CN113186337A - Flanking sequence of glyphosate-resistant corn GG2 exogenous insert and application thereof - Google Patents
Flanking sequence of glyphosate-resistant corn GG2 exogenous insert and application thereof Download PDFInfo
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Abstract
The invention relates to a flanking sequence of a transgenic glyphosate-tolerant corn GG2 exogenous insert and application thereof. The invention screens a transgenic event GG2 with remarkable glyphosate-tolerant effect from corn plants with GR79 and GAT genes; the left border flanking sequence and the right border flanking sequence are obtained by a chromosome walking method, the left border flanking sequence is shown as 1-440 of SEQ ID NO.3, and the right border flanking sequence is shown as 6556-7873 of SEQ ID NO. 3. The boundary sequences at the two ends can be used as specific detection sequences of the transformation event, primers are designed through the two boundary sequences, the specific detection of the transgenic event GG2 can be realized, and the method is applied to the development of detection kits.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a transgenic glyphosate-tolerant corn plant and detection thereof.
Background
Corn (Zea mays L.) is a bulk grain crop and also is an important feed and industrial raw material, weeds not only compete with corn for water, fertilizer, light, space and the like, but also are easy to breed diseases and insect pests, the growth and development of the crops are seriously influenced, the yield of the crops is reduced, and the quality of the crops is reduced, so that the weed control is an important link in the corn production. The main weeds in farmlands in China exceed 250, the distribution area reaches 4000 hectares, and the farmlands with 1000 hectares are seriously damaged. The crop yield reduction caused by the weeds in China is about 13% on average every year, and the direct economic loss accounts for 10% -20% of the total crop yield. The use of herbicides to control weeds has become an indispensable part of modern agriculture because of the high labor consumption, increased planting costs, high mechanical intertillage weeding costs, and susceptibility to soil loss and hardening. But the non-selective characteristic of the herbicide inevitably influences the growth and development of crops during use. With the rapid development of the biological genetic engineering technology and the research progress on the molecular level of the herbicide resistance mechanism of plants, the herbicide resistance traits of crops can be obtained by transgenic technology in the 80 s of the 20 th century. The first herbicide resistant crop, tobacco, was published in 1983, which marks that the research in this field is successful from exploration, and various herbicide tolerant transgenic crops, such as corn, soybean, cotton, rape and the like, have been commercially planted so far, which generates huge social and economic benefits and has a great promoting effect on improving the utilization efficiency of cultivated land and improving the artificial efficiency.
Glyphosate is a broad-spectrum biocidal and systemic conductive herbicide, and has become the most widely applied pesticide in the world due to the advantages of broad spectrum, high efficiency, environmental friendliness and the like. The shikimic acid pathway is an important pathway for the synthesis of aromatic amino acids in plants and microorganisms. 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) is one of the key enzymes in the biosynthesis of phenylalanine, tyrosine and tryptophan among the aromatic amino acids in higher plants. EPSPS catalyses the condensation of phosphoenolpyruvate (PEP) with shikimate-3-phosphate (S3P) in the shikimate metabolic pathway. The glyphosate is a competitive analog of PEP, and the action mechanism of the glyphosate is that stable complex EPSPS-S3P-glyphosate is formed with EPSPS and S3P, the activity of EPSPS is competitively inhibited, shikimic acid-3-phosphate is blocked to be converted into 5-enolpyruvyl-3-phosphoshikimic acid, the formation of aromatic amino acid compounds is blocked, shikimic acid is massively accumulated, hormones and key metabolites such as flavonoid and phenolic compounds are disturbed, and the normal nitrogen metabolism of organisms is disturbed, so that the organisms are killed. According to the action mechanism of glyphosate herbicide, there are currently 3 main ways to cultivate glyphosate-resistant transgenic crops: (1) overexpresses EPSPS protein to compensate for the loss of EPSPS protein by glyphosate; (2) transferring EPSPS/aroA and other genes to generate EPSPS protein insensitive to glyphosate, such as transgenic corn NK603 and transgenic soybean 40-3-2 developed by using EPSPS gene of agrobacterium strain cp4 by Monsanto company; (3) transferring glyphosate-N-acetyltransferase GAT to directly inactivate glyphosate.
The research and development work of herbicide-tolerant transgenic corn starts earlier and develops very rapidly abroad. In 2017, the planting area of the transgenic corn in the whole world reaches 5970 ten thousand hectares, wherein the planting area of the herbicide-tolerant corn (containing composite characters) accounts for more than 90 percent. The currently commercialized herbicide-tolerant transgenic maize varieties are all developed by foreign species companies, such as "NK 603" herbicide-tolerant maize (containing cp4-epsps gene), "GA 21" herbicide-tolerant maize (containing mepsps gene), and "TC 1507" insect-resistant herbicide-tolerant maize (containing cry1Fa2 and pat gene), developed by DuPont Dow, and "Bt 11" insect-resistant herbicide-tolerant maize (containing cry1Ab and pat gene), and "Bt 176" insect-resistant herbicide-tolerant maize (containing cry1Ab and bar gene), developed by Ministra. The research and development of the herbicide-tolerant transgenic corn in China are late, the development is relatively slow, and no herbicide-tolerant corn variety which is industrially applied exists at present. In 2019-2020, China approved the production application safety certificate of 'DBN 9936' insect-resistant herbicide-tolerant corn (containing cry1Ab and epsps genes) and 'DBN 9858' herbicide-tolerant corn (containing epsps and pat genes) which are developed by Beijing Dabei agricultural biotechnology limited, and 'Ruifeng 125' insect-resistant herbicide-tolerant corn (containing cry1Ab/cry2Aj and g10evo-epsps genes) which is jointly developed by Hangzhou Ruifeng biological technology limited and Zhejiang university. The method has great inspiring and promoting effects on the research and development work of the transgenic corn in China.
The GR79 and GAT genes were isolated and cloned from the metagenome of glyphosate-severely contaminated soil bacteria. The GR79 gene codes EPSPS enzyme, glyphosate can not block the reaction of the GR79-EPSPS synthase catalyzing PEP and S3P to generate EPSP, so that the anabolism of aromatic amino acid and other compounds of plants is continued, the plants obtain the herbicide resistance, and the normal growth of the plants is protected. The GAT gene codes glyphosate-N-acetyltransferase, and the glyphosate N-acetyltransferase provides a brand-new action mechanism different from GR79-EPSPS path for resisting glyphosate of crops. Under the action of glyphosate-N-acetyltransferase, acetyl coenzyme A is used as acetyl donor, and secondary amine of glyphosate molecule is used as acetyl receptor, so that glyphosate is acetylated to lose herbicide activity, transgenic plant obtains herbicide resistance, and plant is protected from normal development and growth.
