Single-copy millet gene and method for detecting the number of inserted copies of foreign genes in millet using the single-copy millet gene

Abstract

The present invention provides a single-copy millet gene and a method for detecting the number of inserted copies of an exogenous gene in millet using the same, belonging to the field of biotechnology. The single-copy millet gene is SD1 Gene; A method for detecting the number of inserted copies of foreign genes in millet using the same is based on SD1 The gene was used as the internal reference gene. Cozy The screening marker gene is used as an exogenous gene, and the primer pair SD1-F / SD1-R and the primer pair Hyg-F / Hyg-R are used to detect the genomic DNA of the transgenic millet, and the number of inserted copies of the exogenous gene in the transgenic millet is determined. The present invention provides a method for high-throughput detection of the number of inserted copies of exogenous genes in transgenic millet using digital PCR or fluorescent quantitative PCR technology, which has the characteristics of being simple and rapid, having low sample requirements, being highly efficient, and having high throughput, and can improve the efficiency of large-scale screening of transgenic strains.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and specifically relates to a single-copy broomcorn millet gene and a method for detecting the number of inserted copies of exogenous genes in broomcorn millet using the same, and in particular relates to a single-copy broomcorn millet gene capable of detecting the number of inserted copies of exogenous genes in broomcorn millet and a method for detecting the number of inserted copies of exogenous genes in transgenic broomcorn millet using digital PCR and fluorescent quantitative PCR. Background Art

[0002] Transgenic plants are plants that contain exogenous genes in their genomes. Among them, the number of copies of exogenous genes integrated into the genome of the recipient plant will affect the expression of the exogenous target gene in the recipient plant and its genetic stability. When the exogenous gene is inserted into the genome of the recipient in a single low copy (1~2), it can generally be expressed stably and efficiently, while when the exogenous gene is integrated into the genome of the recipient in multiple copies, the expression level may be low or even gene silencing, so that transgenic plants expressing the target traits cannot be obtained. Therefore, when selecting transgenic T0 generation strains, researchers often choose transgenic strains with single low copy integration for subsequent research. However, to select transgenic T0 generation strains with single low copy integration, it is often necessary to identify and screen from dozens or even hundreds of transgenic T0 generation strains. If the traditional insertion copy number detection method is used, the workload is large, time-consuming and costly. Therefore, a simple, fast, efficient and high-throughput method for detecting the number of inserted copies of exogenous genes is very important for transgenic plant breeding research.

[0003] The traditional method for detecting the number of inserted copies of exogenous genes in transgenic plants is Southern hybridization. This method has strong specificity and high sensitivity, and its accuracy has been widely recognized. However, it has high requirements for the amount and purity of sample DNA, high cost, and long cycle, which makes it difficult to meet the needs of high-throughput detection of the number of inserted copies of exogenous genes. Fluorescence quantitative PCR is also commonly used to detect the number of inserted copies of exogenous genes in transgenic plants, which can be divided into fluorescent dye method and probe method. Compared with the dye method, the probe method can more accurately quantify the low-copy target DNA fragments, but the probe is expensive and costly. Although the dye method is slightly less accurate than the probe method, its accuracy in detecting a single low-copy exogenous gene is sufficient to meet the needs of breeding work, and its cost is low, which is more suitable for high-throughput detection. At present, the determination of the number of inserted copies of exogenous genes by fluorescence quantitative PCR has been successfully applied to the detection of the number of copies of transgenic crops such as rice and cotton. Digital PCR technology is a technological innovation based on fluorescence quantitative PCR technology. Its analysis results can directly obtain the number of DNA molecules and perform absolute quantification of the starting sample. Compared with fluorescent PCR, digital PCR has better measurement independence and does not require any calibrants, and has higher sensitivity, specificity and accuracy. Studies have shown that digital PCR can simply and accurately determine the number of inserted copies of foreign genes in rice, citrus, potato, corn, tomato and wheat.

