A primer set, a kit and its application for detecting stress-resistant cotton ZA102-9 based on KASP technology

Abstract

The present invention discloses a primer set, a kit and a method for detecting gene-edited stress-resistant cotton ZA102-9. This high-throughput detection system based on KASP technology has good specificity, strong repeatability and high accuracy, and can quickly and accurately identify cotton samples ZA102-9 in biological materials and their processed products, providing methods and tools for monitoring and high-throughput detection technology of gene-edited products.

Description

Technical Field

[0001] The present invention belongs to the field of molecular biotechnology, and in particular relates to a primer set, a kit and a method for high-throughput detection of gene-edited stress-resistant cotton ZA102-9. Background Art

[0002] GDP-L-galactose phosphorylase (GGP) in plants can control the synthesis of vitamin C and regulate the stress resistance of plants. The cotton transformant ZA102-9 is a gene-edited material with enhanced stress resistance. Using the editing tool Cas12i, the researchers completed a single-base mutation in the pre-promoter region sequence of the GhVCT2 gene encoding GGP, and improved the drought resistance of cotton by regulating the synthesis of vitamin C.

[0003] The main detection methods for transformant specificity are conventional PCR and real-time fluorescence PCR. The former has low sensitivity, cumbersome operation, and cannot monitor the PCR process in real time, making it difficult to carry out high-throughput detection of a large number of samples. Although the latter has a higher detection sensitivity, the detection fee is expensive and the cost is high.

[0004] Competitive allele specific PCR (KASP) is a genotyping technology based on fluorescence detection. During the test, each reaction well uses dual-color fluorescence (such as FAM and VIC) to detect two possible genotypes at a certain site on the sample genome, and can perform high-precision detection of SNP sites in various genomic nucleic acid samples. After the PCR amplification reaction is completed, there is no need for electrophoresis, staining and band analysis, only the scanning program needs to be run with the help of a fluorescent quantitative PCR instrument. KASP technology is simple to operate, the analysis is stable and accurate, and the cost is low. At this stage, this technology is mainly used in SNP genotyping research, and it is the main means of molecular marker-assisted breeding, fine positioning of trait genes, and seed resource identification. However, there is no report on the use of KASP technology for transformant-specific detection of gene-edited crops.

[0005] The establishment of transformant-specific detection methods can provide an effective detection means for the identification and supervision of biological breeding materials, and provide technical support for the biosafety management of agricultural genetic modification and gene editing. Summary of the invention

[0006] To solve the above problems, the present invention provides a KASP primer set, a kit and a method for detecting nucleic acid samples. The above nucleic acid samples are derived from cotton transformant ZA102-9, or derivative strains of the above transformant, or mixed biological materials containing the above transformant, or various products obtained by processing the above samples as raw materials. Since the sequence shown in SEQ ID NO.3 is a specific sequence for confirming the identity information of cotton transformant ZA102-9, any sample containing the nucleic acid molecule of the sequence shown in SEQ ID NO.3 can be detected by the method provided by the present invention.

[0007] The present invention can accurately identify the cotton transformant ZA102-9 by designing a specific primer set, and can separate it from other cotton materials that do not contain ZA102-9. The detection method has the advantages of high specificity, sensitivity and convenient operation, and makes up for the shortcomings of the conventional PCR method, such as cumbersome process, low detection sensitivity and high cost of real-time fluorescence PCR.

[0008] The present invention provides a primer set for detecting stress-resistant cotton transformant ZA102-9, characterized in that the primer set comprises a first forward primer with a nucleotide sequence of 5'-gagaaactccgtgtatagccgtgaT-3', a second forward primer with a nucleotide sequence of 5'-gagaaactccgtgtatagccgtgaG-3', and a reverse primer with a nucleotide sequence of 5'-GCGTGGAGGTTGGGTATTTGGTTAAGGTA-3';

[0009] The first forward primer and the second forward primer are modified with different labels.

[0010] The above-mentioned marker includes a fluorescent group or a label sequence; the fluorescent group or the label sequence includes any one of FAM, TET, HEX, CY3, JOE, VIC, ROX, CY5, TAMRA or Texas.

