CN113046471A - Method for identifying single-copy transgenic plant based on competitive PCR technology - Google Patents

Method for identifying single-copy transgenic plant based on competitive PCR technology Download PDF

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CN113046471A
CN113046471A CN202110534903.8A CN202110534903A CN113046471A CN 113046471 A CN113046471 A CN 113046471A CN 202110534903 A CN202110534903 A CN 202110534903A CN 113046471 A CN113046471 A CN 113046471A
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王丹阳
樊华
黄留园
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Abstract

The invention discloses a method for identifying single-copy transgenic plants based on competitive PCR technology, which is suitable for transgenic plants containing at least one of the three resistance genes; requires that transgenic plants to be tested and public competitive strains BHK are taken‑1 hybridization was performed to ensure that all diploid genome components of antibiotic-screened F1 plants consisted of one gene from BHK‑And another haploid genome from a transgenic plant. After the genomic DNA of the F1 plants is amplified by competitive PCR, whether the transgenic plants contain single T-DNA copies can be easily judged according to the gray scale ratio of PCR products. The method is simple to operate and high in repeatability, and can be used as a substitute method of Southern blot to detect the single copy of the T-DNA insert in the transgenic plant.

Description

Method for identifying single-copy transgenic plant based on competitive PCR technology
Technical Field
The invention belongs to the technical field of biomolecule detection, and particularly relates to a method for identifying single-copy transgenic plants based on a competitive PCR technology.
Background
The number of mutated genes is closely related to the number of inserted T-DNA on the host chromosome. Southern blot analysis enables detection of a single copy of the T-DNA insertion by hybridizing signal bands [ Southern,1975 ]. Southern blot analysis involves a number of experimental steps such as extraction and digestion of the genome, agarose gel electrophoresis, membrane transfer and hybridization, a series of which makes the experiment very complex [ Southern,1975 ]. In particular, in hybridization, a labeled probe capable of annealing to a target is required. At present, two methods, namely an isotope method and a digoxin method, are mainly adopted for preparing the probe. Isotopically labeled probes make visualization of hybridization signals relatively simple, but because of their radioactivity, the experimenter must consider isotopic contamination. To this end, digoxigenin-labeled probes were developed [ Martin et al, 1990; solanas and Esicrich, 1997 ]. Although digoxigenin is safe, digoxigenin-labeled probes are subsequently required to use immunological affinity methods, which further complicates the visualization of hybridization signals.
Disclosure of Invention
The invention aims to provide a method for identifying single-copy transgenic plants based on a competitive PCR technology aiming at the defects of the prior art.
The purpose of the invention is realized by the following technical scheme: a method for identifying single-copy transgenic plant based on competitive PCR technology is based on competitive PCR and single-copy universal competitor BHK-1, comprising the following steps:
(1) the transgenic plant to be tested and the universal competitive transgenic plant BHK are used-1 hybridization was performed to ensure that all diploid genome components of antibiotic-screened F1 plants consisted of one gene from BHK-And another haploid genome from a transgenic plant;
(2) after the genomic DNA of the F1 plants is amplified by competitive PCR, whether the transgenic plants contain single T-DNA copies is judged according to the gray scale ratio of PCR products.
Further, the transgenic plant contains at least one of the three resistance genes.
Further, the plant is Arabidopsis thaliana.
The invention has the beneficial effects that: compared with the complexity and high pollution of Southern blot, the method is essentially based on quantitative analysis and only requires PCR amplification; the single ratio in the obtained experimental results is almost consistent with the average ratio, which shows that the single ratio is strong in consistency, stable, reliable and high in repeatability; the method is simple to operate and easy to master, and can be used for well replacing Southern blot to identify whether the transgenic plant contains single copy.
