Method for detecting the copy number of inserted foreign genes in Setaria viridis or Setaria italica by fluorescence quantitative PCR or digital PCR

Abstract

The present invention provides a method for detecting the insertion copy number of foreign genes in Setaria viridis or foxtail millet by fluorescence quantitative PCR or digital PCR, belonging to the field of biotechnology. The method uses RA1 gene as an internal reference gene and Hyg screening marker gene as a foreign gene, and uses primer pairs RA1-F / RA1-R and primer pairs Hyg-F / Hyg-R to detect the genomic DNA of transgenic Setaria viridis or transgenic foxtail millet, and determine the insertion copy number of foreign genes in transgenic Setaria viridis or transgenic foxtail millet. The present invention provides a method for detecting the insertion copy number of foreign genes in Setaria viridis or foxtail millet by fluorescence quantitative PCR or digital PCR, which has the characteristics of simplicity, rapidity, low sample consumption, high efficiency, high throughput, etc., can improve the efficiency of screening transgenic lines of Setaria viridis or foxtail millet with low-copy insertions, and provides a new option for the detection method of the insertion copy number of foreign genes in the research of transgenic Setaria viridis or 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 exogenous gene insertion in Setaria viridis or Setaria italica 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 class Poaceae. It is a new type of transgenic model plant, which has the advantages of short growth cycle, short plant height, easy to grow, small genome, diploid, and can produce a large number of self-crossed seeds compared with the dicotyledonous model plant Arabidopsis thaliana. It is an excellent monocotyledonous model plant. Since it originated in the tropics and has a C4 photosynthesis system, it is closely related to Setaria italica, Zea mays, Sorghum bicolor, Saccharum officinarum, 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 research institutions in China have begun to pay attention to it, especially breeders of Setaria italica ( Setaria italica ). Setaria italica 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 Setaria italica. The karyotypes of Setaria italica 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 Setaria italica in Setaria viridis can quickly verify their functions and accelerate the research process of Setaria italica breeding. Therefore, Setaria viridis can be used as an important transgenic model plant for studying the gene functions of Setaria italica.

[0003] In the research of transgenic breeding of plants, the expression and genetic stability of exogenous genes are affected by the copy number of their integration into the genome of the recipient plant. When the exogenous 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 research of transgenic plant breeding, transgenic lines with low-copy insertion of exogenous genes are usually selected. However, transgenic lines with low-copy insertion generally need to be screened from a large number of T0 transgenic lines, which takes a lot of time. Therefore, a simple, rapid, efficient, and high-throughput method for detecting the copy number of exogenous gene insertion is crucial for transgenic plant breeding research.

[0004] At present, the traditional method for detecting the copy number of exogenous 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 exogenous 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 exogenous gene insertions in transgenic Setaria viridis using these two PCR methods. Therefore, there is an urgent need to establish a method for quickly detecting the copy number of exogenous gene insertions in transgenic Setaria viridis 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 exogenous gene insertions in Setaria viridis or Setaria italica using fluorescence quantitative PCR or digital PCR.

[0006] To achieve the above object, the technical solution adopted by the present invention is as follows:

[0007] A method for detecting the copy number of exogenous gene insertions in Setaria viridis or Setaria italica using fluorescence quantitative PCR uses RA1 the gene as the reference gene and Hyg the selectable marker gene as the exogenous 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 Setaria viridis or transgenic Setaria italica is detected, and the copy number of exogenous gene insertions in transgenic Setaria viridis or transgenic Setaria italica is judged;

[0008] Among them, RA1 the gene (Sevir.2G209800) is a single-copy gene in Setaria viridis, the corresponding gene number of this gene in Setaria italica is Seita.2G201700, and it is also a single-copy gene in Setaria italica. The two gene sequences are exactly the same, and the primer pair sequences of the two are also exactly the same; the RA1 gene (Sevir.2G209800) sequence in Setaria viridis is as shown in SEQ ID NO: 7; the RA1 gene (Seita.2G201700) sequence in Setaria italica is as shown in SEQ ID NO: 8;

