CN116397040B - Single copy papaya gene and method for detecting copy number of exogenous gene in transgenic papaya by using same - Google Patents

Single copy papaya gene and method for detecting copy number of exogenous gene in transgenic papaya by using same Download PDF

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CN116397040B
CN116397040B CN202211329253.4A CN202211329253A CN116397040B CN 116397040 B CN116397040 B CN 116397040B CN 202211329253 A CN202211329253 A CN 202211329253A CN 116397040 B CN116397040 B CN 116397040B
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夏启玉
赵辉
谢秀菊
张雨良
郭安平
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Sanya Research Institute Chinese Academy Of Tropical Agricultural Sciences
Institute of Tropical Bioscience and Biotechnology Chinese Academy of Tropical Agricultural Sciences
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Institute of Tropical Bioscience and Biotechnology Chinese Academy of Tropical Agricultural Sciences
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Abstract

The invention discloses a single-copy papaya gene and a method for detecting the copy number of an exogenous gene in transgenic papaya by using digital PCR and fluorescent quantitative PCR, belonging to the field of biotechnology, wherein the single-copy gene is Cpa03g018830 or Cpa03g018770 and is derived from a papaya chr_03 chromosome, the forward detection primer sequence of the gene Cpa03g018830 is shown as SEQ No.1, the reverse detection primer sequence is shown as SEQ No.2, the forward detection primer sequence of the gene Cpa03g018770 is shown as SEQ No.4, and the reverse detection primer sequence is shown as SEQ No. 5; the application is to use the gene Cpa03g018830 or Cpa03g018770 as an internal reference gene for detecting the copy number of an exogenous gene in transgenic papaya.

Description

Single copy papaya gene and method for detecting copy number of exogenous gene in transgenic papaya by using same
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a method for detecting the copy number of an exogenous gene in transgenic papaya by using digital PCR and fluorescent quantitative PCR and application thereof.
Background
The transgenic crop industry since the first 1996Global transgenic technology research and industrial applications have rapidly evolved for chemical applications. Transgenic plants are plants containing exogenous genes in the genome, and are possible to change certain genetic characteristics of the plants and cultivate new crop varieties with high yield, high quality, virus resistance, insect resistance, cold resistance, drought resistance, waterlogging resistance, salt and alkali resistance, herbicide resistance and the like. The integration of an exogenous gene into the genome of a recipient plant is critical to the acquisition of a trait of interest in a transgenic plant, wherein the copy number of the exogenous gene integrated into the genome of the recipient plant affects the expression of the exogenous gene of interest and its genetic stability. When the exogenous gene is integrated into the receptor genome at 1-2 copy numbers, stable and efficient expression can be generally achieved, and when the exogenous gene is integrated into the receptor genome at multiple copy numbers, low expression levels and even gene silencing may occur, so that transgenic plants expressing the desired traits cannot be obtained. Thus, researchers are screening for transgenic T 0 Single low copy integrated transgenic lines are often selected for the generation lines because of the higher genetic stability, gene expression levels, and survival of the single low copy integrated transgenic plant progeny. However, single low copy integrated transgene T 0 The selection of the generation lines often requires the selection from hundreds of lines, and the acquisition of homozygous transgenic plants also requires T isolated from the trait 1 And screening the transgenic plants. Therefore, the detection of the copy number of the exogenous gene is an important link of transgenic plant breeding, and a simple, rapid, efficient and high-throughput exogenous gene copy number detection method is very important.
The traditional method for detecting the copy number of the exogenous gene in the transgenic plant is Southern hybridization, has strong specificity and high sensitivity, has obtained general acceptance on the accuracy, but has higher requirements on the quantity and purity of sample DNA, has high cost and long period, and is difficult to meet the requirement of high-throughput exogenous gene copy number detection. Fluorescent quantitative PCR is also commonly used for detecting the copy number of exogenous genes in transgenic plants, and can be divided into a fluorescent dye method and a probe method, wherein the probe method can more accurately perform quantitative analysis on target DNA fragments with low copy number compared with the dye method, but the probe is more expensive and has high cost. Although the dye method has slightly lower accuracy compared with the probe method, the dye method has lower cost and is more suitable for high-throughput detection, and the accuracy in detecting single low copy exogenous genes is enough to meet the requirements of breeding work. Currently, fluorescent quantitative PCR (polymerase chain reaction) determination of exogenous gene copy number has been successfully applied to copy number detection of transgenic rice, cotton and other crops. The digital PCR technology is a technological innovation based on the fluorescent quantitative PCR technology, and the analysis result can directly obtain the number of DNA molecules to absolutely quantify the initial sample. Compared with fluorescent PCR, the digital PCR has better measurement independence, does not need any calibrator, and has higher sensitivity, specificity and accuracy. Research shows that digital PCR can simply and accurately determine the copy number of exogenous genes in rice, citrus, potato, corn, tomato and wheat.
