Method for detecting exogenous gene insertion copy number in green bristlegrass or millet by adopting digital PCR (Polymerase Chain Reaction)
Through digital PCR technology, the Sevir.3G057200 gene in the genus scattered grass is used as the internal reference gene, and the marker gene is screened in Hyg, and the insertion copy number of foreign genes in the transgenic genus scattered grass or millet was detected, solving the problem of difficulty in detection in the existing technology and achieving a fast and efficient detection effect.
Patent Information
- Application Number
- CN202510529805.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-25
- Publication Date
- 2025-05-27
- Estimated Expiration
- 2045-04-25
AI Technical Summary
The prior art is difficult to quickly, simply and effectively detect the number of exogenous gene insertion copies in transgenic terrestrial grass or millet, especially when screening transgenic lines on a large scale.
Using digital PCR technology, the Sevir.3G057200 gene in the genus scattered grass was used as the internal reference gene, combined with Hyg screening marker genes, and the insertion copy number of foreign genes in the transgenic genus scattered grass or millet was determined by specific primers and probe sequences.
It has achieved rapid and high-throughput detection of exogenous gene insertion copy number in transgenic dogtail grass or millet, which has the characteristics of simple and fast, low sample requirements, high efficiency and high throughput, which can improve the efficiency of large-scale screening of transgenic lines.
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Figure CN120041606A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the field of biotechnology, and particularly relates to a method for detecting the copy number of foreign gene insertions in Setaria viridis or foxtail millet by digital PCR, and more particularly to a single-copy foxtail millet gene capable of detecting the copy number of foreign gene insertions in foxtail millet and a method for detecting the copy number of foreign gene insertions in transgenic foxtail millet by digital PCR. Background Art
[0002] Setaria viridis ( Setaria viridis ) belongs to the genus Setaria in the subfamily Panicoideae of the monocotyledonous family Poaceae. It is a new transgenic model plant. Compared with the dicotyledonous model plant Arabidopsis thaliana, it has the advantages of a short growth cycle, short plant height, easy cultivation, a small genome, diploidy, and the ability to produce a large number of self-crossed seeds. It is an excellent monocotyledonous model plant. Since it originated in the tropics and has a C4 photosynthesis system, it is closely related to foxtail millet, maize, sorghum, sugarcane, Coix lacryma-jobi, and important energy grasses, and is also an important C4 plant model. At present, the whole-genome sequencing and resequencing data of Setaria viridis ME34 and A10 have been published, and more and more domestic research institutions have begun to pay attention to it, especially foxtail millet ( Setaria italica ) breeders. Foxtail millet is also an annual plant in the genus Setaria of the subfamily Panicoideae of the Poaceae family, and Setaria viridis is the wild ancestor of foxtail millet. The karyotypes of foxtail millet and Setaria viridis are basically the same, the banding patterns are similar, and the genome sizes are both about 510 Mb. Setaria viridis has a short growth cycle and high genetic transformation efficiency. Overexpressing or knocking out candidate genes of foxtail millet in Setaria viridis can quickly verify their functions and accelerate the research process of foxtail millet breeding. Therefore, Setaria viridis can be used as an important transgenic model plant for studying the gene functions of foxtail millet.
[0003] In the research of transgenic breeding of plants, the expression and genetic stability of foreign genes are affected by the copy number of their integration into the genome of the recipient plant. When a foreign gene is inserted into the genome of the recipient plant at a low copy number (1-2 copies), it generally can be highly expressed and stably inherited. Therefore, in the research of transgenic plant breeding, transgenic lines with low-copy insertions of foreign genes are usually selected. However, transgenic lines with low-copy insertions generally need to be screened from a large number of T 0 -generation transgenic lines, which takes a lot of time. Therefore, a simple, rapid, efficient, and high-throughput method for detecting the copy number of foreign gene insertions is crucial for transgenic plant breeding research.