[0009] The primer pair sequence for detecting the reference gene RA1 :

[0010] The forward primer RA1-F is: 5’- CCTAGTCGCGCAGTTGTTGA-3’, as shown in SEQ ID NO: 1;

[0011] The reverse primer RA1-R is: 5’- ATGCCAAGGAGAAACGGCAG-3’, as shown in SEQ ID NO: 2;

[0012] Detection Hyg Primer pair sequences for screening marker genes:

[0013] The forward primer Hyg-F is: 5’-GTCAAGACCAATGCGGAGCA-3’ , as shown in SEQ ID NO: 3;

[0014] The reverse primer Hyg-R is: 5’-CCCAATACGAGGTCGCCAAC-3’ , as shown in SEQ ID NO: 4;

[0015] During the detection by fluorescence quantitative PCR, the method for judging the insertion copy number of foreign genes in transgenic Setaria viridis or transgenic foxtail millet is as follows:

[0016] 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 foxtail millet has a single-copy insertion;

[0017] 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 foxtail millet has a double-copy insertion;

[0018] 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 foxtail millet has a triple-copy insertion;

[0019] 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 foxtail millet has a quadruple-copy insertion;

[0020] And so on for other insertion copy numbers of transgenic Setaria viridis or transgenic foxtail millet, such as five-copy insertion, six-copy insertion, etc.

[0021] Furthermore, during the detection by fluorescence quantitative PCR, using the genomic DNA of transgenic Setaria viridis or transgenic foxtail millet as the DNA template, and using the primer pairs RA1-F / RA1-R and primer pairs Hyg-F / Hyg-R for fluorescence quantitative PCR detection.

[0022] Furthermore, during the detection by fluorescence quantitative PCR, amplification RA1The reaction system of the gene was: 2 x qPCR Master 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 ddH2O 7.00μL, with a total volume of 20.00μL.

[0023] 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 ddH2O 7.00μL, with a total volume of 20.00μL.

[0024] 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.

[0025] 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.

[0026] 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;

[0027] Detection of internal reference genes RA1 Primer pairs and probe sequences:

[0028] The forward primer RA1-F is: 5'-CCTAGTCGCGCAGTTGTTGA-3', as shown in SEQ ID NO: 1;

[0029] The reverse primer RA1-R is: 5’- ATGCCAAGGAGAAACGGCAG-3’, as shown in SEQ ID NO: 2;

[0030] The probe RA1-P is: VIC-TCATGAGCTCAGGTTGTCGCGCGCA-MGB, as shown in SEQ ID NO: 5;

[0031] Detection Hyg Primer pairs and probe sequences for screening marker genes:

[0032] The forward primer Hyg-F is: 5’-GTCAAGACCAATGCGGAGCA-3’, as shown in SEQ ID NO: 3;

[0033] The reverse primer Hyg-R is: 5’-CCCAATACGAGGTCGCCAAC-3’, as shown in SEQ ID NO: 4;

[0034] The probe Hyg-P is: FAM-TCGAAGTAGCGCGTCTGCTGCTCCA-BHQ1, as shown in SEQ ID NO: 6;

[0035] During the digital PCR detection process, the method for determining the copy number of foreign gene insertions in transgenic Setaria viridis or transgenic Setaria italica is as follows:

[0036] 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), the transgenic Setaria viridis or transgenic Setaria italica has a single-copy insertion;

[0037] When the copy number ratio of the foreign gene to the reference gene is approximately 1 (when 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;

[0038] When the copy number ratio of the foreign gene to the reference gene is approximately 1.5 (when 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;

[0039] When the copy number ratio of the foreign gene to the reference gene is approximately 2 (when 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;

[0040] 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.

[0041] Furthermore, during the digital PCR detection, the genomic DNA of transgenic Setaria viridis or transgenic Setaria italica was used as the DNA template, and primer pairs RA1-F / RA1-R and primer pair Hyg-F / Hyg-R were used for digital PCR detection.

[0042] Furthermore, the reaction system for digital PCR was 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.