Papaya is one of the four major tropical and subtropical fruits in the world. The biggest limitation on papaya production is papaya virus disease, under the condition that disease-resistant hybridization breeding is difficult to develop, transgenic technology is introduced into papaya breeding, the earliest development is transgenic antiviral disease breeding, the first transgenic papaya variety is obtained by carrying out gene gun transformation by using embryogenic callus of a variety Sunset, the variety is transferred into a mild Coat Protein (CP) gene of Caulophyllum virens (papaya ringspot virus, PRSV) 55-1 of Caulophyllum virens, and the field resistance performance is quite good, so that the strain is commercially applied in Hawaii in 1997. Papaya line X17-2 transformed with PRSV X17-2 line CP gene was commercially used in the university of Florida U.S.A. in 2008. The research of papaya transgenic technology in China starts from 1996, the first transgenic papaya 'Huanong No. 1' is developed by the agricultural university of south China, and the national issued papaya agricultural transgenic biological safety certificate is obtained in 2010, so that the commercial planting of the transgenic papaya is allowed to be formally introduced. In recent years, researchers are still continuously researching new papaya transgenic breeding, and the traditional Southern hybridization method is difficult to meet the requirement of detecting the copy number of the exogenous genes in a large number of transgenic lines, so a simple, rapid, efficient and high-throughput method for detecting the copy number of the transgenic papaya is needed.
Disclosure of Invention
In order to solve the technical problems, the invention provides a single-copy papaya gene and a method for detecting the exogenous transgene copy number of papaya by utilizing the single-copy gene through combining with digital PCR and fluorescent quantitative PCR technologies.
The technical scheme of the invention is as follows: the invention provides two papaya single-copy genes as reference genes for copy number detection, wherein the most commonly used screening marker gene NPTII (neomycin phosphotransferase) of transgenic papaya is used as an exogenous target gene, and the rapid high-throughput detection of the exogenous gene copy number in the transgenic papaya is realized by utilizing digital PCR and fluorescent quantitative PCR technologies. Specific:
a single copy of papaya gene, cpa03g018830 or Cpa03g018770, both derived from the chromosome of papaya chr_03, wherein the forward detection primer sequence of Cpa03g018830 is shown in SEQ No.1, the reverse detection primer sequence is shown in SEQ No.2, the forward detection primer sequence of Cpa03g018770 is shown in SEQ No.4, and the reverse detection primer sequence is shown in SEQ No. 5.
Use of a single copy of a papaya gene, cpa03g018830 or Cpa03g018770, as an internal reference gene for detecting the copy number of an exogenous gene in transgenic papaya.
Furthermore, the transgenic papaya takes NPT II screening marker gene as an exogenous target gene, the forward detection primer sequence of the NPT II screening marker gene is shown as SEQ No.9, and the reverse detection primer sequence is shown as SEQ No. 10.
A method for detecting the copy number of exogenous gene in transgenic papaya by digital PCR features that the genomic DNA of papaya sample is detected by the forward and reverse detection primers of Cpa03g018830 or Cpa03g018770 and the forward and reverse detection primers of NPT II screening marker gene by digital PCR, and the copy number of exogenous gene in the sample is determined by the ratio of exogenous gene to internal gene.
Further, using a Quantum 3D AnalysisSuiteTM digital PCR apparatus (Semer Feishul technology Co., ltd.), the conditions of the reaction system when the digital PCR technique is used for detection are as follows: 2 XddPCR Master Mix 7.30. Mu.L, 1.30. Mu.L each of forward primer and reverse primer (10. Mu.M) for internal gene and foreign gene, 0.36. Mu.L each of probe (10. Mu.M), 1. Mu.L of DNA template (10 ng/. Mu.L), 0.28. Mu.L of nucleic-free water, and 14.50. Mu.L total volume. The amplification procedure was as follows: 96 ℃ for 10min; 2min at 60 ℃, 30s at 98 ℃ and 39 cycles; and 60 ℃ for 2min.
Further, the method for judging the copy number of the exogenous target gene in the transgenic papaya genome is that the copy number ratio of the exogenous target gene to the internal reference gene is single copy when the ratio is 0.5, double copy when the ratio is 1, three copies when the ratio is 1.5, and the copy number of other multi-copy exogenous genes is similar. Specific: after the digital PCR reaction is finished, placing the chip into a digital PCR reader to read FAM and VIC fluorescent signals, and performing fluorescent data analysis by using Quantum 3D AnalysisSuiteTM Software to obtain copy numbers (copy/mu L) of the exogenous target gene and the internal reference gene in the detected sample DNA, calculating the copy number ratio of the exogenous target gene to the internal reference gene in the sample, and judging the copy number of the exogenous target gene in the transgenic papaya genome according to the ratio. The papaya is diploid and the reference gene is single-copy in the papaya, so that the ratio of the copy number of the single-copy integrated exogenous gene fragment to the copy number of the reference gene is about 0.5, the ratio of the copy number of the double-copy integrated exogenous gene fragment to the copy number of the reference gene is about 1, the ratio of the copy number of the three-copy integrated exogenous gene fragment to the copy number of the reference gene is about 1.5, the integrated copy number of the four-copy, five-copy and other multi-copy exogenous gene fragments is similar.
