Method for detecting exogenous gene insertion copy number in green bristlegrass or millet by adopting fluorescent quantitative PCR (Polymerase Chain Reaction) or digital PCR

Abstract

The invention provides a method for detecting the insertion copy number of an exogenous gene in green bristlegrass or millet by adopting fluorescent quantitative PCR (Polymerase Chain Reaction) or digital PCR, and belongs to the technical field of biology.According to the method, the insertion copy number of the exogenous gene in green bristlegrass or millet is detected by taking an RA1 gene as a reference gene, taking a Hyg selection marker gene as an exogenous gene and utilizing a primer pair RA1-F / RA1-R and a primer pair Hyg-F / Hyg-R; and detecting the genome DNA of the transgenic green bristlegrass or the transgenic foxtail millet, and judging the insertion copy number of the exogenous gene in the transgenic green bristlegrass or the transgenic foxtail millet. The invention provides a method for detecting the exogenous gene insertion copy number in green bristlegrass or millet by adopting fluorescent quantitative PCR or digital PCR, has the characteristics of simplicity, rapidness, low sample dosage, high efficiency, high throughput and the like, and can improve the efficiency of screening low-copy inserted green bristlegrass or millet transgenic strains; and a new choice is provided for the detection method of the insertion copy number of the exogenous gene in the research of the transgenic green bristlegrass or the transgenic foxtail millet.

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 insertion in Setaria viridis or foxtail millet by fluorescence quantitative PCR or 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, which has the advantages of short growth cycle, short plant height, easy cultivation, small genome, diploid, and ability to produce a large number of self-crossed line seeds compared with the dicotyledonous model plant Arabidopsis thaliana. It is an excellent monocotyledonous model plant. Due to its origin in the tropics and having 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 re-sequencing 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 transgenic breeding research 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 can generally be highly expressed and stably inherited. Therefore, in the transgenic plant breeding research, transgenic lines with low-copy insertion of foreign genes are usually selected. However, transgenic lines with low-copy insertion 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 insertion 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, fluorescence quantitative PCR (qPCR) and digital PCR (dPCR) have been successfully applied to the detection of the copy number of foreign gene insertions in a variety of transgenic crops due to their simple, rapid, and efficient characteristics. However, there are still no reports on the detection of the copy number of foreign gene insertions in transgenic foxtail millet using these two PCR methods. Therefore, there is an urgent need to establish a method for quickly detecting the copy number of foreign gene insertions in transgenic foxtail millet using qPCR and dPCR. Summary of the Invention

[0005] In view of the above problems, the present invention provides a method for detecting the copy number of foreign gene insertions in foxtail millet or common millet using fluorescence quantitative PCR or digital PCR.

[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 foxtail millet or common millet using fluorescence quantitative PCR uses RA1 the gene as an internal reference gene and Hyg the selectable marker gene as a foreign gene, that is, the selectable marker gene hygromycin phosphotransferase gene (hygromycin phosphotransferase, Hyg ). Using the primer pairs RA1-F / RA1-R and Hyg-F / Hyg-R, the genomic DNA of transgenic foxtail millet or transgenic common millet is detected, and the copy number of foreign gene insertions in transgenic foxtail millet or transgenic common millet is judged; Among them, RA1 the gene (Sevir.2G209800) is a single-copy gene in foxtail millet, the corresponding gene number in common millet is Seita.2G201700, and it is also a single-copy gene in common millet. The two gene sequences are exactly the same, and the primer pair sequences of the two are also exactly the same; RA1 the sequence of the gene (Sevir.2G209800) in foxtail millet is as shown in SEQ ID NO: 7; RA1 the sequence of the gene (Seita.2G201700) in common millet is as shown in SEQ ID NO: 8; The primer pair sequence for detecting the internal reference gene RA1 : The forward primer RA1-F is: 5’- CCTAGTCGCGCAGTTGTTGA-3’, as shown in SEQ ID NO: 1; The reverse primer RA1-R is: 5’- ATGCCAAGGAGAAACGGCAG-3’, as shown in SEQ ID NO: 2; Detection Hyg Primer pair sequences for screening marker genes: The forward primer Hyg-F is: 5’-GTCAAGACCAATGCGGAGCA-3’, as shown in SEQ ID NO: 3; The reverse primer Hyg-R is: 5’-CCCAATACGAGGTCGCCAAC-3’, as shown in SEQ ID NO: 4; During the detection by fluorescence quantitative PCR, the method for judging the copy number of foreign gene insertion in transgenic Setaria viridis or transgenic Setaria italica is as follows: When the relative copy number of the foreign gene is about 0.5 (i.e., when the relative copy number of the foreign gene is 0.26 - 0.74), the transgenic Setaria viridis or transgenic Setaria italica has a single-copy insertion; When the relative copy number of the foreign gene is about 1 (i.e., when the relative copy number of the foreign gene is 0.75 - 1.25), the transgenic Setaria viridis or transgenic Setaria italica has a double-copy insertion; When the relative copy number of the foreign gene is about 1.5 (the relative copy number of the foreign gene is 1.26 - 1.74), the transgenic Setaria viridis or transgenic Setaria italica has a triple-copy insertion; When the relative copy number of the foreign gene is about 2 (the relative copy number of the foreign gene is 1.75 - 2.25), the transgenic Setaria viridis or transgenic Setaria italica has a quadruple-copy insertion; And so on for other insertion copy numbers of transgenic Setaria viridis or transgenic Setaria italica, such as five-copy insertion, six-copy insertion, etc.