[0004] At present, the traditional method for detecting the copy number of foreign gene insertions in transgenic plants is Southern hybridization, which has high accuracy, but also high costs, a long cycle, and it is difficult to simply and quickly detect a large number of samples. In recent years, digital PCR (dPCR) has been successfully applied to the detection of the copy number of foreign gene insertions in various transgenic crops due to its simple, fast, and efficient characteristics. However, there are still no reports on the detection of the copy number of foreign gene insertions in transgenic Setaria viridis using this PCR method. Therefore, there is an urgent need to establish a method for quickly detecting the copy number of foreign gene insertions in transgenic Setaria viridis using dPCR. Summary of the Invention
[0005] In view of the above problems, the present invention provides a single-copy Setaria viridis gene and a method for detecting the copy number of foreign gene insertions in Setaria viridis using digital PCR technology.
[0006] To achieve the above object, the technical solution adopted by the present invention is as follows: A method for detecting the copy number of foreign gene insertions in Setaria viridis or Setaria italica using digital PCR uses the Sevir.3G057200 gene as an internal reference gene and Hyg the selectable marker gene as the foreign gene, that is, the selectable marker gene hygromycin phosphotransferase gene ( Hyg ), and uses the primer pairs 200-F / 200-R and Hyg-F / Hyg-R to detect the genomic DNA of transgenic Setaria viridis or transgenic Setaria italica, and to determine the copy number of foreign gene insertions in transgenic Setaria viridis or transgenic Setaria italica; Among them, the Sevir.3G057200 gene is a single-copy gene in Setaria viridis, the corresponding gene number of this gene in Setaria italica is Seita.3G056300, and it is also a single-copy gene in Setaria italica. The homology of the nucleotide sequences of the two genes is 99.3%, and the primer pair sequences of the two genes are exactly the same; the sequence of the Sevir.3G057200 gene in Setaria viridis is as shown in SEQ ID NO: 7; the sequence of the Seita.3G056300 gene in Setaria italica is as shown in SEQ ID NO: 8; The primer pair sequence for detecting the internal reference gene Sevir.3G057200: The forward primer 200-F is: 5’-GTTCCGCACCAACATCTACTCCTAC-3’, as shown in SEQ ID NO: 1; The reverse primer 200-R is: 5’-GTTGATGACGCTGCTCCCTTCC-3’, as shown in SEQ ID NO: 2; The probe 200-P is: VIC-CCTACTTCCTGGTGACCAAGCACGC-MGB, as shown in SEQ ID NO: 3; Detection Hyg Primer pairs and probe sequences for screening marker genes: The forward primer Hyg-F is: 5’-GTCAAGACCAATGCGGAGCA-3’, as shown in SEQ ID NO: 4; The reverse primer Hyg-R is: 5’-CCCAATACGAGGTCGCCAAC-3’, as shown in SEQ ID NO: 5; The probe Hyg-P is: FAM-TCGAAGTAGCGCGTCTGCTGCTCCA-BHQ1, as shown in SEQ ID NO: 6; The method for determining the copy number of foreign gene insertion in transgenic Setaria viridis or transgenic Setaria italica is: When the copy number ratio of the foreign gene to the reference gene is about 0.5 (i.e., when the copy number ratio of the foreign gene to the reference gene is 0.26 - 0.74), the transgenic Setaria viridis or transgenic Setaria italica has a single-copy insertion; When the copy number ratio of the foreign gene to the reference gene is about 1 (the copy number ratio of the foreign gene to the reference gene is 0.75 - 1.25), the transgenic Setaria viridis or transgenic Setaria italica has a double-copy insertion; When the copy number ratio of the foreign gene to the reference gene is about 1.5 (the copy number ratio of the foreign gene to the reference gene is 1.26 - 1.74), the transgenic Setaria viridis or transgenic Setaria italica has a triple-copy insertion; When the copy number ratio of the foreign gene to the reference gene is about 2 (the copy number ratio of the foreign gene to the reference gene is 1.75 - 2.25), the transgenic Setaria viridis or transgenic Setaria italica has a quadruple-copy insertion; And so on for other copy numbers of foreign gene insertion in transgenic Setaria viridis or transgenic Setaria italica, such as five-copy insertion, six-copy insertion, etc.
