo on a co-culture medium, sealing the young embryo with a ventilation tape, and culturing the young embryo in dark at 20 ℃ for 3 days.
4. After the step 3 is completed, young embryo of the maize inbred line Zheng 58 is taken and placed in a resting medium for 7 days at 28 ℃.
5. After the step 4 is completed, young embryo of the maize inbred line Zheng 58 is firstly placed on a selection medium 1 and is cultivated for 14 days at 28 ℃; then placing the obtained product on a selective medium 2, and culturing the obtained product at 28 ℃ for 14 days to obtain the resistant callus.
6. After the step 5 is completed, the resistant callus is taken and placed in a regeneration culture medium 1 for dark culture at 25 ℃ for 14 days, and mature somatic embryos are obtained.
7. After the step 6 is completed, transferring the mature somatic embryo to a regeneration culture medium 2, and alternately culturing at 25 ℃ and in the dark for 7-10 days to obtain the resistant seedling. The light and dark alternate culture is 16h light culture and 8h dark culture, and the light intensity during light culture is 80-100 mu E/m2/s.
8. And (3) after the step 7 is completed, transplanting the resistant seedlings into soil to obtain the T 0 generation of quasi-transgenic SbSNAC1 gene corn.
Co-culture medium, resting medium, selection medium 1 (selection medium containing 1.5mg/L dipropylamine phosphine), selection medium 2 (selection medium containing 3mg/L dipropylamine phosphine), regeneration medium 1 and regeneration medium 2 are all described in the following documents: frame et al, 2011.
5. Identification of T 0 generation of maize with to-be-transferred SbSNAC1 gene
1. Spraying Basta
Basta (concentration of 2% per mill) was sprayed on leaves of plants of the T 0 generation to be transformed with SbSNAC1 gene maize, respectively, and the leaves were observed after 5-7 days. If the leaves of the T 0 -generation SbSNAC 1-gene-to-be-transferred corn do not wither, the T 0 -generation SbSNAC 1-gene-to-be-transferred corn is preliminarily identified as the T 0 -generation SbSNAC 1-gene-transferred corn.
7 Lines of maize, which were initially identified as T 0 generations of transgenic SbSNAC1 by spraying Basta, were designated SbSNC-382, sbSNAC1-383, sbSNAC1-389, sbSNAC1-466, sbSNAC1-467, sbSNAC1-471 and SbSNAC1-474, respectively.
2. PCR detection of SbSNAC1 Gene
(1) Genomic DNA of leaves of T 0 generation transgenic SbSNAC1 corn (SbSNC-382, sbSNAC1-383, sbSNAC1-389, sbSNAC1-466, sbSNAC1-467, sbSNAC1-471 or SbSNAC 1-474) is extracted respectively and used as a template, and PCR amplification is carried out by using a specific primer pair (consisting of SbSNAC1-F:5'-GACCGCAAGTACCCAAACGG-3' and SbSNAC1-R:5'-CACCCAGTCATCCAGCCTGAG-3', wherein SbSNAC1-F spans two exons) for amplifying the SbSNAC1 gene, thereby obtaining a PCR amplification product.
According to the above procedure, the template was replaced with genomic DNA from leaf of Zea mays inbred line Zheng 58, and the other procedures were unchanged as a negative control.
The template was replaced with water according to the procedure described above, with the other steps unchanged as water controls.
According to the steps, the template is replaced by a recombinant plasmid 35S, sbSNAC1, and other steps are unchanged and serve as positive control.
Reaction conditions: 95 ℃ for 5min;95 ℃ 30s,60 ℃ 30s,72 ℃ 30s,34 cycles; 72 ℃ for 5min; preserving at 15 ℃.
(2) Each PCR amplification product was subjected to 1% (m/v) agarose gel electrophoresis, and the following determination was made based on the result of the electrophoresis: if the PCR amplification product of a strain contains a DNA fragment of about 249bp, the strain is identified as a T 0 -generation SbSNAC1 gene-transferred corn.
Agarose gel electrophoresis results are shown in FIG. 2 (Marker is DNA MARKER,1 to 7 are SbSNC-382, sbSNAC1-383, sbSNAC1-389, sbSNAC1-466, sbSNAC1-467, sbSNAC1-471 and SbSNAC1-474 in order).