[0007] Furthermore, during the detection by fluorescence quantitative PCR, using the genomic DNA of transgenic Setaria viridis or transgenic Setaria italica as the DNA template, fluorescence quantitative PCR detection is carried out using the primer pairs RA1-F / RA1-R and Hyg-F / Hyg-R.

[0008] Furthermore, during the detection by fluorescence quantitative PCR, amplification RA1 The reaction system for the gene is: 2 x qPCRMaster Mix 10.00μL, Low Rox dye 0.20μL, RA1-F with a concentration of 10.00µM 0.40μL, RA1-R with a concentration of 10.00µM 0.40μL, DNA template with a concentration of 30.00ng / µL 2.00μL, and ddH 2 O 7.00μL, with a total volume of 20.00µL.

[0009] Furthermore, in the process of fluorescence quantitative PCR detection, the amplification Hyg The reaction system of the gene was: 2 x qPCR Master Mix 10.00μL, Low Rox dye 0.20μL, Hyg-F 0.40μL with a concentration of 10.00μM, Hyg-R 0.40μL with a concentration of 10.00μM, DNA template 2.00μL with a concentration of 30.00ng / μL, and ddH 2 O 7.00μL, total volume 20.00µL.

[0010] Furthermore, in the process of fluorescence quantitative PCR detection, the amplification RA1 Gene and Hyg The gene amplification program was as follows: pre-denaturation at 95°C for 30 s; denaturation at 95°C for 10 s, annealing at 62°C for 30 s, and 40 cycles; melting curve was performed at 95°C for 15 s, 62°C for 60 s, and 95°C for 15 s.

[0011] Furthermore, the relative copy number of the exogenous gene was calculated by using the known double-copy transgenic foxtail grass or transgenic millet as a reference factor. The method is used to analyze the multiples of the copy number of the tested transgenic Setaria viridis or the tested transgenic millet relative to the reference factor.

[0012] A method for detecting the number of inserted copies of exogenous genes in Setaria or millet using digital PCR is based on RA1 The gene was used as an internal reference gene. Hyg Screening marker genes as exogenous genes, using primer pairs RA1-F / RA1-R and primer pairs Hyg-F / Hyg-R to detect genomic DNA of transgenic Setaria or transgenic millet, and determining the number of inserted copies of the exogenous genes in the transgenic Setaria or transgenic millet; Detection of internal reference genes RA1 Primer pairs and probe sequences: The forward primer RA1-F is: 5'-CCTAGTCGCGCAGTTGTTGA-3', as shown in SEQ ID NO: 1; The reverse primer RA1-R is: 5'-ATGCCAAGGAGAAACGGCAG-3', as shown in SEQ ID NO: 2; Probe RA1-P is: VIC-TCATGAGCTCAGGTTGTCGCGCGCA-MGB, as shown in SEQ ID NO: 5; 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: 3; The reverse primer Hyg-R is: 5’-CCCAATACGAGGTCGCCAAC-3’, as shown in SEQ ID NO: 4; The probe Hyg-P is: FAM-TCGAAGTAGCGCGTCTGCTGCTCCA-BHQ1, as shown in SEQ ID NO: 6; During the digital PCR detection, the method for determining the copy number of the foreign gene inserted in transgenic Setaria viridis or transgenic Setaria italica is as follows: When the copy number ratio of the foreign gene to the reference gene is approximately 0.5 (i.e., when the copy number ratio of the foreign gene to the reference gene is 0.26 - 0.74), 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 approximately 1 (the copy number ratio of the foreign gene to the reference gene is 0.75 - 1.25), 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 approximately 1.5 (the copy number ratio of the foreign gene to the reference gene is 1.26 - 1.74), 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 approximately 2 (the copy number ratio of the foreign gene to the reference gene is 1.75 - 2.25), transgenic Setaria viridis or transgenic Setaria italica has a quadruple-copy insertion; And so on for other insertion copy numbers of transgenic Setaria viridis or transgenic Setaria italica, such as five-copy insertion, six-copy insertion, etc.