[0004] Millet Panicum miliaceum L.) belongs to the genus millet of the Poaceae family. It originated in the Yellow River Basin of my country and is one of the earliest grain crops successfully domesticated by humans. It has strong drought resistance, short growth period and high medicinal value. It is an important crop in arid and semi-arid areas. The protein content in millet is about 13%, which is higher than that of wheat, rice and corn. It is rich in various amino acids, vitamins and minerals, and has the functions of health care and medicine and food. Millet is divided into glutinous and glutinous. The glutinous one is called millet, which is mainly distributed in North China and Northeast China, and the glutinous one is called millet or millet, which is mainly distributed in Northwest China. At present, millet breeding in my country lacks the discovery, creation and utilization of key germplasm, and lacks the application and guidance of modern biological breeding, making it difficult to achieve precision breeding. In recent years, the development of genetic engineering technology has provided a fast and effective way for the breeding and variety improvement of millet. Chinese invention patent application 202111531566.3 discloses a genetic transformation method for millet plants, which uses gene gun bombardment for genetic transformation to obtain transgenic millet. Chinese invention patent 202311246503.2 discloses an Agrobacterium-mediated efficient genetic transformation method for millet, and obtained multiple transgenic millet strains. In recent years, with the development of millet genetic transformation technology, more and more researchers have begun to conduct transgenic millet biological breeding research for the purpose of trait improvement. In order to quickly screen out transgenic millet strains with single low-copy insertion integration from a large number of transgenic millet strains for subsequent breeding research, it is urgent to establish a simple, rapid and efficient method for detecting the number of inserted copies of exogenous genes in transgenic millet, and preliminarily screen single low-copy transgenic millet strains. Summary of the invention

[0005] In view of the above problems, the present invention provides a single-copy millet gene and a method for detecting the number of inserted copies of exogenous genes in millet by combining the single-copy millet gene with digital PCR and fluorescent quantitative PCR techniques.

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

[0007] A single-copy millet gene, the single-copy millet gene is used to detect the number of inserted copies of exogenous genes in transgenic millet SD1 The gene is PM13G15420, which comes from the chromosome chr_13 of millet;

[0008] In the process of detecting the number of inserted copies of foreign genes in transgenic millet, the SD1 Gene as internal reference gene, detection SD1 The sequence of the forward primer SD1-F of the gene is shown in SEQ ID NO: 1, and the sequence of the reverse primer SD1-R is shown in SEQ ID NO: 2.

[0009] Furthermore, in the process of detecting the number of inserted copies of foreign genes in transgenic millet,Hyg Screening marker genes as exogenous genes, detection Hyg The sequence of the forward primer Hyg-F for screening marker gene is shown in SEQ ID NO:4, and the sequence of the reverse primer Hyg-R is shown in SEQ ID NO:5.

[0010] Further, in the process of detecting the number of inserted copies of the exogenous gene in the transgenic millet, the method for determining the number of inserted copies of the exogenous gene in the transgenic millet is as follows:

[0011] When the copy number ratio of the exogenous gene to the internal reference gene or the relative copy number of the exogenous gene is about 0.5, the transgenic millet is a single copy insertion (that is, when the copy number ratio of the exogenous gene to the internal reference gene or the relative copy number of the exogenous gene is 0.26-0.74, the transgenic millet is a single copy insertion);

[0012] When the copy number ratio of the exogenous gene to the internal reference gene or the relative copy number of the exogenous gene is about 1, the transgenic millet has a double copy insertion (i.e., when the copy number ratio of the exogenous gene to the internal reference gene or the relative copy number of the exogenous gene is 0.75-1.25, the transgenic millet has a double copy insertion);

[0013] When the copy number ratio of the exogenous gene to the internal reference gene or the relative copy number of the exogenous gene is about 1.5, the transgenic millet has three copies inserted (that is, when the copy number ratio of the exogenous gene to the internal reference gene or the relative copy number of the exogenous gene is 1.26-1.74, the transgenic millet has three copies inserted);

[0014] The same can be said for other insertion copy numbers of transgenic millet, such as four-copy insertion, five-copy insertion, etc.

[0015] A method for detecting the number of inserted copies of foreign genes in millet using the above-mentioned single-copy millet gene, the method is based on SD1 The gene was used as the internal reference gene. Hyg The screening marker gene was used as the exogenous gene, and the genomic DNA of the transgenic foxtail millet was detected using the primer pair SD1-F / SD1-R and the primer pair Hyg-F / Hyg-R. The copy number of the exogenous gene insertion in the transgenic foxtail millet was determined based on the copy number ratio of the exogenous gene to the internal reference gene or the relative copy number of the exogenous gene.

[0016] Furthermore, the genomic DNA of the transgenic millet is detected using digital PCR technology or fluorescent quantitative PCR technology.

[0017] Furthermore, in the process of digital PCR detection, the method for determining the number of inserted copies of exogenous genes in transgenic millet is as follows:

[0018] Since millet is an allotetraploid and the internal reference gene is a single copy in the millet genome, when the copy number ratio of the exogenous gene to the internal reference gene is about 0.5, the transgenic millet is a single copy insertion (that is, when the copy number ratio of the exogenous gene to the internal reference gene is 0.26-0.74, the transgenic millet is a single copy insertion);

[0019] When the copy number ratio of the exogenous gene to the internal reference gene is about 1, the transgenic millet has a double copy insertion (i.e., when the copy number ratio of the exogenous gene to the internal reference gene is 0.75-1.25, the transgenic millet has a double copy insertion);

[0020] When the copy number ratio of the exogenous gene to the internal reference gene is about 1.5, the transgenic millet has three copies inserted (that is, when the copy number ratio of the exogenous gene to the internal reference gene is 1.26-1.74, the transgenic millet has three copies inserted);

[0021] The same can be said for other insertion copy numbers of transgenic millet, such as four-copy insertion, five-copy insertion, etc.