3. PCR detection of Bar gene
(1) Extracting genomic DNA of leaves of T 0 generation transgenic SbSNAC1 corn (SbSNC-382, sbSNAC1-383, sbSNAC1-389, sbSNAC1-466, sbSNAC1-467, sbSNAC1-471 or SbSNAC 1-474) and using the genomic DNA as a template, and carrying out PCR amplification by adopting a specific primer pair (composed of 5'-GAAGTCCAGCTGCCAGAAAC-3' and 5'-GTCTGCACCATCGTCAACC-3') for amplifying Bar genes to obtain PCR amplification products.
According to the above procedure, the template was replaced with genomic DNA from leaf of Zea mays inbred line Zheng 58, and the other procedures were unchanged as a negative control.
The template was replaced with water according to the procedure described above, with the other steps unchanged as a blank.
According to the steps, the template is replaced by a recombinant plasmid 35S, sbSNAC1, and other steps are unchanged and serve as positive control.
Reaction conditions: 95 ℃ for 5min;95 ℃ 30s,60 ℃ 30s,72 ℃ 30s,34 cycles; 72 ℃ for 5min; preserving at 15 ℃.
(2) Each PCR amplification product was subjected to 1% (m/v) agarose gel electrophoresis, and the following determination was made based on the result of the electrophoresis: if the PCR amplification product of a strain contains a DNA fragment of about 444bp, the strain is identified as a T 0 -generation SbSNAC1 gene-transferred corn.
The results of partial agarose gel electrophoresis are shown in FIG. 3 (Marker: DNA MARKER, lanes 1 to 7: sbSNC-382, sbSNAC1-383, sbSNAC1-389, sbSNAC1-466, sbSNAC1-467, sbSNAC1-471 and SbSNAC1-474 in this order, lane 11: positive control, and lane 12: negative control).
The results show that SbSNC-382, sbSNAC1-383, sbSNAC1-389, sbSNAC1-466, sbSNAC1-467, sbSNAC1-471 and SbSNAC1-474 are all T 0 -generation transgenic SbSNAC1 corn.
6. Acquisition of the T 1 -T 5 -SbSNAC 1-382
And (3) taking seeds of the T 0 -generation SbSNAC 1-gene-transferred corn, and selfing to obtain the seeds of the T 1 -generation SbSNAC 1-transferred corn. The seeds of the T 1 -generation SbSNAC 1-gene-transformed corn are continuously selfed to sequentially obtain the seeds of the T 2 -generation SbSNAC 1-gene-transformed corn, the seeds of the T 3 -generation SbSNAC 1-gene-transformed corn, the seeds of the T 4 -generation SbSNAC 1-gene-transformed corn and the seeds of the T 5 -generation SbSNAC 1-gene-transformed corn.
And selecting the T 1 -generation SbSNAC1 gene-transgenic corn-T 5 -generation SbSNAC1 gene-transgenic corn for subsequent experiments.
7. Drought resistance identification in greenhouse of T 1 -T 5 -generation SbSNAC1 gene-transferred corn
The corn to be tested is T 1 -T 5 -generation SbSNAC 1-gene-transformed corn (SbSNC-382, sbSNAC1-383, sbSNAC1-389, sbSNAC1-466, sbSNAC1-467, sbSNAC1-471 or SbSNAC 1-474) or corn inbred line Zheng 58. The maize inbred line Zheng 58 served as a control.
(1) 3 Corn to be detected are planted in the flowerpot, and 5 repeats are planted in each plant line; then placed in a greenhouse for routine management.
(2) After the step (1) is completed, stopping watering when the corn to be tested grows until the visible leaves of the corn reach 4 leaves and 1 heart, and continuously drought for 21 days. And observing the growth state of the corn to be detected.
The growth state of part of the corn to be tested is shown in FIG. 4 (SbSNC-382 are T 5 generation SbSNC1-382, zheng 58 is maize inbred Zheng 58). The results show that leaf curl and wilting of maize inbred line Zheng 58 occurs, while leaf stretch of T 1 -T 5 -transformed SbSNAC1 gene maize remains green. Therefore, compared with the maize inbred line Zheng 58, the drought resistance of the T 1 -T 5 -generation SbSNAC1 gene-transformed maize is obviously improved.
8. Identification of drought resistance in field of T 3 generation-T 5 generation SbSNC-382
In 2015, the drought resistance of the T 3 generation SbSNC-382 and the maize inbred line Zheng 58 are identified.
In 2016, the drought resistance of the T 4 generation SbSNC-382 and the maize inbred line Zheng 58 in the field is identified.
In 2017, the drought resistance of the T 5 generation SbSNC-382 and the maize inbred line Zheng 58 are identified.
