CN105112534B - Primer pair and method for identifying copy numbers of internal and external genes of chrysanthemum through fluorescent quantitative PCR - Google Patents

Primer pair and method for identifying copy numbers of internal and external genes of chrysanthemum through fluorescent quantitative PCR Download PDF

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CN105112534B
CN105112534B CN201510587171.3A CN201510587171A CN105112534B CN 105112534 B CN105112534 B CN 105112534B CN 201510587171 A CN201510587171 A CN 201510587171A CN 105112534 B CN105112534 B CN 105112534B
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陈发棣
种昕冉
王海滨
张飞
蒋甲福
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Nanjing Agricultural University
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Abstract

The invention discloses a primer pair and a method for identifying the copy number of endogenous and exogenous genes in chrysanthemum by fluorescent quantitative PCR. The application of the PGK gene as an internal reference gene in the identification of the copy number of the internal and external genes of the chrysanthemum by fluorescent quantitative PCR. An endogenous gene amplification primer pair applied to fluorescent quantitative PCR identification of the copy number of endogenous and exogenous genes in chrysanthemum, wherein a forward primer sequence is shown as SEQ ID NO.1, and a reverse primer sequence is shown as SEQ ID NO. 2. The method can quickly, accurately and efficiently identify the copy number of the endogenous gene of the chrysanthemum and the copy number of the transgenic chrysanthemum transformed into the endogenous gene and the exogenous gene. According to the detection primer and the method provided by the invention, chrysanthemum and transgenic chrysanthemum are used as experimental materials, and the copy number of the endogenous gene of the chrysanthemum and the transgenic copy number of the transgenic chrysanthemum can be obtained by comparative analysis according to the CT values of the gene of which the copy number is to be detected and the endogenous reference gene PGK. The detection method provides a new idea for identifying the copy number of the complex genome gene, and greatly improves the efficiency of detecting the copy number of the gene.

Description

Primer pair and method for identifying copy numbers of internal and external genes of chrysanthemum through fluorescent quantitative PCR
Technical Field
The invention belongs to the technical field of biology, and relates to a primer pair and a method for identifying the copy numbers of internal and external genes of chrysanthemum by fluorescent quantitative PCR.
Background
Chrysanthemum morifolium (Chrysanthemum morifolium) is native to China, is one of ten traditional famous flowers and four cut flowers in the world in China, has ornamental, edible and medicinal values, has a cultivation history of more than 1600 years to date, and is prepared by natural hybridization and artificial long-term breeding of Maohua, chrysanthemum indicum, chrysanthemum marianum and the like. The chrysanthemum flower type, the flower color, the plant type and the like are extremely rich, are important flower types applied to potted plants, cut flowers and garden lands, have high ornamental and application values, and occupy very important positions in flower production.
The Asteraceae family is the first major family of angiosperms, the most evolved group of dicotyledons, with about 1100 genera and over 30000 species. Recent studies have shown that at least 70% of angiosperms are polyploids, undergoing two or more hybridization polyploidization processes. Asteraceae plants generally undergo three polyploidization events, whereas polyploid species are more prevalent within the genus Chrysanthemum and its related species. While chrysanthemum, which is representative of plants of the Asteraceae family, is typically an allohexaploid and an aneuploid thereof. However, because the genome is doubled many times, the genome structure is relatively complex along with the change of the number of genes, and compared with the original genome, the gene expression level and the genome composition are significantly changed, such as chromosome recombination, elimination of parent sequences, gene silencing, homeotic conversion, etc., so that a large number of homologous genes are generated and the copy number of the genes is increased, which leads to gene redundancy, original gene sub-functionalization and even silencing, and greatly increases the difficulty of screening low-copy or single-copy target genes and cloning genes.