[0043] Furthermore, the amplification program for digital PCR was: 95°C for 10 min; 95°C for 30 s, 62°C for 1 min, for 45 cycles; 98°C for 10 min;

[0044] After the digital PCR reaction was completed, the chip was placed in a biochip analyzer to read the FAM and VIC fluorescence signals, and the fluorescence data was analyzed using QuantDrop Software to obtain the copy numbers (copy / μL) of the foreign gene and the reference gene in the DNA template, and calculate the ratio of the copy numbers of the foreign gene to the reference gene in transgenic Setaria viridis or transgenic Setaria italica, and judge the inserted copy number of the foreign gene in the genome of transgenic Setaria viridis or transgenic Setaria italica through the ratio.

[0045] The beneficial effects of a method for detecting the inserted copy number of a foreign gene in Setaria viridis or Setaria italica using fluorescence quantitative PCR or digital PCR of the present invention are as follows:

[0046] 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 inserted copy number of a foreign gene in transgenic Setaria viridis or transgenic Setaria italica, and the most commonly used selection marker in transgenic Setaria viridis or transgenic Setaria italica Hyg The gene is a foreign gene, and the digital PCR or fluorescence quantitative PCR technology is used to achieve rapid and high-throughput detection of the inserted copy number of the foreign gene in transgenic Setaria viridis or transgenic Setaria italica;

[0047] 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 or quantitative fluorescence PCR technology. The method is characterized by being simple, rapid, having low sample requirements, being efficient, and high-throughput. It can improve the efficiency of large-scale screening of transgenic lines and provide a new option for the detection of the copy number of foreign gene insertions in transgenic Setaria viridis or transgenic foxtail millet breeding research;

[0048] A method for detecting the copy number of foreign gene insertions in Setaria viridis or foxtail millet using quantitative fluorescence PCR provided by the present invention can be used for all transgenic Setaria viridis with Hyg as a screening marker for copy number detection; the RA1 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 a screening marker; the method has the advantages of being simple, rapid, low-cost, and having a small sample requirement. Its accuracy is sufficient to meet the breeding requirements for detecting single or low copies, and it is more suitable for batch detection;

[0049] A method for detecting the copy number of foreign gene insertions in Setaria viridis or foxtail millet using digital PCR provided by the present invention can be used for all transgenic Setaria viridis or transgenic foxtail millet with Hyg as a screening marker for copy number detection; the RA1 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 a screening marker; the method has the advantages of absolute quantification, high accuracy, being simple, rapid, having a small sample requirement, and being able to perform batch detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 is the alignment result of the Setaria viridis gene RA1 in the genome in Example 1 of the present invention;

[0051] Figure 2 is the result of exploring 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 respectively; 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;

[0052] Figure 3 It 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; the labels 1 to 24 represent transgenic Setaria viridis SV-1 to SV-24 in sequence; "-" represents Hyg negative control, and "+" represents Hyg positive control;

[0053] Figure 4 It 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 the non-signal pore numbers;

[0054] Figures 5 to 8 It is in Example 5 of the present invention RA1 gene and Hyg gene fluorescence quantitative primer verification graph; among them, Figure 5 is RA1 gene amplification curve graph; Figure 6 is RA1 gene melting curve graph; Figure 7 is Hyg gene amplification curve graph; Figure 8 is Hyg gene melting curve graph;

[0055] Figure 9 It is the result of fluorescence quantitative PCR copy number detection of 17 transgenic positive plants in Example 5 of the present invention; among them, the abscissa is the number of transgenic positive plants, and the ordinate is the relative copy number, and all take the transgenic positive plant SV-3 as the reference sample. Detailed implementation manners

[0056] 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 extensions without departing from the connotation of the present invention, so the present invention is not limited by the specific embodiments disclosed below. The present invention will be further described in detail below with specific embodiments for those skilled in the art to understand.

[0057] In addition, for those specific embodiments disclosed below where specific technologies or conditions are not indicated, they shall be in accordance with the technologies or conditions described in the literature in this field (for example, referring to "Molecular Cloning: A Laboratory Manual" by Sambrook et al., translated by Huang Peitang et al., Third Edition, Science Press) or in accordance with the product instructions. For reagents whose manufacturers are not indicated, they are all conventional products that can be purchased.

