Primer combination for detecting soybeans without beany flavor, detection product and application thereof

Abstract

The invention discloses a primer combination for detecting soybeans without beany flavor, a detection product and application of the primer combination and the detection product, and relates to the technical field of biological detection. The invention provides a primer combination for detecting soybeans without beany flavor. The primer combination comprises a primer LOX3-F, a primer LOX3-H and a primer LOX3-C1, the nucleotide sequence of the primer LOX3-F is as shown in SEQ ID NO.4, the nucleotide sequence of the primer LOX3-H is as shown in SEQ ID NO.5, and the nucleotide sequence of the primer LOX3-C1 is as shown in SEQ ID NO.6; a first fluorescent tag sequence is added to the 5'end of the primer LOX3-F, and a second fluorescent tag sequence is added to the 5 'end of the primer LOX3-H; the first fluorescent label sequence and the second fluorescent label sequence are different fluorescent labels. By utilizing the primer combination, the soybean variety which is edited by the gene GmLox3 and has no beany flavor can be effectively screened out. The detection efficiency of the primer combination is high and is 14-56 times that of fluorescent quantitative PCR, the cost is low, the primer combination is suitable for high-throughput and automatic detection, and the error probability is effectively reduced.

Description

Technical Field

[0001] The present invention relates to the field of biological detection technology, and in particular to a primer combination for detecting soybeans without beany smell, a detection product and application thereof. Background Art

[0002] Gene editing technologies include ZFN (zinc finger ribonuclease technology), TANLEN (transcription activator-like effector nucleases) and CRISPR. CRISPR technology has become more and more mainstream due to its simple method and low cost, and has been successfully applied to different species of animals, plants and microorganisms. Gene editing technology can not only break through the genetic barriers that are difficult to solve in traditional breeding technology, but also achieve precise changes in specific traits, subverting the existing animal and plant genetic improvement technology paths and breeding efficiency. With the continuous development of gene editing technology and its wide application in animals and plants, researchers around the world have made a large number of edits to the genomes of different species of animals and plants, created different animal and plant species transformants, and promoted the progress of animal and plant breeding.

[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 PCR (Kompetitive Allele Specific PCR, KASP) is a genotyping technology based on fluorescence detection. At present, this technology is mainly used in SNP or InDel genotyping research, and is gradually becoming the main technical means for molecular assisted breeding, fine positioning of trait genes, and seed resource identification. This technology has two major advantages: first, as long as two universal fluorescent probes, two universal quenching probes, and multiple SNP PCR primers targeting specific sites are synthesized, many sites can be measured; second, due to the high cost of fluorescent probes and quenching probes, the KASP method can replace the fluorescent probes targeting the site with universal fluorescent probes compared to the Taqman method, which greatly saves costs. At present, there is no method for detecting the gene-edited soybean transformant 28-8 using KASP technology. Summary of the invention

[0005] The present invention provides a primer combination, a detection product and an application thereof for detecting soybeans without beany smell. For soybean variety transformants with a specific mutation gene GmLOX3 through gene editing technology, the present invention provides a primer combination capable of specifically detecting such soybean variety transformants based on KASP technology, which can quickly, efficiently and accurately identify gene-edited soybeans. This is specifically achieved through the following technologies.

[0006] In the first aspect, the present invention provides a primer combination for detecting soybeans without beany smell, comprising primer LOX3-F, primer LOX3-H and primer LOX3-C1; the nucleotide sequence of the primer LOX3-F is shown in SEQ ID NO.4, the nucleotide sequence of the primer LOX3-H is shown in SEQ ID NO.5, and the nucleotide sequence of the primer LOX3-C1 is shown in SEQ ID NO.6; a first fluorescent label sequence is added to the 5' end of the primer LOX3-F, and a second fluorescent label sequence is added to the 5' end of the primer LOX3-H; the first fluorescent label sequence and the second fluorescent label sequence are different fluorescent labels.

[0007] Furthermore, the first fluorescent label sequence is selected from any one of the fluorescent label sequences of FAM, HEX, TET, VIC, JOE, Cy3, Cy3.5, NED, TAMRA, ROX, Texas Red, Cy5, Cy5.5 and Quasar670; the second fluorescent label sequence is selected from any one of the fluorescent label sequences of FAM, HEX, TET, VIC, JOE, Cy3, Cy3.5, NED, TAMRA, ROX, Texas Red, Cy5, Cy5.5 and Quasar670.

[0008] Furthermore, the first fluorescent label sequence is a sequence of a fluorescent label FAM, and the second fluorescent label sequence is a sequence of a fluorescent label HEX.