The drought resistance identification is set with two treatments of water and drought, each material is planted with 6 rows, the row length is 5 meters, the steps are repeated for 3 times, the water treatment is drip-irrigated for 7 times, the water quantity is 350 square/mu, and the drought treatment is watered for 150 square/mu.
The corns for drought resistance identification are planted in Xinjiang Uruku Muzian Ning channel test field. The growth state and yield during the grouting period were observed.
The test results are as follows:
1. The grouting period observations show (FIG. 5, sbSNC1-382 is T 3 th generation SbSNC-382): in the drought region, compared with the plants of the generation T 3, the generation SbSNC, the generation T 4, the generation SbSNC, the generation T 5 and the generation SbSNC, the plant height of the corn inbred line Zheng 58 is obviously reduced, and the leaf color is obviously green and yellow;
2. Post-harvest yield measurements (Table 1 and FIG. 6) showed a significant increase in yield in arid regions, either T 3 generation SbSNC1-382, T 4 generation SbSNC-382, or T 5 generation SbSNC1-382 over maize inbred line Zheng 58.
TABLE 1 average individual yield of SbSNAC1-382 in Water and Dry regions
Note that: data in the table are mean ± standard error; the average individual yield was the kernel yield (moisture 14%).
The test result shows that compared with the maize inbred line Zheng 58, the drought resistance of the T 3 generation SbSNC-382, the T 4 generation SbSNC1-382 or the T 5 generation SbSNC1-382 is obviously improved.
Example 2 genetic stability test of T 3 generation-T 5 generation SbSNAC1-382
The schematic diagram of the recombinant plasmid 35S is shown in FIG. 7. The size of the recombinant plasmid 35S is 10.24kb, and the vector has only one restriction enzyme HindIII and one EcoRI restriction enzyme site, so that the two enzymes are used for single restriction, and a 10.24kb linear fragment can be obtained. In addition, there are no restriction sites for the restriction enzymes BglII and DraI on the vector.
Genomic DNA of SbSNC.sup.1-382 events or maize inbred line Zheng 58 was singly digested with the restriction enzymes HindIII and EcoRI, respectively, and hybridized using probes specific for the SbSNAC1 gene and the Bar gene, respectively. The results show that the SbSNAC1-382 event contains 2 copies of the SbSNAC1 gene and the Bar gene, and that both copies can be stably inherited at both transgene T 3、T4 and T 5 generations. Thus, these 2 copies are likely to be inserted at the same location in the maize genome. To verify this hypothesis, the number of insertion sites (the number of integration sites of T-DNA in the maize genome) was determined by digestion of the genomic DNA of the SbSNAC1-382 event and maize inbred line Zheng 58, respectively, with the restriction enzymes BglII and DraI, which do not have a cleavage site within the insertion sequence (T-DNA). Southern hybridization results showed that the T-DNA in the SbSNAC1-382 event (i.e., the T 3 generation-T 5 generation SbSNAC 1-382) was a single site on the maize genome with 2 copies of the SbSNAC1 and Bar genes inserted and was stably inherited from generation to generation. The specific results are as follows:
1. Results of Southern hybridization Using SbSNAC1 Gene probes, restriction endonucleases HindIII and EcoRI
1. The recombinant plasmid 35S is SbSNAC1 as a template and 5'-CGCGTGGGGTCAAGACGGACTG-3' and 5'-GGGAACGAGTCCAGCTCCGGGAAC-3' as primers are used for PCR amplification to obtain a DNA fragment of about 386 bp. The fragment is the SbSNAC1 gene probe.
2. The genomic DNA of the generation T 3 SbSNAC1-382, the generation T 4 SbSNAC1-382, the generation T 5 SbSNAC1-382 or the maize inbred line Zheng 58 is digested with restriction enzymes HindIII and EcoRI, respectively, and the SbSNAC1 gene probe obtained in the step 1 is hybridized.
The recombinant plasmid 35S, sbSNAC1, was digested with restriction enzyme HindIII, and hybridized with the SbSNAC1 gene probe obtained in step 1. As a positive control.