Because the position of the exogenous gene integrated on the corn genome can influence the expression of the exogenous gene, if a transgenic plant with high expression quantity and good herbicide tolerance effect is to be obtained, herbicide tolerance corn with industrialization prospect can be obtained only by screening from a large number of transformation events and carrying out multi-generation genetic stability detection, meanwhile, a boundary sequence of the transgenic corn event inserted into the corn genome can be used as an identity label of the transgenic material, an insertion site of a chromosome can be used as an independent transformation event, and the herbicide tolerance corn can be detected by utilizing a specific primer. The GAT and GR79 transgenic glyphosate-resistant corn GG2 obtained by the invention is a novel transformation event, the position of the event inserted into the corn genome is different from other transgenic events, and the border sequence of the event can be used as an identity tag for specific identification.
Disclosure of Invention
The invention constructs a plant expression vector pCGG containing GR79 and GAT genes, obtains a transgenic corn plant containing GR79 and GAT genes, and screens a transgenic event GG2 with high glyphosate resistance; the left border flanking sequence and the right border flanking sequence of the maize genome inserted therein are obtained by a chromosome walking method, the flanking sequences at the two ends of the border can be used as specific detection sequences of the transformation event, and primers are designed by the two border flanking sequences, so that the specific detection of the transgenic event GG2 can be realized.
The left border flanking sequence of the exogenous insert of the transgenic glyphosate-tolerant maize GG2 is shown as position 269-462 in SEQ ID NO. 1.
The right border flanking sequence of the exogenous insert of the transgenic glyphosate-tolerant maize GG2 is shown in SEQ ID NO.2 at position 456-1773.
And a specific primer pair for PCR reaction detection is designed according to the left border flanking sequence.
The specific primer pair of the left border flanking sequence is as follows:
GG2-Left-F3：5'-GGAGCAAGGAAGCGGACTAC-3'，
GG2-Left-R1：5'-CCCCACATCCTGATGTACAAG-3'。
and a specific primer pair for PCR reaction detection is designed according to the flanking sequence of the right boundary.
The specific primer pair of the right border flanking sequence is as follows:
GG2-Ubi-F1：5'-ATGATTCTCTAAAACACTG-3'，
GG2-Right-R1：5'-GCGAACATAGCGTCTTAC-3'。
the PCR reaction detection method of the transgenic glyphosate-tolerant corn GG2 is characterized in that: the primers in the PCR reaction are the specific primer pair.
The specific primer pair is as follows:
GG2-Left-F3：5'-GGAGCAAGGAAGCGGACTAC-3'，
GG2-Left-R1：5'-CCCCACATCCTGATGTACAAG-3'。
the size of the fragment obtained by the PCR reaction is 734 bp; or
The specific primer pair is as follows:
GG2-Ubi-F1：5'-ATGATTCTCTAAAACACTG-3'，
GG2-Right-R1：5'-GCGAACATAGCGTCTTAC-3'。
the size of the fragment obtained by the PCR reaction is 1773 bp.
A kit for detecting glyphosate-tolerant corn is characterized by comprising a specific primer pair of a left border flanking sequence or/and a specific primer pair of a right border flanking sequence.
The specific primers of the left border flanking sequence are as follows:
GG2-Left-F3：5'-GGAGCAAGGAAGCGGACTAC-3'，
GG2-Left-R1：5'-CCCCACATCCTGATGTACAAG-3'。
the specific primers of the right border flanking sequence are as follows:
GG2-Ubi-F1：5'-ATGATTCTCTAAAACACTG-3'，
GG2-Right-R1：5'-GCGAACATAGCGTCTTAC-3'。
the flanking sequence, the specific primer of the flanking sequence and the application of the glyphosate-tolerant corn detection kit in the detection of transgenic corn.
The invention transforms the vector pCGG into maize immature embryo by agrobacterium-mediated method, and the specific method is shown in the patent application of 'expression vector and application of glyphosate resistance gene GR79 and GAT' (application number: 202110376902.5)). And (3) obtaining 103T 0 transformed plants in total through transformation, and detecting positive plants to be 76 plants through PCR. All positive transformed plants were transferred to the greenhouse and seeds were harvested. The method comprises the steps of planting T1 generation transgenic corn in a field, manually spraying glyphosate (the spraying amount is 1 time, 2 times and 4 times of the medium dosage of a pesticide registration label, adding water to 450L/hectare, the medium dosage of Roundup (trade name of glyphosate pesticide) is 1230g of effective components/hectare, the same is true below) when the corn grows to four-six leaf stage, screening transgenic glyphosate-resistant corn GG2 (preservation number CGMCC No.20132) for multiple generations without being damaged by the glyphosate, obtaining a left boundary flanking sequence and a right boundary flanking sequence by a chromosome step transfer method, using the flanking sequences at two ends of the boundary as specific detection sequences of the transformation event, designing primers by using the two flanking sequences of the boundary, specifically detecting the transgenic event GG2, and applying the primers to the development of a detection kit.
Transgenic maize GG2, classified and named maize, Zea mays
The preservation number is CGMCC No.20132
The preservation date is as follows: 14 days 1 month in 2021
The preservation unit: china general microbiological culture Collection center
And (4) storage address: xilu No.1 Hospital No.3 of Beijing, Chaoyang, China academy of sciences, microbial research institute, postal code 100101
Drawings
FIG. 1 shows PCR detection of GAT gene (A) and GR79 gene (B) in transgenic maize of the T1 generation;
wherein M is DNA molecular weight standard Super Marker; CK +: plasmid pCGG is taken as a template amplification product; CK-: using non-transgenic corn genome DNA as a template amplification product; 0: blank, using water as template amplification product; 1-14: the T1 generation GG1-GG14 transgenic corn genome DNA is used as a template amplification product;
FIG. 2 is a field glyphosate tolerance assay of GG2 transgenic corn;
wherein, the graph A is 4 times glyphosate sprayed on BC4 GG2, and the graph B is 4 times glyphosate sprayed on BC5 GG 2;
FIG. 3 shows the southern blot hybridization results of transgenic maize GG 2;
wherein, the detection result of the GAT probe is shown in a graph A, and the detection result of the GR79 probe is shown in a graph B. The Marker is a DNA molecular weight standard and consists of 7 DNA fragments, and the sizes of the bands are 23,130bp, 9,416bp, 6,557bp, 4,361bp, 2,322bp, 2,027bp and 564bp from top to bottom respectively; CK +: cutting pCGG plasmid/HindIII enzyme; CK-: carrying out enzyme digestion on non-transgenic corn genome DNA/HindIII;
FIG. 4 is a schematic diagram of the restriction enzyme site of the insert fragment and the expected size of the southern blot hybridization band;
FIG. 5 is a chromosome walking map of the left flanking sequence of transgenic maize GG 2;
m is Trans5K DNA Marker, 1stAs a result of 1 st round PCR, 2ndAs a result of the 2 nd round PCR, 3rdIs the result of the 3 rd round PCR;
FIG. 6 is a PCR electropherogram specific to the right border (A) and the left border (B) of transgenic maize GG2 of T2-T4 generation;
Marker：Trans5K DNA Marker，CK1(H2o): with H2O as template as blank, CK2 (pCGG): plasmid pCGG as template amplification product, CK3 (sitter event GG 3): is an amplified product by taking transformant GG3 genome DNA as a template, CK4 (site event GG 4): so as to makeTransformant GG4 genomic DNA was the product of template amplification, CK5 (reporter B104): is an amplified product using transformation receptor B104 genomic DNA as a template, CK6 (Z58): the product amplified by using backcross transfer receptor Z58 genome DNA as a template, T2: the T2 generation transformant GG2 genome DNA is used as a template amplification product, T3: the T2 generation transformant GG2 genome DNA is used as a template amplification product, T4: using T2 generation transformant GG2 genome DNA as template amplification product;
FIG. 7 is a depiction of the location of a transgenic maize GG2 insert in the maize genome.