[0007] Furthermore, the method uses the genomic DNA of transgenic Setaria viridis or transgenic Setaria italica as the DNA template, and uses the primer pairs RA1-F / RA1-R and primer pairs Hyg-F / Hyg-R for digital PCR detection.
[0008] Furthermore, the reaction system of digital PCR is: reaction premix, 200-F, 200-R, Hyg-F, Hyg-R, probe 200-P, probe Hyg-P, DNA template, and nuclease-free water.
[0009] Furthermore, the reaction premix is the reaction premix for droplet digital PCR.
[0010] Furthermore, the reaction system for digital PCR is as follows: 10.00 μL of reaction premix, 1.80 μL of 200-F1 with a concentration of 10.00 μM, 1.80 μL of 200-R with a concentration of 10.00 μM, 1.80 μL of Hyg-F with a concentration of 10.00 μM, 1.80 μL of Hyg-R with a concentration of 10.00 μM, 0.50 μL of probe 200-P with a concentration of 10.00 μM, 0.50 μL of probe Hyg-P with a concentration of 10.00 μM, 1 μL of DNA template with a concentration of 30.00 ng / μL, and 0.80 μL of nuclease-free water, with a total volume of 20.00 μL.
[0011] Furthermore, the amplification program for digital PCR is as follows: 95°C for 10 min; 95°C for 30 s, 56.4 - 69.6°C for 1 min, 45 cycles; 98°C for 10 min.
[0012] Furthermore, the amplification program for digital PCR is as follows: 95°C for 10 min; 95°C for 30 s, 62°C for 1 min, 45 cycles; 98°C for 10 min.
[0013] Furthermore, after the digital PCR reaction is completed, the copy numbers of the foreign gene and the reference gene are obtained, and the ratio of the copy number of the foreign gene to the copy number of the reference gene in transgenic Setaria viridis or transgenic Setaria italica is calculated, and the insertion copy number of the foreign gene in the genome of transgenic Setaria viridis or transgenic Setaria italica is judged by the ratio.
[0014] Furthermore, the copy numbers of the foreign gene and the reference gene are obtained by placing the chip in a biochip analyzer to read the FAM and VIC fluorescence signals and using QuantDrop Software for fluorescence data analysis.
[0015] Furthermore, the genomic DNA of transgenic Setaria viridis or transgenic Setaria italica is obtained by extraction using the MEGA Plant Genomic DNA Kit.
[0016] The beneficial effects of a method for detecting the insertion copy number of a foreign gene in Setaria viridis or Setaria italica using digital PCR according to the present invention are as follows: The single-copy Setaria viridis gene or Setaria italica gene provided by the present invention can be used as a reference gene for detecting the insertion copy number of a foreign gene in transgenic Setaria viridis or transgenic Setaria italica, with the most commonly used selection marker in transgenic Setaria viridis or transgenic Setaria italica Hyg as the foreign gene, and the rapid and high-throughput detection of the insertion copy number of the foreign gene in transgenic Setaria viridis is realized by using digital PCR technology; The present invention provides a method for high-throughput detection of the copy number of foreign gene insertions in transgenic Setaria viridis or transgenic foxtail millet using digital PCR technology. It has the characteristics of being simple, rapid, having low sample requirements, being efficient, and high-throughput, and can improve the efficiency of large-scale screening of transgenic lines, providing a new option for the detection of the copy number of foreign gene insertions in transgenic Setaria viridis or transgenic foxtail millet breeding research; The method provided by the present invention for detecting the copy number of foreign gene insertions in Setaria viridis or foxtail millet using digital PCR can be used for all transgenic Setaria viridis or transgenic foxtail millet with Hyg as a selection marker for copy number detection; the nucleotide sequence homology between the Sevir.3G057200 gene in Setaria viridis and its corresponding gene Seita.3G056300 in foxtail millet in the present invention is 99.3%, and the primer pair sequences of the two are exactly the same; therefore, the method provided by the present invention is also applicable to transgenic foxtail millet with Hyg as a selection marker for copy number detection; this method has the advantages of absolute quantification, high accuracy, being simple and rapid, having a small sample requirement, and being able to detect in batches. BRIEF DESCRIPTION OF THE DRAWINGS