Further, when the gene Cpa03g018830 is used as the reference gene, the probe sequence is shown in SEQ No.3, and when the gene Cpa03g018770 is used as the reference gene, the probe sequence is shown in SEQ No.6, and the probe sequence of the NPT II screening marker gene is shown in SEQ No. 12.
A method for detecting the copy number of exogenous gene in transgenic papaya by fluorescent quantitative PCR features that the forward and reverse detection primers of Cpa03g018830 or Cpa03g018770 gene and the forward and reverse detection primers of NPT II screening marker gene are used to test the genome D of papayaNA was detected by fluorescent quantitative PCR technique using 2 -ΔΔCt Analyzing the relative copy number of the NPTII screening marker gene in the transgenic papaya by the method, and judging the copy number of the exogenous target gene in the genome of the transgenic papaya by the relative quantitative value.
Furthermore, when the fluorescent quantitative PCR technology is used for detection, the homozygous single-copy transgenic papaya strain is used as a reference template, and the reaction system conditions are as follows: 2x Q3 SYBR qPCR Master Mix 10. Mu.L each of the forward primer and the reverse primer (10. Mu.M) was 0.4. Mu.L, and Template DNA (50 ng/. Mu.L) was 1. Mu.L, ddH 2 O9.2. Mu.L, total volume 20. Mu.L. The amplification procedure was: pre-denaturation at 95 ℃ for 30s; denaturation at 95℃for 10s, annealing at 58℃for 30s,40 cycles; melting curves were 95℃15s,60℃60s,95℃15s. Each sample was repeated 3 times.
Further, the method for judging the copy number of the exogenous target gene in the transgenic papaya genome comprises the following steps: the known single copy homozygous transgenic plant is used as a reference factor, namely 1, and the copy number of each sample relative to the reference factor is multiplied by 2 -ΔΔCt The relative expression amount of single copy plants is 0.5, the relative expression amount of double copy plants is 1, the relative expression amount of three copy plants is 1.5, the integrated copy number of multi-copy exogenous genes is similar. Specific:
after the fluorescent quantitative PCR reaction is finished, the method is used96SW 1.1 System for data analysis, 2 -ΔΔCt Analyzing the relative copy number of NPTII gene in transgenic plant by using method, defining known single copy homozygote transgenic plant as reference factor, i.e. 1, and the multiple of copy number of each sample relative to reference factor is 2 -ΔΔCt . Due to T 0 The plant of the generation is inserted into heterozygous type, so that the relative quantitative value (RQ) of the single copy plant is 1/2 of that of the homozygous single copy reference, and the relative expression quantity is about 0.5; the RQ value of the double-copy plant is equal to that of the homozygous single-copy control, and the relative expression quantity is about 1; the relative quantitative RQ of the three-copy plants should be 3/2 of that of the homozygous single-copy control, and the relative expression amount is about 1.5; integration of copy number of multiple copies of exogenous gene to facilitate such。
The beneficial effects of the invention are as follows:
the invention aims to provide a method for detecting the exogenous gene copy number of transgenic papaya by utilizing digital PCR and fluorescent quantitative PCR technology with high flux, which has the characteristics of simplicity, rapidness, low sample requirement, high efficiency, high flux and the like, improves the efficiency of large-scale transgenic strain screening, and can provide a new choice for the detection method of the exogenous gene copy number in the breeding research of transgenic papaya.
The 2 papaya single copy genes provided by the invention can be used as internal reference genes for detecting the copy number of exogenous genes of transgenic papaya; the method for detecting the copy number of the exogenous gene in the transgenic papaya by utilizing the digital PCR technology provided by the invention can be used for detecting the copy number of all transgenic papaya taking NPTII as a screening mark. The method is absolute quantitative, high in accuracy, simple and rapid, small in sample demand and capable of detecting in batches; the method for detecting the copy number of the exogenous gene in the transgenic papaya by utilizing the fluorescent quantitative PCR technology provided by the invention can be used for detecting the copy number of all transgenic papaya taking NPTII as a screening mark. The method is simple and quick, low in cost and small in sample demand, and the accuracy is enough to meet the breeding demand of single-copy detection, so that the method is more suitable for batch detection.
Drawings
FIG. 1 is 21 plants of CP transgenic papaya T 1 And (5) detecting the PCR result of the young seedling. M is DL 2000Marker, the unit is bp, and the molecular weight is 2000, 1000, 750, 500, 250 and 100 from top to bottom. 1-21 is transgenic CP papaya T 1 Seedlings were replaced, K+ was positive control for transgenic CP papaya and K-was water control.