[0009] Specifically, the nucleotide sequence of the first fluorescent tag sequence is shown as SEQ ID NO.9, and the nucleotide sequence of the second fluorescent tag sequence is shown as SEQ ID NO.10.

[0010] Furthermore, the soybean without beany smell is obtained by performing site-directed replacement / insertion mutation on the gene GmLox3 in the wild-type soybean genome using the endonuclease protein Cas9 and sgRNA-GmLox3; the nucleotide sequence of the sgRNA-GmLox3 is shown in SEQ ID NO.3.

[0011] When using the above-mentioned primer combination provided by the present invention for detection, two forward primers with fluorescent label sequences and one universal reverse primer need to be added to the reaction system. Therefore, how to ensure the specific amplification of these three primers is the first technical key of the present invention. How to ensure that the above-mentioned three primers do not interfere with each other in the same reaction system to ensure the accuracy and reliability of the test results is the second technical key of the present invention. In order to test the detection effect and accuracy of several alternative primer combinations, the present invention performs specific detection on the gene-edited soybean 28-8 sample without beany smell, and then compares the detection results with the genotype information of the sample itself, and finally screens out the primer combination that meets the above-mentioned effects at the same time.

[0012] In a second aspect, the present invention further provides a product for detecting soybeans without beany smell, comprising any one of the above primer combinations.

[0013] Furthermore, the product for detecting soybeans without beany smell is a detection kit or a detection combination reagent.

[0014] In a third aspect, the present invention further provides a use of any one of the above primer combinations, or any one of the above products for detecting soybeans without beany smell, in breeding soybeans without beany smell.

[0015] In a fourth aspect, a method for breeding soybeans without beany smell, comprising extracting genomic DNA of soybeans to be tested, and performing PCR amplification using any of the primer combinations described above, or using any of the products described above for detecting soybeans without beany smell;

[0016] If the fluorescent tag luminescence signal corresponding to the second fluorescent tag sequence is detected, and the fluorescent tag luminescence signal corresponding to the first fluorescent tag sequence is not detected, then the soybean to be tested is a soybean variety without beany smell;

[0017] If the fluorescent tag luminescence signal corresponding to the first fluorescent tag sequence is detected, and the fluorescent tag luminescence signal corresponding to the second fluorescent tag sequence is not detected, then the soybean to be tested is a wild-type soybean variety;

[0018] If the fluorescent tag luminescence signals corresponding to the first fluorescent tag sequence and the second fluorescent tag sequence are not detected, re-detection is required.

[0019] Furthermore, the reaction system of PCR amplification is shown in Table 1 below.

[0020] Table 1 PCR amplification reaction system

[0021] Reagents 2μL system 10μL system KASP Primer mix 1μL 5μL KASP Master mix 0.1μL 0.5μL DNA template 5-50ng 25-250ng

[0022] The KASP Primer mix includes the primer LOX3-F, the primer LOX3-H and the primer LOX3-C1;

[0023] The KASP Master mix includes universal FRET cassette fluorescent primers, ROX internal reference dye, Taq DNA polymerase, dNTP and MgCl2.

[0024] Furthermore, the reaction procedure of PCR amplification is: pre-denaturation at 90-95°C for 10-20 min; performing the first step of amplification reaction, denaturation at 90-95°C for 10-30 s, annealing and extension for 30-90 s, and 5-20 touchdown cycles, wherein the temperature of annealing and extension is reduced by 0.1-3.0°C in each cycle; performing the second step of amplification reaction, denaturation at 90-95°C for 10-30 s, annealing and extension at 55-60°C for 30-90 s, and 20-30 cycles.

[0025] Preferably, the starting temperature of annealing is 62-68°C, and the ending temperature of annealing is 55-59°C; the temperature of each cycle of annealing and extension decreases by 0.15-2.6°C.

[0026] More preferably, the reaction procedure of PCR amplification is: pre-denaturation at 95°C for 10 min; performing the first step of amplification reaction, denaturation at 95°C for 15 s, annealing and extension at 61-55°C for 60 s, and 10 touchdown cycles, with the temperature of annealing and extension decreasing by 0.6°C in each cycle; performing the second step of amplification reaction, denaturation at 95°C for 15 s, annealing and extension at 55°C for 60 s, and 35 cycles.

[0027] Compared with the prior art, the present invention is beneficial in that: the present invention designs several KASP primer combinations at the SNP editing site of the target gene of the gene-edited soybean transformant without beany odor, and obtains a set of primer combinations after testing and screening. The primer combination can effectively distinguish the variant site, thereby efficiently and accurately identifying the gene-edited soybean 28-8.