Low copy number genes are an effective means of analyzing plant heteromultiploid phylogeny. The method can be used for discussing the system evolution relationship among different ploidy levels (or different species with the same ploidy level), the genetic structure of communities, plant pedigree geography and the like, and can also be used for researching the origin and evolution history of different genes. Therefore, single-copy or low-copy genes are essential for research in phylogeny and the like. In addition, the transgenic copy number of the transgenic plant is an important factor influencing the expression level and genetic stability of a target gene, so that the key step in transgenic research is to detect the copy number of endogenous and exogenous genes so as to screen out transgenic plants with few copy numbers or single copy for further research or breeding utilization.
The traditional copy number detection is mainly a Southern hybridization method, but the method has the disadvantages of large workload, more required DNA materials, time and labor waste, and frequent use of potentially dangerous drugs such as radioactive isotopes. Therefore, how to quickly, accurately and efficiently identify the copy number of the endogenous gene of the chrysanthemum and the copy number of the transgenic chrysanthemum transformed with the endogenous gene and the exogenous gene becomes a key problem for identifying the copy number of the gene of the chrysanthemum.
Disclosure of Invention
The invention aims to solve the problems of complex identification method, large workload and more required DNA materials of the copy number of the genes of the polyploid plant chrysanthemum and the copy number of the internal and external genes of the transgenic chrysanthemum in the prior art, and provides a primer and a method for identifying the copy number of the internal and external genes of the chrysanthemum based on fluorescence quantitative PCR.
The purpose of the invention is realized by the following technical scheme:
the application of the PGK gene in the identification of the copy number of the internal and external genes of the chrysanthemum by fluorescent quantitative PCR, wherein the PGK gene is used as an internal reference gene. The accession number of the PGK gene in genbank is KJ524576.
The chrysanthemum comprises transgenic chrysanthemum or non-transgenic chrysanthemum.
An endogenous gene amplification primer pair applied to the fluorescent quantitative PCR identification of the copy number of endogenous and exogenous genes in chrysanthemum is characterized in that primer premier 5.0 software is adopted to design a fluorescent quantitative primer of an internal reference gene PGK, the length of the primer is generally 17-25bp, the difference between an upstream primer and a downstream primer cannot be too large, the length of a fluorescent quantitative product is 80-150bp, and the longest length is 300bp; the Tm value is between 55 and 65 ℃, wherein the difference between the Tm values of the front primer and the Tm value of the rear primer is 4 ℃; the product cannot form secondary structures.
The primer pair is preferably as follows: the length of each primer is 24bp, the length of a primer amplification fragment is 225 ℃, the Tm value is 58 DEG C
The primer pair is further preferably: the sequence of the forward primer is shown as SEQ ID NO.1, and the sequence of the reverse primer is shown as SEQ ID NO. 2.
A method for identifying the copy number of an endogenous gene of a non-transgenic chrysanthemum by fluorescent quantitative PCR comprises the following steps:
(1) Extracting DNA of chrysanthemum;
(2) Measuring the quality and concentration of DNA of a sample, uniformly diluting the sample into the same concentration of 50ng/ul, designing and synthesizing a primer according to the DNA fragment of the endogenous gene of the chrysanthemum to be measured, wherein the design parameters of the primer are that the length of the primer is 18-25bp, the temperature is Tm55-65 ℃, the Tm value difference of the front primer and the rear primer is 4 ℃, and the size of a product is 100-300bp;
(3) Carrying out fluorescent quantitative PCR reaction on a Mastercycler ep Realplex type fluorescent quantitative instrument, and carrying out PCR reaction on internal reference gene PGK and a gene to be detected on each sample, wherein the primer pair shown in SEQ ID NO.1/SEQ ID NO.2 is used for the fluorescent quantitative PCR reaction of the internal reference gene PGK;
(4) The uniqueness of the gene is determined by the dissolution curve analysis, whether the gene dissolution curve is of a single peak type or not can be seen by the dissolution curve analysis, if the gene dissolution curve is of the single peak type, the non-specific amplification does not occur in the PCR amplification process, and therefore the data obtained by quantitative PCR amplification is reliable;
(5) Analyzing fluorescence quantitative data, and determining the copy number of the gene: and according to the comparative analysis of the CT values of the endogenous gene and the PGK of the reference gene of the chrysanthemum to be detected, the CT value is the number of cycles of the fluorescence signal in each PCR reaction tube when the fluorescence signal reaches a set threshold value. The larger the initial copy number, the smaller the CT value. PGK is a single copy gene, the copy number of the gene to be detected is 2 times of the copy number of the PGK if the gene to be detected has one CT value less than the PGK, and if the CT average value of the gene to be detected is n less than the PGK, the copy number of the endogenous gene to be detected of the chrysanthemum can be determined to be 2 n 。
Preferably, the method is performed in 3 replicates per sample, the PCR assay is repeated 3 times, and the CT value is averaged over three determinations.
