Sweet potato gene copy number detection method and application thereof

Abstract

The invention belongs to the technical field of gene detection and the field of crop molecular genetic breeding, and particularly relates to a sweet potato gene copy number detection method and application thereof. Specifically, according to the technology, transgenic sweet potatoes or arabidopsis thaliana carrying target genes (exogenous genes such as HPT, 35S, IbMYB1-4 and the like which are cloned to expression vectors, namely external reference genes) are taken as control materials, the control materials and sweet potato cultivated species needing to be analyzed are mixed according to a preset mass ratio, the target genes HPT of the transgenic materials are taken as the external reference genes on the basis of detection of the qPCR technology, and the sweet potato cultivated species needing to be analyzed are obtained. Or IbTUA, IbActin and the like in the screened sweet potato cultivar genome are taken as reference genes, and the relative copy number of the target gene in the genome of a sample to be detected can be quickly obtained by calculating the relative copy number between the target gene to be detected and the reference gene, so that data reference is provided for subsequent genetic research.

Description

Technical Field

[0001] The invention belongs to the technical field of gene detection and the field of molecular genetic breeding of crops, and specifically relates to a method for detecting the copy number of sweet potato genes and an application thereof. Background Art

[0002] The gene dosage effect refers to a genetic phenomenon in which the gene dosage of an organism (i.e., the number of copies of the target gene in the genome) changes, leading to changes in phenotype. The gene dosage effect in the polyploid crop genome is an extremely complex phenomenon. An increase in genome dosage may lead to a dosage effect in gene expression. When the expression level of the corresponding gene is proportional to the increase in the number of homologous chromosomes, it is called a positive dosage effect gene, and vice versa. It is called a negative dosage effect gene. This gene dosage effect not only affects crop growth and yield, such as polyploidy leading to the enlargement of cells and seeds, but may also affect crop traits such as stress resistance and quality. Therefore, in-depth research on the gene dosage effect in the polyploid genome of crops is of great significance for improving crop yield and quality.

[0003] The technologies currently used for detecting gene copy number variation mainly include Southern hybridization, real-time fluorescence quantitative PCR (qPCR), digital PCR (dPCR), whole genome sequencing (WGS), etc. These technologies have different focuses in terms of accuracy, experimental operation, time, cost, efficiency, etc. qPCR technology is a method that monitors the total amount of specific products after each amplification cycle in real time in a DNA-specific amplification reaction, and quantitatively analyzes the specific DNA sequence in the sample to be tested through internal or external reference methods. This method has many advantages such as high detection sensitivity, good compatibility with various detection schemes, simple operation technology, and low equipment purchase and maintenance costs. It has been widely used in biology and medicine.

[0004] Sweet potato (Ipomoea batatas (L.) Lam.) is the world's main food, feed and industrial raw material crop. There are many types of sweet potato related wild species in nature, such as diploid, tetraploid, hexaploid, etc., but the cultivated sweet potato is a typical highly heterozygous hexaploid crop (chromosomes are 2n=6x=90). Its complex genetic background has caused the application of sweet potato genetic breeding technology to lag behind other food crops. Studies have shown that the dosage effect difference caused by gene copy number variation in polyploid crops cannot be ignored. For example, there is an obvious gene dosage effect in the vernalization of wheat in the polyploid genome; the allelic variation and allelic copy number of key enzyme genes in the starch synthesis pathway of sweet potato SSII, GBSS, SBEI directly affect the physical and chemical factors such as starch gelatinization temperature and branching structure of tubers; the research of this patent found that the accumulation of sweet potato anthocyanins, carotene and other metabolites also has a significant corresponding relationship with the copy number of key regulatory genes in the corresponding synthesis pathway. Therefore, clarifying the gene dosage effect caused by the difference in the copy number of key genes in germplasm resources has great reference value for genetic breeding.

[0005] In recent years, thanks to the advancement of sweet potato genome sequencing and assembly, scientists have obtained relatively complete sweet potato genome assembly information and completed the fine assembly of each haplotype sequence of the hexaploid genome, providing accurate reference information for the development of modern sweet potato genetic breeding. However, the current methods for sweet potato gene copy number analysis are mostly based on high-throughput sequencing technology, and there are still great limitations in the detection of target gene copy number. Summary of the invention

[0006] In order to solve the above technical problems, the present invention provides the following technical solutions:

[0007] A primer pair for detecting the copy number of a sweet potato gene based on an internal reference gene, the primer pair comprising a specific detection primer pair Primer-T1 for an internal reference gene IbTUA, a specific detection primer pair Primer-T2 for an internal reference gene IbActin, and a primer pair Primer-T n ; The nucleotide sequence of the internal reference gene IbTUA is shown in SEQ ID NO:1, and the nucleotide sequence of the internal reference gene IbActin is shown in SEQ ID NO:2.