[0058] Example 1 Determination of reference gene

[0059] RA1 Bioinformatics analysis of the copy number of genes in the Setaria viridis genome and the foxtail millet genome specifically includes the following steps:

[0060] 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 . From the alignment result, it can be seen that RA1 the 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 Setaria viridis.

[0061] RA1 The corresponding gene number of the gene in foxtail millet is Seita.2G201700. 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 sequence of the RA1 gene (Sevir.2G209800) in Setaria viridis is as shown in SEQ ID NO: 7; the sequence of the RA1 gene (Seita.2G201700) in foxtail millet is as shown in SEQ ID NO: 8.

[0062] Since the RA1 gene (Sevir.2G209800) in Setaria viridis and the RA1 gene (Seita.2G201700) in foxtail millet have exactly the same gene sequences and are both single-copy genes, therefore, the primers and probes designed based on the RA1 gene in Setaria viridis are also equally applicable to amplifying and detecting the RA1 gene in foxtail millet (that is, the primer pair sequence is exactly the same as the sequence in the corresponding Seita.2G201700 gene in foxtail millet).

[0063] At the same time, since the commonly used selection marker genes in transgenic Setaria viridis and transgenic foxtail millet are both HygGenes can all Hyg Taking the gene as an exogenous screening marker gene, and since both foxtail millet and green foxtail are diploid plants, foxtail millet is also an annual plant in the genus Setaria of the Panicoideae subfamily of the Poaceae family, and green foxtail is the wild ancestor of foxtail millet. The karyotypes of foxtail millet and green foxtail are basically the same, the banding patterns are similar, and the genome sizes are both about 510M. Therefore, the method for detecting the copy number of exogenous gene insertions in green foxtail of the present invention can be directly applied to detecting the copy number of exogenous gene insertions in foxtail millet. In summary, the method for detecting the copy number of exogenous gene insertions in green foxtail by digital PCR and the method for detecting the copy number of exogenous gene insertions in green foxtail by fluorescence quantitative PCR of the present invention can both be directly used to detect the copy number of exogenous gene insertions in transgenic foxtail millet.

[0064] Example 2 Exploration of PCR amplification conditions

[0065] Since the commonly used screening marker gene in transgenic green foxtail is Hyg gene, so select Hyg the screening marker gene as the exogenous gene in transgenic green foxtail, and use RA1 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 exogenous gene includes the following specific steps:

[0066] 1) Primer sequences

[0067] The primer pair sequences for detecting the internal reference gene RA1 include:

[0068] The forward primer RA1-F is: 5’- CCTAGTCGCGCAGTTGTTGA-3’, as shown in SEQ ID NO: 1;

[0069] The reverse primer RA1-R is: 5’- ATGCCAAGGAGAAACGGCAG-3’, as shown in SEQ ID NO: 2;

[0070] The primer pair sequences for detecting Hyg the screening marker gene include:

[0071] The forward primer Hyg-F is: 5’-GTCAAGACCAATGCGGAGCA-3’, as shown in SEQ ID NO: 4;

[0072] The reverse primer Hyg-R is: 5’-CCCAATACGAGGTCGCCAAC-3’, as shown in SEQ ID NO: 5;

[0073] 2) RA1 The exploration of the PCR amplification conditions for the gene primers and Hyg the gene primers

[0074] Using the DNA of one transgenic Setaria viridis plant as a template, gradient PCR reactions were carried out using the primer pairs RA1-F / RA1-R and Hyg-F / Hyg-R, respectively.

[0075] The PCR reaction system for detecting the reference gene RA1 was as follows: Green Master Mix 12.50 µL, RA1-F (10.00 µM) 1.00 µL, RA1-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.

[0076] The PCR reaction system for detecting the foreign gene 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.