[0028] The primer combination and detection method provided by the present invention use a universal fluorescent probe, and do not need to synthesize a specific probe, which greatly reduces the cost compared with the probe method fluorescence quantitative PCR. The fluorescence scanning of this method is performed at the reaction end point, so PCR amplification of a large number of samples can be performed first, and then fluorescence scanning detection can be performed centrally after the reaction is completed, thereby achieving high-throughput detection. The primer combination provided by the present invention has a detection efficiency that is 14-56 times that of fluorescence quantitative PCR. After the reaction is completed, gel electrophoresis is not required, which is suitable for automated detection, can save a lot of labor costs, and effectively reduce the probability of error. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1This is a sequence alignment of the gene editing regions of wild-type HC6 and gene-edited material 28-8.

[0030] Figure 2 The typing results of KASP reverse universal primer LOX3-C2 / C3 and the first and second forward primers LOX3-F and LOX3-H.

[0031] Figure 3 The typing results are shown for the KASP universal reverse primer LOX3-C1 and the first and second forward primers LOX3-F and LOX3-H. DETAILED DESCRIPTION

[0032] The technical solution of the present invention will be described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0033] Example 1: Obtaining gene-edited soybean transformant 28-8

[0034] The present invention uses the gene-edited soybean transformant 28-8 cultivated by the applicant as the detection object.

[0035] The gene-edited soybean transformant 28-8 is a soybean without bean smell developed by Fujian Agriculture and Forestry University and the applicant of the present invention (Wuhan Aidijing Biotechnology Co., Ltd.) using gene editing technology.

[0036] The gene editing and screening and breeding methods of gene-edited soybean transformant 28-8 have been disclosed in paragraphs 27-88 of the specification of Chinese patent CN110684796B. Specifically, the CRISPR-Cas9 gene editing technology is used to perform specific gene editing on the gene Glyma.15g026300 (GmLox3) expressing soybean lipoxygenase of wild-type soybean Huachun No. 6 (HC6), and the gene is obtained after breeding. The present invention also adopts the same gene editing method as the Chinese patent CN110684796B to breed and obtain the gene-edited soybean transformant 28-8; the sgRNA-GmLox3 sequence used is 5'-cctttccttatcctcgtaggggg-3', as shown in SEQ ID NO.3.

[0037] The nucleic acid sequence of GmLOX3 of wild-type soybean HC6 is shown in SEQ ID NO.1. After gene editing, GmLOX3 of gene-edited soybean transformant 28-8 is shown in SEQ ID NO.2. The comparison results of GmLOX3 gene between gene-edited soybean 28-8 and wild-type soybean HC6 are shown in Figure 1 As shown, it can be seen that the gene GmLOX3 of the edited soybean transformant 28-8 is missing 8 bases compared with the wild-type soybean HC6.

[0038] After cultivation, it was found that soybean 28-8 had excellent agronomic traits and its yield was comparable to that of wild-type soybean HC6.

[0039] Example 2: Design and screening of KASP primer sets for gene GmLOX3

[0040] 1. Using the sequence of the target gene at the editing site of the gene-edited soybean transformant 28-8 before and after editing (the wild type is shown in SEQ ID NO.1, and the transformant is shown in SEQ ID NO.2) as a template, the first forward primer LOX3-F and the second forward primer LOX3-H were designed respectively, and the 3' end of the primer was the SNP mutation site.

[0041] The first forward primer LOX3-F: 5'-ggaaatgacacctttccttatc-3', shown in SEQ ID NO.4.

[0042] The second forward primer LOX3-H: 5'-ggaaatgacacctttccttagg-3', SEQ ID NO.5.

[0043] 2. According to the template sequence, the first forward primer and the second forward primer sequence, a reverse universal primer sequence is designed. In the present invention, a total of 3 reverse universal primers are designed.

[0044] Reverse universal primer LOX3-C1: 5'-gctaaaacaatataccaattcaa-3', shown in SEQ ID NO.6.

[0045] Reverse universal primer LOX3-C2: 5'-acagttaaagtttaatgaaatgg-3', shown in SEQ ID NO.7.

[0046] Reverse universal primer LOX3-C3: 5'-atagcagaacttattagcaacaa-3', shown in SEQ ID NO.8.