[0008] SEQ ID NO:1

[0009]

[0010] SEQ ID NO:2

[0011] Tggcagatgctgaggatattcagccacttgtctgtgacaatggaactggaatggtgaaggctggatttgctggtgatgatgctccaagagcagtgtttcccagtattgtgggtcgcccccgtcacactggtgttatggttgggatgggacagaaggatgcctatgtgggtgatgaagcccagtctaagagaggtatcttgaccttgaaataccccattgagcatggtattgttagcaactgggatgatatggaaaagatctggcatcacactttctacaacgagcttcgtgttgcacctgaggagcaccctgtccttcttaccgaggcacccctcaacccaaaagcaaacagagagaagatgacacaaatcatgtttgagacatttaatgttcctgcaatgtatgttgctattcaggctgtgctatccttgtatgccagtggacgtacaactggtattgtgctggattctggtgatggtgtgagccatactgtacccatctatgaaggttatgcccttcctcatgccattcttcgtttggaccttgctggccgtgacctcaccgatcaccttatgaagatcctaactgagagaggttacatgttcactactactgctgagcgggaaattgtccgtgacatgaaggaaaagcttgcatatgtggcccttgactatgagcaggagcttgaaactgcaaagagcagctcctctgttgagaagaactaa

[0012] Preferably, the specific detection primer pair Primer-T 1、 The specific detection primer pair Primer-T2 and the primer pair Primer-T n have the same working conditions.

[0013] Preferably, the nucleotide sequence of the specific detection primer pair Primer-T1 is shown as SEQ ID NO:3-4; the nucleotide sequence of the specific detection primer pair Primer-T2 is shown as SEQ ID NO:5-6.

[0014] A kit for detecting the copy number of a sweet potato gene, the kit comprising a primer pair for detecting the copy number of a sweet potato gene based on an internal reference gene, the primer pair comprising a specific detection primer pair Primer-T1 of an internal reference gene IbTUA, a specific detection primer pair Primer-T2 of an internal reference gene IbActin, and a primer pair Primer-T n ; The nucleotide sequence of the internal reference gene IbTUA is shown in SEQ ID NO: 1, and the nucleotide sequence of the internal reference gene IbActin is shown in SEQ ID NO: 2.

[0015] A primer pair for detecting the copy number of a sweet potato gene based on an internal reference gene or a kit containing the primer pair is used in detecting the copy number of a sweet potato target gene and analyzing the ploidy of the genome in sweet potato closely related wild species.

[0016] A method for detecting the copy number of a sweet potato gene, the method comprising: performing qPCR detection on the DNA of a sample to be tested using a primer pair for detecting the copy number of a sweet potato gene based on an internal reference gene or a kit containing the primer pair to obtain Ct value data of the sample to be tested;

[0017] Preferably, the DNA in the samples to be tested are respectively transgenic CK n and mixed n .

[0018] Preferably, the transgenic CK n The preparation method is as follows: IbMYB1 -4::CDS IbMYB1 The expression vector of ::NosTerminator::pCIAMBIA1300 was transferred into wild-type Arabidopsis or sweet potato cultivar "Xushu 29", and DNA was extracted from young leaves of normally growing transgenic plants to obtain transgenic CK n ; The mixed sample MIX n The preparation method is to transfer the transgenic CK n Mixed with the DNA extracted from the sweet potato leaves to be tested to obtain a mixed sample MIX n .

[0019] Preferably, when the transgenic CK n Arabidopsis CK n When the target gene relative copy number C value is: C = 6 × 2^ ΔCt ; ΔCt=(CtX1+CtX2) / 2-CtXn ;

[0020] When transgenic CK n Transgenic sweet potato CK n When the target gene relative copy number C value is: C = C N =6×2^ ΔCtN And at the same time meet n = (2^ ΔCtK -2^ ΔCtX ) / (2^ ΔCtX -2^ ΔCtN ); ΔCtK=(CtK1+CtK2) / 2-CtK n ; ΔCtX=(CtX1+CtX2) / 2-CtX n ; ΔCtN=(CtN1+CtN2) / 2-CtN n .