[0017] Figure 1 is the alignment result of the Setaria viridis gene Sevir.3G057200 in the genome in Example 1 of the present invention; Figure 2 is the exploration result of the PCR amplification conditions of the reference gene and foreign gene in Setaria viridis in Example 2 of the present invention; in the figure, M is DL 2000 Marker, with the unit of bp, and the molecular weights from top to bottom are 2000, 1000, 750, 500, 250, and 100; labels 1 to 8 respectively represent the PCR products when the annealing temperatures are 56.4 °C, 58.0 °C, 60.0 °C, 62.0 °C, 64.0 °C, 66.0 °C, 68.0 °C, and 69.6 °C; Figure 3 is the PCR identification result of transgenic Setaria viridis in Example 3 of the present invention; in the figure, M is DL 2000 Marker, with the unit of bp, and the molecular weights from top to bottom are 2000, 1000, 750, 500, 250, and 100; labels 1 to 24 represent transgenic Setaria viridis SV-1 to SV-24 in sequence; "-" represents Hyg negative control, and "+" represents Hyg positive control; Figure 4 is the digital PCR scatter plot of the Sevir.3G057200 gene and Hyg gene in Example 4 of the present invention; the blue scatter points represent HygNumber of fluorescence pores of the gene; the green scatter points represent the number of fluorescence pores of the Sevir.3G057200 gene; the red scatter points represent the number of fluorescence pores of the Sevir.3G057200 gene and Hyg the number of fluorescence pores of the gene, and the gray scatter points represent the number of pores without signal. Specific implementation mode
[0018] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Many specific details are set forth in the following description in order to fully understand the present invention, but the present invention can also be implemented in other ways different from those described herein. Those skilled in the art can make similar promotions without departing from the connotation of the present invention. Therefore, the present invention is not limited by the specific embodiments disclosed below. The present invention will be further described in detail below in conjunction with specific embodiments for those skilled in the art to understand.
[0019] In addition, for those not specifying specific technologies or conditions in the specific embodiments disclosed below, they shall be carried out according to the technologies or conditions described in the literature in the field (for example, referring to "Molecular Cloning: A Laboratory Manual" written by Sambrook et al. and translated by Huang Peitang et al., the third edition, Science Press) or according to the product instructions. For reagents not indicating the manufacturer, they are all conventional products that can be purchased.
[0020] Example 1 Determination of reference gene Bioinformatics analysis of the copy number of the Sevir.3G057200 gene in the foxtail millet genome specifically includes the following steps: On the Phytozome website (https: / / phytozome-next.jgi.doe.gov / ), using the nucleotide sequence of the Sevir.3G057200 gene as the query sequence to query in the foxtail millet genome, the alignment result is obtained, as shown in Figure 1 . It can be seen from the alignment result that the Sevir.3G057200 gene is a single-copy gene and there are no homologous genes with similar sequences, so it can be used as a reference gene for detecting the insertion copy number of foreign genes in transgenic foxtail millet.
[0021] The gene number corresponding to the Sevir.3G057200 gene in foxtail millet is Seita.3G056300; the sequence homology between the Sevir.3G057200 gene in foxtail millet and the Seita.3G056300 gene in foxtail millet is 99.3%. Among them, the sequence of the Sevir.3G057200 gene in foxtail millet is as shown in SEQ ID NO: 7; the sequence of the Seita.3G056300 gene in foxtail millet is as shown in SEQ ID NO: 8.
[0022] Since the gene sequence homology between Sevir.3G057200 gene in Setaria viridis and Seita.3G056300 gene in Setaria italica is as high as 99.3%, and both are single-copy genes, therefore, the primers and probes designed based on the Sevir.3G057200 gene of Setaria viridis are also applicable to amplify and detect the Seita.3G056300 gene of Setaria italica (that is, the primer pair sequence is exactly the same as the sequence in the corresponding Seita.3G056300 gene in Setaria italica).