The experimental results are shown in FIG. 8 (lane 1 is the genomic DNA of the restriction enzyme HindIII-cut T 3 -generation SbSNAC1-382, lane 2 is the genomic DNA of the restriction enzyme HindIII-cut T 4 -generation SbSNAC1-382, lane 3 is the genomic DNA of the restriction enzyme HindIII-cut T 5 -generation SbSNAC1-382, lane 4 is the genomic DNA of the restriction enzyme HindIII-cut maize inbred line Zheng 58, lane 5 is the genomic DNA of the restriction enzyme EcoRI-cut T 3 -generation SbSNAC1-382, lane 6 is the genomic DNA of the restriction enzyme EcoRI-cut T 4 -generation SbSNAC1-382, lane 7 is the genomic DNA of the restriction enzyme EcoRI-cut T 5 -generation SbSNAC1-382, lane 8 is the genomic DNA of the restriction enzyme I-cut maize inbred line Zheng 58, and lane 9 is the restriction enzyme EcoRI-cut plasmid 35S; sbSNAC1, lane 10 Takara DL15kb DNA MARKER). The result shows that the SbSNAC1 hybridizes with a 10.24kb fragment; after cleavage with restriction endonuclease HindIII, the generation T 3 -generation T 5 SbSNAC1-382 has 2 specific hybridization bands between 2.5 and 5kb relative to maize inbred line Zheng 58, maize inbred line Zheng 58 has endogenous background hybridization bands, these signals are generated by nonspecific hybridization of SbSNAC1 gene probe with homologous sequences in the maize genome, and the same hybridization bands are also observed in SbSNAC1-382 events, regardless of the insertion, and thus are endogenous background hybridization bands; after cleavage with the restriction endonuclease EcoRI, the generation T 3 -generation T 5 SbSNAC1-382 has 2 specific hybridization bands between 2.5 and 5kb relative to maize inbred line Zheng 58, and maize inbred line Zheng 58 has endogenous background hybridization bands, which are generated by nonspecific hybridization of the SbSNAC1 gene probe with homologous sequences in the maize genome, and the same hybridization bands are also observed in the SbSNAC1-382 event regardless of the insertion, and are thus endogenous background hybridization bands. Because the recombinant plasmid 35S is that the SbSNAC1 has only one restriction enzyme HindIII and one EcoRI restriction enzyme site, the obtained specific strip algebra of the two enzymes for restriction hybridization of the transgenic corn genome DNA respectively represents the inserted copy number. The hybridization results showed that the insertion sequence of the SbSNAC1-382 event had 2 copies of the SbSNAC1 gene.
2. Southern hybridization results Using Bar Gene Probe, restriction endonucleases HindIII and EcoRI
1. The recombinant plasmid 35S is SbSNAC1 as a template and 5'-ATGAGCCCAGAACGACGCCCG-3' and 5'-TCAAATCTCGGTGACGGGCAGGAC-3' as primers are used for PCR amplification to obtain a DNA fragment of about 552 bp. The fragment is the Bar gene probe.
2. The genome DNA of the generation T 3 SbSNAC1-382, the generation T 4 SbSNAC1-382, the generation T 5 SbSNAC1-382 or the maize inbred line Zheng 58 is respectively subjected to enzyme digestion by restriction enzymes HindIII and EcoRI, and the Bar gene probe obtained in the step 1 is hybridized.
The recombinant plasmid 35S, sbSNAC1, was digested with restriction enzyme HindIII, and hybridized with the Bar gene probe obtained in step 1. As a positive control.
The experimental results are shown in FIG. 9 (lane 1 is the genomic DNA of the restriction enzyme HindIII-cut T 3 -generation SbSNAC1-382, lane 2 is the genomic DNA of the restriction enzyme HindIII-cut T 4 -generation SbSNAC1-382, lane 3 is the genomic DNA of the restriction enzyme HindIII-cut T 5 -generation SbSNAC1-382, lane 4 is the genomic DNA of the restriction enzyme HindIII-cut maize inbred line Zheng 58, lane 5 is the genomic DNA of the restriction enzyme EcoRI-cut T 3 -generation SbSNAC1-382, lane 6 is the genomic DNA of the restriction enzyme EcoRI-cut T 4 -generation SbSNAC1-382, lane 7 is the genomic DNA of the restriction enzyme EcoRI-cut T 5 -generation SbSNAC1-382, lane 8 is the genomic DNA of the restriction enzyme I-cut maize inbred line Zheng 58, and lane 9 is the restriction enzyme EcoRI-cut plasmid 35S; sbSNAC1, lane 10 Takara DL15kb DNA MARKER). The result shows that SbSNAC1 hybridizes with a 10.24kb fragment (the hybridization band is weaker due to lower plasmid loading) by using the recombinant plasmid 35S digested with restriction endonuclease HindIII; after cleavage with restriction endonuclease HindIII, the generation T 3 -generation T 5 SbSNAC1-382 had one band around 2.5kb and 4kb, respectively, and no band appeared in maize inbred line Zheng 58; after restriction enzyme EcoRI is used for cleavage, the T 3 generation-T 5 generation SbSNAC1-382 has a band around 4kb and 5kb respectively, and no band appears in the maize inbred line Zheng 58; because the recombinant plasmid 35S is that the SbSNAC1 has only one restriction enzyme HindIII and one EcoRI restriction enzyme site, the obtained specific strip algebra of the two enzymes for restriction hybridization of the transgenic corn genome DNA respectively represents the inserted copy number. The hybridization results showed that the insertion sequence of the SbSNAC1-382 event had 2 copies of the Bar gene.