[0021] Beneficial effects of the present invention:

[0022] The present invention develops a simple method for detecting the copy number of sweet potato genes based on qPCR technology. IbTUA, IbActin and the like in the genome of screened sweet potato cultivars are used as internal reference genes. The relative copy number between the target gene to be detected and the reference gene is calculated, so that the relative copy number of the target gene in the genome of the sample to be detected can be quickly obtained, thereby providing data reference for subsequent genetic research. The method has the advantages of low requirements on the experimental environment, low detection cost, and large detection sample throughput. The method is not only suitable for copy number analysis of any target gene, but also suitable for ploidy analysis of the sweet potato genome. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The relative copy number detection results of candidate reference genes in the genomes of 58 sweet potato cultivars;

[0024] Figure 2 is the relative copy number of genes IbCHI, IbCHS, IbANS, IbPSY, IbLCYα and IbCHYβ. DETAILED DESCRIPTION

[0025] In order to make the purpose, technical solutions and advantages of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in combination with the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0026] The specific implementation process of the technical solution of the present invention is:

[0027] 1. Preparation of standard samples and external reference samples

[0028] 1.1 Build Pro IbMYB1-4 ::CDS IbMYB1 The expression vector of ::NOS Terminator::pCIAMBIA1300, in which the promoter sequence and CDS of the IbMYB1-4 gene are sequences unique to some purple germplasm resources of cultivated sweet potato varieties, have been disclosed in the description of the Chinese invention patent "A method for creating sweet potato colored germplasm resources using IbMYB1-4 purple gene loci" (patent number ZL20221 1470759.7) in paragraphs 9-10 and mentioned in the NCBI database GenBank: PP731796.1. The constructed expression vector was transformed into DH5α Escherichia coli competent cells, positive strains were screened on LB medium containing 75 mg / L kanamycin, positive colonies were picked for bacterial amplification, and the expression vector was extracted from the bacterial solution as a standard for qPCR absolute quantification.

[0029] 1.2 Pro Build IbMYB1-4 ::CDS IbMYB1 The expression vector of ::Nos Terminator::pCIAMBIA1300 was transferred into Agrobacterium tumefaciens EHA105, and then into wild-type Arabidopsis thaliana with a Col genetic background according to the pollen tube pathway method to obtain transgenic positive strains in which the purple trait of the plant was linked to the IbMYB1 gene and the F2 generation no longer separated. The rosette leaves of the transgenic plants that grew normally for 30d-60d were taken for use.

[0030] 1.3 Pro IbMYB1-4 ::CDS IbMYB1 The expression vector of ::Nos Terminator::pCIAMBIA1300 was transferred into Agrobacterium tumefaciens EHA105, and then transferred into the sweet potato cultivar "Xu Shu 29" by the embryonic callus infection method. The transgenic positive callus was selected and induced to differentiate into regenerated plants. The young leaves of the normally growing transgenic plants were taken for use.

[0031] 2. Preparation of sweet potato samples to be tested

[0032] The transgenic Arabidopsis leaves prepared in 1.2 or the transgenic sweet potato plant samples prepared in 1.3 were mixed with the young leaves of the sweet potato to be tested at a mass ratio of 1:n, and the DNA of the mixed leaves was extracted, which was named MIX. n The DNA of the sweet potato leaves to be tested was extracted separately and named SIN n The DNA of the leaves of the transgenic material was extracted separately and named CK n Use an ultra-micro nucleic acid analyzer or 1% agarose gel electrophoresis to determine the DNA quality and set aside.

[0033] 3. Quantification of the copy number of the external reference gene in the standard sample (optional)

[0034] The actual molar concentration of the standard prepared in step 1.1 for absolute quantification by qPCR was detected using an ultra-micro nucleic acid analyzer. The standard was diluted in a 10-fold dilution to obtain standard concentrations of 1, 10, and 10, respectively. -1 times, 10 -2 times, 10 -3 times, 10 -4 times, 10 -5 The standard samples with multiple times concentration were detected and the amplification curves and high-resolution melting curves of the standards with different concentration gradients were obtained using a qPCR instrument and supporting reagents, using the IbMYB1 gene specific binding primer pair Primer-C1 (MYB1F / NOSR) and primer pair Primer-C2 (BlackF1 / MYB1R) and HPT (hygromycin phosphotransferase gene) specific binding primer pair Primer-C3 (HptF / HptR). The primer combinations and working conditions are shown in Table 1.

