CN105087759B

CN105087759B - Identify copy number of target genes and the method for screening low-copy plant in genetically modified plants

Info

Publication number: CN105087759B
Application number: CN201410188109.2A
Authority: CN
Inventors: 王绪霞; 滕晓璐; 黄炜; 陈凯; 李晨; 刘博林; 马崇烈
Original assignee: Sub-Group Co ltd Of China Seed
Current assignee: Syngenta Group Inc
Priority date: 2014-05-06
Filing date: 2014-05-06
Publication date: 2019-08-02
Anticipated expiration: 2034-05-06
Also published as: CN105087759A

Abstract

The present invention provides copy number of target genes and the method for screening low-copy plant in a kind of high throughput identification genetically modified plants, using Real-Time Real-Time Fluorescent Quantitative PCR Technique, choose suitable Species Normal gene (i.e. reference gene), the detection of relative quantification is carried out with foreign gene, pass through the amplification curve of the two, copy number of foreign gene is calculated to be corrected the copy number of acquisition in conjunction with traditional Southern hybridization technique.According to the demand to genetically modified crops difference copy number, by this method, quick, high-throughput detection and it can screen and copy plant needed for genetically modified crops.

Description

Method for identifying copy number of target gene in transgenic plant and screening low-copy plant

Technical Field

The invention relates to the technical field of transgenic plant detection and the field of crop breeding, in particular to a method for identifying the copy number of a target gene in a transgenic plant at high flux and screening a low-copy plant.

Background

At present, the mature transformation methods applied in the field of plant transgenosis include a gene gun method and an agrobacterium-mediated transformation method. In both methods, the foreign gene is inserted randomly into the plant genome, usually one or more copies of the foreign gene are inserted into the chromosome. A large number of experiments show that the phenomenon of exogenous gene expression silencing of a transformed plant is often caused by multi-copy insertion, and the high-level expression of the exogenous gene in the transgenic plant is often only caused by single-copy or double-copy insertion. In addition, for the transgenic breeding industry, T0 generation exogenous gene single copy inserted plants can be obtained by screening, so that the subsequent screening of exogenous gene mRNA and protein layers is more reliable, and meanwhile, the method is more favorable for obtaining stably inherited transgenic homozygous plants by screening, and the plant breeding process is accelerated.

Disclosure of Invention

The invention aims to provide a method for identifying the copy number of a target gene in a transgenic plant at high flux and screening low-copy plants.

The invention adopts Real-Time fluorescence quantitative PCR technology, selects proper species standardized gene (namely internal reference gene), carries out relative quantitative detection with exogenous gene, calculates the copy number of the exogenous gene through the amplification curve of the two genes, and corrects the obtained copy number by combining the traditional Southern hybridization technology. According to the requirements of different copy numbers of the transgenic crops, the method can quickly and high-flux detect and screen the copy plants needed in the transgenic crops.

In order to achieve the object of the present invention, the present invention provides a method for high-throughput identification of copy number of a target gene in a transgenic plant and screening of low-copy plants, comprising the steps of:

1) extracting the genome DNA of the transgenic plant to be detected;

2) screening a target gene fluorescent quantitative PCR primer and an internal reference gene fluorescent quantitative PCR primer to ensure that the amplification efficiency of the target gene and the internal reference gene is consistent;

3) taking genome DNA of a transgenic plant as a template, and respectively carrying out fluorescent quantitative PCR by using a target gene primer and an internal reference gene primer; appointing a fluorescence signal threshold value, and determining the copy number of the target gene in the transgenic plant to be detected according to the difference value of the cycle times of the target gene and the internal reference gene;

4) screening low-copy transgenic plants;

5) the obtained copy number was corrected by Southern hybridization technique.

The transgenic plant involved in the invention is transgenic rice or corn and the like.