[0077] The PCR reaction conditions were all as follows: pre-denaturation at 94 °C for 5 min; denaturation at 94 °C for 30 s, annealing for 30 s, extension at 72 °C for 30 s, for a total of 35 cycles; finally, extension at 72 °C for 7 min and preservation at 4 °C; among which 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.

[0078] After the PCR amplification reaction was completed, 5 µL of the corresponding PCR products obtained were taken for electrophoresis detection on a 2% agarose gel. If RA1 and Hyg the PCR products of showed bands of 111 bp and 143 bp 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 RA1 gene and the Hyg gene.

[0079] As shown in the electrophoresis results in Figure 2 , when the annealing temperatures were 56.4 °C, 58.0 °C, 60.0 °C, and 62.0 °C respectively, RA1 the gene and Hyg the gene could both obtain the target amplified bands, but the band was the brightest at the annealing temperature of 62.0 °C and there were no non-specific bands. RA1 The gene and Hyg the PCR products of the gene were sequenced and compared with the sequences of the Setaria viridis RA1 gene and Hyg gene, and they were all consistent. Therefore, the primer pair RA1-F / RA1-R can be used for Setaria viridis RA1Detection of genes, Hyg-F / Hyg-R can be used for exogenous genes Hyg Detection of genes, and the annealing temperature was determined to be 62.0 °C for all.

[0080] Example 3 PCR identification of transgenic Setaria viridis positive plants

[0081] Using the Hyg gene primers to perform PCR identification on transgenic Setaria viridis plants, which specifically includes the following steps:

[0082] 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 negative control, and use the genomic DNA of transgenic Setaria viridis containing Hyg gene as Hyg positive control, and perform PCR experiments on Hyg gene 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 are the same as those for detecting Hyg gene in Example 2, and the annealing temperature is 62.0 °C.

[0083] After the PCR amplification reaction, 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, 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; 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.

[0084] Example 4 Digital PCR detection

[0085] The method for detecting the copy number of exogenous gene insertion in Setaria viridis by digital PCR includes the following specific steps:

[0086] 1) Digital PCR primers and probes

[0087] Primer pairs and probe sequences for detecting the internal reference gene RA1 :

[0088] The forward primer RA1-F is: 5’- CCTAGTCGCGCAGTTGTTGA-3’, as shown in SEQ ID NO: 1;

[0089] The reverse primer RA1-R is: 5’- ATGCCAAGGAGAAACGGCAG-3’, as shown in SEQ ID NO: 2;

[0090] The probe RA1-P is: VIC-TCATGAGCTCAGGTTGTCGCGCGCA-MGB, as shown in SEQ ID NO: 5;

[0091] Detection Hyg Primer pairs and probe sequences for screening marker genes:

[0092] The forward primer Hyg-F is: 5’-GTCAAGACCAATGCGGAGCA-3’, as shown in SEQ ID NO: 3;

[0093] The reverse primer Hyg-R is: 5’-CCCAATACGAGGTCGCCAAC-3’, as shown in SEQ ID NO: 4;

[0094] The probe Hyg-P is: FAM-TCGAAGTAGCGCGTCTGCTGCTCCA-BHQ1, as shown in SEQ ID NO: 6.

[0095] 2) DNA template extraction

[0096] Nine plants (SV-1~SV-6 and SV-8~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.

[0097] 3) Detection of the copy number of foreign gene insertions in the test samples by digital PCR

[0098] Using RA1 the gene as the internal 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.

[0099] 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.

[0100] 4) Result analysis

[0101] 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 reference gene in the corresponding DNA template, calculate the copy number ratio of the exogenous gene and the reference gene in the DNA template, and judge the copy number of the exogenous gene in the Setaria viridis genome through the ratio.

[0102] 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 (that is, 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 (that is, 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 (that is, 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 (that is, 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.

[0103] The digital PCR scatter plots of 9 transgenic positive Setaria viridis plants in this example are shown in Figure 4 , and 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 insertion copy number of foreign genes in transgenic Setaria viridis.