A method for identifying the copy number of transgenic chrysanthemum transformed into endogenous and exogenous genes based on fluorescence quantitative PCR comprises the following steps:
(1) Respectively extracting DNA of wild chrysanthemum and transgenic chrysanthemum;
(2) Measuring the quality and concentration of sample DNA, diluting uniformly to the same concentration of 50ng/ul, designing primers according to the trans-internal and external genes or the carrier fragment containing the trans-internal and external genes, wherein the design parameters of the primers are that the length of the primers is 18-25bp, the Tm is 55-65 ℃, the Tm value difference of the front and rear primers is 4 ℃, and the size of the product is 100-300bp;
(3) Carrying out fluorescent quantitative PCR reaction on a Mastercycler ep Realplex type fluorescent quantitative instrument, and carrying out PCR reaction of an internal reference gene PGK and a gene to be detected or a vector fragment for each sample (if the internal reference gene is transferred, a wild type gene to be detected needs to be detected), wherein the primer pair shown in SEQ ID NO.1/SEQ ID NO.2 is used for the fluorescent quantitative PCR reaction of the internal reference gene PGK;
(4) The uniqueness of the gene is determined by the analysis of the dissolution curve, whether the gene dissolution curve is unimodal or not can be seen by the analysis of the dissolution curve, and if the gene dissolution curve is unimodal, the non-specific amplification does not occur in the PCR amplification process, so that the data obtained by quantitative PCR amplification is inferred to be reliable;
(5) Analyzing fluorescence quantitative data, determining the copy number of the gene:
(A) When a primer is designed for a vector fragment containing the trans-internal and exogenous genes to carry out fluorescence quantitative PCR, CT values of an internal reference gene PGK and a gene to be detected of the primer designed according to the vector fragment are respectively obtained and then are compared and analyzed, and the larger the initial copy number is, the smaller the CT value is. PGK is a single copy gene, every less one CT value of the gene to be detected represents that the copy number of the gene to be detected is 2 times of the copy number of the PGK, if the CT value of fluorescence quantification performed by a primer designed according to the gene carrier to be detected is the same as the CT value of the PGK gene, the transgenic chrysanthemum to be detected is converted into an endogenous gene or an exogenous gene as the single copy gene; if the CT value of the gene to be detected is n less than the CT value of PGK, the copy number of the transgenic chrysanthemum to be detected to the endogenous gene or the exogenous gene can be determined to be 2 n ；
(B) When a primer is designed for the exogenous gene per se to carry out fluorescence quantitative PCR, CT values of an internal reference gene PGK and the exogenous gene are respectively obtained, then the CT value of the exogenous gene is compared and analyzed with the CT value of a reference gene PGK, the PGK is a single copy gene, each less CT value of the gene to be detected represents 2 times of the copy number of the gene to be detected, if the CT value of the fluorescence quantitative primer designed according to the gene to be detected per se is the same as the value of the PGK, the exogenous gene to be detected is the single copy gene, and if the CT value of the fluorescence quantitative primer designed according to the gene to be detected per se is less than the value of the PGK, the exogenous gene to be detected is the single copy gene, and if the fluorescence quantitative primer designed according to the gene to be detected per se is less than the value of the PGK, the CT value of the reference gene is less than the value of the PGKn, the copy number of the gene to be detected in the chrysanthemum can be determined to be 2 n ；
(C) When a primer is designed for the endogenous gene per se to carry out fluorescence quantitative PCR, the reference gene PGK in the transgenic chrysanthemum and the wild chrysanthemum and the CT value of the endogenous gene to be detected are respectively obtained, and if the CT value of the endogenous gene to be detected is converted 1 Average value is n less than PGK 1 Then, the total copy number of the endogenous gene in the transgenic chrysanthemum can be determined to be 2 n1 (ii) a CT of wild type gene 2 Comparing the value with the CT value of the reference gene PGK, and determining that the number of the genes is n less than that of the PGK 2 If the number of copies of the endogenous gene of the wild chrysanthemum is 2 n2 Then the copy number of the transferred endogenous gene is 2 n1 -2 n2 。
The method preferably comprises the following steps: 3 replicates per sample were tested in 3 replicates and the PCR assay was repeated 3 times and the CT values averaged over the three assays.