3. Results of Southern hybridization Using SbSNAC1 Gene Probe, restriction endonucleases BglII and DraI
1. The same as 1 in the first step.
2. And (2) respectively carrying out enzyme digestion on the genomic DNA of the generation T 3 SbSNAC1-382, the generation T 4 SbSNAC1-382, the generation T 5 SbSNAC1-382 or the maize inbred line Zheng 58 by using restriction endonucleases BglII and DraI, and hybridizing the SbSNAC1 gene probes obtained in the step (1).
And (2) carrying out enzyme digestion on the recombinant plasmid 35S by using restriction enzyme DraI, and hybridizing the SbSNAC1 gene probe obtained in the step (1). As a positive control.
The experimental results are shown in FIG. 10 (lane 1 is the genomic DNA of the T 3 generation SbSNAC1-382 digested with restriction enzyme BglII, lane 2 is the genomic DNA of the T 4 generation SbSNAC1-382 digested with restriction enzyme BglII, lane 3 is the genomic DNA of the T 5 generation SbSNAC1-382 digested with restriction enzyme BglII, lane 4 is the genomic DNA of the maize inbred line Zheng 58 digested with restriction enzyme BglII, lane 5 is the genomic DNA of the T 3 generation SbSNAC1-382 digested with restriction enzyme DraI, lane 6 is the genomic DNA of the T 4 generation SbSNAC1-382 digested with restriction enzyme DraI, lane 7 is the genomic DNA of the T 5 generation SbSNAC1-382 digested with restriction enzyme DraI, lane 8 is the genomic DNA of the maize inbred line Zheng 58 digested with restriction enzyme DraI, lane 9 is the genomic DNA of the Takara 1-10, and lane 6 is the plasmid Takara 1, 10 is cut by restriction enzyme Takara. The result shows that the SbSNAC1 hybridizes with a 10.24kb fragment; after cleavage with the restriction endonuclease BglII, the generation T 3 -generation T 5 SbSNAC1-382 has 1 specific hybridization band at a position greater than 15kb relative to maize inbred line Zheng 58, maize inbred line Zheng 58 has endogenous background hybridization bands, these signals are generated by nonspecific hybridization of the SbSNAC1 gene probe with homologous sequences in the maize genome, and the same hybridization band is also observed in the SbSNAC1-382 event, irrespective of the insertion, and thus is an endogenous background hybridization band; after cleavage with the restriction endonuclease DraI, the T 3 -T 5 -generation SbSNAC1-382 has 1 specific hybridization band between 10-15kb relative to maize inbred line Zheng 58, which has endogenous background hybridization band to maize inbred line Zheng 58, these signals are generated by nonspecific hybridization of SbSNAC1 gene probe with homologous sequences in the maize genome, and the same hybridization band is also observed in SbSNAC1-382 events, irrespective of the insertion, and thus is endogenous background hybridization band. Hybridization results showed that 2 copies of the SbSNAC1 gene were inserted at a single site on the genome for the SbSNAC1-382 events.
4. Results of Southern hybridization Using Bar Gene Probe, restriction endonucleases BglII and DraI
1. And 1 in the same step two.
2. And (2) respectively carrying out enzyme digestion on the genomic DNA of the generation T 3 SbSNAC1-382, the generation T 4 SbSNAC1-382, the generation T 5 SbSNAC1-382 or the maize inbred line Zheng 58 by using restriction endonucleases BglII and DraI, and hybridizing the Bar gene probes obtained in the step (1).
The recombinant plasmid 35S, sbSNAC1, was digested with restriction enzyme HindIII, and hybridized with the Bar gene probe obtained in step 1. As a positive control.
