CN116219065A - Primer group, kit and method for identifying grass-resistant agent corn - Google Patents
Primer group, kit and method for identifying grass-resistant agent corn Download PDFInfo
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Abstract
The invention provides a primer group, a kit and a method for identifying grass-resistant corn. The primer set includes: at least one pair of a first primer pair and a second primer pair, wherein the first primer pair comprises a first primer and a second primer, and the sequence of the first primer is shown as SEQ ID NO in a sequence table: 1, wherein the sequence of the second primer is shown as SEQ ID NO:2, wherein the second primer pair comprises a third primer and a fourth primer, and the sequence of the third primer is shown as SEQ ID NO:3, the sequence of the fourth primer is shown as SEQ ID NO: 4. The embodiment of the invention provides a primer group, a kit and a method for identifying herbicide-resistant corn, wherein the primer group and the kit have higher sensitivity and specificity for detecting the herbicide-resistant corn, and the identification method can accurately identify the herbicide-resistant corn and is simple in detection process and high in efficiency.
Description
Technical Field
The invention relates to the field of biotechnology, in particular to a primer group, a kit and a method for identifying grass-resistant corn.
Background
Corn is one of the most widely planted crops in the world. Corn is also the crop with the widest planting area in China. With the development of plant genetic engineering, genetic engineering by introducing exogenous genes has become one of the main means of genetic breeding. The herbicide-resistant corn can greatly reduce the agricultural labor force required by weed control, reduce the investment of the labor force and reduce the influence of the weed on the corn yield. As people pay more attention to health, people pay more attention to whether crops contain transgenic components. The am79epsps gene can confer tolerance to the herbicide glyphosate to plants. The herbicide-resistant corn WYN17132 inserted with the am79epsps gene is prepared by optimizing the am79epsps gene according to the codon preference characteristics of corn, and the obtained herbicide-resistant corn has excellent herbicide tolerance performance.
The design of transformant specific assays based on flanking sequences of the transgene insert is currently the most effective method of transgene detection. Common methods for detecting sequences flanking exogenous insertion vectors in transgenic plants include exogenous adaptor-mediated PCR. However, this method is complicated in a cycle process, thereby reducing detection efficiency and being disadvantageous for detection.
Disclosure of Invention
In order to solve the technical problems, the invention provides a primer group, a kit and a method for identifying grass-resistant corn, and the method can accurately identify grass-resistant corn, and has the advantages of simple detection process and high efficiency.
The embodiment of the invention provides a primer group for identifying grass-resistant corn, which comprises the following components: at least one pair of a first primer pair and a second primer pair, wherein the first primer pair comprises a first primer and a second primer, and the sequence of the first primer is shown as SEQ ID NO in a sequence table: 1, wherein the sequence of the second primer is shown as SEQ ID NO:2, wherein the second primer pair comprises a third primer and a fourth primer, and the sequence of the third primer is shown as SEQ ID NO:3, the sequence of the fourth primer is shown as SEQ ID NO: 4.
In another aspect, embodiments of the present invention provide a kit for identifying herbicide tolerant corn, the kit comprising: the primer set of claim 1, the kit further comprising: DNA polymerase and dntps.
Specifically, the final concentrations of the first primer, the second primer, the third primer, and the fourth primer are all 0.25. Mu.M.
In yet another aspect, embodiments of the present invention provide a method of identifying grass-resistant corn, the method comprising:
obtaining genome DNA of corn to be tested;
performing enzyme digestion on the genome DNA to obtain enzyme-digested fragments;
cyclizing the enzyme section to obtain cyclized genome DNA;
taking the circularized genome DNA as a template, and adopting an inverse PCR primer group to carry out 2 rounds of PCR amplification to obtain an inverse PCR amplification product;
sequencing the inverse PCR product, comparing the inverse PCR product with a reference genome, determining the insertion site of the exogenous insertion sequence, and determining the flanking sequences on both sides of the exogenous insertion sequence;
amplifying by using the genome DNA as a template and adopting a sequencing primer to obtain a PCR amplification product, sequencing the product, and verifying whether the product sequence is consistent with the flanking sequence and the exogenous insertion sequence;
if the product sequence is consistent with the flanking sequence and the exogenous insertion sequence, amplifying the genomic DNA as a template according to the flanking sequence by using the primer set according to claim 1 to obtain a PCR product, comparing the PCR product with the expected DNA fragment, and if the PCR product is consistent with the expected DNA fragment, identifying as grass-resistant corn WYN17132.