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FIG. 1 is a schematic representation of the real-time fluorescent quantitative PCR detection of the ratio of copies of ProHTR10 to copies of GFP in seb 19-L; wherein A comprises the T-DNA integration site of seb19-L (line 1), the corresponding fragment of chromosome 5 (line 2) of a wild-type plant, the HTR10 promoter (ProHTR10) in chromosome 1 (line 3) and the partial structure of plasmid pGreen-ProHTR10: GFP (line 4); b is PCR identification of a heterozygote plant seb 19-L; c is three experimental ratios of ProHTR10 copy to GFP copy;
FIG. 2 is a schematic diagram showing the evaluation of the sensitivity of competitive PCR for plasmids DMG & DG and 2 XDMG & DG; wherein A comprises the structures of fragments DMG, DG, DMG & DG and 2 XDMG & DG; b is 5 groups of products with the plasmids DMG & DG and 2 XDMG & DG as the gray ratio (upper limit and lower limit) of the template products;
FIG. 3 is a schematic diagram of the evaluation of the sensitivity of genome competitive PCR of transgenic plants; wherein, A comprises 35S: CpB-mCherry (B) and 35S: MT-mCherry T-DNA partial structure; b comprises transgenic plants B5, B8, M2, M3T-DNA insertion position schematic diagram; c is PCR detection of transgenic plants B5, B8, M2 and M3 (upper half part); d comprises double hybrid mutants B5/- & M2/-and B8/- & M3/-, simple and double mutants B5/B5& M2/-and B8/B8& M3/-PCR detection; e is a competitive PCR with the DNA genomes of the plants B5/- & M2/-, B8/- & M3/-, B5/B5& M2/-and B8/B8& M3/-as templates, with the average from 5 independent measurements;
FIG. 4 shows the construction of a widely used competitor BHK-And testing the availability schematic diagram; wherein A is fragment BHK-、BHK+、BHK-&BHK+And BHK-&2×BHK+Structure; bar, Hyg and Kan represent the entire herbicide, hygromycin and kanamycin genes, respectively, Bar-，Hyg-And Kan-Represents herbicide, hygromycin and kanamycin genes with fragment deletion respectively; target BHK+Composed of Bar, Hyg and Kan, competes for template BHK-By Bar-，Hyg-And Kan-Composition is carried out; b is plasmid BHK-&BHK+And BHK-&2×BHK+Competitive PCR results, mean from 5 independent measurements;
FIG. 5 is a single copy universal competitor BHK-The transgenic plant BHK--1, a schematic construction diagram; wherein A comprises a competitive template BHK-And fusion gene ProRPS5A H2B-tdTomato T-DNA for transgenic seed screening; b is a fraction containing only one BHK-A process of transgenic plant of the segment; screening of transgenic BHK by fluorescence microscopy-Seeds (left panel); seeds were separated using glass microtubes under an inverted fluorescence microscope (right panel); c only contains one portion of BHK for separation-The process of insertion of (a); detection of the heterozygote genotype of seb19-L, B8 and B8 by PCR amplification and determination of BHK by fluorescence microscopy-The heterozygote genotype of (a), wherein half of the pollen grains do not exhibit red fluorescence in the nucleus; d is T-DNA in BHK-The insertion position in (1); e, F, G confirmed BHK by PCR-The T-DNA insertion position of (1); e represents two ways in which T-DNA may be inserted into the host chromosome in a direct tandem repeat; f indicates that the T-DNA may be inserted into the host chromosome in an inverted tandem repeat; g represents PCR amplification confirmation BHK-The T-DNA of (1) can only be inserted in the D insertion mode, and the insertion mode in E, F does not exist;
FIG. 6 is a flow chart for creating a BHK±1:1,BHK±2:1And BHK±3:1The construction of (a); wherein A is a compound containing 1:1, 2:1 and 3:1 different ratios of BHK+Fragment of (4) and BHK-A fragment; b uses Kan primer pair BHK±1:1,BHK±2:1And BHK±3:1Genomic DNA was used as template and the ratio between PCR products was evaluated.