[0011] In some embodiments, the first forward primer is modified with a FAM tag sequence, and the second forward primer is modified with a VIC tag sequence; the nucleotide sequence of the FAM tag sequence is as follows:

[0012] 5'-GAAGGTGACCAAGTTCATGCT-3'; the VIC tag sequence is shown in

[0013] 5′-GAAGGTCGGAGTCAACGGATT-3′.

[0014] The present invention also provides a detection kit, characterized in that it comprises the above-mentioned primer set.

[0015] In some embodiments, the above detection kit further comprises 2×KASP Master Mix.

[0016] The present invention also provides a detection method for detecting cotton transformant ZA102-9, characterized in that it comprises the following steps:

[0017] 1) Extracting genomic DNA from the sample to be tested;

[0018] 2) using the genomic DNA extracted in step 1) as a template and using the above primer set to perform PCR amplification to obtain a PCR amplification product;

[0019] 3) The PCR amplification product in step 2) is subjected to marker detection. If a VIC fluorescence signal is detected, the sample contains cotton ZA102-9.

[0020] In some embodiments, the reaction system for PCR amplification in the above detection method is 10 μL, wherein the template DNA is 50 ng, the volume of the first forward primer and the second forward primer is 0.1 μL, the volume of the reverse primer is 0.3 μL, 2×KASP MasterMix is ​​5 μL, the final concentration of the first forward primer or the second forward primer is 0.1 μM, and the final concentration of the reverse primer is 0.3 μM.

[0021] In some embodiments, the PCR amplification reaction program of the above detection method is: 95°C for 10 min; 95°C for 15 s, gradient annealing and extension at 59°C to 53°C for 60 s, 10 cycles, the annealing and extension temperature is reduced by 0.6°C in each cycle; 95°C for 15 s, 55°C for 60 s, 30 cycles.

[0022] The present invention also provides the use of the above primer set, kit and detection method in high-throughput detection of cotton transformant ZA102-9; wherein the cotton transformant ZA102-9 contains a nucleic acid molecule with a sequence shown in SEQ ID NO.3.

[0023] The invention has the beneficial effects of designing a KASP primer set at the SNP editing site of the target gene of the stress-resistant cotton transformant, and after testing and screening, a primer set obtained can effectively distinguish the variation site, thereby efficiently and accurately identifying the cotton transformant ZA102-9; the method of the invention adopts an end-point method for detecting the fluorescence of the product, so when the method of the invention is combined with a matching instrument for transformant-specific detection, the detection flux is 14 to 56 times that of fluorescent quantitative PCR; moreover, the method of the invention does not need to synthesize specific probes, which can greatly save the detection cost. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Other features, objects and advantages of the present application will become more apparent by reading the detailed description of non-limiting embodiments made with reference to the following drawings:

[0025] Figure 1 Physical map of C016-Cas12i editing vector

[0026] Figure 2 Results of drought resistance test on transformant ZA102-9 plants

[0027] Figure 3 Peak diagram of sequencing results of the edited body target site (the box marks the deletion of the edited body base A)

[0028] Figure 4 Results of KASP reaction with primer set 1

[0029] Figure 5 Results of KASP reaction with the second primer set

[0030] Figure 6 Sample testing DETAILED DESCRIPTION

[0031] The following definitions and methods are provided to better define this application and guide those of ordinary skill in the art in the practice of this application. Unless otherwise specified, terms are understood according to the conventional usage of those of ordinary skill in the relevant field. All patent documents, academic papers, industry standards and other public publications cited herein are incorporated herein by reference in their entirety.

[0032] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Without departing from the spirit and substance of the present invention, the modifications or replacements made to the inventive method, steps or conditions are within the scope of this application. Unless otherwise specified, the examples are based on conventional experimental conditions or the conditions recommended by the manufacturer's instructions. Unless otherwise specified, the chemical reagents used in the examples are conventional commercial reagents, and the technical means used in the examples are conventional means well known to those skilled in the art.