Specifically, the method of determining the expected DNA fragment comprises: if the primer set is a first primer and the second primer, the expected DNA fragment is a 275bp DNA fragment.
Specifically, if the primer set is the third primer and the fourth primer, the intended DNA fragment is a 467bp DNA fragment.
Specifically, the corn to be tested is herbicide-resistant corn WYN17132.
Specifically, the PCR amplification procedure included 35 cycles, each cycle including: 95 ℃ for 5min,95 ℃ for 30s,58 ℃ for 30s,72 ℃ for 45s, and the cycle is ended; then, the temperature is 72 ℃ for 5min; finally, the temperature was kept at 4 ℃.
Specifically, the final concentrations of the first primer, the second primer, the third primer, and the fourth primer were all 0.25 μm.
The embodiment of the invention provides a primer group, a kit and a method for identifying grass-resistant corn, which can adopt inverse PCR to separate the flanking sequence of an exogenous insert fragment of am79epsps gene grass-resistant corn WYN17132, and the primer group and the kit have higher sensitivity and specificity for detecting grass-resistant corn.
Additional features and advantages of the invention will be set forth in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. The objectives and other advantages of the invention will be realized and attained by the structure particularly pointed out in the written description and claims hereof as well as the appended drawings.
Drawings
The accompanying drawings are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate and do not limit the invention.
FIG. 1 is an integration map of exogenous flanking sequence databases according to a fifth embodiment of the present invention.
FIG. 2 is a schematic diagram of PCR amplification according to the fifth embodiment of the present invention.
FIG. 3 is a diagram showing the amplification of a target band using a first primer pair according to the fifth embodiment of the present invention, wherein lane M is the standard molecular weight; lane 1 is a blank control; lane 2 is non-transgenic corn 4125-2; lane 3 is T4 generation of am79epsps gene transgenic herbicide resistant maize WYN 17132; lane 4 is the result of am79epsps gene transferred herbicide resistant corn non WYN17132 corn DNA amplification; lanes 5-9 are, in order, transgenic maize Bt11, transgenic maize MON863, transgenic maize NK603, transgenic maize TC1507, and transgenic maize GA21.
FIG. 4 is a graph showing the results of primer sensitivity detection using the first primer set according to the fifth embodiment of the present invention, wherein lane M is the standard molecular weight; lanes 1-24 are in turn genomic DNA containing concentrations of 100%,10%,1%,0.5%,0.1%,0.05%,0.01% and 0% WYN17132, each concentration being set to 3 replicates.
Fig. 5 is a diagram of a plant WYN17132 provided in example seven of the present invention without glyphosate sprayed.
Fig. 6 is a diagram of a plant WYN17132 provided in example seven of the present invention sprayed with glyphosate at a concentration of 800 mL/mu.
FIG. 7 is a diagram showing the amplification of a target band using a second primer set according to the sixth embodiment of the present invention, wherein lane M is the standard molecular weight; lane 1 is a blank control; lane 2 is non-transgenic corn 4125-2; lane 3 is T4 generation of am79epsps gene transgenic herbicide resistant maize WYN 17132; lane 4 is the result of am79epsps gene transferred herbicide resistant corn non WYN17132 corn DNA amplification; lanes 5-9 are, in order, transgenic maize Bt11, transgenic maize MON863, transgenic maize NK603, transgenic maize TC1507, and transgenic maize GA21.