[0035] Use the PCR MIX kit for qPCR detection with SYBR Green dye and prepare the PCR working solution according to the instructions. The order of adding samples is 2×PCR MIX, ddH2O, and DNA template. The final concentration of the DNA template in the reaction solution is usually between 10^3 and 10^7 copies / μL. After the PCR working solution is fully mixed, it is divided into a PCR detection system with 3× reaction solution as the unit. Then, the primer combination of the gene to be detected is added to each 3×PCR reaction solution system to ensure that the final working concentration of a single primer is close to 0.4μmol / L. After the primers are added, the 3×PCR reaction solution system is fully mixed and divided into 3 mechanical repeat wells of the qPCR reaction plate.

[0036] Table 1 Primer combinations and working conditions

[0037]

[0038]

[0039]

[0040] Let the actual molar concentration of the target gene in the DNA sample be M, and the number of amplification cycles required for the fluorescence signal of the amplified product to reach the set fluorescence threshold during qPCR detection be Ct. For any two samples tested in the same batch, there should be M1×2 Ct1 =M2×2 Ct2 Therefore, the standard curve between the logarithm of the actual molar concentration of the target gene and the Ct value can be calculated and drawn Ct = -k×log10 (M0)+b, where M0 is the initial molar concentration of the standard, k is the slope of the standard curve, and R is the correlation coefficient of the standard curve. The R value is required to be greater than 0.98, and the slope k of the corresponding standard curve is calculated, and then the amplification efficiency E=10 is further calculated. (-1 / k) -1.

[0041] like Figure 1 As shown, comparative analysis found that the qPCR amplification efficiency of the primer pairs Primer-C1, Primer-C2, and Primer-C3 were all in the range of 90%-110% (3.6>k>3.1), so it can be considered that the three pairs of primers have the same detection efficiency in detecting the copy number of the target gene.

[0042] 4. Detection of target gene copy number

[0043] Design a specific binding primer pair Primer-Tn for target gene copy number detection (primer working conditions are shown in Table 1, including specific detection primers for key internal reference genes IbTUA and IbActin. The working conditions of the primers are required to be basically consistent with the working conditions of Primer-C1, Primer-C2, and Primer-C3 primers. The specificity and amplification efficiency of the primers can be analyzed and verified using the absolute quantitative method described in step 3). According to the instructions for use of the qPCR reagent, use the single DNA sample prepared in step 2 to prepare a PCR detection system without adding primers. Mix the reaction solution thoroughly and then divide it into 4 equal parts. Add the required concentrations of Primer-C1, Primer-C2, Primer-C3, and Primer-Tn primer pairs to the divided reaction system. Mix the reaction solution thoroughly and divide it into 3 adjacent reaction wells (chambers) of the qPCR reaction plate, and ensure that the volume of the reaction system divided into each reaction well (chamber) meets the requirements of qPCR instrument detection. The test was performed according to the reaction conditions in Table 1, and a valid Ct value was obtained according to the standard of 14≤Ct value≤35 and the high-resolution melting curve presenting an obvious single peak curve for subsequent copy number analysis.

[0044] 5. Calculation of relative copy number of target gene

[0045] According to Table 2 below, the corresponding indicators are now tested and the relative copy number of the corresponding samples is calculated. In the genome of the cultivated species, the corresponding sequences of the internal reference genes IbTUA and IbActin selected by this technology have been verified by this technology to be stable 6 copies, that is, there are only 6 alleles of the target gene in the hexaploid genome, and there should be no homologous sequences at other sites in the genome.

[0046] Table 2 Calculation formula for relative copy number of target genes

[0047]

[0048]

[0049] Application Cases

[0050] 1. Analysis of the copy number of candidate reference genes

[0051] According to the analysis results of the sweet potato genome (Ming GaoID, Sara Fuentes Soriano, Qinghe Cao, Xinsun Yang, Guquan Lu. Hexaploid sweetpotato (Ipomoea batatas (L.) Lam.) may not be a truetype to either auto-or allopolyploid. PLoS ONE, 2020, 15 (3): e0229624.), 20 DNA sequences with a certain copy number were randomly selected, and it was ensured that these sequences were distributed on the 15 chromosomes of the sweet potato genome. Specific primers were designed for genes such as IbActin, IbTUA, and IbEF1a as universal internal reference genes.