[0013] Furthermore, during the digital PCR detection, using the genomic DNA of transgenic Setaria viridis or transgenic Setaria italica as the DNA template, digital PCR detection is carried out using the primer pairs RA1-F / RA1-R and Hyg-F / Hyg-R.

[0014] Furthermore, the reaction system for digital PCR is: 10.00 µL of reaction premix for droplet digital PCR, 1.80 µL of RA1-F with a concentration of 10.00 µM, 1.80 µL of RA1-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 the probe RA1-P with a concentration of 10.00 µM, 0.50 µL of the 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.

[0015] Further, the amplification program of 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; After the digital PCR reaction is completed, the chip is placed in a biochip analyzer to read the FAM and VIC fluorescence signals, and the fluorescence data is analyzed using QuantDrop Software to obtain the copy numbers (copy / µL) of the foreign gene and the reference gene in the DNA template, calculate the ratio of the copy numbers of the foreign gene to the reference gene in transgenic Setaria viridis or transgenic foxtail millet, and determine the inserted copy number of the foreign gene in the genome of transgenic Setaria viridis or transgenic foxtail millet through the ratio.

[0016] The beneficial effects of a method for detecting the inserted copy number of foreign genes in Setaria viridis or foxtail millet using fluorescence quantitative PCR or digital PCR according to the present invention are as follows: The single-copy Setaria viridis gene or foxtail millet gene provided by the present invention can be used as a reference gene for detecting the inserted copy number of foreign genes in transgenic Setaria viridis or transgenic foxtail millet, and the most commonly used selection marker Hyg in transgenic Setaria viridis or transgenic foxtail millet is used as the foreign gene, and the rapid and high-throughput detection of the inserted copy number of foreign genes in transgenic Setaria viridis or transgenic foxtail millet is realized by using digital PCR or fluorescence quantitative PCR technology; The present invention provides a method for high-throughput detecting the inserted copy number of foreign genes in transgenic Setaria viridis or transgenic foxtail millet using digital PCR or fluorescence quantitative PCR technology, which has the characteristics of being simple, rapid, low sample requirement, efficient, and high-throughput, can improve the efficiency of large-scale screening of transgenic lines, and can provide a new option for detecting the inserted copy number of foreign genes in transgenic Setaria viridis or transgenic foxtail millet breeding research; A method for detecting the inserted copy number of foreign genes in Setaria viridis or foxtail millet using fluorescence quantitative PCR provided by the present invention can be used for all Hyg transgenic Setaria viridis with RA1 as the selection marker for copy number detection; the Hyg gene of Setaria viridis in the present invention has exactly the same nucleotide sequence as its corresponding gene Seita.2G201700 in foxtail millet, and the primer pair sequences of the two are also exactly the same; therefore, the method provided by the present invention is also applicable to the copy number detection of transgenic foxtail millet with Hyg as the selection marker; this method has the advantages of being simple, rapid, low cost, and low sample requirement, and its accuracy is sufficient to meet the breeding requirements for detecting single and low copies, and is more suitable for batch detection; A method for detecting the inserted copy number of foreign genes in Setaria viridis or foxtail millet using digital PCR provided by the present invention can be used for all HygCopy number detection of transgenic Setaria viridis or transgenic foxtail millet with a screening marker; In the present invention, the nucleotide sequence of the gene in Setaria viridis is exactly the same as that of its corresponding gene Seita.2G201700 in foxtail millet, and the primer pair sequences of the two are also exactly the same; Therefore, the method provided by the present invention is also applicable to the copy number detection of transgenic foxtail millet with RA1 as a screening marker; This method has the advantages of absolute quantification, high accuracy, simplicity and rapidity, small sample requirement, and batch detection. Hyg BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 is the alignment result of the Setaria viridis gene in Example 1 of the present invention RA1 in the genome; Figure 2 is the exploration result of the PCR amplification conditions of the reference gene and the exogenous 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 respectively; Labels 1 to 8 represent the PCR products at annealing temperatures of 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 respectively; 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 respectively; 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 in Example 4 of the present invention RA1 gene and Hyg gene digital PCR scatter plot; The blue scatter points represent Hyg gene fluorescence pore numbers; The green scatter points represent RA1 gene fluorescence pore numbers; The red scatter points represent RA1 gene and Hyg gene fluorescence pore numbers, and the gray scatter points represent signal-free pore numbers; Figures 5 to 8 is in Example 5 of the present invention RA1 gene and Hyg gene fluorescence quantitative primer verification diagram; Among them, Figure 5 is RA1 gene amplification curve diagram; Figure 6 is RA1 gene melting curve diagram; Figure 7 is​Hyg Amplification curve of the gene; Figure 8 is Hyg Melting curve of the gene; Figure 9 It is the result of the fluorescence quantitative PCR copy number detection of 17 transgenic positive plants in Example 5 of the present invention; wherein, the abscissa is the number of transgenic positive plants, and the ordinate is the relative copy number, and the transgenic positive plant SV-3 is used as the reference sample. Detailed implementation manners