When the transgenic plant is transgenic rice, the target gene is a glyphosate-resistant gene Epsps-CP4(SEQ ID No.2), and the reference gene is a rice Actin7 gene (SEQ ID No. 7). The target gene fluorescent quantitative PCR primer comprises a forward primer and a reverse primer, the nucleotide sequences of the primers are respectively shown as SEQ ID No.5 and SEQ ID No. 6, and the internal reference gene fluorescent quantitative PCR primer comprises a forward primer and a reverse primer, the nucleotide sequences of the primers are respectively shown as SEQ ID No.9 and SEQ ID No. 10.

When the fluorescent quantitative PCR amplification is carried out on the transgenic rice, the reaction system is as follows: FastStart Universal SYBRGreen Master [ ROX ] 5. mu.l, forward and reverse primers of 300nM each 1.25. mu.l, template DNA 2.5. mu.l; the reaction procedure is as follows: 10min at 95 ℃; 95 ℃ 10sec, 60 ℃ 30sec, 28 cycles total.

When the transgenic plant is transgenic corn, the target gene is green fluorescent protein gene GFP (SEQ ID No.1), and the reference gene is corn Ivr1 gene (SEQ ID No. 8). The target gene fluorescent quantitative PCR primer comprises a forward primer and a reverse primer, the nucleotide sequences of the primers are respectively shown as SEQ ID No.3 and SEQ ID No. 4, and the internal reference gene fluorescent quantitative PCR primer comprises a forward primer and a reverse primer, the nucleotide sequences of the primers are respectively shown as SEQ ID No.11 and SEQ ID No. 12.

When the fluorescent quantitative PCR amplification is carried out on the transgenic corn, the reaction system is as follows: FastStart Universal SYBRGreen Master [ ROX ] 5. mu.l, forward and reverse primers of 300nM each 1.25. mu.l, template DNA 2.5. mu.l; the reaction procedure is as follows: 10min at 95 ℃; 32 cycles of 95 ℃ 10sec, 60 ℃ 30 sec.

In the method, the threshold value of the fluorescence signal in the step 3) is 0.2, and the RQ value is 0.1-1.5.

In the method, the DNA template used for screening the target gene fluorescent quantitative PCR primer and the reference gene fluorescent quantitative PCR primer in the step 2) is the genome DNA of the transgenic plant to be detected.

The requirements and the principle of the method in the technical scheme of the invention are as follows:

RQ value of 2^-(CtA-CtB)

In an ideal PCR reaction, PCR product amplification proceeds exponentially with increasing cycle number.

X_n＝X₀*2ⁿ

Wherein,

X_n: product quantity after n cycle

X₀: initial template amount

n: number of PCR cycles

Thus, X₀＝X_n/2ⁿ

For two genes subjected to fluorescent quantitative PCR, target gene a and reference gene B:

A₀＝A_n/2ⁿ

B₀＝B_n/2ⁿ

when PCR amplification is specific, the fluorescent signal is exactly equal to the amount of PCR product. A threshold fluorescence signal is assigned, and the amount of product is recorded as T. The number of cycles that elapse when the fluorescence signal just reaches the set threshold is called the Ct value, then:

A₀＝T/2^CtA

B₀＝T/2^CtB

RQ＝A₀/B₀＝(T/2^CtA)/(T/2^CtB)＝(1/2^CtA)/(1/2^CtB)＝2^CtB/2^CtA＝2^CtB-^CtA＝2^-(CtA-CtB)

in the case of a diploid species, the selected reference gene is single-copy on the genome, and an RQ value of 0.5 indicates that the foreign gene of the sample to be tested is single-copy on the genome, and an RQ value of 1 indicates double-copy. The transgenic plant, rice and corn are both diploid, and the reference gene is single copy on the genome.

The quantitative PCR of the invention adopts a 384-well plate module and a 10 mul amplification system, each gene is doubly repeated, the amplification is completed in about 30 cycles, and the method is accurate and efficient. Using the method of the present invention, each person can test 1000 samples per day.

The method is simple and feasible, can detect and screen the transgenic plant containing the low copy target gene in high flux, and provides an identification basis for efficient large-scale commercial production of high-quality transgenic plants.