The SYBR Green II real-time fluorescent quantitative PCR method adopted in the method is to add SYBR Green II fluorescent dye into a PCR reaction system, wherein the dye can be combined with all DNA double helix minor groove regions and has the capability of generating Green fluorescence by excitation. When the fluorescent probe is combined with double-stranded DNA synthesized by PCR, fluorescence is generated under the irradiation of exciting light, and the increase of the fluorescence signal intensity is completely synchronous with the increase of a PCR product, so that PCR amplification data can be indirectly obtained by detecting the fluorescence signal intensity in the PCR reaction process in real time. The transgene copy number is obtained by comparing and analyzing CT values of transgenic plants of endogenous and exogenous genes and standard endogenous reference genes of chrysanthemum.
Has the advantages that:
in recent years, the detection of transgene copy number by high-throughput, rapid and sensitive quantitative PCR methods is becoming favored by researchers. However, since the chrysanthemum is hexaploid, there is no reference genome, the background is very complex, and finding haploid genes is very difficult, so far, there is no report of an analysis method for identifying the copy number of chrysanthemum gene by fluorescent quantitative PCR, and the copy number of chrysanthemum gene still remains in the Southern hybridization method. A large number of experiments discover a single copy gene in chrysanthemum, and a primer suitable for fluorescent quantitative PCR is developed for the gene. By using the gene as an internal reference gene, the copy numbers of the internal and external genes of the chrysanthemum can be successfully, quickly and accurately identified through fluorescent quantitative PCR. The invention has the following advantages:
1. the pair of detection primers is utilized, so that the copy number of the chrysanthemum endogenous gene can be rapidly and accurately identified.
2. The invention can rapidly identify the copy number of the transgenic chrysanthemum transformed into endogenous and exogenous genes by using the detection primer and the method, improves the detection efficiency of detecting the transgenic copy number of the transgenic plant and saves the DNA dosage.
3. The detection primer provides an idea for identifying the gene copy number of the polyploid of the complex genome, the result is real and reliable, and the detection efficiency is greatly improved.
4. The detection primer and the detection method have the advantages of small amount of required experimental materials, small workload, time saving and accurate and reliable result, and have important theoretical and practical significance for guiding the identification of the gene copy number of the allopolyploid plant, especially the detection of the gene copy number of chrysanthemum.
Drawings
FIG. 1 Southern hybridization plot of PGK gene. The first lane is Hind III band color map, the second lane is EcoR I band color map. Note: hindIII enzyme has no enzyme cutting site in PGK gene, and EcoR I enzyme has one enzyme cutting site in PGK gene.
FIG. 2 lysis curves of the reference genes PGK and CmPT 2.