[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 with reference to 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" by Sambrook et al., translated by Huang Peitang et al., the third edition, Science Press) or according to the product specifications. For the reagents not indicating the manufacturer, they are all conventional products that can be purchased.

[0020] Example 1 Determination of reference gene RA1 Bioinformatics analysis of the copy number of the gene in the Setaria viridis genome and the foxtail millet genome specifically includes the following steps: On the Phytozome website (https: / / phytozome-next.jgi.doe.gov / ), using RA1 the nucleotide sequence of the gene (Sevir.2G209800) as the query sequence to query in the Setaria viridis genome, and obtaining the alignment result, as shown in Figure 1 . It can be seen from the alignment result that RA1 the gene is a single-copy gene and there are no other homologous genes with similar sequences, so it can be used as a reference gene for detecting the inserted copy number of foreign genes in transgenic Setaria viridis.

[0021] RA1 The gene number corresponding to the gene in foxtail millet is Seita.2G201700, and the RA1 gene (Sevir.2G209800) in Setaria viridis and the RA1 gene (Seita.2G201700) in foxtail millet have exactly the same gene sequences. Among them, the RA1The sequence of the gene (Sevir.2G209800) is as shown in SEQ ID NO: 7; in foxtail millet RA1 the sequence of the gene (Seita.2G201700) is as shown in SEQ ID NO: 8.

[0022] Since the RA1 gene (Sevir.2G209800) in Setaria viridis is RA1 identical to the two-gene sequence of the gene (Seita.2G201700) in foxtail millet, and both are single-copy genes. Therefore, the primers and probes designed based on the RA1 gene in Setaria viridis are also applicable to amplify and detect the RA1 gene in foxtail millet (i.e., the primer pair sequence is identical to the sequence in the corresponding Seita.2G201700 gene in foxtail millet).

[0023] Meanwhile, since the commonly used selection marker genes in transgenic Setaria viridis and transgenic foxtail millet are both Hyg genes, and both can Hyg use the

[0024] Example 2 Exploration of PCR amplification conditions Since the commonly used selection marker gene in transgenic Setaria viridis is Hyg gene, therefore, Hyg the selection marker gene is used as the foreign gene in transgenic Setaria viridis, and RA1 gene is used 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 RA1 include: The forward primer RA1-F is: 5’- CCTAGTCGCGCAGTTGTTGA-3’, as shown in SEQ ID NO: 1; The reverse primer RA1-R is: 5'-ATGCCAAGGAGAAACGGCAG-3', as shown in SEQ ID NO: 2; Detection Hyg The primer pair sequences for screening marker genes include: 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; 2) RA1 Gene primers and Hyg Exploration of PCR amplification conditions for gene primers Using the DNA of a transgenic Setaria viridis plant as template, gradient PCR reactions were performed using the RA1-F / RA1-R primer pair and the Hyg-F / Hyg-R primer pair.

[0025] The PCR reaction system for detecting the internal reference gene RA1 was: Green Master Mix 12.50µL, RA1-F (10.00µM) 1.00µL, RA1-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 exogenous genes was as follows: Green Master Mix 12.50 µL, Hyg-F (10.00 µM) 1.00 µL, Hyg-R (10.00 µM) 1.00 µL, DNA template (30.00 ng / µL) 1.00 µL, and nuclease-free water 9.50 µL, with a total volume of 25.00 µL.