Detailed Description
The present invention will be described in further detail with reference to examples.
Example 1 acquisition of transgenic maize event GG2
1. Transforming maize young embryo with agrobacterium to obtain transformed plant
The invention transforms the vector pCGG into maize immature embryos by an agrobacterium-mediated method, and the specific method is shown in the patent application of 'expression vectors and applications of glyphosate resistance genes GR79 and GAT' (application number: 202110376902.5)), and 103T 0 generation transformed plants are obtained by transformation.
2. PCR detection of transformed plants
2.1 Small extraction of maize genomic DNA (CTAB method):
(1) taking 100mg of plant leaves, grinding the plant leaves into powder by using liquid nitrogen, adding 1mL of CTAB buffer, and fully and uniformly mixing;
(2) after water bath at 65 ℃ for 10min, adding 1mL of chloroform, and uniformly mixing the mixture by using a vortex mixer to obtain emulsion;
(3) centrifuging at room temperature of 13,000g for 10min, collecting 900 μ L of supernatant, adding 600 μ L of isopropanol, and mixing by turning upside down;
(4) centrifuging at room temperature at 13,000g for 10min, removing supernatant, and washing precipitate with 70% ethanol;
(5) removing ethanol, air drying at room temperature, precipitating, adding appropriate amount of TE or ddH2O, dissolving, and storing at-20 deg.C.
2.2 PCR detection of the GAT Gene
GAT-F1:5'-TCGACGTGAACCCGATCAAC-3'
GAT-R1:5'-TCTGCTCCCTGTAGCCCTCC-3'
Size of the target fragment: 249bp
The PCR reaction system is as follows:
the amplification conditions were as follows: pre-denaturation at 95 ℃ for 3 min; denaturation at 95 ℃ for 20s, annealing at 58 ℃ for 15s, extension at 72 ℃ for 20s, and amplification cycle number of 30; finally, extension is carried out for 5min at 72 ℃.
2.3 PCR detection of GR79 Gene
GR79-F1：5'-TCAGCAGGGCGAGTGGA-3'
GR79-R1：5'-TCGTCGTGCGGGTTCAG-3'
Size of the target fragment: 831bp
The PCR reaction system is as follows:
the amplification conditions were as follows: pre-denaturation at 95 ℃ for 3 min; denaturation at 95 ℃ for 20s, annealing at 59.4 ℃ for 15s, extension at 72 ℃ for 40s, and amplification cycle number of 30; finally, extension is carried out for 5min at 72 ℃.
The positive plants are transgenic plants detected by PCR, and the detection result is shown in figure 1.
The PCR detection shows that 76 GAT and GR79 gene double positive plants are obtained. All positive transformed plants were transferred to the greenhouse and seeds were harvested.
3. Screening of high glyphosate-resistant transgenic material
Example 2 field glyphosate tolerance identification of transgenic corn event GG2
According to the environmental safety detection of transgenic plants and products thereof, part 1 of the herbicide-resistant corn: herbicide tolerance (bulletin No. 953-11.1-2007, Ministry of agriculture) the field herbicide tolerance of transgenic maize was examined.
The test material is GG2 transgenic corn and corresponding non-transgenic corn. The herbicide is glyphosate. Block design (not random), 2 replicates, cell area 30m2(5m is multiplied by 6m), the row spacing is 60cm, and the plant spacing is 25 cm. Isolation belts with the width of 1.0m are arranged among the cells. The treatment comprises that the transgenic corn is not sprayed with herbicide, the transgenic corn is sprayed with target herbicide (glyphosate), the non-transgenic corn is not sprayed with herbicide, and the non-transgenic corn is sprayed with target herbicide (glyphosate). The application amount of the herbicide is 1 time, 2 times and 4 times of the dosage (60 g/mu of effective component) in the pesticide registration label respectively. Is applied at the 4-5 leaf stage of the corn. Survival, plant height, phytotoxicity symptoms were investigated and recorded at 1, 2, 4 weeks of application, respectively. Phytotoxicity symptom grading is performed according to GB/T19780.42. Carrying out glyphosate identification on transgenic corn GG2 and corresponding transgenic control and non-transgenic control thereof, wherein the identification result of the glyphosate tolerance in the field of two continuous generations shows that: compared with the transgenic corn which is not sprayed with the herbicide, the transgenic corn GG2 has no damage symptoms in each period after being sprayed with different glyphosate, and has no significant difference in plant height (tables 1 and 2), which shows that the GG2 transgenic corn can resist glyphosate with the medium dose of 4 times or less and has stable inheritance of glyphosate-resistant characters (figure 2).
TABLE 1 Glyphosate-treated GG2 plant height survey
Note: the data in the table are mean ± sd, and the difference between the lower case letters in english after the same column of data indicates significant difference between different treatments (P < 0.05). The same is as follows.
TABLE 2 Glyphosate treatment GG2 damage Rate survey
Example 3 exogenous Gene insertion site analysis of transgenic maize event GG2
1. Bulk extraction of maize genomic DNA (slightly modified CTAB method):
(1) weighing 5g of leaves, fully grinding the leaves into powder (without melting the material) by using liquid nitrogen, and adding the powder into a 50mL centrifuge tube;
(2) adding 15mL of 2 XCTAB buffer solution (Tris 100mM, NaCl 1.4M, 20mM EDTA, 2% CTAB and 0.1% mercaptoethanol), fully mixing, and performing water bath at 65 ℃ for 1 hour;
(3) cooling to room temperature, adding 15mL of chloroform/isoamyl alcohol (24: 1), turning upside down, uniformly mixing to obtain an emulsion, and standing at room temperature for 15-60 minutes;
(4) centrifuging at 12,000rpm for 15min at room temperature;
(5) transferring the supernatant into a clean centrifuge tube, adding 2/3 volumes of isopropanol, inverting for several times, picking out DNA, and putting into a clean 1.5mL centrifuge tube;
(6) washing DNA with 70% ethanol for 2 times, and air-drying for 1 hr;
(7) after DNA was dissolved in 500. mu.L of TE, 5. mu.L of RNase A (10mg/mL) was added and the mixture was dissolved overnight at 4 ℃ or 1 hour at 37 ℃;
(8) adding 500 μ L phenol, reversing, mixing thoroughly, centrifuging at 12,000rpm for 5min, and transferring the supernatant to another centrifuge tube;
(9) adding 250 μ L phenol and chloroform respectively, reversing, mixing well, centrifuging at 12,000rpm for 5min, and transferring the supernatant to another centrifuge tube;
(10) adding 500 μ L chloroform, reversing, mixing well, centrifuging at 12,000rpm for 5min, transferring the supernatant to a 10mL centrifuge tube;
(11) adding TE to 3mL, then adding 2 times of anhydrous ethanol and 1/10 times of 3M NaAc, and reversing the mixture up and down for several times;
(12) washing the precipitated DNA with 70% ethanol for 2 times; the DNA was transferred to a 1.5mL centrifuge tube, air dried and dissolved in 500. mu.L TE and the DNA quantified for use.