FIG. 2 is a digital PCR scattergram of Cpa03g018830 gene and CP gene. A-H are CP1-CP8 respectively; blue dispersion point: CP gene fluorescent pore number; red dispersion point: cpa03g018830 gene fluorescent pore number; green dispersion point: CP gene and Cpa03g018830 gene fluorescent pore number, yellow scatter: no signal hole number.
FIG. 3 is a fluorescent quantitative primer verification chart. A is an amplification curve graph of NPTII gene; b is a melting curve graph of NPTII gene; c is an amplification curve graph of Cpa03g018770 gene; d is the melting curve diagram of Cpa03g018770 gene; e is an amplification plot of Cpa03g018830 gene; f is the melting curve of Cpa03g018830 gene.
FIG. 4 shows the result of detecting the copy number by fluorescence quantitative PCR of HC-Pro-12-HC-Pro-55. HC-Pro-12-HC-Pro-55 is transgenic for HC-Pro gene papaya T 0 Substitution; 830 is Cpa03g018830 as the reference gene; 770 is an internal reference gene Cpa03g 018770.
FIG. 5 is the result of the fluorescent quantitative PCR detection of copy number of Nib-1-Nib-28. Nib-1-Nib-28 is Nib-gene-transferred papaya T 0 Substitution; 830 is Cpa03g018830 as the reference gene; 770 is an internal reference gene Cpa03g 018770.
FIG. 6 is the result of the fluorescent quantitative PCR detection of copy numbers of samples CP1-CP8 and HC-Pro-1-HC-Pro-11. CP1-CP8 is transgenic papaya T 1 Substitution; HC-Pro-1-HC-Pro-11 is transgenic for HC-Pro gene papaya T 0 Substitution; 830 is Cpa03g018830 as the reference gene; 770 is an internal reference gene Cpa03g 018770.
Detailed Description
The present invention is described in further detail below in conjunction with specific embodiments for understanding by those skilled in the art. The specific techniques or conditions are not identified in the examples and are performed according to techniques or conditions described in the literature in this field or according to the product specifications. The reagents used were not manufacturer-identified and were all conventional commercially available products.
Example 1
The detection of the copy numbers of papaya genes Cpa03g018830 and Cpa03g018770 in the genome specifically comprises the following steps:
1) T of transgenic CP gene single copy papaya 1 Molecular characterization of generation plants
By T 0 T of transgenic papaya with single copy verified by Southern hybridization 1 Young seedlings were generated as reference for identification of copy number of reference genes. Since papaya belongs to a diploid plant, T 1 The papaya plant accords with Mendelian genetic law, and the CP is homozygous: CP heterozygosity: the non-rotating ratio should be 1:2:1. t for collecting 21 strains of CP transgenic papaya 1 Leaf of young plant is extracted by using Bomaide plant genome DNA rapid extraction kit (CZ 301-01), and non-transformed T is eliminated by conventional PCR experiment 1 Papaya seedlings were replaced. The PCR detection primers were as follows: the forward primer CP-F is: 5'-TCACGAGCCCTATCAGGTGTCTTT-3' (SEQ No. 7), the reverse primer CP-R is: 5'-TCAACGCCGGAACTAGTGGAACTTPCR-3' (as shown in SEQ No. 8). The PCR reaction system is as follows: green Master Mix 12.5. Mu.L, CP-F (10. Mu.M) 1. Mu.L, CP-R (10. Mu.M) 1. Mu.L, DNA template (50 ng/. Mu.L) 1. Mu.L, nucleic-free water 9.5. Mu.L, and total volume 25. Mu.L. The PCR reaction conditions were: pre-denaturation at 94℃for 3min; denaturation at 94℃for 30s, annealing at 58℃for 30s, extension at 72℃for 45s for 35 cycles; finally, the mixture is extended for 10min at 72 ℃ and stored at 4 ℃. After the PCR amplification reaction is finished, 10 mu L of PCR products are taken to carry out electrophoresis detection on 2% agarose gel, if about 500bp of target bands appear, the target bands are transgenic plants, the target bands are possibly homozygous or heterozygous with CP, and if no target bands exist, the target bands are non-transgenic plants. 21 strain T 1 The PCR detection results of the young seedlings are shown in figure 1, wherein 2, 3, 4, 6, 7, 8, 9, 10, 11, 13, 14, 15, 16, 17, 18, 19 and 21 are transgenic positive plants; 1.5, 12, 20 are non-transgenic plants. The 17 transgenic positive plants can be used as references for identifying copy numbers of internal reference genes.