[0027] The PCR reaction conditions were as follows: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 s, annealing for 30 s, and extension at 72°C for 30 s, for a total of 35 cycles; finally extension at 72°C for 7 min, and storage at 4°C; the annealing temperatures were 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, respectively.

[0028] After the PCR amplification reaction is completed, take 5 μL of the corresponding PCR product and perform electrophoresis on 2% agarose gel. RA1 and Hyg The PCR products of 111 bp and 143 bp were the amplified bands of the target gene. The remaining PCR products were sent for sequencing. The sequencing results were consistent with RA1 Genes andHyg Align the nucleotide sequences of the genes.

[0029] The electrophoresis results are as Figure 2 shown. When the annealing temperatures are 56.4 °C, 58.0 °C, 60.0 °C, and 62.0 °C respectively, RA1 the gene and Hyg the gene can both obtain the target amplification bands, but the band is the brightest at the annealing temperature of 62.0 °C and there are no non-specific bands. RA1 the gene and Hyg the PCR products of the gene are sequenced and aligned with the genes of Setaria viridis RA1 the gene and Hyg the sequences of the genes are all consistent. Therefore, the primer pair RA1-F / RA1-R can be used for the detection of the gene in Setaria viridis, and Hyg-F / Hyg-R can be used for the detection of the exogenous gene RA1 the gene, and it is determined that the annealing temperature is 62.0 °C for both. Hyg the gene.

[0030] Example 3 PCR Identification of Transgenic Setaria viridis Positive Plants Use the Hyg gene primer pair in Example 2 to perform PCR identification on transgenic Setaria viridis plants, which specifically includes the following steps: Collect the leaves of 24 transgenic Setaria viridis plants (sequentially numbered SV-1 to SV-24), 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 the gene as Hyg the positive control. Perform the PCR experiment of the Hyg gene using Hyg-F / Hyg-R by the method in Example 2 to exclude transgenic Setaria viridis plants with negative identification. Among them, the PCR reaction amplification system and reaction conditions are the same as those for detecting the Hyg gene in Example 2, and the annealing temperature is 62.0 °C.

[0031] After the PCR amplification reaction is 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 are 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 are all transgenic negative plants, a total of 6 plants.

[0032] Example 4 Digital PCR Detection A method for detecting the copy number of inserted foreign genes in Setaria viridis by digital PCR includes the following specific steps: 1) Digital PCR primers and probes Primer pair and probe sequence for detecting the reference gene RA1 : Forward primer RA1-F is: 5’- CCTAGTCGCGCAGTTGTTGA-3’, as shown in SEQ ID NO: 1; Reverse primer RA1-R is: 5’- ATGCCAAGGAGAAACGGCAG-3’, as shown in SEQ ID NO: 2; Probe RA1-P is: VIC-TCATGAGCTCAGGTTGTCGCGCGCA-MGB, as shown in SEQ ID NO: 5; Detection Hyg Primer pair and probe sequence for screening marker gene: Forward primer Hyg-F is: 5’-GTCAAGACCAATGCGGAGCA-3’, as shown in SEQ ID NO: 3; Reverse primer Hyg-R is: 5’-CCCAATACGAGGTCGCCAAC-3’, as shown in SEQ ID NO: 4; Probe Hyg-P is: 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 copy number of inserted foreign genes in the test samples by digital PCR Using RA1 the 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 droplet digital PCR system, the reaction system is as follows: 10.00 μL of reaction premix for droplet digital PCR, 1.80 μL of RA1-F with a concentration of 10.00 μM, 1.80 μL of RA1-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 RA1-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.

[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 put into 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 internal reference gene in the corresponding DNA template, calculate the copy number ratio of the exogenous gene and the internal 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 internal reference gene fragment is about 0.5 (that is, the general copy number ratio of the single-copy inserted exogenous gene fragment to the internal reference gene fragment is 0.26 - 0.74), the copy number ratio of the double-copy inserted exogenous gene fragment to the internal reference gene fragment is about 1 (that is, the general copy number ratio of the double-copy inserted exogenous gene fragment to the internal reference gene fragment is 0.75 - 1.25), the copy number ratio of the triple-copy inserted exogenous gene fragment to the internal reference gene fragment is about 1.5 (that is, the general copy number ratio of the double-copy inserted exogenous gene fragment to the internal reference gene fragment is 1.26 - 1.74), the copy number ratio of the quadruple-copy inserted exogenous gene fragment to the internal reference gene fragment is about 2 (that is, the general copy number ratio of the double-copy inserted exogenous gene fragment to the internal 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] The digital PCR scatter plots of 9 transgenic positive Setaria viridis plants in this example are shown in Figure 4, the copy number detection results are shown in Table 1. The copy number ratios of the transgenic positive plants SV-1, SV-4, and SV-5 are close to 0.5, indicating single-copy insertion; the copy number ratios of the 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 the transgenic positive plants SV-6 and SV-10 are close to 2, indicating four-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 Setaria viridis.