2. Preliminary experiments with small restriction of genomic DNA:
uniformly mixing, and performing enzyme digestion at 37 ℃ for 2-3 hr; the cleavage reaction was electrophoretically separated on 0.7% agarose, and the cleavage effect was checked.
3. Bulk digestion of genomic DNA:
genomic DNA 100. mu.g
Enzyme (10U/. mu.L) 5. mu.L
(HindIII and Kpn I were selected for this experiment)
10×Buffer 40μL
Total 400μL
Mixing, and enzyme-cutting at 37 deg.C for 10 hr; taking 2 mu L of enzyme digestion product for electrophoretic separation, and checking the enzyme digestion effect; precipitating the enzyme-digested product after completely digesting, adding 1/10 volume of 3M NaAc and 2 times volume of absolute ethyl alcohol (precooling at-20 ℃), mixing, and standing at-20 ℃ for 2 hr; centrifuging at 4 deg.C for 20min at 12,000rpm, discarding supernatant, adding 1mL 70% ethanol into the precipitate, centrifuging at 12,000rpm for 2min, discarding supernatant, blow drying the precipitate, and dissolving in 30 μ L ddH2And O for later use.
4. Preparation of Probe
Probes were prepared according to the PCR DIG Probe Synthesis Kit instructions.
GAT gene probe primers:
GAT probe-F1:5'-TCGACGTGAACCCGATCAAC-3'，
GAT probe-R1:5'-TCTGCTCCCTGTAGCCCTCC-3'，
the GAT gene probe size is 249 bp.
GR79 gene probe primer:
GR79 probe-F1:5'TCAGCAGGGCGAGTGGA 3'，
GR79 probe-R1:5'TCGTCGTGCGGGTTCAG 3'，
the GR79 gene probe size was 831 bp.
The PCR reaction system is as follows:
the PCR reaction conditions were as follows:
after the PCR is finished, the DIG-labeled probe is electrophoretically detected and the concentration is measured.
5. Southern blot hybridization
(1) Preparing 0.7% agarose gel, adding 6 muL of 6 × loading buffer into 30 muL of genome DNA enzyme digestion products for electrophoretic separation, standing for 10min after sample loading, increasing the voltage to 50V at the beginning by adopting low voltage after the bromophenol blue runs out of a sample loading hole by 2-3 cm, and performing electrophoresis for 5-6 hours;
(2) after electrophoresis, the following treatments were sequentially performed on the gel: soaking the gel in 0.125M hydrochloric acid for 10min to turn bromophenol blue in the gel into yellow; treating gel with distilled water for 5 min; soaking the gel in the neutralization solution for 30 min;
(3) transferring DNA to a nylon membrane by adopting a capillary transfer method (the specific operation is shown in an experimental manual of molecular cloning);
(4) soaking the nylon membrane in 6 times SSC for 5min after the membrane conversion is finished, and drying the nylon membrane in a super clean bench or at room temperature;
(5) drying the membrane for 2hr at 80 deg.C, and fixing DNA sample;
(6) preparation of DIG-labeled probes: GAT and GR79 gene-labeled probes were prepared and the concentration of the probes was determined according to the method for preparing probes in PCR DIG Probe Synthesis Kit (purchased from Roche);
(7) pre-hybridization: carefully loading the nylon membrane into a hybridization tube with forceps, carefully handling without generating air bubbles, then adding 10mL of DIG Easy Hyb hybridization solution (digoxin labeling and detection kit II from Roche) preheated at 42 ℃, and prehybridizing for 3hr at 42 ℃;
(8) and (3) hybridization: the probe treatment was first performed by denaturing the labeled probe at 99 ℃ for 6min and immediately cooling on ice for 2 min. Adding a treated probe (25ng/mL Hyb hybridization solution) into 7mL of DIG Easy Hyb hybridization solution, gently mixing uniformly without generating bubbles, putting the mixture into a hybridization furnace, and hybridizing at 42 ℃ for 16-20 hr;
(9) washing the membrane: first, the cells were washed twice with 50mL of 2 XSSC, 0.1% SDS solution at room temperature for 15min each. Then washed twice with 0.5 XSSC, 0.1% SDS solution at 50mL65 ℃ for 30min each time. Carefully taking out the membrane by using a pair of tweezers, transferring the membrane into a dish filled with 50mL Washing buffer, and Washing for 1-5 min in a shaking way;
(10) incubating with 100ml of 1 × Blocking solution for 60min at room temperature;
(11) incubating with 20ml Antibody solution for 30 min;
(12) washing with 50ml Washing buffer for 2 times, each time for 30 min;
(13) balancing in 20ml Detection buffer for 2-5 min;
(14) flatly placing the film between two layers of preservative films by using tweezers, lifting the upper layer of preservative film, adding 1ml of CSPD substrate, slowly putting down the upper layer of preservative film from one end to uniformly cover the surface of the film, and standing for 5min at room temperature;
(15) removing the excess liquid with a glass rod, sucking the substrate outside the membrane with filter paper, and incubating at 37 deg.C for 10 min;
(16) image analysis was performed using an AI600(GE, USA) automated chemiluminescence imaging analysis system, and the results are shown in FIG. 3.
Performing analysis according to the Southern blot result of the transgenic maize event GG 2: in the T-DNA sequence inserted into the maize genome, there are 1 HindIII site and 1 Kpn I site, which do not affect the identification of the copy number of the GAT and GR79 genes. The schematic diagram of the restriction enzyme sites of the insert and the expected size of the southern blot hybridization band are shown in FIG. 4. The GAT gene fragment and the GR79 gene fragment are used as probes, southern blot is used for detecting the copy number of the GG2 transgenic corn insertion sequence, the result shows that enzyme digestion products of HindIII and Kpn I are hybridized to form 1 band (figure 3), and the GAT and GR79 genes are proved to be inserted into the genome of the corn in 1 copy. The analytical results are shown in Table 3.
TABLE 3 summary of southern hybridization test results of target genes in GG2 transgenic maize
Example 4 obtaining left and right Border flanking sequences of transgenic event GG2 by chromosome walking
As shown in FIG. 4, the exogenous fragment inserted into the maize genome is derived from a maize ubiquitin gene, and the maize genome contains the ubiquitin promoter, so that the right border flanking sequence is not easily obtained by the chromosome walking method, and therefore, primers are designed from the GAT gene, and the left border flanking sequence is amplified from the 5 'end to the 3' end of the GAT gene.