[0023] Meanwhile, since the commonly used selection marker genes in transgenic Setaria viridis and transgenic Setaria italica are both Hyg genes, and both can Hyg use
[0024] Example 2 Exploration of PCR Amplification Conditions Since the commonly used selection marker gene in transgenic Setaria viridis is Hyg gene, therefore, select Hyg the selection marker gene as the foreign gene in transgenic Setaria viridis, and use the Sevir.3G057200 gene as the internal reference gene for detection. The exploration of the PCR amplification conditions for the primers of the internal reference gene and the foreign gene includes the following specific steps: 1) Primer Sequences The primer pair sequences for detecting the internal reference gene Sevir.3G057200 include: Forward primer 200-F: 5’-GTTCCGCACCAACATCTACTCCTAC-3’, as shown in SEQ ID NO: 1; Reverse primer 200-R: 5’-GTTGATGACGCTGCTCCCTTCC-3’, as shown in SEQ ID NO: 2; Detect Hyg The primer pair sequences for the selection marker gene include: Forward primer Hyg-F: 5’-GTCAAGACCAATGCGGAGCA-3’, as shown in SEQ ID NO: 4; The reverse primer Hyg-R is: 5’-CCCAATACGAGGTCGCCAAC-3’, as shown in SEQ ID NO: 5; 2) Exploration of PCR amplification conditions for Sevir.3G057200 gene primers and Hyg gene primers Using the DNA of 1 transgenic Setaria viridis plant as a template, gradient PCR reactions were carried out using the primer pairs 200-F / 200-R and Hyg-F / Hyg-R respectively.
[0025] The PCR reaction system for detecting the reference gene Sevir.3G057200 is: Green Master Mix 12.50µL, 200-F (10.00µM) 1.00µL, 200-R (10.00µM) 1.00µL, DNA template (30.00ng / µL) 1.00µL, and nuclease-free water 9.50µL, with a total volume of 25.00µL.
[0026] The PCR reaction system for detecting the foreign gene is: Green Master Mix 12.50µL, Hyg-F (10.00µM) 1.00µL, Hyg-R (10.00µM) 1.00µL, DNA template (30.00ng / µL) 1.00µL, and nuclease-free water 9.50µL, with a total volume of 25.00µL.
[0027] The PCR reaction conditions are all: pre-denaturation at 94℃ for 5 min; denaturation at 94℃ for 30 s, annealing for 30 s, extension at 72℃ for 30 s, for a total of 35 cycles; finally, extension at 72℃ for 7 min, and preservation at 4℃; among which the annealing temperatures are 56.4℃, 58.0℃, 60.0℃, 62.0℃, 64.0℃, 66.0℃, 68.0℃, and 69.6℃ respectively.
[0028] After the PCR amplification reaction ended, 5µL of the corresponding PCR products obtained were respectively subjected to electrophoresis detection on a 2% agarose gel. If the PCR products of Sevir.3G057200 and Hyg showed bands of 83bp and 143bp respectively, they were the amplified bands of the target gene. The remaining PCR products were sent for sequencing, and the sequencing results were compared with the nucleotide sequences of the Sevir.3G057200 gene and Hyg gene.
[0029] The electrophoresis results are as Figure 2 shown. When the annealing temperatures were 56.4℃, 58.0℃, 60.0℃, and 62.0℃ respectively, the Sevir.3G057200 gene and HygAll genes could obtain the target amplification bands, but the bands were the brightest at the annealing temperature of 62.0 °C and there were no non-specific bands. For the Sevir.3G057200 gene and Hyg The PCR products of the gene were sequenced and aligned, which were consistent with the Sevir.3G057200 gene of Setaria viridis and Hyg the sequences of the gene were all the same. Therefore, the primer pair 200-F / 200-R could be used for the detection of the Sevir.3G057200 gene of Setaria viridis, and Hyg-F / Hyg-R could be used for the detection of the exogenous Hyg gene, and it was determined that the annealing temperature was 62.0 °C.