[0039] Table 1 Results of digital PCR for detecting the inserted copy number of foreign genes in transgenic Setaria viridis

[0040] Example 5 Fluorescent quantitative PCR detection The method for detecting the inserted copy number of foreign genes in Setaria viridis by fluorescent quantitative PCR includes the following specific steps: 1) Fluorescent quantitative PCR primers Primer pair sequences for detecting the reference gene RA1 : The forward primer RA1-F is: 5’-CCTAGTCGCGCAGTTGTTGA-3’, as shown in SEQ ID NO: 1; The reverse primer RA1-R is: 5’-ATGCCAAGGAGAAACGGCAG-3’, as shown in SEQ ID NO: 2; Primer pair sequences for detecting the foreign gene Hyg screening marker gene: The forward primer Hyg-F is: 5’-GTCAAGACCAATGCGGAGCA-3’, as shown in SEQ ID NO: 3; The reverse primer Hyg-R is: 5’-CCCAATACGAGGTCGCCAAC-3’, as shown in SEQ ID NO: 4.

[0041] 2) DNA template extraction Using the 17 transgenic positive plants screened in Example 3 as the test samples, and the numbers of the 18 transgenic positive plants are in sequence: SV-1, SV-2, SV-4~SV-6, SV-8~SV-10, SV-13~SV-19, SV-21, and SV-23. Using the transgenic positive plant SV-3 with double copies detected in Example 4 as a control, and the non-transgenic negative plant SV-7 screened in Example 3 as a negative control, use the MEGA plant genome kit to extract the corresponding genomic DNA as the DNA template.

[0042] 3) Primer verification Select four known transgenic positive Setaria DNA templates for fluorescence quantitative PCR experiments to verify the internal reference gene RA1 and foreign genes Hyg The reaction was carried out in a QuantStudioTM 1 plus real-time fluorescence quantitative PCR instrument (Thermo Fisher (Shanghai) Instrument Co., Ltd.); Amplification RA1 The reaction system of the gene was: 2 x qPCR Master Mix 10.00μL, Low Rox dye 0.20μL, RA1-F 0.40μL at a concentration of 10.00μM, RA1-R 0.40μL at a concentration of 10.00μM, DNA template 2.00μL at a concentration of 30.00ng / μL, and ddH 2 O 7.00μL, total volume 20.00µL; Amplification Hyg The reaction system of the gene was: 2 x qPCR Master Mix 10.00μL, Low Rox dye 0.20μL, Hyg-F 0.40μL with a concentration of 10.00μM, Hyg-R 0.40μL with a concentration of 10.00μM, DNA template 2.00μL with a concentration of 30.00ng / μL, and ddH 2 O 7.00μL, total volume 20.00µL.

[0043] Amplification RA1 Gene and Hyg The amplification procedures of the genes were: 95℃ pre-denaturation for 30s; 95℃ denaturation for 10s, 62℃ annealing for 30s, 40 cycles; melting curve 95℃ 15s, 62℃ 60s, 95℃ 15s. Each group of experiments was repeated 3 times. The results of the amplification curves and melting curves of the two primer pairs are shown in Figures 5 to 8 It can be seen that both pairs of primers have obvious amplification curves, and the melting curves have unique absorption peaks, which can be used as fluorescent quantitative PCR detection primers.

[0044] 4) Fluorescence quantitative PCR detection of the number of copies of exogenous genes in the sample to be tested by RA1 The gene is an internal reference gene. HygThe gene is an exogenous gene, and the double-copy transgenic positive plant SV-3 is used as a control. The insertion copy number of the exogenous gene in the genome of Setaria viridis of all the samples to be tested (i.e., 17 transgenic positive plants) is detected by the above-mentioned fluorescence quantitative PCR experiment. Among them, the reaction system and amplification procedure of the fluorescence quantitative PCR detection are the same as the reaction system and amplification procedure in step 3) of the primer verification of this embodiment, and each sample to be tested is tested 3 times.