1. Left flanking sequence of transgenic event GG2 obtained by chromosome walking
Specific primers for amplification of left border flanking sequences were designed as follows:
GAT-SP 1: 5'-GATGACGCACAATCCCAC-3' (on CaMV 35S promoter)
GAT-SP 2: 5'-CTACGCTGGAGGGCTACA-3' (located on the GAT gene)
GAT-SP 4: 5'-GAGCAGGGCGAGGTGTTC-3' (located on the GAT gene)
A chromosome Walking Kit (Genome Walking Kit, Code No.6108) is purchased from TaKaRa company, 4 degenerate primers are arranged in the Kit, GAT-SP1 and GAT-SP2 are respectively used for carrying out 2 rounds of amplification with 4 degenerate primers (AP1, AP2, AP3 and AP4), finally, an AP4 primer is selected according to the amplification effect, a third round of amplification is carried out with an AP4 primer and GAT-SP4, and the obtained PCR product is sent for sequencing.
(1) 1 st round PCR reaction
Reaction system:
reaction conditions are as follows:
(2) 2 nd round PCR reaction
The PCR reaction product of round 1 was subjected to 5. mu.L electrophoresis (FIG. 5), and the dilution was selected according to the brightness of the band in the first round. Taking 1 μ L of the diluted product of the 1 st round PCR reaction as a template to perform 2 nd round PCR reaction, taking GAT-SP2 as an upstream primer and 4 degenerate primers as downstream primers respectively, taking AP4 as an example, to perform 2 nd round PCR reaction.
Reaction system:
reaction conditions are as follows:
(3) 3 rd round PCR reaction
The 2 nd round PCR reaction product was subjected to 5. mu.L electrophoresis (FIG. 5), and the dilution was selected according to the brightness of the band in the 2 nd round electrophoresis. 1 μ L of the diluted product of the 2 nd round PCR reaction was used as a template for the 3 rd round PCR reaction, and the 3 rd round PCR reaction was performed using GAT-SP4 as the upstream primer and AP4 as the downstream primer.
Reaction system:
reaction conditions are as follows:
(4) 5 mu.L of the 3 rd round PCR reaction product is subjected to electrophoresis by 1% agarose gel, the electrophoresis picture is shown in figure 5, clear electrophoresis bands are recovered by cutting gel, and DNA sequencing is carried out on the 3 rd round PCR product by using GAT-SP4 as a primer.
The sequencing result is shown in SEQ ID NO. 1. The 1-268 site of SEQ ID NO.1 is GG 2T-DNA sequence through sequence alignment, and the 3' end of the left border of the vector sequence is deleted by 22 bp. Position 269-462 of SEQ ID NO.1 is the maize genome chromosome 1 chr1:269325682-269325493(Zea mays (B73-RefGen-v 4)) sequence.
3' -end flanking sequence chromosome walking sequencing result of transgenic glyphosate-resistant corn GG2 exogenous insert
The maize genomic sequence was underlined.
2. Acquisition of the right border flanking sequence of the insert
And searching a known maize genome sequence according to the obtained left boundary flanking sequence, and designing a specific primer on the ubiquitin promoter sequence of the vector and the presumed right boundary flanking sequence for PCR amplification.
Specific primers:
GG 2-Ubi-F1: 5'-ATGATTCTCTAAAACACTG-3' (in the Ubiquitin promoter sequence)
GG 2-Right-R1: 5'-GCGAACATAGCGTCTTAC-3' (on the maize genome)
Size of PCR product: 1773 bp.
Reaction system:
reaction conditions are as follows:
the 2. mu.L PCR product was electrophoretically detected, and the results are shown in FIG. 6. The remaining PCR products were subjected to DNA sequencing.
The sequencing result is shown in SEQ ID NO. 2. Through sequence alignment, 1-435 bit of SEQ ID NO.2 is a T-DNA sequence in the right border of the vector, and 46bp of 5' end in the right border (including the right border) of the vector sequence is deleted. 436-455 are recombination sequences. The 456-1773 locus is the maize genome chromosome 1 chr1:269325753-269326914(Zea mays (B73-RefGen-v 4)) sequence. 5' end flanking sequence specificity PCR sequencing result of transgenic glyphosate-resistant corn GG2 exogenous insert
The maize genomic sequence was underlined.
3. Obtaining the left border flanking sequence of the insert
Since the left border flanking sequence obtained by chromosome walking is too short (less than 300bp), to obtain a longer left border flanking sequence, known maize genomic sequences are retrieved and specific primers are designed for PCR amplification in the GAT gene sequence of the vector and the left border maize genomic reference sequence.
Specific primers:
GG 2-Left-F3: 5'-GGAGCAAGGAAGCGGACTAC-3' (on the maize genome)
GG 2-Left-R1: 5'-CCCCACATCCTGATGTACAAG-3' (in the gat Gene sequence)
Size of PCR product: 734 bp.
Reaction system:
reaction conditions are as follows:
the 2. mu.L PCR product was electrophoretically detected, and the results are shown in FIG. 6. The remaining PCR products were subjected to DNA sequencing.
The position of the insert in maize is shown in FIG. 7, in combination with the sequencing of the sequences flanking the left and right borders. Integration of the GG2 transgenic maize T-DNA sequence into the maize genome at chromosome 1 at the 269325682-269325753 site (Zea mays (B73-RefGen-v 4)) resulted in a 70bp sequence deletion in the maize genome at the insertion site, corresponding to chr1:269325683-269325752(Zea mays (B73-RefGen-v 4)). The exogenous insertion sequence of the transgenic maize GG2 is shown as 441-6535 of SEQ ID NO.3, the left border flanking sequence is shown as 1-440 of SEQ ID NO.3, and the right border flanking sequence is shown as 6556-7873 of SEQ ID NO. 3.
The transgenic corn GG2 is a transgenic corn strain with high glyphosate herbicide tolerance and has important production and application values. The invention discloses flanking sequences at two ends of an exogenous insertion fragment of transgenic glyphosate-tolerant corn GG2 and a specific primer, which can be used as a molecular marker for simply, quickly and accurately detecting a transgenic glyphosate-tolerant corn strain GG2 and derivative materials thereof.