[0033] Example 1 Creation and character identification of stress-resistant cotton transformant ZA102-9

[0034] Vitamin C is an antioxidant widely found in plants. It can help plants resist drought, ozone and ultraviolet rays, and protect plants from harmful substances in photosynthesis. Vitamin C is one of the members of the corresponding protection system against free radical damage in organisms. Therefore, the determination of its content can be used as an important physiological indicator for anti-aging and resistance to adversity. GDP-L-galactose phosphorylase 1 (GGP) in plants can control the synthesis of vitamin C. Therefore, the synthesis of vitamin C can be affected by regulating the expression of GGP, thereby regulating the stress resistance of plants.

[0035] GhVCT2 is the coding gene of cotton GGP. The present invention uses the authorized Cas12i to construct a C016-Cas12i editing vector targeting the promoter region sequence of the cotton GhVCT2 gene (see the vector physical map for details). Figure 1 ), the editing vector contains the Cas12i gene expression cassette, the gRNA1 / gRNA2 expression cassette and the marker gene AADA expression cassette, wherein gRNA1 and gRNA2 target the GhVCT2 gene promoter sequence on cotton D05 and D01, respectively. The recipient Zhongmian Institute 24 was transformed by Agrobacterium-mediated method, and 10 transformants ZA102-1 to ZA102-10 were obtained. After molecular detection, 7 transformants ZA102-3 to ZA102-9 without exogenous vector sequences were screened out from the 10 transformants. By measuring its VC content, it was found that ZA102-9 had the highest vitamin C content, so its target traits (drought resistance) and molecular characteristics were screened and identified.

[0036] 1. Screening of transformants

[0037] The drought resistance test of the transformant ZA102-9 was carried out in the greenhouse. Figure 2 ), under drought conditions, the cotyledons of the control cotton began to wilt first, and then those of ZA102-9. After rehydration, ZA102-9 returned to normal first, indicating that the drought resistance of ZA102-9 was better than that of the control.

[0038] 2. Molecular characterization of transformant ZA102-9

[0039] Primers (shown in Table 1) were designed based on the nucleotide sequences of the target gene GhVCT2 and its promoter on cotton D05 and D01 (SEQ ID NO.1 and SEQ ID NO.4) and PCR amplification was performed.

[0040] SEQ ID NO.1 (the box is gRNA1, and the shaded part is primer F1 / R1):

[0041]

[0042]

[0043]

[0044] SEQ ID NO.4 (the box is gRNA2, and the shaded part is primer F2 / R2):

[0045]

[0046] Table 1 Description of PCR primers and amplified fragments

[0047]

[0048] 1: Indicates the unit bp

[0049] PCR reaction system: 1 μL DNA template (50 ng / μL), 0.5 μL primers F1 and R1, 10 μL PCR mix, and ddH2O to 20 μL.

[0050] The reaction conditions were as follows: denaturation at 95°C for 5 min; denaturation at 95°C for 30 s, annealing at 59°C for 30 s, extension at 72°C for 30 s, 35 cycles; extension at 72°C for 5 min; and storage at 4°C.

[0051] Amplify the target fragment of the expected size. Connect the PCR product to the Peasy-Blunt vector and perform DNA Sanger sequencing analysis. The sequencing results are shown in Figure 3 and Table 2.

[0052] Table 2 Comparison of transformant target gene sequences before and after editing

[0053]

[0054] The sequence in the box is PAM, and “-” indicates a deletion sequence.

[0055] The results showed that the editing enzyme Cas12i in the transformant ZA102-9, under the guidance of gRNA1, caused the deletion of base A at position 472 of the receptor target gene sequence SEQ ID NO.1. The single base mutation in the promoter changed the expression of the gene GhVCT2, thereby improving the drought resistance of cotton plants.