[0045] 5) Result analysis After the reaction, the data were analyzed using the QuantStudio™ Design & Analysis Software v1.5.2 system. Analysis of transgenic positive plants Hyg The relative copy number of the gene is defined as the transgenic Setaria viridis positive plant SV-3 with a known double copy as the reference factor, which is 1. The multiple of the copy number of each sample to be tested relative to the reference factor is , so the single-copy insertion plants Hyg The relative copy number of the gene should be 1 / 2 of the double copy control, that is, about 0.5 (usually 0.26~0.74); Hyg The relative copy number of the gene should be equal to that of the two-copy control, that is, about 1 (usually 0.75~1.25); Hyg The relative copy number of the gene should be 3 / 2 of that of the two-copy control, that is, about 1.5 (usually 1.26~1.74); Hyg The relative copy number of the gene should be 2 of the two-copy control, that is, about 2 (that is, the general copy number ratio of the two-copy inserted exogenous gene fragment to the internal reference gene fragment is 1.75~2.25), and the inserted copy number of five-copy, six-copy and other multi-copy exogenous gene fragments is similar.

[0046] The results of the fluorescence quantitative PCR detection of the copy number of 17 transgenic Setaria positive plants in this example are shown in Figure 9 and Table 2.

[0047] Table 2 Results of fluorescence quantitative PCR detection of the copy number of exogenous genes inserted into transgenic Setaria viridis

[0048] Depend on Figure 9 As can be seen from Table 2, there are 4 transgenic positive plants (SV-1, SV-4, SV-5 and SV-18) Hyg The relative copy number of the gene is about 0.5, which is a single-copy inserted transgenic plant. There are 6 transgenic positive plants (SV-2, SV-8, SV-9, SV-14, SV-15 and SV-19). HygThe relative copy number of the gene is approximately 1, and it is a transgenic plant with double-copy insertion. There are 4 transgenic positive plants (SV-6, SV-10, SV-17, and SV-23) Hyg The relative copy number of the gene is approximately 1.5, and it is a transgenic plant with triple-copy insertion. The remaining samples are all transgenic plants with more than triple copies. Among them, the copy numbers of the 7 transgenic positive plants SV-1, SV-2, SV-3, SV-4, SV-5, SV-8, and SV-9 detected by the fluorescence quantitative PCR in this example are consistent with the results of the copy numbers detected by digital PCR in Example 4, and they are all single-copy or double-copy; the fluorescence quantitative PCR detection results of the 2 transgenic positive plants SV-6 and SV-10 are triple-copy, while the absolute quantification result of digital PCR is quadruple-copy. The above results show that the fluorescence quantitative PCR method in this example has a high accuracy in detecting low-copy insertion of foreign genes in transgenic Setaria viridis, but it is not accurate enough for the detection of multi-copy insertion. However, this method is simple, rapid, low-cost, requires less sample volume, is easy to operate in batches, and transgenic breeding research generally selects transgenic lines with low-copy insertion of foreign genes. Therefore, this method 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.

[0049] Other parts not described in detail are all prior arts. Although the above embodiments have made a detailed description of the present invention, 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 according to this example without creative efforts, and these embodiments all belong to 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 fluorescent quantitative PCR, characterized in that: The method for detecting the number of inserted copies of foreign genes in Setaria viridis or millet by using fluorescent quantitative PCR is as follows: RA1 The gene was used as an internal reference gene. Hyg Screening marker genes as exogenous genes, using primer pairs RA1-F / RA1-R and primer pairs Hyg-F / Hyg-R to detect genomic DNA of transgenic Setaria or transgenic millet, and determining the number of inserted copies of the exogenous genes in the transgenic Setaria or transgenic millet; Detection of internal reference genes RA1 Primer pair sequences: Forward primer RA1-F is shown in SEQ ID NO: 1; The reverse primer RA1-R is shown in SEQ ID NO: 2; Detection Hyg Primer pair sequences for screening marker genes: The forward primer Hyg-F is shown in SEQ ID NO: 3; The reverse primer Hyg-R is shown in SEQ ID NO: 4; In the process of fluorescence quantitative PCR detection, the method for determining the number of inserted copies of the exogenous gene in transgenic Setaria viridis or transgenic millet is as follows: When the relative copy number of the exogenous gene was 0.26-0.74, the transgenic Setaria viridis or transgenic millet was a single copy insertion; When the relative copy number of the exogenous gene is 0.75-1.25, the transgenic Setaria viridis or transgenic millet has double copy insertion; When the relative copy number of the exogenous gene was 1.26-1.74, the transgenic Setaria viridis or transgenic millet had three copies inserted; When the relative copy number of the exogenous gene is 1.75-2.25, the transgenic Setaria viridis or transgenic millet has four copies inserted; The same can be said for other insertion copy numbers of transgenic Setaria viridis or transgenic millet.