FIG. 8 is a graph showing the result of sensitivity detection using the second primer set according to the sixth embodiment of the present invention, wherein lane M is the standard molecular weight; lanes 1-24 are in turn genomic DNA containing concentrations of 100%,10%,1%,0.5%,0.1%,0.05%,0.01% and 0% WYN17132, each concentration being set to 3 replicates.
FIG. 9 is a diagram showing the amplification of a target band using a first primer pair and a second primer pair according to the sixth embodiment of the present invention, wherein lane M is a standard molecular weight; lane 1 is a blank control; lane 2 is non-transgenic corn 4125-2; lane 3 is T4 generation of am79epsps gene transgenic herbicide resistant maize WYN 17132; lane 4 is the result of am79epsps gene transferred herbicide resistant corn non WYN17132 corn DNA amplification; lanes 5-9 are, in order, transgenic maize Bt11, transgenic maize MON863, transgenic maize NK603, transgenic maize TC1507, and transgenic maize GA21.
FIG. 10 is a graph showing the sensitivity test results of equimolar mixing of the first primer pair and the second primer pair according to the seventh embodiment of the present invention, wherein lane M is the standard molecular weight; lanes 1-24 are in turn genomic DNA containing concentrations of 100%,10%,1%,0.5%,0.1%,0.05%,0.01% and 0% WYN17132, each concentration being set to 3 replicates.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. It will be apparent that the described embodiments are some, but not all, embodiments of the invention. All other embodiments, which can be made by a person skilled in the art without creative efforts, based on the described embodiments of the present invention fall within the protection scope of the present invention.
Example 1
The embodiment of the invention provides a primer group for identifying grass-resistant corn, which comprises the following components: the first primer pair comprises a first primer and a second primer, and the sequence of the first primer is shown as SEQ ID NO in a sequence table: 1, the sequence of the second primer is shown as SEQ ID NO: 2. The final concentration of both the first primer and the second primer was 0.25. Mu.M.
Example two
The embodiment of the invention provides a primer group for identifying grass-resistant corn, which comprises the following components: the second primer pair comprises a third primer and a fourth primer, and the sequence of the third primer is shown as SEQ ID NO in a sequence table: 3, the sequence of the fourth primer is shown as SEQ ID NO: 4. The final concentration of both the third primer and the fourth primer was 0.25. Mu.M.
Example III
The embodiment of the invention provides a primer group for identifying grass-resistant corn, which comprises the following components: the sequence of the first primer pair is shown as SEQ ID NO in a sequence table: 1, the sequence of the second primer is shown as SEQ ID NO:2, wherein the second primer pair comprises a third primer and a fourth primer, and the sequence of the third primer is shown as SEQ ID NO:3, the sequence of the fourth primer is shown as SEQ ID NO: 4. The final concentrations of the first primer, the second primer, the third primer and the fourth primer were all 0.25. Mu.M.
Example IV
The embodiment of the invention provides a kit for identifying grass-resistant corn, which comprises the following components: the primer set provided in the third embodiment of the present invention, the kit further includes: DNA polymerase and dntps.
Specifically, the final concentrations of the first primer, the second primer, the third primer and the fourth primer were all 0.25. Mu.M.
Example five
The embodiment of the invention provides a method for identifying grass-resistant corn, which comprises the following steps:
obtaining genome DNA of corn to be tested;
enzyme cutting is carried out on the genome DNA to obtain enzyme cutting sections;
cyclizing the enzyme section to obtain cyclized genome DNA;
taking the circularized genome DNA as a template, and adopting an inverse PCR primer group to carry out 2 rounds of PCR amplification to obtain an inverse PCR amplification product;
sequencing the inverse PCR product, comparing the inverse PCR product with a reference genome, determining the insertion site of the exogenous insertion sequence, and determining the flanking sequences on both sides of the exogenous insertion sequence;
amplifying by using genome DNA as a template and adopting a sequencing primer (shown in table 5) to obtain a PCR amplified product, sequencing the product, and verifying whether the sequence of the product is consistent with a flanking sequence and an exogenous insertion sequence;
if the product sequence is consistent with the flanking sequence and the exogenous insertion sequence, the genomic DNA is taken as a template according to the flanking sequence, a primer group is adopted for amplification to obtain a PCR product, the PCR product is compared with an expected DNA fragment, and if the PCR product is consistent with the expected DNA fragment, the grass-resistant corn WYN17132 is identified.