[0104] Table 1 Results of digital PCR for detecting the insertion copy number of foreign genes in transgenic Setaria viridis

[0105]

[0106] Example 5 Fluorescent quantitative PCR detection

[0107] The method for detecting the insertion copy number of foreign genes in Setaria viridis by fluorescent quantitative PCR includes the following specific steps:

[0108] 1) Fluorescent quantitative PCR primers

[0109] Primer pair sequences for detecting the reference gene RA1 :

[0110] Forward primer RA1-F is: 5’-CCTAGTCGCGCAGTTGTTGA-3’, as shown in SEQ ID NO: 1;

[0111] Reverse primer RA1-R is: 5’-ATGCCAAGGAGAAACGGCAG-3’, as shown in SEQ ID NO: 2;

[0112] Primer pair sequences for detecting the foreign gene Hyg screening marker gene:

[0113] Forward primer Hyg-F is: 5’-GTCAAGACCAATGCGGAGCA-3’, as shown in SEQ ID NO: 3;

[0114] Reverse primer Hyg-R is: 5’-CCCAATACGAGGTCGCCAAC-3’, as shown in SEQ ID NO: 4.

[0115] 2) DNA template extraction

[0116] Using the 17 transgenic positive plants screened in Example 3 as the test samples, the numbers of 18 transgenic positive plants are as follows: SV-1, SV-2, SV-4 to SV-6, SV-8 to SV-10, SV-13 to SV-19, SV-21, and SV-23. Using the transgenic positive plant SV-3 with two copies detected in Example 4 as the control, and the non-transgenic negative plant SV-7 screened in Example 3 as the negative control, the corresponding genomic DNA was extracted using the MEGA Plant Genomic Kit as the DNA template.

[0117] 3) Primer verification

[0118] Select 4 known transgenic positive Setaria viridis DNA templates for fluorescence quantitative PCR experiments to verify the internal reference gene RA1 and the exogenous gene Hyg of the primers. The reaction was carried out in a QuantStudioTM 1 plus real-time fluorescence quantitative PCR instrument (Thermo Fisher Scientific (Shanghai) Instrument Co., Ltd.);

[0119] Amplification RA1 The reaction system for amplifying the

[0120] gene is: 2 x qPCR Master Mix 10.00 μL, Low Rox dye 0.20 μL, RA1-F at a concentration of 10.00 μM 0.40 μL, RA1-R at a concentration of 10.00 μM 0.40 μL, DNA template at a concentration of 30.00 ng / μL 2.00 μL, and ddH2O 7.00 μL, with a total volume of 20.00 μL; Hyg The reaction system for amplifying the

[0121] gene is: 2 x qPCR Master Mix 10.00 μL, Low Rox dye 0.20 μL, Hyg-F at a concentration of 10.00 μM 0.40 μL, Hyg-R at a concentration of 10.00 μM 0.40 μL, DNA template at a concentration of 30.00 ng / μL 2.00 μL, and ddH2O 7.00 μL, with a total volume of 20.00 μL. RA1 The amplification procedures for amplifying the Hyg gene and the Figures 5 to 8 gene are both: pre-denaturation at 95°C for 30 s; denaturation at 95°C for 10 s, annealing at 62°C for 30 s, for 40 cycles; melting curve: 95°C for 15 s, 62°C for 60 s, 95°C for 15 s. Each group of experiments was set with 3 replicates. The amplification curves and melting curves of the two pairs of primers 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 a unique absorption peak, which can be used as primers for fluorescence quantitative PCR detection.

[0122] 4) Detect the copy number of exogenous genes in the test samples by fluorescence quantitative PCR

[0123] Using RA1 gene as the internal reference gene, Hyg gene as the exogenous gene, and using the transgenic positive plant SV-3 with two copies as the control, detect the inserted copy number of the exogenous gene in the genome of Setaria viridis of all test samples (i.e., 17 transgenic positive plants) through the above fluorescence quantitative PCR experiment. Among them, the reaction system and amplification program for fluorescence quantitative PCR detection are the same as those in step 3) primer verification of this example, and each test sample is detected 3 times repeatedly.