[0022] Furthermore, in the process of digital PCR detection, the gene SD1 The probe sequence of the internal reference gene is shown in SEQ ID NO: 3. Hyg The probe sequence of the screening marker gene as an exogenous gene is shown in SEQ ID NO:6.

[0023] Furthermore, the digital PCR technology uses a MicroDrop microdroplet digital PCR system to perform a digital PCR reaction;

[0024] In the process of digital PCR detection, the genomic DNA of transgenic foxtail millet was used as the DNA template. The reaction system was: 10.00µL of premixed solution for droplet digital PCR, 1.80µL of SD1-F with a concentration of 10.00µM, 1.80µL of SD1-R with a concentration of 10.00µM, 1.80µL of Hyg-F with a concentration of 10.00µM, 1.80µL of Hyg-R with a concentration of 10.00µM, 0.50µL of probe SD1-P with a concentration of 10.00µM, 0.50µL of probe Hyg-P with a concentration of 10.00µM, 1µL of DNA template with a concentration of 50.00ng / µL, and 0.80µL of nuclease-free water, with a total volume of 20.00µL.

[0025] The reaction amplification program was: 95°C for 10 min; 95°C for 30 s, 60°C for 1 min, 45 cycles; 98°C for 10 min;

[0026] After the digital PCR reaction is completed, the chip is placed in a biochip analyzer to read the FAM and VIC fluorescence signals, and QuantDrop Software is used to perform fluorescence data analysis to obtain the copy number (copy / µL) of the exogenous gene and the internal reference gene in the detection DNA template, and the copy number ratio of the exogenous gene to the internal reference gene in the transgenic millet is calculated. The insertion copy number of the exogenous gene in the transgenic millet genome is determined by the ratio.

[0027] Furthermore, in the process of using fluorescent quantitative PCR technology to detect, the method for determining the number of inserted copies of exogenous genes in transgenic millet is as follows:

[0028] The data were analyzed using QuantStudio™ Design & Analysis Software v1.5.2. Analysis of transgenic millet Hyg The relative copy number of the screening marker gene, with double-copy transgenic millet as the reference factor, is 1, and the multiple of each transgenic millet copy number relative to the reference factor is ; Since the insertion of T0 generation transgenic millet is heterozygous, the relative quantitative value (RQ) of double-copy transgenic millet should be 1 / 2 of the double-copy reference, that is, when the relative copy number of the exogenous gene is about 0.5, the transgenic millet is a single-copy insertion (that is, when the relative copy number of the exogenous gene is 0.26-0.74, the transgenic millet is a single-copy insertion);

[0029] The RQ value of the double-copy transgenic millet was equal to that of the double-copy control, that is, when the relative copy number of the exogenous gene was about 1, the transgenic millet had a double-copy insertion (that is, when the relative copy number of the exogenous gene was 0.75-1.25, the transgenic millet had a double-copy insertion);

[0030] The relative quantitative value RQ of three-copy transgenic foxtail millet should be 3 / 2 of the two-copy control, that is, when the relative copy number of the exogenous gene is about 1.5, the transgenic foxtail millet has three copies inserted (that is, when the relative copy number of the exogenous gene is 1.26-1.74, the transgenic foxtail millet has three copies inserted);

[0031] The same can be said for other insertion copy numbers of transgenic millet, such as four-copy insertion, five-copy insertion, etc.

[0032] Furthermore, the fluorescence quantitative PCR technology was used to detect the double-copy transgenic millet as the reference template, and the reaction was carried out in the QuantStudioTM 1 plus real-time fluorescence quantitative PCR instrument. Analysis of transgenic millet Hyg Relative copy number of screening marker gene;

[0033] In the process of fluorescence quantitative PCR detection, the genomic DNA of transgenic millet was used as the DNA template to amplify SD1 The reaction system of the gene was: 2 x qPCR Master Mix 10.00μL, Low Rox dye 0.20μL, SD1-F at a concentration of 10.00µM 0.40µL, SD1-R at a concentration of 10.00µM 0.40µL, DNA template at a concentration of 50.00ng / µL 2.00μL and ddH2O 7.00μL, with a total volume of 20.00µL;

[0034] 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 at a concentration of 10.00μM, Hyg-R 0.40μL at a concentration of 10.00μM, DNA template 2.00μL at a concentration of 50.00ng / μL, and ddH2O 7.00μL, with a total volume of 20.00μL;

[0035] Amplification SD1 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 60°C for 30 s, and 40 cycles; melting curve was performed at 95°C for 15 s, 60°C for 60 s, and 95°C for 15 s.