FIG. 3 amplification curves of PGK and CmPT2 gene real-time quantitative PCR of wild chrysanthemum 'Nannongxue Peak' (panel a) and transgenic endogenous gene PT2 chrysanthemum 'Nannongxue Peak' (panel b, c). FIG. 3a: the former amplification curve is the CmPT2 gene, and the average value of three repeated CTs is 22. The later amplification curve is PGK gene, and the average value of three repeated CTs is 24; FIG. 3b: the former amplification curve is CmPT2 gene, and the three repeated CT mean values are 22.04. The latter amplification curve is the PGK gene, and the average value of the CT of the three replicates is 24.36. FIG. 3c: the previous amplification curve is the CmPT2 gene, and the mean value of three repeated CTs is 21. The latter amplification curve is the PGK gene, and the average of three repeated CTs is 23.59.
Detailed Description
The present invention is further illustrated by the following examples, in which experimental procedures not specifically illustrated are generally performed according to methods well known in the art.
Example 1
(one) selection of stably expressed endogenous reference genes:
endogenous reference genes are generally selected from a class of genes that have a low copy number and that have stable sequence structure and copy number between different ecotypes within the same species. The internal reference genes of chrysanthemum are less reported, and firstly, the PGK gene is identified to be a single copy gene in the chrysanthemum by using a traditional Southern hybridization method. As shown in FIG. 1, hindIII without enzyme cutting site and EcoRI with one enzyme cutting site in PGK gene are used to cut PGK gene respectively, and the result of Southern hybridization after cutting with HindIII is found to be one band and that after cutting with EcoRI is two bands, so that PGK gene in chrysanthemum is single copy gene and can be used as internal reference gene for identifying chrysanthemum gene copy number. The accession number of the PGK gene in genbank is KJ524576.
(II) designing a primer of a reference gene for identifying the copy number of the chrysanthemum gene and a gene primer of a PT2 copy number of a transgenic endogenous gene to be detected:
primer premier 5.0 software is adopted to design the internal reference gene PGK and the fluorescence quantitative primer of the trans-endogenous gene CmPT2 with copy number to be identified. The fluorescent quantitative primer of CmPT2 is designed according to the transgenic PT2 (the ORF sequence of the gene is shown as SEQ ID NO. 5). The primer lengths of the internal reference gene PGK are respectively 24bp, the length of the primer amplification fragment is 225, and the Tm value is 58 ℃. The primer length of the endogenous gene CmPT2 is 18-19bp, and the Tm value is 55 ℃. The primer sequences are shown in the following table:
Figure BDA0000802771440000061
Figure BDA0000802771440000071
(III) obtaining a test material:
the test material is a chrysanthemum variety preserved in the China Chrysanthemum germplasm resources preservation center of Nanjing agriculture university: 'south nongxuefeng'. Respectively 1 chrysanthemum 'Nannongxuefeng' (wild type) plant and 8 current generation plants of 'Nannongxuefeng' transformed with endogenous CmPT2 gene. The plant expression vector pMDC43-CmPT2 is transferred into the 'Nannongxuefeng peak' by an agrobacterium-mediated plant transformation method, and the current generation plant of the 'Nannongxuefeng peak' of the transferred endogenous gene CmPT2 is obtained.