[0124] 5) Result analysis

[0125] After the reaction, use the QuantStudio™ Design & Analysis Software v1.5.2 system to analyze the data, and use the method to analyze the relative copy number of the Hyg gene in the transgenic positive plants, and define the transgenic Setaria viridis positive plant SV-3 with known two copies as the reference factor, which is 1, and the multiple of the copy number of each test sample relative to the reference factor is , so the relative copy number of the Hyg gene in the single-copy inserted plants should be 1 / 2 of the two-copy control, that is, about 0.5 (generally 0.26 - 0.74); the relative copy number of the Hyg gene in the two-copy plants should be equal to the two-copy control, that is, about 1 (generally 0.75 - 1.25); the relative copy number of the Hyg gene in the three-copy plants should be 3 / 2 of the two-copy control, that is, about 1.5 (generally 1.26 - 1.74); the relative copy number of the Hyg gene in the four-copy plants should be 2 of the two-copy control, that is, about 2 (that is, the general copy number ratio of the exogenous gene fragment inserted with two copies to the internal reference gene fragment is 1.75 - 2.25), and the inserted copy numbers of multi-copy exogenous gene fragments such as five copies and six copies can be deduced by analogy.

[0126] The results of the fluorescence quantitative PCR detection copy numbers of 17 transgenic Setaria viridis positive plants in this example are shown in Figure 9 and Table 2.

[0127] Table 2 Results of the inserted copy number of the exogenous gene in transgenic Setaria viridis detected by fluorescence quantitative PCR

[0128]

[0129] From Figure 9As can be seen from Table 2, the relative copy numbers of the Hyg gene in 4 transgenic positive plants (SV-1, SV-4, SV-5 and SV-18) are approximately 0.5, which are transgenic plants with single-copy insertion. The relative copy numbers of the Hyg gene in 6 transgenic positive plants (SV-2, SV-8, SV-9, SV-14, SV-15 and SV-19) are approximately 1, which are transgenic plants with double-copy insertion. The relative copy numbers of the Hyg gene in 4 transgenic positive plants (SV-6, SV-10, SV-17 and SV-23) are approximately 1.5, which are transgenic plants with triple-copy insertion. The remaining samples are all transgenic plants with more than triple copies. Among them, the copy numbers of 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 copy number results detected by digital PCR in Example 4, all being single-copy or double-copy; the fluorescence quantitative PCR detection results of 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 high accuracy in detecting low-copy insertion of foreign genes in transgenic Setaria viridis, but is less accurate in detecting multi-copy insertion. However, this method is simple, fast, low-cost, requires less sample volume, is easy for batch operation, 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 lot of time and cost for the breeding of excellent transgenic lines.

[0130] 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 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 inserted copy number of foreign genes in Setaria viridis or Setaria italica by fluorescence quantitative PCR, characterized in that, The method for detecting the copy number of inserted foreign genes in Setaria viridis or Setaria italica by fluorescence quantitative PCR uses RA1 gene as the internal reference gene, and Hyg screening marker gene as the foreign gene. Using primer pairs RA1-F / RA1-R and primer pairs Hyg-F / Hyg-R, fluorescence quantitative PCR is used to detect the genomic DNA of transgenic Setaria viridis or transgenic Setaria italica, and to determine the copy number of inserted foreign genes in transgenic Setaria viridis or transgenic Setaria italica; Reference gene detection RA1 Primer pair sequence: The forward primer RA1-F is shown as SEQ ID NO: 1; The reverse primer RA1-R is shown as SEQ ID NO: 2; Detection Hyg Primer pair sequences for screening marker genes: The forward primer Hyg-F is shown as SEQ ID NO: 3; The reverse primer Hyg-R is shown as SEQ ID NO: 4; During the detection by fluorescence quantitative PCR, the method for judging the copy number of exogenous gene insertion in transgenic Setaria viridis or transgenic foxtail millet is as follows: When the relative copy number of the exogenous gene is 0.26 - 0.74, the transgenic Setaria viridis or transgenic foxtail millet has a single-copy insertion; When the relative copy number of the exogenous gene is 0.75 - 1.25, the transgenic Setaria viridis or transgenic foxtail millet has a double-copy insertion; When the relative copy number of the exogenous gene is 1.26 - 1.74, the transgenic Setaria viridis or transgenic foxtail millet has a triple-copy insertion; When the relative copy number of the exogenous gene is 1.75 - 2.25, the transgenic Setaria viridis or transgenic foxtail millet has a quadruple-copy insertion; And so on for other insertion copy numbers of transgenic Setaria viridis or transgenic foxtail millet.