2) Digital PCR detection primer and probe
Cpa03g018830 gene:
forward primer 830-F was: 5'-TTCGAACGAGTCTGCAGTGG-3' (SEQ No. 1)
Reverse primer 830-R is: 5'-ACCACCTGAACCCAGGCTAA-3' (SEQ No. 2)
Probes 830-P are: VIC-ACGTGGCGGAGGGTGGTGAAGCTGATA-MGB; (as shown in SEQ No. 3)
Cpa03g018770 gene:
the forward primer 770-F is: 5'-ATCCCAATTGCAGCTGGTGG-3' (SEQ No. 4)
The reverse primer 770-R is: 5'-GTGAACCCTGGTGATGCCAA-3' (SEQ No. 5)
The probes 770-P are: VIC-TCCAGTTGAGTTTGGTGGCGGCCACA-MGB (SEQ No. 6).
CP gene:
the forward primer CP-F is: 5'-GCCGTAAAGACTGGCGAACA-3' (SEQ No. 7)
The reverse primer CP-R is: 5'-GCCCTTTGGTCTTCTGAGACTG-3' (SEQ No. 8)
The probe CP-P is: FAM-TCGTCAACATGGTGGAGCACGACACGC-BHQ1 (SEQ No. 9)
3) Detection of Cpa03g018830 and Cpa03g018770 Gene copy numbers
T of papaya from the 17 pieces of CP transgenic papaya 1 8 plants are randomly selected from the generation positive plants as reference for Cpa03g018830 copy number identification, the number is CP1-CP8, the CP gene is used as reference gene, and the copy number of the target gene Cpa03g018830 in the papaya genome is detected through a digital PCR experiment. Using a Quantum studio TM3DAnalysisSuite digital PCR instrument, the reaction system was: 2 XddPCR Master Mix 7.3. Mu.L, forward and reverse primers (10. Mu.M) for the CP gene and Cpa03g018830 gene each 1.3. Mu.L, probes (10. Mu.M) each 0.36. Mu.L, DNA template (10 ng/. Mu.L) 1. Mu.L, nucleic-free water 0.28. Mu.L, and total volume 14.50. Mu.L. The amplification procedure was as follows: denaturation at 96℃for 10min; 2min at 60 ℃, 30s at 98 ℃ and 39 cycles; and 60 ℃ for 2min. After the reaction is finished, placing the chip into a digital PCR reader to read FAM and VIC fluorescent signals, and performing fluorescent data analysis by using corresponding software to obtain copy numbers (copy/mu L) of the CP genes and Cpa03g018830 genes in the detected sample DNA, calculating the copy number ratio of the CP genes to the Cpa03g018830 genes in the sample, and judging the copy numbers of the Cpa03g018830 genes in the papaya genome according to the ratio.
Since papaya belongs to diploid plants, the transgenic CP papaya single copy strain T 1 When the copy number ratio of the CP gene of the generation positive plant to the Cpa03g018830 gene is 1 or 0.5, the Cpa03g018830 gene is a single copy gene. And based on the copy number ratio of the CP gene to the target gene (Cpa 03g018830 gene as the target gene in this example), T of the CPgenic papaya can be obtained 1 The generation positive plants are homozygous or heterozygous, the copy number ratio is 1 is single-copy homozygous CP plants, and the copy number ratio is 0.5 is single-copy heterozygous CP plants. The digital PCR scatter plot of 8 samples is shown in FIG. 2, and the copy number detection results of the digital PCR experiments (Table 1) show that the CP genes of 7 samplesThe copy number ratio to Cpa03g018830 gene was approximately 1 or 0.5, and the copy number ratio of the CP8 sample was 0.12, resulting in abnormality, which was eliminated. Thus, the Cpa03g018830 gene may be a single copy gene in the papaya genome. And the CP3 and CP4 samples are single copy homozygous plants, and the CP1, CP2, CP5, CP6 and CP7 samples are single copy heterozygous plants.
TABLE 1 digital PCR analysis of Cpa03g018830 Gene
The method comprises the steps of selecting a plant CP7 confirmed to be heterozygous in single copy as a reference for identifying the copy number of Cpa03g018770 genes in papaya genome, taking the CP genes as reference genes, detecting the copy number of Cpa03g018770 genes in papaya genome through a digital PCR experiment, and simultaneously verifying the copy number of Cpa03g018770 genes by taking Cpa03g018830 genes as single copy control. The digital PCR experiments for Cpa03g018770 gene and Cpa03g018830 gene were each repeated 3 times. The digital PCR method was the same as that described above for identifying Cpa03g018830 gene, and since CP7 was a single copy heterozygous plant, when the copy number ratio of the CP gene to the target gene (Cpa 03g018830 gene and Cpa03g018770 gene were the target genes in this example) was 0.5, the target gene was a single copy gene. The results of the digital PCR experiments with CP7 (Table 2) showed that the copy number ratio of the CP gene to Cpa03g018770 gene and Cpa03g018830 gene were each approximately 0.5. Therefore, cpa03g018770 gene and Cpa03g018830 gene are single copy genes in papaya genome, and can be used as internal reference genes for detecting the copy number of exogenous genes in transgenic papaya.