And (IV) identifying the copy number of the chrysanthemum endogenous gene and the copy number of the trans-endogenous gene PT2 by using a fluorescent quantitative PCR technology:
1) Referring to the improved CTAB micro-method (Murray and Thompson, 1980), taking 'Nannongxuefruit' (wild type) and transgenic 'Nannongxuefruit' young leaves to extract genome DNA, detecting the quality of the DNA by 1.0% agarose gel electrophoresis of Lambda DNA, then using a nucleic acid and protein quantitative analyzer to measure the quality and concentration of the DNA, and using ddH 2 Diluting O to 50 ng. Mu.L -1 。
2) Quantitative PCR reaction system and program
According to the fluorescent quantitative PCR kit (SYBR Green real PCR Master Mix-Plus (QPK-212))
The instructions set up a 20 μ L reaction: SYBRGreen Realtime PCR Master Mix-Plus 10. Mu.L,
Forward primer(10μM) 1μL
Reverse primer(10μM) 1μL
DNA template 5μL
dd H 2 O 3μL
Total 20μL
the PCR tubes were placed in a fluorescent quantitative PCR instrument (Mastercycler ep Realplex, eppendorf, germany) and the PCR reaction was performed according to the following reaction program: 2min at 95 ℃;95 ℃ 15s,55 ℃ 15s,72 ℃ 30s,40 cycles. During the cycle, the temperature was increased from 55 ℃ at a rate of 0.5 ℃ per second to obtain the necessary denaturation curve, and the purity of the amplification was judged as a single dissolution peak. PCR reaction of reference gene PGK and gene PT2 to be detected is carried out on each sample, each sample is paralleled by 3, and the technology is repeated for 3 times.
And (V) fluorescent quantitative data analysis:
(1) Dissolution Curve analysis
When SYBRGreen II fluorescence chimeric method is adopted for detection, because SYBRGreen II can be chimeric into minor groove regions of all double-stranded DNA and emits fluorescence under excitation, specificity detection of PCR reaction is required, and amplification specificity of the PCR reaction is generally analyzed through a dissolution curve. The ideal dissolution profile should be a single peak profile, if two or more peaks are present, indicating the presence of non-specific amplification such as primer dimers. In this experiment, it can be seen from the analysis of the melting curves of the two genes PGK and PT2 (fig. 2) that the melting curves of both genes are monomodal, indicating that no non-specific amplification occurs during the PCR amplification, and therefore it is concluded that the data obtained by quantitative PCR amplification is reliable.
(2) Analysis of copy number of endogenous gene CmPT2 in chrysanthemum and copy number of endogenous gene CmPT2 in chrysanthemum transformation
According to the comparative analysis of the CT values of the PT2 gene of 'Nannongxuefeng peak' (wild type) and the PGK of the endogenous reference gene, the CT value is the number of cycles that the fluorescence signal passes when reaching the set threshold value in each PCR reaction tube. The CT value of each template has a linear relation with the logarithm of the initial copy number of the template, and the CT value is smaller when the initial copy number is larger. PGK is a single copy gene, and every less CT value of the gene to be detected represents that the copy number of the PT2 gene is 2 times of that of the PGK. The amplification curve of the internal reference gene PGK and the endogenous CmPT2 gene real-time quantitative PCR of the wild chrysanthemum 'Nannongxuefeng' (fig. 3 a) is known, the former amplification curve is the CmPT2 gene, and the average value of three repeated CTs is 22; the later amplification curve is PGK gene, the CT average value of three repeats of the PGK gene is 24, the CT average value of three repeats of CmPT2 is less than 2 than that of PGK, and therefore, the copy number of the endogenous gene PT2 of the chrysanthemum 'Nannongxuefeng' (wild type) can be determined to be 2 2 And 4 in number.
Then according to the comparative analysis of CT values (figure 3 b) of PT2 gene and endogenous reference gene PGK in transgenic chrysanthemum 'Nannongxuefeng peak' of endogenous gene CmPT2, in 4 transgenic endogenous gene CmPT2 strains, the former amplification curve isThe CmPT2 gene of which the average value of three repeated CTs is 22.05; the latter amplification curve is the PGK gene with an average CT value of 24.36 for the three replicates, 2.3 less for all three replicates of CmPT2 than for PGK, the copy number of CmPT2 in the transgenic lines is 2 2.3 And if the number of the transferred endogenous genes PT2 is not less than 5, the number of the transferred endogenous genes PT2 is the number of the chrysanthemum copies of the transgenic CmPT2 minus the number of the chrysanthemum endogenous genes PT2, namely 1, and the number of the transferred endogenous genes PT2 of 4 plants is 1.