[0030] Example 3 PCR identification of transgenic Setaria viridis positive plants Using the Hyg gene primer pair in Example 2 to perform PCR identification on transgenic Setaria viridis plants, which specifically included the following steps: Collect the leaves of 24 transgenic Setaria viridis plants (numbered SV-1 to SV-24 in sequence), extract their genomic DNA using the MEGA Plant Genomic DNA Rapid Extraction Kit (D3187-02) as the DNA template, and use the genomic DNA of wild-type Setaria viridis as Hyg the negative control, and use the genomic DNA of transgenic Setaria viridis containing Hyg gene as Hyg the positive control, and perform Hyg gene PCR experiments through Hyg-F / Hyg-R using the method in Example 2 to exclude transgenic-negative Setaria viridis plants. Among them, the PCR reaction amplification system and reaction conditions were the same as those in the PCR reaction amplification system and reaction conditions for detecting Hyg gene in Example 2, and the annealing temperature was 62.0 °C.
[0031] After the PCR amplification reaction was completed, take 5 μL of the corresponding PCR product and perform electrophoresis detection on a 2% agarose gel. If a target band of about 143 bp appears, it is a transgenic Setaria viridis positive plant; if there is no target band, it is a transgenic negative plant. The PCR detection results of 24 transgenic Setaria viridis plants are shown in Figure 3 , among which, the plants numbered SV-1 to SV-6, SV-8 to SV-10, SV-13 to SV-19, SV-21, and SV-23 were all transgenic positive plants, a total of 18 plants; the plants numbered SV-7, SV-11, SV-12, SV-20, SV-22, and SV-24 were all transgenic negative plants, a total of 6 plants.
[0032] Example 4 Digital PCR detection The method for detecting the copy number of exogenous gene insertion in Setaria viridis by digital PCR includes the following specific steps: 1) Digital PCR primers and probes Primer pair sequence and probe sequence for detecting the reference gene Sevir.3G057200: Forward primer 200-F: 5’-GTTCCGCACCAACATCTACTCCTAC-3’, as shown in SEQ ID NO: 1; Reverse primer 200-R: 5’-GTTGATGACGCTGCTCCCTTCC-3’, as shown in SEQ ID NO: 2; Probe 200-P: VIC-CCTACTTCCTGGTGACCAAGCACGC-MGB, as shown in SEQ ID NO: 3; Detection Hyg Primer pair and probe sequence for screening marker gene: Forward primer Hyg-F: 5’-GTCAAGACCAATGCGGAGCA-3’, as shown in SEQ ID NO: 4; Reverse primer Hyg-R: 5’-CCCAATACGAGGTCGCCAAC-3’, as shown in SEQ ID NO: 5; Probe Hyg-P: FAM-TCGAAGTAGCGCGTCTGCTGCTCCA-BHQ1, as shown in SEQ ID NO: 6.
[0033] 2) DNA template extraction Nine plants (SV-1 to SV-6 and SV-8 to SV-10) were selected from the 18 transgenic positive plants screened in Example 3 as test samples, and the transgenic negative plant SV-7 was used as a negative control. The corresponding genomic DNA was extracted using the MEGA plant genome kit as the DNA template.
[0034] 3) Detecting the inserted copy number of foreign genes in the test samples by digital PCR Using the Sevir.3G057200 gene as the reference gene, HygThe gene is an exogenous gene. The insertion copy number of the exogenous gene in the genome of transgenic Setaria viridis is detected by digital PCR experiment. Three replicates are set for each sample to be tested, and the DNA of transgenic negative plant SV-7 is used as the negative control. Using the MicroDrop microdrop digital PCR system, the reaction system is as follows: 10.00 µL of reaction premix for microdrop digital PCR, 1.80 µL of 200-F (10.00 µM), 1.80 µL of 200-R (10.00 µM), 1.80 µL of Hyg-F (10.00 µM), 1.80 µL of Hyg-R (10.00 µM), 0.50 µL of probe 200-P (10.00 µM), 0.50 µL of probe Hyg-P (10.00 µM), 1 µL of DNA template (30.00 ng / µL), and 0.80 µL of nuclease-free water, with a total volume of 20.00 µL.