[0036] The single-copy millet gene of the present invention and the method for detecting the number of inserted copies of exogenous genes in millet using the single-copy millet gene have the following beneficial effects:

[0037] The present invention provides a single-copy millet gene, which can be used as an internal reference gene for detecting the copy number of foreign gene insertion in transgenic millet, and the most commonly used screening marker hygromycin phosphotransferase gene (hygromycin phosphotransferase, Hyg ) is an exogenous gene, and digital PCR and fluorescence quantitative PCR technology are used to achieve rapid high-throughput detection of the number of inserted copies of exogenous genes in transgenic millet;

[0038] The present invention provides a method for high-throughput detection of the number of inserted copies of exogenous genes in transgenic millet using digital PCR or fluorescent quantitative PCR technology, which has the characteristics of being simple and rapid, having low sample requirements, being highly efficient, and having high throughput, and can improve the efficiency of large-scale screening of transgenic strains, and can provide a new option for detecting the number of inserted copies of exogenous genes in transgenic millet breeding research;

[0039] The present invention provides a method for detecting the number of inserted copies of exogenous genes in transgenic millet using digital PCR technology, which can be used for all Hyg The method is to detect the copy number of transgenic millet with screening markers; the method has the advantages of absolute quantification, high accuracy, simplicity and rapidity, small sample requirement, and batch detection.

[0040] The present invention provides a method for detecting the number of inserted copies of exogenous genes in transgenic millet by using fluorescent quantitative PCR technology, which can be used for all Hyg The method is to detect the copy number of transgenic millet with screening markers; this method has the advantages of being simple, fast, low cost, and requiring few samples. Its accuracy is sufficient to meet the breeding needs of detecting single low copies and is more suitable for batch testing. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 It is the millet gene in Example 1 of the present invention SD1 Alignment results in the genome;

[0042] Figure 2 It is the exploration result of PCR amplification conditions of broomcorn millet internal reference gene and exogenous gene in Example 2 of the present invention; wherein, the left figure is the exploration result of PCR amplification conditions of broomcorn millet exogenous gene, and the right figure is the exploration result of PCR amplification conditions of broomcorn millet internal reference gene; in the figure, M is DL 2000 Marker, the unit is bp, and the molecular weight is 2000, 1000, 750, 500, 250 and 100 from top to bottom; numbers 1 to 10 represent PCR products when annealing temperatures are 52.7°C, 55.6°C, 57.8°C, 60.0°C, 62.2°C, 64.4°C, 66.6°C, 68.4°C, 69.5°C and 70.0°C respectively;

[0043] Figure 3 is the PCR identification result of the transgenic millet in Example 3 of the present invention; in the figure, M is DL 2000 Marker, the unit is bp, and the molecular weights from top to bottom are 2000, 1000, 750, 500, 250 and 100; the numbers 1 to 38 represent the transgenic millet JS1 to JS38 respectively; K- represents Hyg Negative control, K+ represents Hyg Positive control;

[0044] Figure 4 In Example 4 of the present invention SD1 Genes and Hyg Digital PCR scatter plots of genes; Figures A to I are digital PCR scatter plots of transgenic positive plants JS1, JS3, JS4, JS6, JS25, JS27, JS28, JS29 and JS38, respectively; blue dots represent HygNumber of gene fluorescence wells; green scattered dots represent SD1 Gene fluorescence well number; red scattered dots represent SD1 Genes and Hyg The number of gene fluorescence wells, and the gray scattered dots represent the number of wells without signal;

[0045] Figures 5 to 8 In Example 5 of the present invention SD1 Genes and Hyg Fluorescence quantitative primer verification diagram of the gene; among them, Figure 5 for SD1 Gene amplification curve diagram; Figure 6 for SD1 Melting curve plot of the gene; Figure 7 for Hyg Gene amplification curve diagram; Figure 8 for Hyg Melting curve plot of the gene; Figure 6 and Figure 8 The numbers 13, 17, 18, 22, 23, 24, 25 and 33 represent the non-transgenic negative plant JS13 and the transgenic positive plants JS17, JS18, JS22, JS23, JS24, JS25 and JS33, respectively;

[0046] Figure 9 It is the result of fluorescence quantitative PCR copy number detection of 23 transgenic positive plants in Example 5 of the present invention; wherein, the horizontal axis is the number of the transgenic positive plant, the vertical axis is the relative copy number, and the transgenic positive plant JS1 is used as the reference factor. DETAILED DESCRIPTION

[0047] The technical solutions in the embodiments of the present invention are described clearly and completely below. In the following description, many specific details are set forth to facilitate a full understanding of the present invention, but the present invention can also be implemented in other ways different from those described herein, and those skilled in the art can make similar generalizations without violating the connotation of the present invention, so the present invention is not limited by the specific embodiments disclosed below. The present invention is further described in detail below in conjunction with specific embodiments so that those skilled in the art can understand.