TABLE 2 digital PCR analysis of Cpa03g018830 Gene and Cpa03g018770 Gene
Example 2
Detection of transgenic papaya T using digital PCR 0 The method for copy number of exogenous gene of generation specifically comprises the following stepsThe method comprises the following steps:
1) Digital PCR primer and probe
Exogenous gene NPT II:
the forward primer NPTII-F is: 5'-GGAGTTCATTCAGGGCACCG-3' (SEQ No. 10)
The reverse primer NPTII-R is: 5'-TGGGTGGAGAGGCTATTCGG-3' (SEQ No. 11)
The probe NPTII-P is: FAM-ACAGCCGAAACACGGCGGCATCAGA-BHQ1, (SEQ No. 12).
Internal reference gene Cpa03g018830:
forward primer 830-F was: 5'-TTCGAACGAGTCTGCAGTGG-3' the number of the individual pieces of the plastic,
reverse primer 830-R is: 5'-ACCACCTGAACCCAGGCTAA-3' the number of the individual pieces of the plastic,
probes 830-P are: VIC-ACGTGGCGGAGGGTGGTGAAGCTGATA-MGB;
reference gene Cpa03g018770:
the forward primer 770-F is: 5'-ATCCCAATTGCAGCTGGTGG-3' the number of the individual pieces of the plastic,
the reverse primer 770-R is: 5'-GTGAACCCTGGTGATGCCAA-3' the number of the individual pieces of the plastic,
the probes 770-P are: VIC-TCCAGTTGAGTTTGGTGGCGGCCACA-MGB.
2) Sample DNA extraction
T by transforming papaya PRSV virus HC-Pro gene 0 The generation positive papaya seedlings are taken as samples to be detected, leaves of 11 seedlings are collected, the leaves are respectively numbered HC-Pro-1 to HC-Pro-11, and genome DNA of the leaves is extracted by adopting a Bomaide plant genome kit.
3) Digital PCR method for detecting exogenous gene copy number of sample to be detected
The copy number of the exogenous gene in the HC-Pro transgenic papaya genome is detected by a digital PCR experiment by taking Cpa03g018770 gene and Cpa03g018830 gene as internal reference genes and taking NPT II gene sequence as exogenous target genes. Wherein, cpa03g018770 gene is taken as an internal reference gene, 6 papaya seedlings transformed with HC-Pro gene are detected, and the number is HC-Pro-1-HC-Pro-6; all 11 papaya seedlings transformed with HC-Pro gene were tested using Cpa03g018830 gene as an internal reference gene. Using a Quantum studio TM3DAnalysisSuite digital PCR instrument, the reaction system was: 2 XddPCR Master Mix 7.3. Mu.L, forward and reverse primers (10. Mu.M) for NPTII gene and Cpa03g018830 gene each 1.3. Mu.L, probes (10. Mu.M) each 0.36. Mu.L, DNA template (10 ng/. Mu.L) 1. Mu.L, nucleic-free water 0.28. Mu.L, and total volume 14.50. Mu.L. The amplification procedure was as follows: 96 ℃ for 10min; 2min at 60 ℃, 30s at 98 ℃ and 39 cycles; and 60 ℃ for 2min.
4) Analysis of results
After the reaction is finished, placing the chip into a digital PCR reader to read FAM and VIC fluorescent signals, and performing fluorescent data analysis by using corresponding software to obtain copy numbers (copy/mu L) of NPT II genes and Cpa03g018830 genes and Cpa03g018770 genes in the detected sample DNA, calculating the copy number ratio of the NPT II genes to the Cpa03g018830 genes or Cpa03g018770 genes in the sample, and judging the copy number of the NPT II genes in the papaya genome by the ratio. Papaya is a diploid plant, so that the copy number ratio of single-copy integrated exogenous gene fragments to internal reference gene is about 0.5, the copy number ratio of double-copy integrated exogenous gene fragments to internal reference gene is about 1, the copy number ratio of three-copy integrated exogenous gene fragments to internal reference gene is about 1.5, the integrated copy number of four-copy, five-copy and other multi-copy exogenous gene fragments is analogized. ( The method for judging the copy number of the exogenous gene is judged by the copy number ratio of the exogenous target gene and the internal reference gene, and the copy numbers of the exogenous target gene and the internal reference gene are the same in the transgenic papaya because the exogenous target gene is part of the exogenous gene, so the copy number ratio of the exogenous target gene and the internal reference gene can be used for judging the copy number of the exogenous target gene; the following is the same. )
As a result of digital PCR using Cpa03g018770 gene as a reference gene (Table 3), HC-Pro-1, HC-Pro-2, HC-Pro-3, HC-Pro-4 and HC-Pro-5 were detected as single copies, and HC-Pro-6 was detected as five copies. As a result of digital PCR using Cpa03g018830 gene as a reference gene (Table 4), it was revealed that HC-Pro-1, HC-Pro-2, HC-Pro-3, HC-Pro-4, HC-Pro-5 were detected as a single copy, HC-Pro-6 was detected as five copies, HC-Pro-7, HC-Pro-8 and HC-Pro-10 were detected as four copies, and HC-Pro-9 and HC-Pro-11 were detected as six copies. Wherein, the results of HC-Pro-1-HC-Pro-6 detected by the two internal reference genes are consistent.