Sequence listing
<110> institute of biotechnology of Chinese academy of agricultural sciences
<120> flanking sequence of glyphosate-tolerant corn GG2 exogenous insert and application thereof
<141> 2021-06-07
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 462
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
cgcgtgactc gagtttctcc ataataatgt gtgagtagtt cccagataag ggaattaggg 60
ttcctatagg gtttcgctca tgtgttgagc atataagaaa cccttagtat gtatttgtat 120
ttgtaaaata cttctatcaa taaaatttct aattcctaaa accaaaatcc agtactaaaa 180
tccagatccc ccgaattaat tcggcgttaa ttcagtacat taaaaacgtc cgcaatgtgt 240
tattaagttg tctaagcgtc aatttgttat tcttatcatc atgtaaaata cgtacaacac 300
attgcatgac tcgtcatgca cgcacttggg ctgctgctgc tttacatgca cgcgcgcacc 360
gacggccggc cggtggtgct gatcagaaat gtacacgcct gtgaggcagg caggcagaga 420
gagagagaga gagatacata ttcacacacg cacgcacgca cc 462
<210> 2
<211> 1773
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
atgattctct aaaacactga tattattgta gtactataga ttatattatt cgtagagtaa 60
agtttaaata tatgtataaa gatagataaa ctgcacttca aacaagtgtg acaaaaaaaa 120
tatgtggtaa ttttttataa cttagacatg caatgctcat tatctctaga gaggggcacg 180
accgggtcac gctgcactgc aggcatgcaa gcttggcact ggccgtcgtt ttacaacgtc 240
gtgactggga aaaccctggc gttacccaac ttaatcgcct tgcagcacat ccccctttcg 300
ccagctggcg taatagcgaa gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc 360
tgaatggcga atgctagagc agcttgagct tggatcagat tgtcgtttcc cgccttcagt 420
ttaaactatc agtgtgaccg tggaacacga atcgccgaat cggtgcgtgc gtgagtgcgt 480
gcttcatcga ttcagactat cgggcgtgtt cggctggttg caagccgaca ctgttgcagc 540
tgtttggact gctgcagctg caatccatag agagaaaaat actgtagaag ccgcagccgc 600
agccggattg cagccgcagc aagccgcagc gaacaagctg atcgtctacg ctgggtacgt 660
gctggcgact caaatcatcg attggacggt ggcgtcgcgc ggcggggagg cgttgccgtt 720
gggcgatgac gcggacggat gcagatggaa tgacgtcgcg tcgttgcgtg cgttctactt 780
ctataaacta cgccatcgat ctcctgcctg gctggcatcg gtcgccacgc acgcatgttt 840
gtctattctc cgtccgtcac gtcaccctcg cgctgtcgtc gctatgcaca cggtcggcct 900
caccctcgcg gatcacgtcg cagtcgccgt cgccgtcgcc gtcgccacca agtcatcgtc 960
gcgtcggcgc gacagcgagg gcgcacgcgc gccacgctaa gctcagacga gggacacgac 1020
gacctggcct tatcggctct gatagcgtgc ctagccggat cggagagggg cgcagtggcc 1080
agtgtggggg gtgtcactgt cagtcacggt tcttgtccgc ccgatcgcat cccgatagcc 1140
ttctgctcgg agcctctgtc cgcctgtctg tcgtgtgcct gtaaaatcag tgtgggttgg 1200
agtgcgcgcg cgcgtttctg atggctgatg ctgccatcgt taaaatcggt gtgggagatg 1260
ttgagcttga gcgtgcacat gcatgtccag gtccaccatg tatttatttg ccatgctgcg 1320
atgatggccc tttaggcaaa caggttctag agcagttcga ttctgtgtat aaccgagccg 1380
cttaaatttt ttttaaaaaa tatttctcgt agctgctgca tctggcagct tccaaaaata 1440
aggatattct ttatagatgt tgcatctatt ctaggggctc cctacaatcg acagagccta 1500
atgacacgct accggctact ccctcgtccc attcagagct tgttcggtta tttacaatcc 1560
atatggattg gaggggattg atacggattg gagagaattt tgacttacta gggattgaaa 1620
ccccctcaat ccatatggat tgaggtagaa ccgaacaagc cctcaggaca tgttcggtta 1680
caccaatcca gaaggggatt acaatccaga agggtcaatc cccttctgga ttggtgtaac 1740
cgaacaagcc ctcacgtaag acgctatgtt cgc 1773
<210> 3
<211> 7873
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
ggagcaagga agcggactac gcggtggaga gcatcggcgc gctccggcgg gccatcccgg 60
agatctgggg cgaggccgct gaacgctcct ccgaccgcag cagcagcatc gacaagctgc 120
cggtgccggc cgtgagcgtg gtgaggtcga cgacgtccga cctggactcc atcatcatcc 180
agcccacgtc catccacgcc taattaataa cctcatccct agctcccacc ccaatcagaa 240
tcagcgggtg cgtgcgtgcg tgtgtgaata tgtatctctc tctctctctc tctgcctgcc 300
tgcctcacag gcgtgtacat ttctgatcag caccaccggc cggccgtcgg tgcgcgcgtg 360
catgtaaagc agcagcagcc caagtgcgtg catgacgagt catgcaatgt gttgtacgta 420
ttttacatga tgataagaat aacaaattga cgcttagaca acttaataac acattgcgga 480
cgtttttaat gtactgaatt aacgccgaat taattcgggg gatctggatt ttagtactgg 540
attttggttt taggaattag aaattttatt gatagaagta ttttacaaat acaaatacat 600
actaagggtt tcttatatgc tcaacacatg agcgaaaccc tataggaacc ctaattccct 660
tatctgggaa ctactcacac attattatgg agaaactcga gtcacgcgat cctcttgtac 720
atcaggatgt gggggccgac tggtggcgtg tcgaacacct cgccctgctc ggagaagccg 780
agcttcttgt agtagccgct agcggaggtg cgagcgttgc accacaggag gtcagcgccc 840
ctcttgcgca ggatctcctc agcgtgcttg atgaggctgg agccggcctt ctgctccctg 900
tagccctcca gcgtagccat gcccctgagc tggtactgct tctggccctg gagctcgctg 960
tgctcagcct ggtggaagct ggcgatggag atcagcttgc cgccgtagta gccgccgagg 1020
tggaaagcgc ccctcaggag gtcggactcg aacatgcagg cctcgattgg ctggtttggc 1080
ctgaggatgc ggtgcctgag ctcgtaggtg tcctcagcgt tgatcgggtt cacgtcgatc 1140
atggttattg taaatagtaa ttgtaatgtt gtttgttgtt tgttgttgtt ggtaattgtt 1200
gtaaaaatac ccggctcgag agagatagat ttgtagagag agactggtga tttcagcgtg 1260
tcctctccaa atgaaatgaa cttccttata tagaggaagg tcttgcgaag gatagtggga 1320
ttgtgcgtca tcccttacgt cagtggagat atcacatcaa tccacttgct ttgaagacgt 1380
ggttggaacg tcttcttttt ccacgatgct cctcgtgggt gggggtccat ctttgggacc 1440
actgtcggca gaggcatctt gaacgatagc ctttccttta tcgcaatgat ggcatttgta 1500
ggtgccacct tccttttcta ctgtcctttt gatgaagtga cagatagctg ggcaatggaa 1560
tccgaggagg tttcccgata ttaccctttg ttgaaaagtc tcaatagccc tttggtcttc 1620