Detailed Description
The method for identifying the single-copy transgenic plant based on the competitive PCR technology can simply identify whether the transgenic plant contains single T-DNA insertion. The method is based on competitive PCR and universal competitor BHK-(herbicide, hygromycin and kanamycin three fragment deletion resistance gene competition modelA plate). Therefore, the present invention is only applicable to transgenic plants containing at least one of the above three resistance genes.
The invention needs to firstly test the transgenic plant to be tested and the universal competitor BHK--1 and using the genome of the F1 plant as a DNA template for PCR. Ensures that all diploid genomes of F1 plants subjected to antibiotic screening consist of one gene from BHK--1 and another haploid genome from a transgenic plant. After the genomic DNA of the F1 plants is amplified by competitive PCR, whether the transgenic plants contain single T-DNA copies can be easily judged according to the gray scale ratio of PCR products.
Different experimental systems may cause the instability of the ratio, and when the genomic DNA of the F1 plant is amplified, the control strain BHK is used±1:1、BHK±2:1And BHK±3:1The genomic DNA of (3) is simultaneously amplified. The three controls can well indicate the ratio change caused by different PCR systems, thereby being beneficial to obtaining correct conclusion and judging whether a transgenic plant contains a single T-DNA copy.
Description of materials involved in the examples of the present invention:
absolute ethanol, agarose, methanol, isopropanol, carbenicillin disodium salt, 1/2MS medium, agar powder (Dingguo), sucrose (raw), peptone (TRYPTONE), YEAST powder (YEASEXTRACT), NaCl, Kanamycin sulfate (Kanamycin sulfate) (70560-51-9, raw), Gentamycin sulfate (Gentamycin sulfate) (1405-41-0, raw), Rifampicin (Rifamicin) (13292-46-1, raw), hygromycin B (hygromycin B) (31282-04-9, raw), EcoT 14I digest (3401, Takara), DNA purification and recovery kit (DP214-02, TIANGGEN), Nutriticale kit (GVDP 103-02, TIANGGEN), Escherichia coli (Escherichia coli) competence CB 635 (101-a, TIAN02), Agrobacterium (GEN) and Agrobacterium (1, Takara) competence 1
Example 1: real-time PCR does not accurately identify single copy T-DNA insertions in transgenic plants.
To determine whether Real-time PCR could detect a single T-DNA insertion in a transgenic plant, transgenic plant seb19-L was selected as the subject. Southern blot clearly showed that the transgenic plants contained only one single copy of a T-DNA insert containing the fusion gene CDKA: GFP.
A promoter fragment (ProHTR10 which is a single-copy DNA fragment on an endogenous genome) of the HTR10 gene is selected as an internal reference, and the single-copy T-DNA (containing a GFP gene) is inserted into the hybrid transgenic plant seb19-L through Real-time PCR evaluation, and the ratio of the copy numbers of the ProHTR10 and the GFP is obtained. For precise quantification, the plasmid pGreen-ProHTR10 was constructed: GFP (with a strict 1:1 ratio of ProHTR10 to GFP copy number) and serial dilutions of the DNA template were amplified to generate two standard curves for analysis of ProHTR10 and GFP copy number in equivalent genomic DNA samples.
Since the material seb19-L selected for this experiment was a heterozygous transgenic plant, the expected ratio of Pro-HTR10 copy number to GFP copy number would be 2: 1. However, as shown in fig. 1, the quantitative ratios for three independent experiments were 3.74:1, 3.80:1 and 3.02:1, respectively, with a combined average ratio of 3.43: 1. it is difficult to determine from the experimental ratio (3.43:1) whether a T-DNA copy number is present in the seb19-L genome, i.e.whether the T-DNA in the transgenic plant is a single copy insertion, compared to the expected ratio (2: 1). The results thus indicate that Real-time PCR cannot be used to identify single copy T-DNA inserted transgenic plants and that more sensitive methods are required for analysis.