[0048] In addition, if no specific techniques or conditions are specified in the specific examples disclosed below, the techniques or conditions described in the literature in this field (for example, refer to "Molecular Cloning Experiment Guide" written by Sambrook et al., translated by Huang Peitang et al., third edition, Science Press) or the product instructions are used. If the manufacturer of the reagents used is not specified, they are all conventional products that can be purchased.

[0049] Example 1 Determination of internal reference genes

[0050] SD1The bioinformatics analysis of the copy number of the gene in the millet genome specifically includes the following steps:

[0051] Use makeblast Longmi-V2 version of the pep file to build the library, use SD1 The gene sequence is used as the query sequence, and blast is used to compare the query sequence with the sequence in the target database to find the local similarity region between the two, and finally obtain the comparison result. Figure 1 From the comparison results, we can see that SD1 The gene is a single-copy gene and can be used as an internal reference gene for detecting the insertion copy number of exogenous genes in transgenic millet.

[0052] Example 2 Exploration of PCR amplification conditions

[0053] Since the commonly used screening marker gene in transgenic millet is Hyg Genes, therefore selection Hyg Screening marker genes as foreign genes in transgenic millet and using SD1 The gene is used as an internal reference gene for detection, wherein the PCR amplification conditions of the internal reference gene and the exogenous gene primers are explored, including the following specific steps:

[0054] 1) Primer sequence

[0055] Detection of internal reference genes SD1 Primer pair sequences:

[0056] The forward primer SD1-F is: 5′-CAAGCTCCCGTGGAAGG-3′, as shown in SEQ ID NO: 1;

[0057] The reverse primer SD1-R is: 5'-CCCATTGGCTCGAAATCCT-3', as shown in SEQ ID NO: 2;

[0058] Detection Hyg Primer pair sequences for screening marker genes:

[0059] The forward primer Hyg-F is: 5'-GTCAAGACCAATGCGGAGCA-3', as shown in SEQ ID NO: 4;

[0060] The reverse primer Hyg-R is: 5'-CCCAATACGAGGTCGCCAAC-3', as shown in SEQ ID NO: 5;

[0061] 2) SD1 Gene primers and Hyg Exploration of PCR amplification conditions for gene primers

[0062] Leaves of a transgenic millet plant were collected and their genomic DNA was extracted using the MEGA Plant Genomic DNA Rapid Extraction Kit (D3187-02). The leaves were used as DNA templates and gradient PCR reactions were performed using the SD1-F / SD1-R primer pair and the Hyg-F / Hyg-R primer pair, respectively.

[0063] The PCR reaction system for detecting the internal reference gene was: Green Master Mix 12.50 µL, SD1-F (10.00 µM) 1.00 µL, SD1-R (10.00 µM) 1.00 µL, DNA template (50.00 ng / µL) 1.00 µL, and nuclease-free water 9.50 µL, with a total volume of 25.00 µL.

[0064] 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 (50.00 ng / µL) 1.00 µL, and nuclease-free water 9.50 µL, with a total volume of 25.00 µL.

[0065] 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 52.7°C, 55.6°C, 57.8°C, 60.0°C, 62.2°C, 64.4°C, 66.6°C, 68.4°C, 69.5°C and 70.0°C, respectively.

[0066] After the PCR amplification reaction is completed, take 5 μL of the corresponding PCR product and perform electrophoresis on 2% agarose gel. SD1 The PCR products 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 SD1 Genes and Hyg The nucleotide sequences of the genes were compared.

[0067] The electrophoresis results are as follows Figure 2 As shown, when the annealing temperatures are 52.7°C, 55.6°C, 57.8°C, 60.0°C, 62.2°C and 64.4°C respectively, SD1 Genes and Hyg The target amplified bands were obtained for all genes, but the bands were brightest when the annealing temperature was 60.0℃, and there were no non-specific bands. SD1 PCR products of genes and Hyg The PCR products of the genes were sequenced and compared withSD1 The gene sequence and Hyg The sequences of the genes are identical, so the SD1-F / SD1-R primer pair can be used for millet SD1 Gene detection, Hyg-F / Hyg-R can be used for exogenous genes Hyg The annealing temperature of the gene was determined to be 60.0℃.