Specifically, the corn to be tested is herbicide-resistant corn WYN17132.
Specifically, the procedure for PCR amplification included 35 cycles, each cycle including: 95 ℃ for 5min,95 ℃ for 30s,58 ℃ for 30s,72 ℃ for 45s, and the cycle is ended; then, the temperature is 72 ℃ for 5min; finally, the temperature was kept at 4 ℃.
Specifically, the method for extracting genomic DNA of corn (WYN 17132) to be tested comprises the following steps: leaves of transgenic herbicide-resistant corn WYN17132 planted in a greenhouse are extracted by adopting a high-efficiency plant genome DNA extraction kit (with the commodity number of DP 350) of the genome DNA of Tiangen biochemical technology (Beijing) limited company, and the specific method refers to the specification of the kit to obtain the genome DNA of the corn to be detected.
The obtained genomic DNA of corn to be tested was dissolved in 200/. Mu.L of TE buffer (prepared from Tris and EDTA) to obtain a DNA solution, and 5. Mu.L of the DNA solution was taken and subjected to electrophoresis using agarose gel having a concentration of 0.7%. The A260/A280 value and the DNA concentration of the DNA solution were measured by an ultraviolet spectrophotometer. In this example, the DNA solution A260/A280 had a value of about 1.8 at a concentration of 750 ng/. Mu.L, was of relatively high purity and met the requirements of the experiment.
Specifically, the method for the digestion of genomic DNA and the cyclization of the digested fragments comprises the following steps: taking 10 mug of genome DNA of corn to be detected as a template, carrying out enzyme digestion with 15 mug of SacI endonuclease in 50 mug of total reaction volume (10 XL buffer and ddH20 are complemented according to a SacI enzyme digestion kit) overnight, then placing the mixture at 65 ℃ for 15min to obtain a reaction solution, and taking 6 mug of the reaction solution for electrophoresis detection of enzyme digestion effect. After precipitating the remaining reaction solution with 2 times of the volume of ethanol, the precipitate was dissolved in 20. Mu.L of TE buffer to obtain a mixed solution. 5. Mu.L of the mixed solution was taken and ligated with 0.3. Mu. L T4 DNA ligase overnight at 4℃in a total volume of 20. Mu.L, respectively. 2. Mu.L of the reaction solution was taken the next day for inverse PCR amplification
Specifically, in this embodiment, two inverse PCR amplifications are adopted, and the amplification system of each inverse PCR amplification is the same, and only the templates are different, i.e., the template of the first inverse PCR amplification is the genomic DNA of the corn to be circularized, and the template of the second inverse PCR amplification is the amplification product of the first inverse PCR amplification.
2 pairs of reverse PCR primers were designed based on known exogenous T-DNA insert sequences, as shown in Table 1.
Table 1 sets of primers for 2 rounds of inverse PCR amplification
The inverse PCR amplification system is shown in Table 2.
Table 2 shows an inverse PCR amplification system
The inverse PCR amplification procedure comprises a round 1 inverse PCR amplification reaction and a round 2 inverse PCR amplification reaction, and is specifically as follows:
the reaction conditions for round 1 inverse PCR amplification are shown in Table 3.