Example 2: competitive PCR is highly sensitive in DNA samples and detects single-copy T-DNA insertions in DNA samples.
It was evaluated whether the competitive PCR could accurately distinguish between the DNA sample containing the target and competitor templates 1:1 and the DNA sample containing the target and competitor templates 2: 1. To this end, fragments of pGreen-ProDD45: MT-GFP (DMG) and pGreen-ProDD45: GFP (DG) were created and assembled to obtain plasmids of DMG & DG and 2 XDMG & DG. In plasmid DMG & DG the ratio of DMG copies to DG copies was 1:1, in plasmid 2 x DMG & DG the ratio was 2: 1.
The primer pair DD45-S1 and GFP-A147 were used to perform competitive PCR on the assembled DMG & DG plasmid with the target and competitive template at 1:1 and the plasmid with the target and competitive template at 2: 1. As shown in FIG. 2, after subjecting the product to agarose gel electrophoresis, two bands having different molecular sizes were obtained as shown in the previous stage. The upper band is from the target DMG and the lower band is from the competing template DG. When the plasmid DMG & DG is used as a template for PCR amplification, in five groups of single experiments, the gray scale ratio is respectively 0.88: 1. 0.89:1, 0.86:1, 0.85:1, 0.90: 1; the average gray ratio of the upper band to the lower band is 0.88: 1; when the plasmid 2 x DMG & DG was used as a template for competitive PCR amplification, the gray scale ratios in five single experiments were 1.35: 1. 1.35:1, 1.36: 1. 1.33:1, 1.35:1, average gray scale ratio of upper band to lower band of 1.35: 1. these results clearly show that competitive PCR is extremely sensitive enough to distinguish between DNA samples containing the target and competitive template ratios 1:1 and 2:1, and can identify single copy inserted DNA samples.
Example 3: competitive PCR is highly sensitive in transgenic plants and detects single copy T-DNA insertions in transgenic plants.
By hybridization, transgenic lines of the target and competitive templates 1:1(B5/- & M2/-and B8/& M3-) and 2:1(B5/B5& M2-and B8/B8& M3/-) were obtained, and their genomic DNAs were subjected to competitive PCR to determine whether the competitive PCR had sensitivity in transgenic plants. Single and double mutants B5/B5& M2-and B8/B8& M3/plants, double hybrid mutants B5/- & M2/-and B8/- & M3-plants were identified by PCR from the F2 population. In plants B5/- & M2/-and B8/- & M3-, 35s: the ratio of the copy number of CpB-mCherry to the copy number of 35s MT-mCherry is 1:1, and in the plants B5/B5& M2-and B8/B8& M3/-, the copy number of 35s: the ratio of the copy number of CpB-mCherry to the copy number of 35s MT-mCherry is 2: 1. Plants B5/- & M2/-and B8/- & M3-genome DNA are respectively used for carrying out competitive polymerase chain reaction, and the templates in the amplification reaction are the genome DNA of each plant.
As shown in FIG. 3, the gray scale ratio in five single experiments using the plant B5/- & M2/-genome as template was 0.82: 1. 0.82:1, 0.87:1, 0.88:1, 0.93:1, the average ratio obtained being 0.86: 1. the gray ratio in five groups of single experiments with the plant B8/- & M3-genome as a template is 0.83: 1. 0.87:1, 0.86:1, 0.90:1, 0.95:1, average gray ratio of 0.88: 1. in contrast, the gray scale ratio in five groups of single experiments with the genome of the plant B5/B5& M2 as a template is 1.35: 1. 1.35:1, 1.36:1, 1.33:1, 1.35:1, the resulting average ratio being 1.39: 1. The gray ratio in five groups of single experiments with the plant B8/B8& M3/-genome DNA as a template is 1.22: 1. 1.32:1, 1.28:1, 1.26:1, 1.30:1, average ratio 1.28: 1. the ratios obtained in all experiments were almost identical to the amplification results using the corresponding plasmids as templates, indicating that competitive PCR can indeed be used to identify single copy insertions of T-DNA in transgenic plants.