[0068] Example 3 Hyg Gene PCR identification

[0069] Using Example 2 Hyg Gene primer pair Hyg The PCR identification of transgenic millet positive plants by gene analysis specifically includes the following steps:

[0070] The leaves of 38 transgenic foxtail millet plants (numbered JS1 to JS38) were collected and their genomic DNA was extracted using the MEGA Plant Genomic DNA Rapid Extraction Kit (D3187-02) as a DNA template. Hyg Negative control, containing Hyg Genetically modified millet Hyg Positive control, using the method in Example 2 through Hyg-F / Hyg-R Hyg The PCR experiment of the gene was performed to exclude non-transgenic negative millet plants. The PCR reaction amplification system and reaction conditions were the same as those in Example 2. Hyg The PCR reaction amplification system and reaction conditions of the gene were the same, and the annealing temperature was 60.0°C.

[0071] After the PCR amplification reaction was completed, 5 μL of the corresponding PCR product was taken for electrophoresis detection on 2% agarose gel. If a target band of about 143 bp appeared, it was a transgenic millet plant. If no target band was found, it was a non-transgenic negative plant. The PCR detection results of 38 transgenic millet plants are shown in Figure 3 Among them, the plants numbered JS1, JS3, JS4, JS6~JS10, JS14-JS19, JS21~JS25, JS27~JS34 and JS38 were all transgenic positive plants, totaling 28 plants; the plants numbered JS2, JS5, JS11~JS13, JS20, JS26 and JS35~JS37 were all non-transgenic negative plants, totaling 10 plants.

[0072] Example 4 Digital PCR Detection

[0073] The method for detecting the number of inserted copies of exogenous genes in transgenic millet by digital PCR comprises the following specific steps:

[0074] 1) Digital PCR primers and probes

[0075] Detection of internal reference genes SD1 Primer pairs and probe sequences:

[0076] The forward primer SD1-F is: 5'-CAAGCTCCCGTGGAAGG-3', as shown in SEQ ID NO: 1;

[0077] The reverse primer SD1-R is: 5'-CCCATTGGCTCGAAATCCT-3', as shown in SEQ ID NO: 2;

[0078] Probe SD1-P is: VIC-AGACCCTCTCCTTCGGCTTCCA-MGB, as shown in SEQ ID NO: 3;

[0079] Detection Hyg Primer pairs and probe sequences for screening marker genes:

[0080] The forward primer Hyg-F is: 5'-GTCAAGACCAATGCGGAGCA-3', as shown in SEQ ID NO: 4;

[0081] The reverse primer Hyg-R is: 5'-CCCAATACGAGGTCGCCAAC-3', as shown in SEQ ID NO: 5;

[0082] The probe Hyg-P is: FAM-TCGAAGTAGCGCGTCTGCTGCTCCA-BHQ1, as shown in SEQ ID NO:6.

[0083] 2) DNA template extraction

[0084] From the 28 transgenic positive plants screened in Example 3, 9 plants (JS1, JS3, JS4, JS6, JS25, JS27, JS28, JS29 and JS38) were selected as test samples, and the non-transgenic negative plant JS2 was used as a negative control. The corresponding genomic DNA was extracted using the MEGA Plant Genome Kit as a DNA template.

[0085] 3) Digital PCR method to detect the number of inserted copies of exogenous genes in the sample to be tested

[0086] by SD1 The gene is an internal reference gene. HygThe gene is an exogenous gene, and the insertion copy number of the exogenous gene in the transgenic millet genome was detected by digital PCR experiment. Each sample to be tested was repeated 3 times, and the DNA of the non-transgenic negative plant JS2 was used as a negative control. The MicroDrop droplet digital PCR system was used, and the reaction system was: 10.00µL of droplet digital PCR reaction premix, 1.80µL of SD1-F (10.00µM), 1.80µL of SD1-R (10.00µM), 1.80µL of Hyg-F (10.00µM), 1.80µL of Hyg-R (10.00µM), 0.50µL of probe SD1-P (10.00µM), 0.50µL of probe Hyg-P (10.00µM), 1µL of DNA template (50.00ng / µL), and 0.80µL of nuclease-free water, with a total volume of 20.00µL.

[0087] The reaction amplification program was as follows: 95°C for 10 min; 95°C for 30 s, 60°C for 1 min, 45 cycles; 98°C for 10 min.

[0088] 4) Result analysis

[0089] After the reaction is completed, the obtained 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 corresponding DNA template. Hyg Gene and SD1 The copy number of the gene (copy / µL) is calculated in the DNA template Hyg Gene and SD1 The copy number ratio of the gene is used to determine Hyg The copy number of the gene in the millet genome.

[0090] Since millet is an allotetraploid 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), and the insertion copy numbers of quadruple, pentacle and other multi-copy exogenous gene fragments are similar.