[0047] 3. Then, a FAM linker sequence was added to the 5' end of the first forward primer LOX3-F, 5'--3', as shown in SEQ ID NO. 9; and a HEX linker sequence was added to the 5' end of the second forward primer LOX3-H, 5'-gaaggtcggagtcaacggatt-3', as shown in SEQ ID NO. 10. The synthesized primers were used to detect gene-edited soybean 28-8 and wild-type soybean HC6.

[0048] 4. Prepare DNA templates. Prepare DNA templates of gene-edited soybean transformant 28-8 and wild-type soybean HC6 respectively. Use CTAB extraction method to extract genomic DNA from gene-edited soybean 28-8 and wild-type soybean HC6 respectively.

[0049] The first forward primer LOX3-F with added tag sequence, the second forward primer LOX3-H, reverse universal primers (LOX3-C1, LOX3-C2, LOX3-C3), DNA template and KASP reaction solution were used to construct the KASP reaction system, as shown in Table 2. The amount of DNA template in each reaction was about 30 ng.

[0050] Table 2 KASP reaction system

[0051] Components volume KASP Primer mix 5μL 2×KASP Master mix 0.5μL DNA template 30ng Total volume Refill to 10 μL

[0052] The KASP Primer mix contains a first forward primer, a second forward primer and three reverse universal primers, and their concentrations are 30 μM, 12 μM and 12 μM respectively. The KASP Master Mix contains the following components: universal FRETcassette fluorescent primer, ROX internal reference dye, Taq DNA polymerase, dNTP and MgCl2;

[0053] PCR reaction conditions: pre-denaturation at 95°C for 10 min; first step amplification reaction, denaturation at 95°C for 15 s, annealing and extension at 61-55°C for 60 s, 10 touchdown cycles, annealing and extension temperatures decreased by 0.6°C in each cycle; second step amplification reaction, denaturation at 95°C for 15 s, annealing and extension at 55°C for 60 s, for a total of 35 cycles.

[0054] Result analysis: After the reaction is completed, the product is subjected to fluorescence scanning, and the scanning results will appear in the form of a scatter plot. The genotype of the sample is determined based on the scatter plot. The horizontal axis of the graph represents the FAM fluorescence released by the product, and the vertical axis represents the HEX fluorescence released by the product. If only significant HEX fluorescence is detected and no significant FAM fluorescence is detected, it indicates that the sample contains components of the gene-edited transformant 28-8. If only significant FAM fluorescence is detected and no significant HEX fluorescence is detected, it indicates that the sample is wild type. If neither HEX fluorescence nor FAM fluorescence is detected, retesting is required.

[0055] The results are as follows Figure 2-Figure 3 As shown; Figure 2 The typing results of KASP reverse universal primer LOX3-C2 / C3 and the first and second forward primers LOX3-F and LOX3-H. Figure 2In the figure, the blue dots are NTC, and there is no DNA template in the reaction well; the green dots are the mixed fluorescence of FAM and HEX, representing the heterozygous 28-8 genotype. Figure 3 The typing results are shown for the KASP universal reverse primer LOX3-C1 and the first and second forward primers LOX3-F and LOX3-H. Figure 3 In the figure, the blue dots are NTC, and there is no DNA template in the reaction well; the red dots are FAM fluorescence, representing the genotype of 28-8; the orange dots are HEX fluorescence, representing the wild-type genotype.

[0056] from Figure 2 It can be seen that the combination of reverse universal primers LOX3-C2, LOX3-F and LOX3-H, and the combination of LOX3-C3, LOX3-F and LOX3-H were unable to distinguish between the gene-edited soybean 28-8 and the wild type, and both amplified the heterozygous 28-8 genotype. Figure 3 It can be seen that the combination of reverse universal primers LOX3-C1, LOX3-F and LOX3-H can accurately distinguish the edited soybean 28-8 from the wild type.

[0057] Therefore, the combination of KASP primers LOX3-C1, LOX3-F and LOX3-H can distinguish the genotyping of gene-edited soybean 28-8, and can realize the gene-edited soybean 28-8 or its derivative lines, or mixed biological materials containing the above-mentioned transformants, or various products obtained by processing the above-mentioned samples as raw materials.

[0058] The above specific embodiments describe the implementation of the present invention in detail, but the present invention is not limited to the specific details in the above embodiments. Within the scope of the claims and technical concept of the present invention, the technical solution of the present invention can be modified and changed in many simple ways, and these simple modifications all belong to the protection scope of the present invention.