Table 3 shows the reaction conditions for round 1 inverse PCR amplification
And (3) obtaining a round 1 inverse PCR amplification product after the round 1 inverse PCR amplification reaction is finished, taking 2 mu L of the round 1 inverse PCR amplification product, and carrying out a round 2 inverse PCR amplification reaction by using a round 2 inverse PCR amplification primer to obtain a round 2 inverse PCR amplification product. And taking 2-6 mu L of the 2 nd round of inverse PCR amplification products after the 2 nd round of inverse PCR amplification reaction is finished, and detecting the 2 nd round of inverse PCR amplification products by using agarose gel with the concentration of 2%. The PCR amplification product of round 2 was purified and recovered using a PCR product purification kit (the procedure was performed according to the kit instructions) and then sequenced.
The reaction conditions for round 2 inverse PCR amplification are shown in Table 4.
Table 4 shows the reaction conditions for round 2 inverse PCR amplification
Flanking sequence analysis
In the NCBI database, BLAST analysis was performed on the sequencing results. Based on BLAST analysis results, the exogenous fragment of the transgene integration site transformant WYN17132 on chromosome6 was initially determined to be inserted into chromosome6, and the integration site was set in Chr6:31096533-31096601 (Zea mays cultivar B73 chromoname 6, version B73 RefGen_v4). Exogenous flanking sequence database integration alignment is shown in figure 1.
Full-length cloning of herbicide-resistant maize WYN17132 insert by transferring am79epsps gene
The target site is subjected to a main GDB BLASTN to obtain a plant reference genome sequence, a sequencing primer is designed for a region of 640bp upstream and 402bp downstream of 31096534 bp of a chromosome6 (NC_ 024464.2) (Zea mays cultivar B73chromosome6, B73RefGen_v4 edition) 31096534 site sequence of herbicide-resistant corn WYN17132, and the whole T-DNA region is subjected to segmented cloning to obtain a cloned fragment, and the cloned fragment is sequenced. The schematic of PCR amplification is shown in FIG. 2, and the sequencing primers are shown in Table 5.
Table 5 shows sequencing primers
Full-length splice analysis of the inserted sequence of am79epsps gene herbicide resistant corn WYN17132 is specifically as follows:
splicing the obtained clone fragment sequencing results by utilizing snapGene software, wherein the sequence obtained by splicing is shown as a sequence table SEQ ID NO: shown at 5.
The results show that: the target gene was inserted at the position of maize chromosome6 (NC-024464.2:31096534-31096601). 1 am79epsps gene complete expression element is inserted into herbicide resistant corn WYN17132, and 68bp acceptor genome sequence is deleted after the whole expression element is inserted.
Comparing the actual T-DNA sequence obtained by PCR sequencing with the theoretical T-DNA sequence, deleting 9bp left border sequence from the left region of the actual T-DNA sequence, deleting 79bp sequence from the right region, wherein the 79bp sequence comprises: a 16bp nos terminator sequence, a 37bp spacer sequence and a26 bp right border sequence.
Sequence listing SEQ ID NO:5 comprises the total length 3911bp of the sequence integrated into the genome, 640bp of the flanking sequence at the 5 'end of the inserted sequence, 500bp of the flanking sequence at the 3' end of the inserted sequence, 5051bp in total, and specific information of the 5051bp sequence is described from 5 'to 3', and is shown in Table 6.
Table 6 introduction to insertion sequence information
Note that: genome reference version (Zea mays cultivar B73chromosome6, B73RefGen_v4 edition)
Determination of the flanking sequence of am79epsps Gene-transferred herbicide-resistant corn WYN17132
According to the splicing result of 5051bp sequence sequencing, determining that the 5' -end side sequence of the herbicide-resistant corn WYN17132 of the am79epsps gene is as shown in SEQ ID NO:6 (corresponding to positions 141-1140 of SEQ ID NO:5 of the sequence Listing). Wherein, SEQ ID NO:6, the 1 st to 500 th positions are corn genome sequences, and the 501 st to 1100 th positions are sequences of exogenous insertion am79epsps genes.