[0091] The digital PCR scatter plots of the 9 transgenic positive plants in this example are shown in Figure 4The copy number detection results are shown in Table 1. The copy number ratio of the transgenic positive plant JS6 is close to 0.5, which is a single copy insertion; the copy number ratios of the transgenic positive plants JS1, JS3, JS4, JS25, JS27, JS28, JS29 and JS38 are all close to 1, which is a double copy insertion.

[0092] Table 1 Results of digital PCR detection of the number of inserted copies of exogenous genes in transgenic millet

[0093]

[0094] Example 5 Fluorescence quantitative PCR detection

[0095] The method for detecting the number of inserted copies of exogenous genes in transgenic millet plants by using fluorescent quantitative PCR comprises the following specific steps:

[0096] 1) Fluorescence quantitative PCR primers

[0097] Detection of internal reference genes SD1 Primer pairs and probe sequences:

[0098] The forward primer SD1-F is: 5'-CAAGCTCCCGTGGAAGG-3', as shown in SEQ ID NO: 1;

[0099] The reverse primer SD1-R is: 5'-CCCATTGGCTCGAAATCCT-3', as shown in SEQ ID NO: 2;

[0100] Detection Hyg Primer pairs and probe sequences for screening marker genes:

[0101] The forward primer Hyg-F is: 5'-GTCAAGACCAATGCGGAGCA-3', as shown in SEQ ID NO: 4;

[0102] The reverse primer Hyg-R is: 5'-CCCAATACGAGGTCGCCAAC-3', as shown in SEQ ID NO:5.

[0103] 2) DNA template extraction

[0104] The 23 transgenic positive plants screened in Example 3 were used as samples to be tested, wherein the 23 transgenic positive plants were numbered as follows: JS2, JS3, JS4, JS5, JS6, JS7, JS8, JS9, JS10, JS11, JS12, JS13, JS14, JS15, JS16, JS17, JS18, JS19, JS20, JS21, JS22, JS23, JS24, JS25, JS26, JS30, JS31, JS32, JS33, JS34, JS35, JS36 and JS37. The double-copy transgenic positive plant JS1 detected in Example 4 was used as a control, and the non-transgenic negative plant JS2 screened in Example 3 was used as a negative control, and the corresponding genomic DNA was extracted using the MEGA Plant Genome Kit as a DNA template.

[0105] 3) Primer verification

[0106] The genomic DNA of seven transgenic positive plants (JS17, JS18, JS22, JS23, JS24, JS25 and JS33) and one non-transgenic negative plant JS13 were randomly selected as templates for fluorescence quantitative PCR experiments to verify the internal reference gene SD1 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.);

[0107] Amplification SD1 The reaction system of the gene was: 2 x qPCR Master Mix 10.00μL, Low Rox dye 0.20μL, SD1-F (10.00µM) 0.40µL, SD1-R (10.00µM) 0.40µL, DNA template (50.00ng / µL) 2.00μL and ddH2O7.00μL, with a total volume of 20.00µL;

[0108] Amplification Hyg The reaction system of the gene was: 2 x qPCR Master Mix 10.00μL, Low Rox dye 0.20μL, Hyg-F (10.00µM) 0.40µL, Hyg-R (10.00µM) 0.40µL, DNA template (50.00ng / µL) 2.00μL and ddH2O7.00μL, with a total volume of 20.00µL.

[0109] Amplification SD1 Gene and HygThe amplification procedures of the genes were: 95℃ pre-denaturation for 30s; 95℃ denaturation for 10s, 60℃ annealing for 30s, 40 cycles; melting curve 95℃ 15s, 60℃ 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.

[0110] 4) Fluorescence quantitative PCR detection of the number of copies of exogenous genes in the sample to be tested

[0111] by SD1 The gene is an internal reference gene. Hyg The gene is an exogenous gene, and the double-copy transgenic positive plant JS1 is used as a control. The insertion copy number of the exogenous gene in the millet genome of all the samples to be tested (i.e., 23 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.

[0112] 5) Result analysis

[0113] 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, and the transgenic positive plant JS1 with known double copies is defined 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 the double-copy control, which is about 1.5 (generally 1.26~1.74); the copy number of four-copy, five-copy, and other multi-copy exogenous gene insertions is similar.

[0114] The results of the fluorescence quantitative PCR detection of the copy number of 23 transgenic positive plants in this example are shown in Figure 9 and Table 2.