[0035] The reaction amplification program is as follows: 95°C for 10 min; 95°C for 30 s, 62°C for 1 min, 45 cycles; 98°C for 10 min.
[0036] 4) Result analysis After the reaction is completed, the obtained chip is placed in a biochip analyzer to read the FAM and VIC fluorescence signals, and the QuantDrop Software is used for fluorescence data analysis to obtain the copy numbers (copy / µL) of the exogenous gene and the reference gene in the corresponding DNA template, calculate the copy number ratio of the exogenous gene to the reference gene in the DNA template, and judge the copy number of the exogenous gene in the Setaria viridis genome through the ratio.
[0037] Since Setaria viridis is a diploid plant, the copy number ratio of the single-copy inserted exogenous gene fragment to the reference gene fragment is about 0.5 (i.e., the general copy number ratio of the single-copy inserted exogenous gene fragment to the reference gene fragment is 0.26 - 0.74), the copy number ratio of the double-copy inserted exogenous gene fragment to the reference gene fragment is about 1 (i.e., the general copy number ratio of the double-copy inserted exogenous gene fragment to the reference gene fragment is 0.75 - 1.25), the copy number ratio of the triple-copy inserted exogenous gene fragment to the reference gene fragment is about 1.5 (i.e., the general copy number ratio of the double-copy inserted exogenous gene fragment to the reference gene fragment is 1.26 - 1.74), the copy number ratio of the quadruple-copy inserted exogenous gene fragment to the reference gene fragment is about 2 (i.e., the general copy number ratio of the double-copy inserted exogenous gene fragment to the reference gene fragment is 1.75 - 2.25), and so on for the insertion copy numbers of multi-copy exogenous gene fragments such as five-copy and six-copy.
[0038] In this example, the digital PCR scatter plots of 9 transgenic positive Setaria viridis plants are shown in Figure 4, the copy number detection results are shown in Table 1. The copy number ratios of transgenic positive plants SV-1, SV-4, and SV-5 are close to 0.5, indicating single-copy insertion; the copy number ratios of transgenic positive plants SV-2, SV-3, SV-8, and SV-9 are all close to 1, indicating double-copy insertion; the copy number ratios of transgenic positive plants SV-6 and SV-10 are close to 2, indicating quadruple-copy insertion. The results obtained from the two reference genes RA1 and Sevir.3G057200 are consistent, indicating that both of these two genes can be used as reference genes for detecting the inserted copy number of foreign genes in transgenic foxtail millet.
[0039] Table 1 Results of digital PCR for detecting the inserted copy number of foreign genes in transgenic foxtail millet
[0040] From Figure 4 and Table 1, it can be seen that the copy number ratios of 3 transgenic positive plants (SV-1, SV-4, and SV-5) are approximately 0.5, which are transgenic plants with single-copy insertion. The copy number ratios of 4 transgenic positive plants (SV-2, SV-3, SV-8, and SV-9) are approximately 1, which are transgenic plants with double-copy insertion. The copy number ratios of 2 transgenic positive plants (SV-6 and SV-10) are approximately 2, which are transgenic plants with quadruple-copy insertion. The above results show that the method for detecting the inserted copy number of foreign genes in foxtail millet using digital PCR in this example has the advantages of absolute quantification, high accuracy, simplicity and rapidity, low sample requirement, and the ability to perform batch detection. It can be used to quickly screen out plants with low-copy insertion of foreign genes from a large number of transgenic plants, saving a large amount of time and cost for the breeding of excellent transgenic lines.
[0041] Other parts not described in detail are all prior arts. Although the above embodiments have described the present invention in detail, they are only a part of the embodiments of the present invention, not all embodiments. Those of ordinary skill in the art can also obtain other embodiments based on this example without creative efforts, and these embodiments all fall within the protection scope of the present invention.