TABLE 3 results of exogenous gene copy number detection Using Cpa03g018770 as a reference Gene
TABLE 4 results of exogenous gene copy number detection Using Cpa03g018830 as a reference Gene
Example 3
Detection of transgenic papaya T using fluorescent quantitative PCR 0 The method for the copy number of the exogenous gene of the generation plant specifically comprises the following steps:
1) Fluorescent quantitative PCR primer
The method is the same as the digital PCR primer, and specifically comprises the following steps:
exogenous gene NPT II:
the forward primer NPTII-F is: 5'-GGAGTTCATTCAGGGCACCG-3' the number of the individual pieces of the plastic,
the reverse primer NPTII-R is: 5'-TGGGTGGAGAGGCTATTCGG-3' the number of the individual pieces of the plastic,
internal reference gene Cpa03g018830:
forward primer 830-F was: 5'-TTCGAACGAGTCTGCAGTGG-3' the number of the individual pieces of the plastic,
reverse primer 830-R is: 5'-ACCACCTGAACCCAGGCTAA-3' the number of the individual pieces of the plastic,
reference gene Cpa03g018770:
the forward primer 770-F is: 5'-ATCCCAATTGCAGCTGGTGG-3' the number of the individual pieces of the plastic,
the reverse primer 770-R is: 5'-GTGAACCCTGGTGATGCCAA-3' the number of the individual pieces of the plastic,
2) Sample DNA extraction
T of papaya transformed with HC-Pro gene 0 44 plants of the generation positive plants are samples to be tested, and the number is HC-Pro-12-HC-Pro-55; t of papaya transformed with Nib gene 0 The 28 plants of the generation positive are samples to be tested, and the number is Nib-1-Nib-28. Respectively collecting leaves of papaya seedlings, and extracting genome DNA of the papaya seedlings by using a Bomaide plant genome kit. In addition, HC-Pro transgenic papaya T 0 Generation HC-Pro-1-HC-Pro-11 and T of transgenic CP papaya 1 The copy number of the substituted CP1-CP8 samples is also verified again as the samples to be tested. The samples to be tested were 91 parts in total.
3) Primer verification
And (3) taking 2 parts of genome DNA of the transgenic papaya with known copy number as a template, and performing a fluorescent quantitative PCR experiment to verify the effectiveness of primers of Cpa03g018830 and Cpa03g018770 genes of internal reference and NPTII of exogenous gene. Reaction in96 fluorescence analyzer, the reaction system is: 2x Q3 SYBR qPCR Master Mix (Universal) (Uygur autonomous Biotech 22204) 10. Mu.L, forward and reverse primers (10. Mu.M) 0.4. Mu.L each, template DNA (50 ng/. Mu.L) 1. Mu.L, ddH 2 O9.2. Mu.L, total volume 20. Mu.L. The amplification procedure was: pre-denaturation at 95 ℃ for 30s; denaturation at 95℃for 10s, annealing at 58℃for 30s,40 cycles; melting curves were 95℃15s,60℃60s,95℃15s. Experiments were performed in 3 replicates. The amplification curves and melting curves (figure 3) of the three pairs of primers show that the three pairs of primers have obvious amplification curves, and the melting curves have unique absorption peaks and can be used as fluorescent quantitative PCR detection primers.
4) Fluorescent quantitative PCR (polymerase chain reaction) detection of exogenous gene copy number of sample to be detected
The copy numbers of exogenous genes in papaya genomes of all samples to be detected are detected through a fluorescent quantitative PCR experiment by taking Cpa03g018770 gene and Cpa03g018830 gene as internal reference genes, taking NPT II gene as exogenous target genes and taking single copy homozygous transgenic papaya strains as a control. The PCR reaction system and procedure were verified with the primers of 3), and each sample was repeated 3 times.
5) Analysis of results
After the reaction was completed, use was made of96SW 1.1 System for data analysis, 2 -ΔΔCt Analyzing the relative copy number of NPTII gene in transgenic plant by using method, defining known single copy homozygote transgenic plant as reference factor, i.e. 1, and the multiple of copy number of each sample relative to reference factor is 2 -ΔΔCt . Due to T 0 The plant of the generation is inserted into heterozygous type, so that the relative quantitative value (RQ) of the single copy plant is 1/2 of that of the homozygous single copy reference, and the relative expression quantity is about 0.5; the RQ value of the double-copy plant is equal to that of the homozygous single-copy control, and the relative expression quantity is about 1; the relative quantitative RQ of the three-copy plants should be 3/2 of that of the homozygous single-copy control, and the relative expression amount is about 1.5; the integrated copy number of the multi-copy exogenous gene and so on.