The real-time quantitative PCR amplification curves of the internal reference gene PGK and the internal source CmPT2 gene in the other 4 strains of the trans-endogenous gene CmPT2 can be known (figure 3 c), the former amplification curve is the CmPT2 gene, and the average value of three repeated CTs is 20.99. The latter amplification curve is the PGK gene with an average CT value of 23.59 for the three repeats, 2.6 less for all three repeats than the PGK, and the CmPT2 copy number in the transgenic lines is 2 2.6 = 6. Then the copy number of the transferred endogenous gene PT2 is 2 obtained by subtracting the copy number of the chrysanthemum endogenous gene PT2 from the chrysanthemum copy number of the transgenic CmPT 2.
Therefore, the copy number of the endogenous gene PT2 of the chrysanthemum 'Nannongxuefeng' (wild type) is 4. Of the 8 transgenic lines, 4 transgenic lines had a PT2 transgene copy number of 1,4 transgenic lines and 2.
It will be understood that the above-described embodiments are merely illustrative of the principles of the invention, which is not limited thereto, and that various modifications and changes can be made by those skilled in the art without departing from the spirit of the invention, which also falls within the scope of the invention.
Figure IDA0000802771530000011
Figure IDA0000802771530000021

Claims (5)

1.PGKGene identification of internal and external sources of chrysanthemum by fluorescent quantitative PCRUse in gene copy number, characterized in thatPGKThe gene is used as a single copy reference gene; saidPGKThe gene has an accession number KJ524576 in genbank.
2. The use according to claim 1, wherein the chrysanthemum comprises transgenic chrysanthemum and non-transgenic chrysanthemum.
3. A method for identifying the copy number of an endogenous gene of a non-transgenic chrysanthemum based on fluorescence quantitative PCR is characterized by comprising the following steps:
(1) Extracting DNA of chrysanthemum;
(2) Measuring the quality and concentration of DNA of a sample, uniformly diluting the sample into the same concentration of 50ng/ul, designing and synthesizing primers according to the DNA fragment of the endogenous gene of the chrysanthemum to be measured, wherein the design parameters of the primers are that the length of the primers is 18-25bp, the Tm is 55-65 ℃, the difference of Tm values of the front primer and the rear primer is 4 ℃, and the size of a product is 100-300bp;
(3) Performing fluorescent quantitative PCR reaction on a Mastercycler ep Realplex type fluorescent quantitative instrument, and performing PCR reaction on internal reference gene PGK and endogenous gene to be detected for each sample, wherein the primer pair shown in SEQ ID NO.1/SEQ ID NO.2 is used for the fluorescent quantitative PCR reaction of the internal reference gene PGK, and the primer pair is used for the fluorescent quantitative PCR reaction of the internal reference gene PGKPGKThe accession number of the gene in genbank is KJ524576;
(4) The uniqueness of the gene is determined by the dissolution curve analysis, whether the gene dissolution curve is of a single peak type or not can be seen by the dissolution curve analysis, if the gene dissolution curve is of the single peak type, the non-specific amplification does not occur in the PCR amplification process, and therefore the data obtained by quantitative PCR amplification is reliable;
(5) Analyzing fluorescence quantitative data, and determining the copy number of the gene: according to the endogenous gene and the reference gene of the chrysanthemum to be detectedPGKThe CT value of the CT is compared and analyzed, PGKfor single copy genes, the ratio of genes to be testedPGKIf there is one less CT value, it represents that the copy number of the gene to be tested isPGK2 times of copy number, if the CT average ratio of the gene to be testedPGKN less, determining that the copy number of the endogenous gene to be detected in the chrysanthemum is 2 n ;
Wherein each sample is 3 replicates, PCR detection is repeated 3 times, and CT values are averaged over three determinations.