Specific detection of primer pairs
The first primer pair in the primer set is designed and screened according to the 5' -end flanking sequence (SEQ ID NO: 6) of herbicide-resistant corn WYN17132 of the trans-am 79epsps gene obtained in the first embodiment; the second primer set was designed and selected according to the 5' -terminal flanking sequence (SEQ ID NO: 6) of herbicide-resistant maize WYN17132 of the am79epsps gene obtained in example one.
First primer pair for 5' flanking sequence:
Zm132LB-F1：5'-GCTGGTTTGGGGCTGAATTG-3'；
Zm132LB-R1：5'-AAAAACGTCCGCAATGTGTT-3'。
size of target fragment: 275bp.
In theory, the genome DNA of the am79epsps gene herbicide-resistant corn WYN17132 is subjected to PCR amplification by adopting the specific primer pair (Zm 132LB-F1/Zm132 LB-R1), so that a target band with the size of 275bp is obtained, namely, a sequence table SEQ ID NO:6 at positions 286-560.
Sample supply: the T4 generation of transgenic am79epsps gene herbicide resistant corn WYN17132, transgenic am79epsps gene herbicide resistant corn non-WYN 17132 transgenic corn strain, transgenic corn Bt11, transgenic corn MON863, transgenic corn NK603, transgenic corn TC1507, transgenic corn GA21, non-transgenic corn 4125-2, and blank control group (using water as template amplification result) were set. The test samples are all from Zhejiang New safety chemical group Co., ltd.
Extraction of corn genomic DNA
Leaves of the test specimens planted in the greenhouse were taken and referred to a high-efficiency plant genomic DNA extraction kit (cat No. DP 350) from the company of the chemical technology of the root of heaven (beijing). The extracted corn genome DNA is dissolved in 200/mu LTE buffer solution to obtain a mixed solution, and 5 mu L of the mixed solution is taken and subjected to electrophoresis detection by using agarose gel with the concentration of 0.7 percent. The A260/A280 value and the DNA concentration of the DNA solution were measured by an ultraviolet spectrophotometer.
PCR detection
Each sample is tested with the first primer pair to verify the specificity of the first primer pair. The same detection method is adopted for each sample, and the method is as follows:
and taking the genomic DNA extracted from the sample to be tested as a template, and carrying out PCR amplification by using the primer to obtain an amplification product. The specific reaction system and the procedure are as follows:
table 7 shows a PCR amplification system
Table 8 shows the conditions of the amplification reaction
Agarose gel electrophoresis
The PCR amplified products were subjected to electrophoresis using agarose gel having a concentration of 1%, and after the PCR amplified products were recovered, they were subjected to sample transfer and sequencing, and the recovery process was performed by referring to the instructions of DNA GEL KIT gel recovery and PCR product recovery kit, manufactured by doctor company of day Lian Bao.
Results display
The first primer pair is adopted for PCR amplification, one 275bp product band exists in the genomic DNA sample of herbicide resistant corn WYN17132 transferred into am79epsps gene, and the target band is not amplified in other test samples and blank control groups, and the specific scheme is shown in figure 3. Sequencing results of PCR amplification products show that a target band of 275bp is exactly as shown in SEQ ID NO: bits 286-560 as shown in fig. 6.
Therefore, the first primer pair provided by the embodiment of the invention has higher specificity.
Primer pair sensitivity detection
The genome DNA of the herbicide-resistant corn WYN17132 of the am79epsps gene with mass fractions of 100%,10%,1%,0.5%,0.1%,0.05%,0.01% and 0% (w/w) is adopted, and the specific concentrations of the genome DNA corresponding to the percentages are as follows: 100 ng/. Mu.L, 10 ng/. Mu.L, 1 ng/. Mu.L, 0.5 ng/. Mu.L, 0.1 ng/. Mu.L, 0.05 ng/. Mu.L and 0 ng/. Mu.L. The first primer pair is used for PCR amplification by taking genome DNA as a template, and the amplification system and the amplification reaction conditions are referred to above.
The results showed that the use of the above primer pair for sensitivity detection, combined with that shown in FIG. 4, was able to detect as low as 0.05% (0.05 ng) of transgenic maize WYN17132 genomic DNA.