The result of fluorescence quantitative PCR detection of the copy number of the sample HC-Pro-12-HC-Pro-55 is shown in FIG. 4, wherein 25 of the samples are single copy transgenic plants, 1 of the samples are double copy transgenic plants, 2 of the samples are three copy transgenic plants, and the rest of the samples are more than three copy transgenic plants. The result of the fluorescent quantitative PCR detection of the copy number of the sample Nib-1-Nib-28 is shown in FIG. 5, wherein 5 are single-copy transgenic plants, 2 are double-copy transgenic plants, 6 are three-copy transgenic plants, and the rest are more than three-copy transgenic plants.
The results of the fluorescence quantitative PCR detection of the sample CP1-CP8 and HC-Pro-1-HC-Pro-11 are shown in FIG. 6, the samples CP1, CP2, CP5, CP6 and CP7 are single copy heterozygous plants, the samples CP3, CP4 and CP8 are single copy homozygous plants, the digital PCR result except the sample CP8 is abnormal, and the rest is consistent with the copy number result of the digital PCR detection. HC-Pro-1, HC-Pro-2, HC-Pro-3, HC-Pro-4 and HC-Pro-5 are single-copy transgenic plants, HC-Pro-7, HC-Pro-8 and HC-Pro-10 are four-copy transgenic plants, HC-Pro-6 is five-copy transgenic plants, and the copy number detection results of the plants are consistent with the detection results of digital PCR; HC-Pro-9 and HC-Pro-11 were detected as fifteen copies and five copies by fluorescence quantitative PCR, however, the detection results of digital PCR were six copies, and the results were inconsistent, probably because fluorescence quantification has higher detection sensitivity for single-low copy integration of the foreign gene, and low detection sensitivity for multiple-copy integration.
The results show that the fluorescent quantitative PCR method has higher accuracy in detecting single low copy integration of the exogenous gene in the transgenic papaya, but has insufficient accuracy in detecting multi-copy integration, is not as accurate as digital PCR, has lower cost, is simple and quick, and is suitable for initially screening single low copy integration plants in a large number of transgenic plants.
Other parts not described in detail are prior art. Although the foregoing embodiments have been described in some, but not all, embodiments of the invention, it should be understood that other embodiments may be devised in accordance with the present embodiments without departing from the spirit and scope of the invention.

Claims (3)

1. A method for detecting the copy number of exogenous gene in transgenic papaya by digital PCR features that the forward and reverse detection primers of papaya gene Cpa03g018830 or Cpa03g018770 are used to detect the copy number of exogenous gene in transgenic papayaNPTⅡThe forward and reverse detection primers of the screening marker genes detect the genome DNA of the papaya of the sample by utilizing a digital PCR technology, and the copy number of the exogenous target gene in the sample is judged according to the copy number ratio of the exogenous target gene to the internal reference gene; the gene Cpa03g018830 or Cpa03g018770 is derived from papaya chr_03 chromosome, the forward detection primer sequence of the gene Cpa03g018830 is shown as SEQ No.1, the reverse detection primer sequence is shown as SEQ No.2, the forward detection primer sequence of the gene Cpa03g018770 is shown as SEQ No.4, the reverse detection primer sequence is shown as SEQ No.5,NPTⅡthe forward detection primer sequence of the screening marker gene is shown as SEQ No.10, and the reverse detection primer sequence is shown as SEQ No. 11;
the method for judging the copy number of the exogenous target gene in the genome of the transgenic papaya comprises the following steps: the copy number ratio of the exogenous target gene to the internal reference gene is 0.5, the ratio is four copies at 2.02, the ratio is five copies at 2.22, and the ratio is 6 copies at 2.92.
2. The method for detecting the copy number of an exogenous gene in transgenic papaya according to claim 1, wherein the digital PCR technique is performed under the following conditions: 2x ddPCR Master Mix 7.30 mu L, each of the forward primer and the reverse primer of the reference gene and the exogenous target gene 1.30 mu L, each of the probes 0.36 mu L, the DNA template 1 mu L, the nucleic-free water 0.28 mu L, and the total volume 14.50 mu L; the amplification procedure was as follows: 96 ℃ for 10min;60 ℃ for 2min,98 ℃ for 30s and 39 cycles; and 60 ℃ for 2min.
3. The method for detecting copy number of exogenous gene in transgenic papaya according to claim 2, wherein the probe sequence is shown in SEQ No.3 when Cpa03g018830 gene is used as an internal reference gene, the probe sequence is shown in SEQ No.6 when Cpa03g018770 gene is used as an internal reference gene,
NPTⅡthe probe sequence of the screening marker gene is shown in SEQ No. 12.
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