tgagactgta tctttgatat tcttggagta gacgagagtg tcgtgctcca ccatgttatc 1680
acatcaatcc acttgctttg aagacgtggt tggaacgtct tctttttcca cgatgctcct 1740
cgtgggtggg ggtccatctt tgggaccact gtcggcagag gcatcttgaa cgatagcctt 1800
tcctttatcg caatgatggc atttgtaggt gccaccttcc ttttctactg tccttttgat 1860
gaagtgacag atagctgggc aatggaatcc gaggaggttt cccgatatta ccctttgttg 1920
aaaagtctca atagcccttt ggtcttctga gactgtatct ttgatattct tggagtagac 1980
gagagtgtcg tgctccacca tgttggcaag ctgctctagc caatacgcaa accgcctctc 2040
cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca ggtttcccga ctggaaagcg 2100
ggcagtgagc gcaacgcaat taatgtgagt tagctcactc attaggcacc ccaggcttta 2160
cactttatgc ttccggctcg tatgttgtgt ggaattgtga gcggataaca atttcacaca 2220
ggaaacagct atgaccatga ttacgaattc ccgatctagt aacatagatg acaccgcgcg 2280
cgataattta tcctagtttg cgcgctatat tttgttttct atcgcgtcgc gtattaaatg 2340
tataattgcg ggactctaat cataaaaacc catctcataa ataacgtcat gcattacatg 2400
ttaattatta catgcttaac gtaattcaac agaaattata tgataatcat cgcaagaccg 2460
gcaacaggat tcaatcttaa gaaactttat tgccaaatgt ttgaacgatc ggggaaattc 2520
gagctcttta aatttttttt tttttttttt tttttttgtt aaattttttt tttttttttt 2580
tttttttgtt aaattttttt tttttttttt ttttttttgt taacttgatg tccgaaaaca 2640
aaactgaaag aacacagtaa attacaagca gaacaatggc ttttccaatg ccataatact 2700
caaagttaac cttactcagc ctcgaggggg ggcccggtac ctcagttgta ctccacgtgg 2760
atgccgaact tctggagctc ctcgaagtac gctgggcacg tcttggagac gcagccgggg 2820
tccacgatcc tgatgtgcgg gaccttcacg cccaggaggc cgaagaccat ggcgttgcgg 2880
tggtcgtcgt gcgggttcag cgtggtgccc actggctggc ctgggtagat ggtgaagccg 2940
tcctccctct cctcgacctg cacgcccatc tgctggaggc tggagcagat gacggcgatc 3000
ctgtcgctct cgtgagccct gatgtgagcg acgttcgtca ccctgattgg agcgtcagcg 3060
aatggagcca gagcgccgat ggtgagggcc tggtcggaca tgggcttcat gtcgacctcg 3120
aagccgccct tcagcctcgt tgggccggtc acctccagga aggactcgtt cttgatgacc 3180
tcgcagccca tctgctccag cacgtcgatg aagcgagcgt ctggctggta gctgtggtag 3240
ccgacgttct tcacctggat cgtgccgccg gtgagagcag ccagggagag gaagtagcaa 3300
gccgtgctag cgtcagcctc caggatcgtg tccctgccct ggtagccggt ggggtagacc 3360
ttgaagaggg agtagtcctc gttgtgctcc accttggcgc cgaactcgcg catcagctgg 3420
atggtgatcg cgatgtagct cggctgcacg aggccgttga tgacctcgat ggacacggcc 3480
tcgctagcgt atggggaagc gatcaggagg ccggacagga actggctgga gacgttgccc 3540
gggaccctca cgtgctggcc gctcaggccg gcgcccttca ccctcagtgg gaggcctggg 3600
tgctccgtga ggtactcgat cctgccgccc agctgggtga gagcgtccac cagtggcttg 3660
agcggcctct ccctcagctg ggggacgccg tccacgatcc actcgccctg ctgagcgaca 3720
gccagagcgc ctgggaggaa gcgggcgatg gtgccagcag cgccgatgaa gagctcagcg 3780
gactgcactg gccacttgcc gccgcagccg tggatcgtga cggtctcctc ggccacctcg 3840
atcttgatgc ccagcctcct gagagcgtcg atgcaccagt agctgtcgtc ggacttcagg 3900
atgcccttga gcgtgctggt gccctcagcc agagcagcga tgatgagagc cctgttggtg 3960
tagctcttgg agccggggac gaagatctcg ccgttgatct tgttggacgt cggggtcacg 4020
agggcctcgt ggtactccgt agccttgctc catgggctgc gggaggtgct gtgggacatg 4080
cacctgatcc ttccgccgtt gctgacgttg ccgaggcttc tggaggagcg gcgggcgacg 4140
gggaggctgg cggtggactt gagcccctgg aacggagcga cggcggtggc cgacgaggcc 4200
atcatcacgg tgggcgccat ggttattgta aatagtaatt gtaatgttgt ttgttgtttg 4260
ttgttgttgg taattgttgt aaaaataccc ggggatcctc tagagtcgac ctgcagaagt 4320
aacaccaaac aacagggtga gcatcgacaa aagaaacagt accaagcaaa taaatagcgt 4380
atgaaggcag ggctaaaaaa atccacatat agctgctgca tatgccatca tccaagtata 4440
tcaagatcaa aataattata aaacatactt gtttattata atagataggt actcaaggtt 4500
agagcatatg aatagatgct gcatatgcca tcatgtatat gcatcagtaa aacccacatc 4560
aacatgtata cctatcctag atcgatattt ccatccatct taaactcgta actatgaaga 4620
tgtatgacac acacatacag ttccaaaatt aataaataca ccaggtagtt tgaaacagta 4680
ttctactccg atctagaacg aatgaacgac cgcccaacca caccacatca tcacaaccaa 4740
gcgaacaaaa agcatctctg tatatgcatc agtaaaaccc gcatcaacat gtatacctat 4800
cctagatcga tatttccatc catcatcttc aattcgtaac tatgaatatg tatggcacac 4860
acatacagat ccaaaattaa taaatccacc aggtagtttg aaacagaatt ctactccgat 4920
ctagaacgac cgcccaacca gaccacatca tcacaaccaa gacaaaaaaa agcatgaaaa 4980
gatgacccga caaacaagtg cacggcatat attgaaataa aggaaaaggg caaaccaaac 5040
cctatgcaac gaaacaaaaa aaatcatgaa atcgatcccg tctgcggaac ggctagagcc 5100
atcccaggat tccccaaaga gaaacactgg caagttagca atcagaacgt gtctgacgta 5160
caggtcgcat ccgtgtacga acgctagcag cacggatcta acacaaacac ggatctaaca 5220
caaacatgaa cagaagtaga actaccgggc cctaaccatg gaccggaacg ccgatctaga 5280
gaaggtagag aggggggggg ggggaggacg agcggcgtac cttgaagcgg aggtgccgac 5340
gggtggattt gggggagatc tggttgtgtg tgtgtgcgct ccgaacaaca cgaggttggg 5400
gaaagagggt gtggaggggg tgtctattta ttacggcggg cgaggaaggg aaagcgaagg 5460
agcggtggga aaggaatccc ccgtagctgc cgtgccgtga gaggaggagg aggccgcctg 5520
ccgtgccggc tcacgtctgc cgctccgcca cgcaatttct ggatgccgac agcggagcaa 5580
gtccaacggt ggagcggaac tctcgagagg ggtccagagg cagcgacaga gatgccgtgc 5640
cgtctgcttc gcttggcccg acgcgacgct gctggttcgc tggttggtgt ccgttagact 5700
cgtcgacggc gtttaacagg ctggcattat ctactcgaaa caagaaaaat gtttccttag 5760
tttttttaat ttcttaaagg gtatttgttt aatttttagt cactttattt tattctattt 5820
tatatctaaa ttattaaata aaaaaactaa aatagagttt tagttttctt aatttagagg 5880