4. A method for identifying the copy number of transgenic chrysanthemum transformed into endogenous and exogenous genes based on fluorescence quantitative PCR is characterized by comprising the following steps:
(1) Respectively extracting DNA of wild chrysanthemum and transgenic chrysanthemum;
(2) Measuring the quality and concentration of sample DNA, diluting uniformly to the same concentration of 50ng/ul, designing primers according to the trans-internal and external genes or the carrier fragment containing the trans-internal and external genes, wherein the design parameters of the primers are that the length of the primers is 18-25bp, the Tm is 55-65 ℃, the Tm value difference of the front and rear primers is 4 ℃, and the size of the product is 100-300bp;
(3) Carrying out fluorescent quantitative PCR reaction on a Mastercycler ep Realplex type fluorescent quantitative instrument, and carrying out PCR reaction on internal reference gene PGK and a gene or a vector fragment to be detected on each sample, wherein the primer pair shown in SEQ ID NO.1/SEQ ID NO.2 is used for the fluorescent quantitative PCR reaction of the internal reference gene PGK; if the gene is endogenous gene, a gene to be detected of wild chrysanthemum needs to be determined, wherein the gene to be detected is the gene which is endogenous gene transferredPGKThe accession number of the gene in genbank is KJ524576;
(4) The uniqueness of the gene is determined by the analysis of the dissolution curve, whether the gene dissolution curve is unimodal or not can be seen by the analysis of the dissolution curve, and if the gene dissolution curve is unimodal, the non-specific amplification does not occur in the PCR amplification process, so that the data obtained by quantitative PCR amplification is inferred to be reliable;
(5) Analyzing fluorescence quantitative data, and determining the copy number of the gene:
(A) When designing primers for vector fragments containing the trans-internal and exogenous genes to carry out fluorescent quantitative PCR, respectively obtaining reference genesPGKAnd the CT value of the gene to be tested of the primer is designed according to the carrier fragment for comparative analysis, PGKfor single copy gene, every less one CT value of the gene to be tested represents the copy number of the gene to be testedPGK2 times of copy number, CT value and the ratio of fluorescence quantification if designing primer according to gene vector to be detectedPGKIf the CT values of the genes are the same, the transgenic chrysanthemum to be detected is converted into an endogenous gene or an exogenous gene as a single copy gene; if it isCT value ratio of gene to be testedPGKIf the CT value is n less, the copy number of the transgenic chrysanthemum to be detected to be transferred to the endogenous gene or the exogenous gene can be determined to be 2 n ；
(B) When primers are designed for exogenous genes to carry out fluorescence quantitative PCR, reference genes are respectively obtainedPGKAnd the CT value of the foreign gene, the CT value of the foreign gene and the reference genePGKThe CT value of the CT is compared and analyzed,PGKfor single copy gene, every CT value of the gene to be tested represents the gene to be testedPGKCopy number is 2 times, if the CT value of fluorescence quantification performed by a primer designed according to the gene to be detected is the same as the value of PGK, the exogenous gene to be detected is a single copy gene, and if the ratio is equal to the ratioPGKN less, the copy number of the gene to be detected in the chrysanthemum can be determined to be 2 n ；
(C) When a primer is designed aiming at the endogenous gene to carry out fluorescence quantitative PCR, the reference genes in the transgenic chrysanthemum and the wild chrysanthemum are respectively obtainedPGKAnd the CT value of the endogenous gene to be tested, if the CT value of the endogenous gene to be tested is converted 1 Ratio of mean valuesPGKN is less 1 Then, the total copy number of the endogenous gene in the transgenic chrysanthemum can be determined to be 2 n1 (ii) a CT of wild type of the gene 2 Value and reference genesPGKThe CT value of (1) is compared and analyzed, if isPGKN is less 2 If the number of copies of the endogenous gene of the wild chrysanthemum is 2 n2 Then the copy number of the transferred endogenous gene is 2 n1 -2 n2 ；
3 replicates per sample, PCR assays were repeated 3 times, and CT values were averaged over three assays.
5. The application of the primer pair shown in SEQ ID NO.1/SEQ ID NO.2 in identifying the copy number of internal and external genes of chrysanthemum.
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