Example 4: construction of the Universal competitor BHK-. Competitive PCR is widely used to detect single copy T-DNA insertions in transgenic plants, provided it possesses a universal competitor for most transgenic plants.
A common competition template is created. Considering that most transgenic plants carry kanamycin, hygromycin or herbicide resistance genes, the construction is named as BHK-The resistance competitor comprising the deletion of the above three resistance gene fragments, BHK of interest comprising three complete resistance genes+As a control. Competitive template BHK-And target BHK+The Collection of (1) creates the plasmid BHK-&BHK+And BHK-&2×BHK+It contains the target and competitive templates respectively as 1:1 and 2:1 ratio. Using plasmid BHK-&BHK+(ratio of target to competing template 1: 1) and BHK-&2×BHK+(ratio of target to competitor template 2:1) for competitive PCR.
As shown in FIG. 4, the Bar primer pair amplifies plasmid BHK-&BHK+Then, the upper and lower band gray scale ratios of five independent experiments were 0.82:1, 0.81:1, 0.80:1, 0.76:1, 0.82:1, average ratio 0.81: 1; amplification plasmid BHK-&2×BHK+In this case, the average ratio of the upper and lower band gray scales of five independent experiments is 1.33:1, 1.30:1, 1.24:1, and 1.46:1, respectively, is 1.33: 1. hyg primer pair ampliconsPellet BHK-&BHK+Then, the upper and lower band gray scale ratios for five independent experiments were 0.83:1, 0.81:1, 0.80:1, 0.77:1, 0.78:1, respectively, with an average ratio of 0.80: 1; amplification plasmid BHK-&2×BHK+In time, the upper band brightness for five independent experiments was 1.22:1, 1.28:1, 1.34:1, 1.32:1, 1.38: 1, average ratio 1.31: 1. kan primer pair amplification plasmid BHK-&BHK+Then, the upper band brightness for five independent experiments was 0.85:1, 0.82:1, 0.80:1, 0.77:1, 0.79: 1, average ratio 0.81: 1. amplification plasmid BHK-&2×BHK+In time, the upper band brightness for five independent experiments was 1.35: 1. 1.35: 1. 1.41:1, 1.39:1, 1.34:1, average ratio 1.37: 1.
in conclusion, plasmid BHK-&BHK+And BHK-&2×BHK+The gray scale ratio of (A) is 0.8-0.9: 1 and 1.2 to 1.3: 1, it can be well distinguished whether there is a single copy, indicating BHK-Can be used as a general competitor.
Example 5: construction of BHK containing Single copy Universal competitor-The transgenic plant BHK--1。
In order to obtain a universal competitive transgenic plant suitable for most transgenic plants, the invention constructs a competitive transgenic plant containing BHK-&ProRPS 5A-T-DNA plasmid of H2B-tdTomato fusion fragment.
Mixing BHK--1 and BHK-And-2, infecting wild arabidopsis thaliana with agrobacterium liquid to obtain a positive plant, and screening a transgenic seed with red fluorescence by using a fluorescence microscope by using a fusion gene ProRPS5A: H2B-tdTomato as a reporter gene (ProRPS5A: H2B-tdTomato fusion gene enables the nucleus of the transgenic plant to show red fluorescence) because the transgene has no resistance. Selecting positive seeds by a fluorescence microscope, spreading the seeds in an antibiotic-free culture medium to be germinated and transplanted into soil for culture, and obtaining a plurality of stably transformed plants BHK-。
These transgenic lines were crossed with seb19-L containing a single copy of the Kan resistance gene, and BHK carrying only a single copy was isolated-Inserted transgeneCausal line (BHK)-&ProRPS5A: H2B-tdTomato), named BHK--1. By pairing BHK-Plants obtained by hybridization with seb19-L were subjected to competitive PCR to obtain five groups of individual experimental gray scale ratios of 0.88: 1. 0.83:1, 0.87: 1. 0.86: 1. Since the Kan primer pair mediated competitive PCR yielded 0.86:1, the plant was confirmed to be a single copy of BHK-And (4) inserting.