2. The method for detecting the number of inserted copies of foreign genes in Setaria viridis or millet by using fluorescent quantitative PCR according to claim 1, characterized in that: In the process of fluorescence quantitative PCR detection, the genomic DNA of transgenic Setaria viridis or transgenic millet was used as a DNA template, and the primer pair RA1-F / RA1-R and the primer pair Hyg-F / Hyg-R were used for fluorescence quantitative PCR detection.

3. The method for detecting the number of inserted copies of foreign genes in Setaria viridis or millet by using fluorescent quantitative PCR according to claim 2, characterized in that: During the fluorescence quantitative PCR detection process, the amplification RA1 The reaction system of the gene is: 2 x qPCR Master Mix, Low Rox dye, RA1-F, RA1-R, DNA template and ddH2O.

4. The method for detecting the number of inserted copies of exogenous genes in Setaria viridis or millet by using fluorescent quantitative PCR according to claim 2 or 3, characterized in that: During the fluorescence quantitative PCR detection process, the amplification Hyg The reaction system of the gene was: 2 xqPCR Master Mix, Low Rox dye, Hyg-F, Hyg-R, DNA template and ddH2O.

5. The method for detecting the number of inserted copies of foreign genes in Setaria viridis or millet by using fluorescent quantitative PCR according to any one of claims 1 to 3, characterized in that: During the fluorescence quantitative PCR detection process, the amplification RA1 Gene and Hyg The gene amplification program was as follows: pre-denaturation at 95°C for 30 s; denaturation at 95°C for 10 s, annealing at 62°C for 30 s, and 40 cycles; melting curve was performed at 95°C for 15 s, 62°C for 60 s, and 95°C for 15 s.

6. The method for detecting the number of inserted copies of foreign genes in Setaria viridis or millet by using fluorescent quantitative PCR according to any one of claims 1 to 3, characterized in that: The relative copy number of the exogenous gene was calculated by using the known double-copy transgenic foxtail grass or transgenic millet as the reference factor. The method is used to analyze the multiples of the copy number of the tested transgenic Setaria viridis or the tested transgenic millet relative to the reference factor.

7. A method for detecting the number of inserted copies of exogenous genes in Setaria or millet using digital PCR, characterized in that: The method for detecting the number of inserted copies of exogenous genes in Setaria or millet by using digital PCR is as follows: RA1 The gene was used as an internal reference gene. Hyg Screening marker genes as exogenous genes, using primer pairs RA1-F / RA1-R and primer pairs Hyg-F / Hyg-R to detect genomic DNA of transgenic Setaria or transgenic millet, and determining the number of inserted copies of the exogenous genes in the transgenic Setaria or transgenic millet; Detection of internal reference genes RA1 Primer pairs and probe sequences: Forward primer RA1-F is shown in SEQ ID NO: 1; The reverse primer RA1-R is shown in SEQ ID NO: 2; Probe RA1-P is as shown in SEQ ID NO: 5; Detection Hyg Primer pairs and probe sequences for screening marker genes: The forward primer Hyg-F is shown in SEQ ID NO: 3; The reverse primer Hyg-R is shown in SEQ ID NO: 4; The probe Hyg-P is shown in SEQ ID NO: 6; In the process of digital PCR detection, the method for determining the number of inserted copies of the exogenous gene in the transgenic foxtail grass or transgenic millet is as follows: That is, when the copy number ratio of the exogenous gene to the internal reference gene is 0.26-0.74, the transgenic Setaria viridis or transgenic millet is 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.

8. The method for detecting the number of inserted copies of exogenous genes in Setaria or millet using digital PCR according to claim 7, characterized in that: In the digital PCR detection process, the genomic DNA of transgenic Setaria viridis or transgenic millet is used as a DNA template, and the primer pair RA1-F / RA1-R and the primer pair Hyg-F / Hyg-R are used for digital PCR detection.

9. The method for detecting the number of inserted copies of exogenous genes in Setaria or millet using digital PCR according to claim 8, characterized in that: The reaction system of digital PCR is: reaction premix, RA1-F, RA1-R, Hyg-F, Hyg-R, probe RA1-P, probe Hyg-P, DNA template and nuclease-free water.

10. The method for detecting the number of inserted copies of exogenous genes in Setaria viridis or millet using digital PCR according to any one of claims 7 to 9, 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.

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