2. The method for detecting the copy number of exogenous gene insertion in Setaria viridis or Setaria italica by fluorescence quantitative PCR according to claim 1, wherein During the detection by fluorescence quantitative PCR, using the genomic DNA of transgenic Setaria viridis or transgenic foxtail millet as the DNA template, fluorescence quantitative PCR detection is carried out using the primer pairs RA1-F / RA1-R and the primer pair Hyg-F / Hyg-R.

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

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

6. The method for detecting the inserted copy number of exogenous genes in Setaria viridis or foxtail millet by fluorescence quantitative PCR according to any one of claims 1-3, characterized in that, The relative copy number of the exogenous gene uses the known transgenic green foxtail or transgenic millet with two copies as a reference factor, and adopts method to analyze the multiple of the copy number of the transgenic green foxtail to be tested or the transgenic millet to be tested relative to the reference factor.

7. A method for detecting the copy number of inserted foreign genes in Setaria viridis or Setaria italica by digital PCR, characterized in that, The method for detecting the copy number of inserted foreign genes in Setaria viridis or foxtail millet by digital PCR uses RA1 gene as an internal reference gene, and Hyg screening marker gene as the foreign gene. Using primer pairs RA1-F / RA1-R and primer pair Hyg-F / Hyg-R, digital PCR is used to detect the genomic DNA of transgenic Setaria viridis or transgenic foxtail millet, and to determine the copy number of inserted foreign genes in transgenic Setaria viridis or transgenic foxtail millet; Reference gene detection RA1 Primer pairs and probe sequences: The forward primer RA1-F is shown as SEQ ID NO: 1; The reverse primer RA1-R is shown as SEQ ID NO: 2; The probe RA1-P is shown as SEQ ID NO: 5; Detection Hyg Primer pairs and probe sequences for screening marker genes: The forward primer Hyg-F is shown as SEQ ID NO: 3; The reverse primer Hyg-R is shown as SEQ ID NO: 4; The probe Hyg-P is shown as SEQ ID NO: 6; During the detection by digital PCR, the method for judging the copy number of exogenous gene insertion in transgenic Setaria viridis or transgenic foxtail millet is as follows: That is, when the copy number ratio of the exogenous gene to the reference gene is 0.26 - 0.74, the transgenic Setaria viridis or transgenic foxtail millet has a single-copy insertion; When the copy number ratio of the exogenous gene to the reference gene is 0.75 - 1.25, the transgenic Setaria viridis or transgenic foxtail millet has a double-copy insertion; When the copy number ratio of the exogenous gene to the reference gene is 1.26 - 1.74, the transgenic Setaria viridis or transgenic foxtail millet has a triple-copy insertion; When the copy number ratio of the exogenous gene to the reference gene is 1.75 - 2.25, the transgenic Setaria viridis or transgenic foxtail millet has a quadruple-copy insertion; And so on for other insertion copy numbers of transgenic Setaria viridis or transgenic foxtail millet.

8. The method for detecting the copy number of exogenous gene insertion in Setaria viridis or Setaria italica by digital PCR according to claim 7, wherein During the detection by digital PCR, using the genomic DNA of transgenic Setaria viridis or transgenic foxtail millet as the DNA template, digital PCR detection is carried out using the primer pairs RA1-F / RA1-R and the primer pair Hyg-F / Hyg-R.

9. The method for detecting the copy number of exogenous gene insertion in Setaria viridis or Setaria italica by 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 copy number of exogenous gene insertion in Setaria viridis or Setaria italica by digital PCR according to any one of claims 7-9, characterized in that, The amplification program for digital PCR is: 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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