The experimental results are shown in FIG. 11 (lane 1 is the genomic DNA of the restriction enzyme BglII-digested T 3 -generation SbSNAC1-382, lane 2 is the genomic DNA of the restriction enzyme BglII-digested T 4 -generation SbSNAC1-382, lane 3 is the genomic DNA of the restriction enzyme BglII-digested T 5 -generation SbSNAC1-382, lane 4 is the genomic DNA of the restriction enzyme BglII-digested maize inbred line Zheng 58, lane 5 is the genomic DNA of the restriction enzyme DraI-digested T 3 -generation SbSNAC1-382, lane 6 is the genomic DNA of the restriction enzyme DraI-digested T 4 -generation SbSNAC1-382, lane 7 is the genomic DNA of the restriction enzyme DraI-digested T 5 -generation SbSNAC1-382, lane 8 is the genomic DNA of the restriction enzyme DraI-digested maize inbred line Zheng 58, lane 9 is the restriction enzyme Takara 1-digested plasmid 10, and lane 10 is the restriction enzyme Takara 4, restriction enzyme 10 is shown in FIG. 4.
The result shows that the SbSNAC1 hybridizes with a 10.24kb fragment; after cleavage with the restriction endonuclease BglII, the T 3 generation-T5 generation SbSNAC1-382 had 1 specific hybridization band at a position of more than 15kb relative to maize inbred line Zheng 58, which was consistent with the result of hybridization using the SbSNAC1 gene probe (the specific band size for hybridization using the SbSNAC1 gene probe and the Bar gene probe was consistent); after cleavage with the restriction endonuclease DraI, the T 3 generation-T5 generation SbSNAC1-382 had 1 specific hybridization band between 10-15kb relative to maize inbred line Zheng 58, consistent with the result of hybridization using the SbSNAC1 gene probe (the specific band size for hybridization using the SbSNAC1 gene probe and the Bar gene probe). Hybridization results showed that 2 copies of the Bar gene from SbSNAC1-382 event were inserted at a single site on the genome.
The Southern hybridization results show that the SbSNAC1-382 event T-DNA fragment is inserted at a single site on the corn genome, and 2 copies of the SbSNAC1 gene and the Bar gene are arranged at the insertion site, so that stable inheritance can be realized between different generations.
The SbSNAC1-382 event is preserved in China general microbiological culture collection center (CGMCC) with the preservation number of CGMCC No.17493 in the 04 th month of 2019 in the Ganyang area North Star West-way No.1 of Beijing city. The SbSNAC1-382 event is collectively referred to as corn Zea mays SbSNAC1-382CGMCC No.17493, abbreviated as SbSNAC1-382 event.
Example 3 determination of 5 'and 3' flanking sequences of exogenous insert fragment of SbSNAC1-382 event insertion site
The flanking sequences of a particular transgenic event are specific. Thus, the use of flanking sequences allows specific detection of transgenic events. Such as hybridization with a probe comprising at least a portion of the flanking sequence and at least a portion of the exogenous insert, or designing primers for specific amplification comprising at least a portion of the flanking sequence and at least a portion of the exogenous insert, PCR amplification, detection of specific bands, etc. The upstream specific primer can be designed according to the 5' flanking sequence, the downstream specific primer can be designed according to the exogenous insert fragment, and the specific fragment can be amplified; or the upstream specific primer can be designed according to the exogenous insert fragment, the downstream specific primer can be designed according to the 3' -end flanking sequence, and the specific fragment can be amplified.
1. Acquisition and validation of 5' flanking sequences
1. Extracting genomic DNA of the T 5 SbSNAC1-382 leaves and taking the genomic DNA as a template, wherein the genomic DNA is prepared by using specific primers GSP1:5'-TATCCCTGGCTCGTCGCCGA-3' and GSP2:5'-AGGGCTTCAAGAGCGTGGTCGCT-3', specific primer GSP3:5'-CCGTCACCGAGATTTGACTCGAGTTTC-3' and random primers (assemblies in Genome WALKING KIT; genome WALKING KIT is a product of TaKaRa company under the trade name 6108) to obtain the sequence of the exogenous gene at the left boundary of the maize Genome integration site. The sequence is 933bp long, and is specifically shown as sequence 3 in a sequence table. Wherein, the 1 st to 451 st positions from the 5' end in the sequence 3 in the sequence table are corn genome sequences, and the 452 th to 933 rd positions are vector sequences.
Designing and synthesizing a specific upstream primer P1 according to the DNA molecule shown in the 1st to 451 st positions from the 5' end of the sequence 3 in the sequence table: 5'-AGAATCATACACCAGTAACAAGCC-3' and P3:5'-GGAATGAACCTCATCCCAATGA-3'. Designing and synthesizing a downstream identification primer P2 according to the DNA molecule shown in 452-933 from the 5' end of the sequence 3 in the sequence table: 5'-CAGTACATTAAAAACGTCCGCA-3' and P4:5'-ACTAAAATCCAGATCCCCCGAA-3'.