[0052] According to the method described in the present technology, the leaves of the sweet potato strain OE carrying the transgenic HPT element and the sweet potato variety to be tested were mixed and sampled at an equal ratio of 1:1 and DNA was extracted. The Ct values ​​of the external reference genes in all mixed sample DNAs were detected by qPCR, and the non-mixed transgenic material OE samples were used as positive controls to detect all candidate internal reference genes. According to the screening and copy number calculation method of the internal reference genes in Table 2 of step 6, the equal mixing n=1, so the relative copy number of the candidate internal reference gene of the sweet potato sample to be tested relative to the transgenic sweet potato is C N =6x2 ^(ΔCtK-ΔCtX) -1.

[0053] The relative copy numbers of each target gene in different cultivars are as follows Figure 1 As shown, the primer sequences of the target genes and their primer combinations Primer-Tn are recorded in Table 1. Comparative analysis found that the 20 randomly selected single copies had large differences in copy number in the genomes of different cultivars, and among the commonly used internal reference genes, only the two genes IbTUA and IbActin had constant copy numbers in the genomes of all the cultivars tested, and thus could be used as internal reference genes for this technology.

[0054] 2. Analysis of genome ploidy in wild sweet potato relatives

[0055] Taking transgenic sweet potato OE as the positive control, Ipomoea trifida (2×), Ipomoea triloba (4×), Ipomoea trifida (6×) and cultivated variety Xiangshu 20 were selected. The mixed sample DNA (n=1 in the mixing ratio) and the DNA of the single sample were extracted according to the method described in this technology, and the qPCR method was used to detect the copy numbers of the external reference gene HPT and the internal reference genes IbTUA, IbActin, Ib18S, and IbEF1a, respectively.

[0056] After judging that each detection index meets the quality control conditions of copy number analysis, it was found that the relative copy numbers of the reference gene IbTUA in OE (6×), 2×trifida, 4×triloba, 6×trifida, Heiye (6×), and Xiang 20 (6×) were 6.21, 2.06, 3.9, 5.78, and 5.94, and the relative copy numbers of the reference gene IbActin were 5.84, 2.11, 4.06, 5.94, 6.11, and 5.89, which were close to the expected copy numbers of 6, 2, 4, 6, and 6. This indicates that the reference genes IbTUA and IbActin can be used for rapid analysis of sweet potato genome ploidy.

[0057] Table 3 Relative copy numbers of the reference genes IbTUA and IbActin in OE (6×), 2×trifida, 4×triloba, 6×trifida, Heiye (6×), and Xiang 20 (6×)

[0058]

[0059] 3. Copy number analysis of key genes in the anthocyanin and carotene synthesis pathways of sweet potato

[0060] According to the method described in the present technology, the key enzyme genes IbCHI, IbCHS, IbANS in the anthocyanin synthesis pathway and the key enzyme genes IbPSY, IbLCYα, IbCHYβ in the carotene synthesis pathway were selected, and 20 cultivars were randomly selected, including 10 purple sweet potatoes and 10 non-purple sweet potatoes. The DNA of the mixed samples and the single samples were extracted respectively according to the method described in the present technology, and the corresponding external references, internal references and target genes were detected by qPCR. After judging that each detection index meets the quality control conditions of copy number analysis, the relative copy number of the target gene in different cultivars was calculated, and the corresponding relationship between the copy number of each target gene and the accumulation of anthocyanins in sweet potatoes was compared and analyzed. It was found that the relative copy numbers of the selected IbCHI, IbCHS, and IbCHYβ genes were close to 6, while the relative copy numbers of the IbANS, IbPSY, and IbLCYα genes varied greatly in the genomes of different cultivars. The results are as follows Figure 2This difference in gene copy number reflects, to a certain extent, the gene dosage effect of the secondary metabolite regulation involved in the corresponding gene, indicating that this technology can be used to analyze the dosage effect of gene inheritance in polyploid genomes.