[0056] Example 2 Application of KASP technology to detect stress-resistant cotton transformant ZA102-9

[0057] 1. Design and screening of KASP primer sets

[0058] The sequence of the target gene at the editing site before and after editing of the stress-resistant cotton transformant (the receptor is SEQ ID NO.2, and the transformant is SEQ ID NO.3) is used as a template to design the first forward primer and the second forward primer respectively, requiring the primer length to be 25bp, and the last base at the 3' end of the primer to be the SNP mutation site; then the template sequence is input into the primer design software (such as ABIPrimer Express 3.0), the forward primer sequence is limited, and the 1) PCR product length is required to be 80-150bp, and the 2) reverse primer length is 29bp, and the reverse primer sequence is obtained by the software. The present invention obtains 2 primer sets in total. Among them, each primer set contains 2 forward primers and 1 reverse primer. Finally, a FAM linker sequence is added to the 5' end of the first forward primer, and a VIC linker sequence is added to the 5' end of the second forward primer. The primer sequences are shown in Table 3.

[0059] Table 3 Description of KASP primer sets and amplified fragments

[0060]

[0061]

[0062] 1: Indicates the unit bp

[0063] 2. Primer set synthesis and screening

[0064] The two primer sets shown in Table 3 were synthesized. The genomic DNA of the recipient Zhongmiansuo 24 and the transformant ZA102-9 extracted by CTAB method was used as template, ddH2O was used as control, and the KASP reaction system shown in Table 4 and the reaction program shown in Table 5 were used to screen the specificity of the primers.

[0065] Table 4 KASP reaction system

[0066]

[0067] Table 5 KASP reaction procedure

[0068]

[0069] Result analysis: The reaction results will appear in the form of a scatter plot, and the scatter plot can be used to determine whether the sample contains transformant components. The horizontal axis of the graph represents the FAM fluorescence released by the product, and the vertical axis represents the VIC fluorescence released by the product. If significant FAM fluorescence is detected from the product of a sample, it indicates that the sample contains recipient cotton; if significant VIC fluorescence is detected from the product of a sample, it indicates that the sample contains transformant components (positive); if no fluorescence is detected from a sample, it indicates that the sample is a control ddH2O or there is a problem with the experimental design and operation, and retesting is required. At this time, the corresponding data point appears near NTC (no DNA template).

[0070] like Figure 4 As shown in the figure, the results of the KASP reaction using the first set of primers showed that significant FAM fluorescence was detected in the recipient cotton, significant VIC fluorescence was detected in the transformant ZA102-9, and no fluorescence was detected in the control ddH2O, indicating that the first set of primers has good specificity and can distinguish the transformant and recipient genomic DNA. Figure 5 As shown, the results of the KASP reaction using the second set of primers showed that no fluorescence was detected in the recipient, transformant, and control, indicating that the second set of primers had poor specificity and could not distinguish between the transformant and recipient genomic DNA.

[0071] Therefore, the first set of primers was selected as the primer set for detecting transformant ZA102-9, and the specific positions of the primers are shown below (boxes indicate primer positions).

[0072] SEQ ID NO.2 (receptor target site sequence)

[0073]

[0074] SEQ ID NO.3 (Editor target site sequence)

[0075]

[0076] 3. Optimize KASP reaction procedure

[0077] After obtaining a specific primer set, whether the KASP reaction can have a better amplification efficiency is also affected by various factors in the reaction program, including the annealing temperature. Therefore, in order to obtain accurate identification results, the PCR reaction program needs to be further optimized.

[0078] Three different annealing temperatures (55°C, 59°C and 62°C) were tested for amplification. The results showed that the reaction program with an annealing temperature of 55°C had the best amplification efficiency and the best typing effect on receptors and transformants.

[0079] Therefore, the optimized KASP reaction procedure of the present invention is shown in Table 6.

[0080] Table 6 Optimized KASP reaction program

[0081]

[0082] 4. Sample-specific detection

[0083] The samples to be tested were the recipient Zhongmian Institute 24 (samples 1-5), the transformant ZA102-9 (samples 6-10), and the cotton mixed material containing ZA102-9 and Zhongmian Institute 24 (samples 11-15). The genomic DNA of samples 1-15 extracted by the CTAB method was used as a template, ddH2O was used as a control, the first primer set, the reaction system shown in Table 4, and the optimized reaction procedure (Table 6) were used for KASP detection.