Therefore, the first primer pair provided by the embodiment of the invention has good sensitivity.
Identification of herbicide-resistant positive plants by primer group
By taking a plant without glyphosate spraying as a control, and adopting 800 mL/mu of glyphosate spraying to the corn WYN17132 plant to be tested provided by the embodiment, and comparing with fig. 5 and 6, the corn plant identified by the primer group provided by the embodiment of the invention has good glyphosate tolerance.
Example six
The difference between this embodiment and the fifth embodiment is that: when the genomic DNA is used as a template and the primer set is used for amplification according to the flanking sequence, the second primer pair (third primer and fourth primer) provided in the second example is used for amplification in place of the first primer pair. The specific amplification system is shown in Table 9.
Table 9 shows a PCR amplification system
For the third primer and the fourth primer:
Zm132LB-F2：5'-ATCGGGAATCGCTCACCAGA-3'；
Zm132LB-R2：5'-AGCGTCAATTTGTTTACACCACA-3'。
size of target fragment: 467bp.
The third primer is according to the sequence table SEQ ID NO:6, and the fourth primer is obtained according to the design of the 1 st to 500 th positions of the sequence table SEQ ID NO: the 501 th to 1000 th bit of 6 is obtained.
The genomic DNA of the am79epsps gene herbicide resistant corn WYN17132, amplified by the second primer pair, had a 467bp product band, but was not amplified to the target band in other test samples and blank control groups, as shown in FIG. 7. Sequencing results of the PCR products show that the target band of 467bp is exactly as shown in SEQ ID NO:6 at positions 61-527. Therefore, the second primer pair provided by the second embodiment of the invention has higher specificity.
Primer pair sensitivity detection
The genome DNA of the herbicide-resistant corn WYN17132 of the am79epsps gene with mass fractions of 100%,10%,1%,0.5%,0.1%,0.05%,0.01% and 0% (w/w) is adopted, and the specific concentrations of the genome DNA corresponding to the percentages are as follows: 100 ng/. Mu.L, 10 ng/. Mu.L, 1 ng/. Mu.L, 0.5 ng/. Mu.L, 0.1 ng/. Mu.L, 0.05 ng/. Mu.L and 0 ng/. Mu.L. PCR amplification was performed using the genomic DNA as template and the second primer, respectively, and the reaction was submitted and the procedure was as described in example five.
The results showed that the second primer pair provided in example two was used to detect sensitivity, and in combination with the second primer pair shown in fig. 8, transgenic maize WYN17132 genomic DNA as low as 0.05% (0.05 ng) could be detected. Therefore, the second primer pair provided by the second embodiment of the invention has good sensitivity.
Example seven
The difference between this embodiment and the fifth embodiment is that: when the genomic DNA is used as a template and the primer set is used for amplification according to the flanking sequences, the primer set (the first primer set and the second primer set) provided in the third embodiment is added for amplification, and amplification is performed respectively. Specific amplification systems are shown in Table 10.
Table 10 shows a PCR amplification system
PCR amplification was performed using the first primer pair and the second primer pair, and one 275bp product band was found in the genomic DNA sample of herbicide-resistant maize WYN17132, into which the am79epsps gene was transferred, whereas no target band was amplified in the other test samples and the blank. Sequencing results of PCR amplification products show that a target band of 275bp is exactly as shown in SEQ ID NO: bits 286-560 as shown in fig. 6. The genomic DNA of am79epsps gene-transgenic herbicide-resistant corn WYN17132 had a 467bp product band, but was not amplified to the target band in other test samples and blank groups, as shown in FIG. 9. Sequencing results of the PCR products show that the target band of 467bp is exactly as shown in SEQ ID NO:6 at positions 61-527. Therefore, the primer group provided by the third embodiment of the invention has higher specificity when in use.