Claims

1. A primer combination for detecting soybeans without beany smell, characterized in that: It includes primer LOX3-F, primer LOX3-H and primer LOX3-C1; the nucleotide sequence of primer LOX3-F is shown in SEQ ID NO.4, the nucleotide sequence of primer LOX3-H is shown in SEQ ID NO.5, and the nucleotide sequence of primer LOX3-C1 is shown in SEQ ID NO.6; a first fluorescent label sequence is added to the 5' end of the primer LOX3-F, and a second fluorescent label sequence is added to the 5' end of the primer LOX3-H; the first fluorescent label sequence and the second fluorescent label sequence are different fluorescent labels.

2. The primer combination for detecting soybeans without beany smell according to claim 1, characterized in that: The first fluorescent label sequence is selected from any one of FAM, HEX, TET, VIC, JOE, Cy3, Cy3.5, NED, TAMRA, ROX, Texas Red, Cy5, Cy5.5 and Quasar670; the second fluorescent label sequence is selected from any one of FAM, HEX, TET, VIC, JOE, Cy3, Cy3.5, NED, TAMRA, ROX, Texas Red, Cy5, Cy5.5 and Quasar670; Furthermore, the nucleotide sequence of the first fluorescent tag sequence is shown as SEQ ID NO.9, and the nucleotide sequence of the second fluorescent tag sequence is shown as SEQ ID NO.

10.

3. The primer combination for detecting soybeans without beany odor according to claim 1, characterized in that: The soybean without beany smell is obtained by performing site-directed replacement / insertion mutation on the gene GmLox3 in the wild-type soybean genome using the endonuclease protein Cas9 and sgRNA-GmLox3; the nucleotide sequence of the sgRNA-GmLox3 is shown in SEQ ID NO.

3.

4. A product for detecting soybeans without beany smell, characterized in that: The invention comprises the primer combination according to any one of claims 1 to 3.

5. The product for detecting soybeans without beany smell according to claim 4, characterized in that: It is a detection kit or a detection combination reagent.

6. Use of the primer combination according to any one of claims 1 to 3, or the product for detecting soybeans without beany smell according to any one of claims 4 to 5 in breeding soybeans without beany smell.

7. A method for breeding soybeans without beany smell, characterized in that: Extracting genomic DNA of the soybean to be tested, and performing PCR amplification using the primer combination described in any one of claims 1 to 3, or using the product for detecting soybeans without beany odor described in any one of claims 4 to 5; If the fluorescent tag luminescence signal corresponding to the second fluorescent tag sequence is detected, and the fluorescent tag luminescence signal corresponding to the first fluorescent tag sequence is not detected, then the soybean to be tested is a soybean variety without beany smell; If the fluorescent tag luminescence signal corresponding to the first fluorescent tag sequence is detected, and the fluorescent tag luminescence signal corresponding to the second fluorescent tag sequence is not detected, then the soybean to be tested is a wild-type soybean variety; If the fluorescent tag luminescence signals corresponding to the first fluorescent tag sequence and the second fluorescent tag sequence are not detected, re-detection is required.

8. The method for breeding soybeans without beany smell according to claim 7, characterized in that: The reaction system for PCR amplification includes: The KASP Primer mix includes the primer LOX3-F, the primer LOX3-H and the primer LOX3-C1; The KASP Master mix includes universal FRET cassette fluorescent primers, ROX internal reference dye, Taq DNA polymerase, dNTP and MgCl2.

9. The method for breeding soybeans without beany smell according to claim 8, characterized in that: The reaction procedure of PCR amplification is as follows: pre-denaturation at 90-95°C for 10-20 min; the first step of amplification reaction is denaturation at 90-95°C for 10-30 s, annealing and extension for 30-90 s, and 5-20 touchdown cycles, wherein the temperature of annealing and extension is reduced by 0.1-3.0°C in each cycle; the second step of amplification reaction is denaturation at 90-95°C for 10-30 s, annealing and extension at 55-60°C for 30-90 s, and 20-30 cycles.

10. The method for breeding soybeans without beany smell according to claim 9, characterized in that: In the first step of amplification reaction, the starting temperature of annealing is 62-68°C, and the ending temperature of annealing is 55-59°C; the temperature of annealing and extension is reduced by 0.15-2.6°C in each cycle; Preferably, the reaction procedure of PCR amplification is: pre-denaturation at 95°C for 10 min; performing the first step of amplification reaction, denaturation at 95°C for 15 s, annealing and extension at 61-55°C for 60 s, and 10 touchdown cycles, with the temperature of annealing and extension decreasing by 0.6°C in each cycle; performing the second step of amplification reaction, denaturation at 95°C for 15 s, annealing and extension at 55°C for 60 s, and 35 cycles.

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