[0084] The test results of the samples are as follows Figure 6 As shown, samples 1-5 detected significant FAM fluorescence, and the identification result was China Cotton Research Institute 24; samples 6-10 detected significant VIC fluorescence, and the identification result was transformant ZA102-9; samples 11-15 detected both FAM and VIC fluorescence, and the identification result was cotton mixed material. The experimental results show that the detection method of the present invention can accurately identify the transformant ZA102-9 with high throughput.

[0085] The above is only a preferred embodiment of the present invention, and does not limit the present invention in any form. Although the present invention has been disclosed as a preferred embodiment as above, it is not used to limit the present invention. Any technician familiar with this profession can use the technical content disclosed above to make changes or modifications to equivalent embodiments of equivalent changes without departing from the scope of the technical solution of the present invention. However, any simple modifications, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solution of the present invention are still within the scope of the technical solution of the present invention.

Claims

1. A primer set, characterized in that: The primer set includes a first forward primer having a nucleotide sequence of 5'-gagaaactccgtgtatagccgtgaT-3', a second forward primer having a nucleotide sequence of 5'-gagaaactccgtgtatagccgtgaG-3', and a reverse primer having a nucleotide sequence of 5'-GCGTGGAGGTTGGGTATTTGGTTAAGGTA-3'; The first forward primer and the second forward primer are modified with different labels.

2. The primer set according to claim 1, characterized in that: The marker includes a fluorescent group or a tag sequence.

3. The primer set according to claim 2, characterized in that: The fluorescent group or label sequence includes any one of FAM, TET, HEX, CY3, JOE, VIC, ROX, CY5, TAMRA or Texas.

4. The primer set according to claim 3, characterized in that: The first forward primer was modified with a FAM tag sequence, and the second forward primer was modified with a VIC tag sequence; The nucleotide sequence of the FAM tag sequence is shown as 5'-GAAGGTGACCAAGTTCATGCT-3'; The nucleotide sequence of the VIC tag sequence is shown as 5'-GAAGGTCGGAGTCAACGGATT-3'.

5. A detection kit, characterized in that: The detection kit comprises the primer set according to any one of claims 1 to 4.

6. The detection kit according to claim 5, characterized in that: The detection kit also includes 2×KASP Master Mix.

7. A detection method, characterized in that The following steps are involved: 1) Extracting genomic DNA from the sample to be tested; 2) using the genomic DNA extracted in step 1) as a template, and using the primer set according to any one of claims 1 to 4 to perform PCR amplification to obtain a PCR amplification product; 3) performing marker detection on the PCR amplification product in step 2); if a VIC fluorescence signal is detected, the sample contains cotton ZA102-9; Among them, the cotton transformant ZA102-9 contains the nucleic acid molecule shown in SEQ ID NO.

3.

8. The detection method according to claim 7, characterized in that: The reaction system for PCR amplification is 10 μL, wherein the template DNA is 1 μL, the first forward primer and the second forward primer are 0.1 μL, the reverse primer is 0.3 μL, and 2×KASP MasterMix is ​​5 μL. The final concentration of the first forward primer or the second forward primer is 0.1 μM, and the final concentration of the reverse primer is 0.3 μM.

9. The detection method according to any one of claims 7 or 8, characterized in that: The reaction program of the PCR amplification is: 95°C for 10 min; 95°C for 15 s, gradient annealing and extension at 59°C to 53°C for 60 s, 10 cycles, the annealing and extension temperature is reduced by 0.6°C in each cycle; 95°C for 15 s, 55°C for 60 s, 30 cycles.

10. Use of the primer set according to any one of claims 1 to 4, the detection kit according to any one of claims 5 to 6, and the detection method according to any one of claims 7 to 9 in high-throughput detection of cotton transformant ZA102-9; in, Cotton transformant ZA102-9 contains the nucleic acid molecule shown in SEQ ID NO.3.

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