2. PCR amplification is carried out by taking genomic DNA of SbSNAC1-382 leaves, water, genomic DNA of corn inbred line Zheng 58 leaves or genomic DNA of SbSNAC1-383 leaves as templates and 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 PCR amplification products.
The reaction system was 20. Mu.L, consisting of 2. Mu.L of 10 XPCR buffer, 0.5. Mu.L of dNTP (concentration: 10 mmol/L), 0.5. Mu.L of Taq enzyme (concentration: 5U/. Mu.L), 1.0. Mu.L of template (concentration: 50 ng/. Mu.L in the case of genomic DNA of maize leaves), 0.5. Mu.L of upstream primer (concentration: 10. Mu.mol/L), 0.5. Mu.L of downstream primer (concentration: 10. Mu.mol/L) and 15. Mu.L of ddH 2 O.
The reaction procedure is: 95 ℃ for 5min;95℃30s,60℃30s,72℃1min,35 cycles; 72 ℃ for 5min; preserving at 15 ℃.
3. The PCR amplified product was subjected to 1% (m/v) agarose gel electrophoresis.
The agarose gel electrophoresis results are shown in FIG. 12 (lanes 5 to 8 are primer set A, lanes 1 to 4 are primer set B, lanes 9 to 12 are primer set C, lanes 1, 5, 9 are genomic DNA of SbSNAC1-382 leaves, lanes 2,6, 10 are water, lanes 3, 7, 11 are genomic DNA of maize inbred line Zheng 58 leaves, and lanes 4, 8, 12 are genomic DNA of SbSNAC1-383 leaves). The result shows that about 311bp DNA fragment can be obtained by using the primer pair A and about 242bp DNA fragment can be obtained by using the primer pair B and about 350bp DNA fragment can be obtained by using the primer pair C by using the genomic DNA of the SbSNAC1-382 leaves as a template.
2. Acquisition and validation of 3' flanking sequences
1. Genomic DNA of the T 5 generation SbSNAC1-382 leaves was extracted to construct a Fosmid library (Takara Biotechnology (Dalian) Co., ltd.) and then PCR amplification was performed with SbSNAC1 gene-specific primers 5'-GACCGCAAGTACCCAAACGG-3' and 5'-CACCCAGTCATCCAGCCTGAG-3' (reaction conditions: 95 ℃,5min;95 ℃ denaturation 30s,60 ℃ annealing 30s,72 ℃ extension 30s,34 cycles; 72 ℃ extension 5min;15 ℃ preservation), positive monoclonal was selected and sequenced using PacBio RSII (Wuhan future group Biotechnology Co.). Based on the sequencing result, the sequence of the exogenous gene at the right boundary of the corn genome integration site is obtained. The sequence is 547bp long, and is specifically shown as sequence 4 in a sequence table. Wherein, the 1 st to 352 th positions from the 5' end of the sequence 4 in the sequence table are corn genome sequences, and the 353 th to 547 th positions are vector sequences.
According to the DNA molecule shown in the 1 st to 352 th positions from the 5' end of the sequence 4 in the sequence table, a specific primer S1 is designed and synthesized: 5'-AGTGCACATTGCAATCCTACAAGC-3' and S2:5'-CCTAAGTTCATGCAACTAGAGGTTTCA-3'. Designing and synthesizing S3 according to DNA molecules shown in 353-547 positions from the 5' end of the sequence 4 in the sequence table: 5'-GGTTTCGCTCATGTGTTGAGC-3' and S4:5'-TCCAGATCCCCCGAATTAATTCG-3'.
2. PCR amplification was performed using genomic DNA from SbSNAC1-382 leaves as a template, using primer set 1 (composed of S1 and S3), primer set 2 (composed of S2 and S3), primer set 3 (composed of S1 and S4), primer set 4 (composed of S2 and S4), or primer set 5 (composed of 5'-GACCGCAAGTACCCAAACGG-3' and 5'-CACCCAGTCATCCAGCCTGAG-3'), to obtain PCR amplification products.
And (3) carrying out 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 reaction system was 20. Mu.L, consisting of 2. Mu.L of 10 XPCR buffer, 0.5. Mu.L of dNTP (concentration: 10 mmol/L), 0.5. Mu.L of Taq enzyme (concentration: 5U/. Mu.L), 1.0. Mu.L of template (concentration: 50 ng/. Mu.L in the case of genomic DNA of maize leaves), 0.5. Mu.L of upstream primer (concentration: 10. Mu.mol/L), 0.5. Mu.L of downstream primer (concentration: 10. Mu.mol/L) and 15. Mu.L of ddH 2 O.