ctaaaataga ataaaataga tgtactaaaa aaattagtct ataaaaacca ttaaccctaa 5940
accctaaatg gatgtactaa taaaatggat gaagtattat ataggtgaag ctatttgcaa 6000
aaaaaaagga gaacacatgc acactaaaaa gataaaactg tagagtcctg ttgtcaaaat 6060
actcaattgt cctttagacc atgtctaact gttcatttat atgattctct aaaacactga 6120
tattattgta gtactataga ttatattatt cgtagagtaa agtttaaata tatgtataaa 6180
gatagataaa ctgcacttca aacaagtgtg acaaaaaaaa tatgtggtaa ttttttataa 6240
cttagacatg caatgctcat tatctctaga gaggggcacg accgggtcac gctgcactgc 6300
aggcatgcaa gcttggcact ggccgtcgtt ttacaacgtc gtgactggga aaaccctggc 6360
gttacccaac ttaatcgcct tgcagcacat ccccctttcg ccagctggcg taatagcgaa 6420
gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc tgaatggcga atgctagagc 6480
agcttgagct tggatcagat tgtcgtttcc cgccttcagt ttaaactatc agtgtgaccg 6540
tggaacacga atcgccgaat cggtgcgtgc gtgagtgcgt gcttcatcga ttcagactat 6600
cgggcgtgtt cggctggttg caagccgaca ctgttgcagc tgtttggact gctgcagctg 6660
caatccatag agagaaaaat actgtagaag ccgcagccgc agccggattg cagccgcagc 6720
aagccgcagc gaacaagctg atcgtctacg ctgggtacgt gctggcgact caaatcatcg 6780
attggacggt ggcgtcgcgc ggcggggagg cgttgccgtt gggcgatgac gcggacggat 6840
gcagatggaa tgacgtcgcg tcgttgcgtg cgttctactt ctataaacta cgccatcgat 6900
ctcctgcctg gctggcatcg gtcgccacgc acgcatgttt gtctattctc cgtccgtcac 6960
gtcaccctcg cgctgtcgtc gctatgcaca cggtcggcct caccctcgcg gatcacgtcg 7020
cagtcgccgt cgccgtcgcc gtcgccacca agtcatcgtc gcgtcggcgc gacagcgagg 7080
gcgcacgcgc gccacgctaa gctcagacga gggacacgac gacctggcct tatcggctct 7140
gatagcgtgc ctagccggat cggagagggg cgcagtggcc agtgtggggg gtgtcactgt 7200
cagtcacggt tcttgtccgc ccgatcgcat cccgatagcc ttctgctcgg agcctctgtc 7260
cgcctgtctg tcgtgtgcct gtaaaatcag tgtgggttgg agtgcgcgcg cgcgtttctg 7320
atggctgatg ctgccatcgt taaaatcggt gtgggagatg ttgagcttga gcgtgcacat 7380
gcatgtccag gtccaccatg tatttatttg ccatgctgcg atgatggccc tttaggcaaa 7440
caggttctag agcagttcga ttctgtgtat aaccgagccg cttaaatttt ttttaaaaaa 7500
tatttctcgt agctgctgca tctggcagct tccaaaaata aggatattct ttatagatgt 7560
tgcatctatt ctaggggctc cctacaatcg acagagccta atgacacgct accggctact 7620
ccctcgtccc attcagagct tgttcggtta tttacaatcc atatggattg gaggggattg 7680
atacggattg gagagaattt tgacttacta gggattgaaa ccccctcaat ccatatggat 7740
tgaggtagaa ccgaacaagc cctcaggaca tgttcggtta caccaatcca gaaggggatt 7800
acaatccaga agggtcaatc cccttctgga ttggtgtaac cgaacaagcc ctcacgtaag 7860
acgctatgtt cgc 7873
Claims (10)
1. The left border flanking sequence of the exogenous insertion fragment of transgenic glyphosate-tolerant corn GG2 is shown in positions 1-440 of SEQ ID NO. 3.
2. The right border flanking sequence of the exogenous insertion fragment of the transgenic glyphosate-tolerant maize GG2 is shown in 6556-7873 of SEQ ID NO. 3.
3. The specific primer pair for PCR reaction detection designed according to the left border flanking sequence of claim 1.
4. The primer pair specific for the left border flanking sequence of claim 3 is:
GG2-Left-F3：5'-GGAGCAAGGAAGCGGACTAC-3'，
GG2-Left-R1：5'-CCCCACATCCTGATGTACAAG-3'。
5. the specific primer for PCR reaction detection designed according to the right border flanking sequence of claim 2.
6. The primers specific for the right border flanking sequence of claim 5 are:
GG2-Ubi-F1：5'-ATGATTCTCTAAAACACTG-3'，
GG2-Right-R1：5'-GCGAACATAGCGTCTTAC-3'。
7. the PCR reaction detection method of the transgenic glyphosate-tolerant corn GG2 is characterized in that: the primer pair in the PCR reaction is the specific primer pair of any one of claims 3-6.
8. The PCR reaction detecting method according to claim 7, wherein:
the specific primer pair is as follows:
GG2-Left-F3：5'-GGAGCAAGGAAGCGGACTAC-3'，
GG2-Left-R1：5'-CCCCACATCCTGATGTACAAG-3'。
the size of the fragment obtained by the PCR reaction is 734 bp; or
The specific primer pair is as follows:
GG2-Ubi-F1：5'-ATGATTCTCTAAAACACTG-3'，
GG2-Right-R1：5'-GCGAACATAGCGTCTTAC-3'。
the size of the fragment obtained by the PCR reaction is 1773 bp.
9. A kit for detecting glyphosate-tolerant maize comprising a primer pair specific for the left border flanking sequence of claim 3 or 4, or/and a primer pair specific for the right border flanking sequence of claim 5 or 6.
10. The use of the flanking sequence of claim 1 or 2, the primer pair specific for the flanking sequence of any one of claims 3 to 6, or the kit for detecting glyphosate-tolerant maize of claim 9 for detecting transgenic maize.
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| CN111057707A (en) * | 2019-12-09 | 2020-04-24 | 中国农业科学院生物技术研究所 | Flanking sequence of exogenous insert of transgenic insect-resistant corn 2HVB4 and its detection process |
| CN112852867A (en) * | 2021-04-08 | 2021-05-28 | 中国农业科学院生物技术研究所 | Expression vector of glyphosate resistance genes GR79 and GAT and application thereof |
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| CN112852867A (en) * | 2021-04-08 | 2021-05-28 | 中国农业科学院生物技术研究所 | Expression vector of glyphosate resistance genes GR79 and GAT and application thereof |
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