[0115] Table 2 Results of fluorescence quantitative PCR detection of the copy number of exogenous genes inserted into transgenic millet

[0116]

[0117] Depend on Figure 9 As can be seen from Table 2, there are 3 transgenic positive plants (JS6, JS15 and JS21) Hyg The relative copy number of the gene is about 0.5, which is a single-copy inserted transgenic plant. There are 7 transgenic positive plants (JS3, JS4, JS9, JS14, JS18, JS23 and JS25). Hyg The relative copy number of the gene is about 1, which is a transgenic plant with double copy insertion, and the other samples are transgenic plants with three copies or more. Among them, the copy number of the transgenic positive plants JS3, JS4, JS6 and JS25 detected by the fluorescent quantitative PCR of this embodiment is consistent with the copy number detected by digital PCR in Example 4. The above results show that the fluorescent quantitative PCR method in this embodiment has high accuracy, low cost, simple and fast when detecting the single low copy integration of exogenous genes in transgenic millet, and is suitable for initial screening of single low copy integration plants in a large number of transgenic plants.

[0118] Although the above embodiments have been described in detail, they are only a part of the embodiments of the present invention, rather than all the embodiments. A person skilled in the art can also obtain other embodiments based on the embodiments without inventiveness, 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 millet using digital PCR or fluorescent quantitative PCR, characterized in that: The method is to SD1 Gene as internal reference gene, SD1 The gene is PM13G15420, Hyg Screening marker genes as exogenous genes, using primer pairs SD1-F / SD1-R and primer pairs Hyg-F / Hyg-R to detect genomic DNA of transgenic millet, and determining the number of inserted copies of the exogenous genes in the transgenic millet; Detection SD1 The forward primer SD1-F sequence of the gene is shown in SEQ ID NO: 1, and the reverse primer SD1-R sequence is shown in SEQ ID NO: 2; Detection Hyg The forward primer Hyg-F sequence of the screening marker gene is shown in SEQ ID NO: 4, and the reverse primer Hyg-R sequence is shown in SEQ ID NO: 5; In the digital PCR detection process, the gene SD1 The sequence of the probe SD1-P used as the internal reference gene is shown in SEQ ID NO:

3. Hyg The screening marker gene is used as a probe Hyg-P sequence of the exogenous gene as shown in SEQ ID NO: 6; In the process of digital PCR detection, the method for determining the number of inserted copies of exogenous genes in transgenic millet is as follows: When the copy number ratio of the exogenous gene to the internal reference gene was 0.26-0.74, the transgenic millet had a single copy insertion; When the copy number ratio of the exogenous gene to the internal reference gene was 0.75-1.25, the transgenic millet had 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 millet had three copies inserted; The same goes for other insertion copy numbers of transgenic millet; In the process of fluorescence quantitative PCR detection, the method for determining the number of inserted copies of exogenous genes in transgenic millet is as follows: When the relative copy number of the exogenous gene was 0.26-0.74, the transgenic millet had a single copy insertion; When the relative copy number of the exogenous gene was 0.75-1.25, the transgenic millet had a double copy insertion; When the relative copy number of the exogenous gene was 1.26-1.74, the transgenic millet had three copies inserted; The same goes for other insertion copy numbers of transgenic millet.

2. The method according to claim 1, characterized in that In the digital PCR detection process, the genomic DNA of transgenic foxtail millet was used as the DNA template. The reaction system was: reaction premix 10.00µL, SD1-F 1.80µL with a concentration of 10.00µM, SD1-R 1.80µL with a concentration of 10.00µM, Hyg-F 1.80µL with a concentration of 10.00µM, Hyg-R 1.80µL with a concentration of 10.00µM, probe SD1-P 0.50µL with a concentration of 10.00µM, probe Hyg-P 0.50µL with a concentration of 10.00µM, DNA template 1µL with a concentration of 50.00ng / µL, and nuclease-free water 0.80µL, with a total volume of 20.00µL; The reaction amplification program was: 95°C for 10 min; 95°C for 30 s, 60°C for 1 min, 45 cycles; 98°C for 10 min.

3. The method according to claim 1, characterized in that In the process of fluorescence quantitative PCR detection, the genomic DNA of transgenic millet was used as the DNA template to amplify SD1 The reaction system for the gene was: 2 x qPCR Master Mix 10.00μL, Low Rox dye 0.20μL, SD1-F at a concentration of 10.00µM 0.40µL, SD1-R at a concentration of 10.00µM 0.40µL, DNA template at a concentration of 50.00ng / µL 2.00μL, and ddH2O 7.00μL, with a total volume of 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 at a concentration of 10.00μM, Hyg-R 0.40μL at a concentration of 10.00μM, DNA template 2.00μL at a concentration of 50.00ng / μL, and ddH2O 7.00μL, with a total volume of 20.00μL; Amplification SD1 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 60°C for 30 s, and 40 cycles; melting curve was performed at 95°C for 15 s, 60°C for 60 s, and 95°C for 15 s.

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