The reaction procedure is: 95 ℃ for 5min;95℃30s,60℃30s,72℃1min,35 cycles; 72 ℃ for 5min; preserving at 15 ℃.
3. The PCR amplified product was subjected to 1% (m/v) agarose gel electrophoresis.
The agarose gel electrophoresis results are shown in FIG. 13 (lanes 1 to 5 are genomic DNA of SbSNAC1-382 leaves, lane 6 is water, lanes 1 and 6 are primer pair 1, lane 2 is primer pair 2, lane 3 is primer pair 3, lane 4 is primer pair 4, and lane 5 is primer pair 5). The result shows that the genome DNA of the SbSNAC1-382 leaves is used as a template, a DNA fragment of about 503bp can be obtained by using a primer pair 1, a DNA fragment of about 547bp can be obtained by using a primer pair 2, a DNA fragment of about 392bp can be obtained by using a primer pair 3, a DNA fragment of about 436bp can be obtained by using a primer pair 4, and a DNA fragment of about 249bp can be obtained by using a primer pair 5.
<110> Institute of crop science at national 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
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<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
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<213> Sorghum bicolor（L.）Moench
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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
100 105 110
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
260 265 270
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
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<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 (2)

1. A method for identifying whether a test plant sample is derived from a SbSNAC1-382 event or progeny thereof, comprising the steps of: detecting whether the genome 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 DNA fragment A and/or DNA fragment B, the plant sample to be tested is derived from SbSNAC1-382 event or its progeny; 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 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 was maize Zea mays SbSNAC1-382 CGMCC 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), S2) or S3):
s1) direct sequencing;
S2) carrying out PCR amplification on genomic DNA of the plant sample to be detected by using the primer pair X and/or the primer pair Y, and then judging as follows: if the target amplification product is obtained, the plant sample to be detected is derived from SbSNAC1-382 event or the progeny thereof; if the target amplification product is not obtained, the plant sample to be tested is not derived from the SbSNAC1-382 event or the progeny thereof;
The primer pair X is at least one of a primer pair X1, a primer pair X2 and a primer pair X3; primer pair X1 consists of primer P1 and primer P2; primer pair X2 consists of primer P3 and primer P4; primer pair X3 consists of primer P1 and primer P4; the target amplification product of the primer pair X is DNA molecule X;
the nucleotide sequence of the primer P1 is shown as a sequence 5 in a sequence table;
the nucleotide sequence of the primer P2 is shown as a sequence 6 in a sequence table;
the nucleotide sequence of the primer P3 is shown as a sequence 7 in a sequence table;
The nucleotide sequence of the primer P4 is shown as a sequence 8 in a 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; primer pair Y1 consists of primer S1 and primer S3; primer pair Y2 consists of primer S2 and primer S3; primer pair Y3 consists of primer S1 and primer S4; primer pair Y4 consists of primer S2 and primer S4; the target amplification product of the primer pair Y is a DNA molecule Y;
the nucleotide sequence of the primer S1 is shown as a sequence 9 in a 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 a sequence table;
The nucleotide sequence of the primer S4 is shown as a sequence 12 in a sequence table;
the nucleotide sequence of the target amplification product of the primer pair X1 is shown as 215-525 bits from the 5' end of a sequence 3 in a sequence table;
the nucleotide sequence of the target amplification product of the primer pair X2 is shown as 323 rd to 564 th from the 5' end of a sequence 3 in a sequence table;
The nucleotide sequence of the target amplification product of the primer pair X3 is shown as 215-564 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 Y1 is shown in the 45 th to 547 th positions from the 5' end of the sequence 4 in the sequence table;
the nucleotide sequence of the target amplification product of the primer pair Y2 is shown in the 1 st to 547 th positions from the 5' end of the sequence 4 in the sequence table;
the nucleotide sequence of the target amplification product of the primer pair Y3 is shown as 215-436 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 in the 1 st-436 th positions from the 5' tail end of the sequence 4 in the sequence table;
S3) carrying out Southern hybridization on 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 judging as follows: if the hybrid fragment is obtained, the plant sample to be tested is derived from the SbSNAC1-382 event or a progeny thereof; if no hybrid fragment is available, the plant sample to be tested is not derived from the SbSNAC1-382 event or its progeny.
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