To further confirm the above results, BHK was used-Conventional crossing of-1 with B8 to obtain the hybrid F1 plant B8/-&BHK--1/-. As expected, the hybrid plant B8/-&BHK-When competitive PCR amplification is performed with-1/-genome as template, as shown in FIG. 5, the gray scale ratios of five separate experiments are 0.90:1, 0.89:1, 0.84:1, 0.89:1, and 0.89:1, respectively, and the gray scale ratio between the two PCR products obtained in the final experiment is 0.88:1, and the gray scale ratio is 0.88:1 with plasmid BHK-&BHK+1And BHK-&2×BHK+As a control, it is evident that the heterozygote F1 plant B8/-&BHK--1 and plasmid BHK±1:1Similarly. In addition, when F2 plants (B8/B8)&BHK--1/-) when amplified by the same method, five separate experimental gray scale ratios were 1.22:1, 1.25:1, 1.34:1, 1.36:1, 1.37:1, respectively, and they were combined with plasmid BHK-&BHK+And BHK-&2×BHK+As a control, the F2 plant (B8/B8) was clearly seen&BHK--1/-) and plasmid BHK-&2×BHK+The gray scale ratio of (a) is similar, and the gray scale ratio between the two PCR products obtained thereby is amplified. Taken together, these results confirm BHK-BHK containing only one competitive template-The plant of (1).
Example 6: construction of control transgenic lines BHK±1:1,BHK±2:1And BHK±3:1
To eliminate the difference in the ratio between the two PCR products under different experimental conditions, the target BHK was generated+And competitor BHK-The proportion is 1:1,2: 1,3: 1 control transgenic plant BHK±1:1,BHK±2:1,BHK±3:1. Kanamycin-mediated screeningThe obtained transgenic line BHK±1:1,BHK±2:1And BHK±3:1Using transgenic lines BHK respectively±1:1,BHK±2:1And BHK±3:1And (3) carrying out competitive PCR on the genome DNA, wherein the template in the amplification reaction is the genome DNA of each plant. By comparing the ratio of the two competing PCR products between these control transgenic plants and the test plants, the number of T-DNA insertions in the test plants can be determined more accurately.
As shown in fig. 6, when the Kan primer pair-mediated PCR amplification was performed, the average ratios of the two PCR products were 0.89:1, 1.51:1, 2.07:1, respectively; performing competitive PCR with bar primer pair, wherein the average ratio of the two PCR products is 0.89:1, 1.51:1 and 2.07: 1; competitive PCR was performed with Hyg primer pairs, with the average ratio of the two PCR products being 0.89:1, 1.51:1, 2.07:1, respectively. Taken together, these lines are shown to be effective controls for the present invention.

Claims (3)

1. A method for identifying single-copy transgenic plants based on competitive PCR technology is characterized in that the method is based on competitive PCR and single-copy universal competitor BHK-1, comprising the following steps:
(1) the transgenic plant to be tested and the universal competitive transgenic plant BHK are used-1 hybridization was performed to ensure that all diploid genome components of antibiotic-screened F1 plants consisted of one gene from BHK-And another haploid genome from a transgenic plant.
(2) After the genomic DNA of the F1 plants is amplified by competitive PCR, whether the transgenic plants contain single T-DNA copies is judged according to the gray scale ratio of PCR products.
2. A method for identifying a single copy transgenic plant based on competitive PCR technology as claimed in claim 1 wherein the transgenic plant contains at least one of the three resistance genes.
3. A method for identifying single copy transgenic plants based on competitive PCR technology as claimed in claim 1 wherein the plants are Arabidopsis thaliana and the like.
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