Primer pair sensitivity detection
The genome DNA of the herbicide-resistant corn WYN17132 of the am79epsps gene with mass fractions of 100%,10%,1%,0.5%,0.1%,0.05%,0.01% and 0% (w/w) is adopted, and the specific concentrations of the genome DNA corresponding to the percentages are as follows: 100 ng/. Mu.L, 10 ng/. Mu.L, 1 ng/. Mu.L, 0.5 ng/. Mu.L, 0.1 ng/. Mu.L, 0.05 ng/. Mu.L and 0 ng/. Mu.L. PCR amplification was performed using the primer set provided in example III, respectively, using genomic DNA as a template, and the reaction was submitted and the procedure was as described in example five.
The results showed that the sensitivity test was performed using the primer set provided in example three, and as low as 0.05% (0.05 ng) of transgenic maize WYN17132 genomic DNA was tested using the primer set provided in example three of the present invention, as shown in fig. 10. Therefore, the primer group provided by the third embodiment of the invention has good sensitivity.
Although the embodiments of the present invention are described above, the embodiments are only used for facilitating understanding of the present invention, and are not intended to limit the present invention. Any person skilled in the art can make any modification and variation in form and detail without departing from the spirit and scope of the present disclosure, but the scope of the present disclosure is to be determined by the appended claims.
Claims (8)
1. A primer set for identifying grass-resistant corn, said primer set comprising: at least one pair of a first primer pair and a second primer pair, wherein the first primer pair comprises a first primer and a second primer, and the sequence of the first primer is shown as SEQ ID NO in a sequence table: 1, wherein the sequence of the second primer is shown as SEQ ID NO:2, wherein the second primer pair comprises a third primer and a fourth primer, and the sequence of the third primer is shown as SEQ ID NO:3, the sequence of the fourth primer is shown as SEQ ID NO: 4.
2. A kit for identifying grass-resistant corn, said kit comprising: the primer set of claim 1, the kit further comprising: DNA polymerase and dntps.
3. The kit of claim 2, wherein the final concentration of each of the first primer, the second primer, the third primer, and the fourth primer is 0.25 μm.
4. A method of identifying grass tolerance corn, comprising:
obtaining genome DNA of corn to be tested;
performing enzyme digestion on the genome DNA to obtain enzyme-digested fragments;
cyclizing the enzyme section to obtain cyclized genome DNA;
taking the circularized genome DNA as a template, and adopting an inverse PCR primer group to carry out 2 rounds of PCR amplification to obtain an inverse PCR amplification product;
sequencing the inverse PCR product, comparing the inverse PCR product with a reference genome, determining the insertion site of the exogenous insertion sequence, and determining the flanking sequences on both sides of the exogenous insertion sequence;
amplifying by using the genome DNA as a template and adopting a sequencing primer to obtain a PCR amplification product, sequencing the product, and verifying whether the product sequence is consistent with the flanking sequence and the exogenous insertion sequence;
if the product sequence is consistent with the flanking sequence and the exogenous insertion sequence, amplifying the genomic DNA as a template according to the flanking sequence by using the primer set according to claim 1 to obtain a PCR product, comparing the PCR product with the expected DNA fragment, and if the PCR product is consistent with the expected DNA fragment, identifying as grass-resistant corn WYN17132.
5. The method of claim 4, wherein the method of determining the expected DNA fragment comprises: if the primer set is a first primer and the second primer, the expected DNA fragment is a 275bp DNA fragment.
6. The method according to claim 4, wherein if the primer set is the third primer and the fourth primer, the intended DNA fragment is a bp DNA fragment.
7. The method of claim 4, wherein the PCR amplification procedure comprises 35 cycles, each cycle comprising: 95 ℃ for 5min,95 ℃ for 30s,58 ℃ for 30s,72 ℃ for 45s, and the cycle is ended; then, the temperature is 72 ℃ for 5min; finally, the temperature was kept at 4 ℃.
8. The method of claim 4, wherein the final concentration of each of the first primer, the second primer, the third primer, and the fourth primer is 0.25 μm.
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