[0061] 4. Analysis of the dosage effect of sweet potato purple gene IbMYB1

[0062] Previous studies have shown that the purple-black trait of the stems and leaves of the sweet potato germplasm resource "Heiye" is controlled by the dominant gene IbMYB1-4. Directed hybridization was performed with "Heiye" as the male parent and the cultivated variety "Xiangshu 99" as the female parent, and a segregation population of 83 self-pollinated progenies was obtained. Molecular marker detection was performed on the purple trait of each individual plant of the hybrid progeny and each copy of the IbMYB1 gene. It was found that the purple trait of the progeny stem was linked to IbMYB1-4 and its segregation ratio was 78:5, which was close to the expected hybrid segregation ratio A_____: aaaaaa=19:1 for the AAAaaa×aaaaaa genotype. Therefore, it was inferred that the copy number of the IbMYB1-4 gene in the "Heiye" genome should be 3.

[0063] The relative copy number of the IbMYB1 gene in its hybrid progeny was detected using the technology described in this patent, and it was verified that the relative copy number of the IbMYB1-4 gene in the "Black Leaf" genome should be 3, and the relative copy number of the IbMYB1-4 gene in the "Xiangshu 99" genome should be 0. The relative copy number of the IbMYB1-4 gene can be detected in all hybrid progenies with purple vines, indicating that the regulation of the IbMYB1-4 gene on the purple trait of the stem should be dominant. The leaves of some hybrid progenies of the Aaaaaa genotype (relative copy number of 1) are green, the leaves of the hybrid progenies of the AAAaaa genotype (relative copy number of 3) are all purple, and most of the leaves of the hybrid progenies of the AAAaaa genotype (relative copy number of 2) are purple, and a small part are green, indicating that the purple trait of the leaves is also related to the copy number of the IbMYB1-4 gene, that is, it is related to the dosage of the IbMYB1-4 gene in the genome.

Claims

1. A primer pair for detecting the copy number of sweet potato genes based on an internal reference gene, characterized in that: The primer pair comprises a specific detection primer pair Primer-T1 for the internal reference gene IbTUA, a specific detection primer pair Primer-T2 for the internal reference gene IbActin, and a primer pair Primer-T n ; The nucleotide sequence of the internal reference gene IbTUA is shown in SEQ ID NO:1, and the nucleotide sequence of the internal reference gene IbActin is shown in SEQ ID NO:

2.

2. The primer pair according to claim 1, characterized in that The specific detection primer pair Primer-T1, the specific detection primer pair Primer-T2 and the primer pair Primer-T n The working conditions are the same.

3. The primer pair according to claim 1, characterized in that The nucleotide sequence of the specific detection primer pair Primer-T1 is shown in SEQ ID NO:3-4; the nucleotide sequence of the specific detection primer pair Primer-T2 is shown in SEQ ID NO:5-6.

4. A kit for detecting the copy number of sweet potato genes, characterized in that: The kit comprises the primer pair described in any one of claims 1-3.

5. The primer pair described in any one of claims 1 to 3 or the kit described in claim 4 is used in detecting the copy number of sweet potato target genes and analyzing the genome ploidy in sweet potato related wild species.

6. A method for detecting the copy number of sweet potato genes, characterized in that: The method comprises: performing qPCR detection on the DNA of the sample to be tested using the primer pair described in any one of claims 1 to 3 or the kit described in claim 4 to obtain Ct value data of the sample to be tested.

7. The method according to claim 6, characterized in that The DNA of the samples to be tested are respectively transgenic CK n and mixed n .

8. The method according to claim 7, characterized in that The transgenic CK n The preparation method is as follows: IbMYB1-4 ::CDS IbMYB1 The expression vector of ::Nos Terminator::pCIAMBIA1300 was transferred into wild-type Arabidopsis or sweet potato cultivar "Xushu 29", and DNA was extracted from young leaves of normally growing transgenic plants to obtain transgenic CK n ; The mixed sample MIX n The preparation method is to transfer the transgenic CK n Mixed with the DNA extracted from the sweet potato leaves to be tested to obtain a mixed sample MIX n .

9. The method according to claim 8, characterized in that When transgenic CK n Arabidopsis CK n When the target gene relative copy number C value is: C = 6 × 2^ ΔCt ; ΔCt=(CtX1+CtX2) / 2-CtX n ; When transgenic CK n Transgenic sweet potato CK n When the target gene relative copy number C value is: C = C N =6×2^ ΔCtN And at the same time meet n = (2^ ΔCtK -2^ ΔCtX ) / (2^ ΔCtX -2^ ΔCtN ); ΔCtK=(CtK1+CtK2) / 2-CtK n ; ΔCtX=(CtX1+CtX2) / 2-CtX n ; ΔCtN=(CtN1+CtN2) / 2-CtN n .

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