Claims
1. A method for detecting the number of inserted copies of exogenous genes in Setaria or millet using digital PCR, characterized in that: The method uses Sevir.3G057200 gene as an internal reference gene. Hyg The screening marker gene is used as an exogenous gene, and the genomic DNA of the transgenic Setaria viridis or transgenic millet is detected using 200-F / 200-R and the primer pair Hyg-F / Hyg-R, and the number of inserted copies of the exogenous gene in the transgenic Setaria viridis or transgenic millet is determined; Primer pair sequence for detecting the internal reference gene Sevir.3G057200: Forward primer 200-F is shown in SEQ ID NO: 1; Reverse primer 200-R is shown in SEQ ID NO: 2; Probe 200-P is shown in SEQ ID NO: 3; Detection Hyg Primer pairs and probe sequences for screening marker genes: The forward primer Hyg-F is shown in SEQ ID NO: 4; The reverse primer Hyg-R is shown in SEQ ID NO: 5; The probe Hyg-P is shown in SEQ ID NO: 6; The method for determining the number of inserted copies of exogenous genes in transgenic foxtail grass or transgenic millet is as follows: When the copy number ratio of the exogenous gene to the internal reference gene was 0.26-0.74, the transgenic Setaria viridis or transgenic millet was a single copy insertion; When the copy number ratio of the exogenous gene to the internal reference gene is 0.75-1.25, the transgenic Setaria viridis or transgenic millet has a double copy insertion; When the copy number ratio of the exogenous gene to the internal reference gene was 1.26-1.74, the transgenic Setaria viridis or transgenic millet had three copies inserted; When the copy number ratio of the exogenous gene to the internal reference gene was 1.75-2.25, the transgenic Setaria viridis or transgenic millet had four copies inserted; The same can be said for other insertion copy numbers of transgenic Setaria viridis or transgenic millet.
2. The method according to claim 1, characterized in that: The method uses the genomic DNA of transgenic foxtail grass or transgenic millet as a DNA template and uses a primer pair RA1-F / RA1-R and a primer pair Hyg-F / Hyg-R to perform digital PCR detection.
3. The method according to claim 2, characterized in that The reaction system of digital PCR is: reaction premix, 200-F, 200-R, Hyg-F, Hyg-R, probe 200-P, probe Hyg-P, DNA template and nuclease-free water.
4. The method according to claim 3, characterized in that: The reaction premix is a reaction premix for droplet digital PCR.
5. The method according to any one of claims 2 to 4, characterized in that: The reaction system of digital PCR was as follows: 10.00µL of reaction premix, 1.80µL of 200-F with a concentration of 10.00µM, 1.80µL of 200-R with a concentration of 10.00µM, 1.80µL of Hyg-F with a concentration of 10.00µM, 1.80µL of Hyg-R with a concentration of 10.00µM, 0.50µL of probe 200-P with a concentration of 10.00µM, 0.50µL of probe Hyg-P with a concentration of 10.00µM, 1µL of DNA template with a concentration of 30.00ng / µL, and 0.80µL of nuclease-free water, with a total volume of 20.00µL.
6. The method according to any one of claims 1 to 4, characterized in that The amplification program of digital PCR was: 95°C for 10 min; 95°C for 30 s, 56.4-69.6°C for 1 min, 45 cycles; 98°C for 10 min.
7. The method according to any one of claims 1 to 4, characterized in that The amplification program of digital PCR was: 95°C for 10 min; 95°C for 30 s, 62°C for 1 min, 45 cycles; 98°C for 10 min.
8. The method according to any one of claims 1 to 4, characterized in that After the digital PCR reaction is completed, the copy numbers of the exogenous gene and the internal reference gene are obtained, and the copy number ratio of the exogenous gene to the internal reference gene in the transgenic Setaria viridis or transgenic millet is calculated. The insertion copy number of the exogenous gene in the genome of the transgenic Setaria viridis or transgenic millet is determined by the ratio.
9. The method according to any one of claims 1 to 4, characterized in that: The copy numbers of the exogenous gene and the internal reference gene are obtained by placing the chip in a biochip analyzer to read the fluorescence signal and perform fluorescence data analysis.
10. The method according to any one of claims 1 to 4, characterized in that The genomic DNA of the transgenic foxtail grass or transgenic millet is extracted using a plant genome kit.
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Microdroplet digital PCR detection method for simultaneously detecting penicillium and fusarium
CN112852988A