Compared with the traditional copy number identification method, the method has the advantages of simplicity, convenience, rapidness and accuracy. The method does not need to draw a standard curve, only needs to calculate the RQ value, adopts the description steps of the invention, and simplifies the whole process; by adopting the fluorescent quantitative PCR technology, rice only needs 28 reaction cycles, corn only needs 32 cycles, and the quantitative reaction speed is obviously improved; the copy number result obtained by the method is further verified by Southern hybridization identification.

The method provided by the invention can be used for stably detecting the copy number of the inserted genes of various transgenic crops in a large scale with high flux, can establish a whole set of high-efficiency and reliable transgenic crop screening industrial standard, has high accuracy and universality, and is particularly suitable for biotechnological enterprises and molecular breeding units for detecting transgenic samples in a large scale.

Drawings

FIG. 1 is a graph showing the detection of the amplification efficiency of a rice reference gene Actin7 and a foreign gene Epsps-CP4 in example 1 of the present invention.

FIG. 2 is a graph showing the examination of the amplification efficiency of maize reference gene Ivr1 and exogenous gene GFP in example 2 of the present invention.

FIG. 3 is a Southern hybridization assay of the exogenous gene Epsps-CP4 of rice in example 1 of the present invention; wherein, M: MarkerIII molecular weight standard; 1-7: the transgenic plants prepared by the invention are numbered; n: and (5) negative control.

FIG. 4 is a Southern hybridization assay of exogenous gene GFP in maize in example 2 of the present invention; wherein, M: MarkerIII molecular weight standard; 1-7: the transgenic plants prepared by the invention are numbered; n: and (5) negative control.

Detailed Description

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. Unless otherwise indicated, the examples follow conventional experimental conditions, such as the Molecular cloning handbook, Sambrook et al (Sambrook J & Russell DW, Molecular cloning: a laboratory manual,2001), or the conditions suggested by the manufacturer's instructions.

Example 1 identification of copy number of target Gene in transgenic Rice and screening of Low copy plants

High-flux extraction of rice genome DNA

Transgenic rice (containing the target gene Epsps-CP 4) in this example was obtained from China seed group, Inc.

1. Respectively putting about 0.5g of young leaves of the transgenic plants into 96-hole sampling tubes, and storing at-70 ℃ for later use;

2. adding 2 steel balls into each sampling tube, then adding 300 ul of 2 xCTAB DNA extraction buffer solution (see the reagent preparation method), tightly covering the tube cap, placing the whole plate sample on an upper sample grinder for sampling at 1400rpm for 90 s;

3. checking the crushing condition of the sample, placing the crushed sample in a water bath kettle or an oven, and carrying out water bath for 30-60m at 65 ℃;

4. taking down the tube cover, adding 300 mul chloroform into each tube of sample, tightly covering the sampling tube cover, and shaking for 10min at 200rpm on a decoloring shaker;

5. centrifuging at 4000rpm and 4 deg.C for 5 min;

6. preparing a 96-well PCR plate, adding 70 mul of isopropanol, carefully taking 70 mul of supernatant from a centrifuged sampling tube, adding the supernatant into the PCR plate, slightly sucking and beating the supernatant for 2 to 3 times by a gun head, standing the supernatant for more than 30min at the temperature of minus 20 ℃, or precipitating the supernatant overnight;

7. centrifuging at 4000rpm and 4 deg.C for 30 min;

8. carefully discarding the supernatant, blotting the residual liquid on the surface of the 96-well PCR plate by absorbent paper, and adding 50 μ l of 75% ethanol;

9. centrifuging at 4000rpm and 4 deg.C for 20-30min, removing supernatant, and air drying;

10. add 70. mu. lddH to each well sample₂O dissolves the DNA and is stored at-20 ℃.

Second, screening the primers of the target gene and the reference gene

In the operation of detecting copy number by using fluorescence quantitative PCR, one or two internal reference genes are selected as internal standard genes of species to carry out normalized analysis on quantitative PCR data. In this example, the rice Actin7 gene was selected as an internal reference gene. The copy number of the target gene is finally analyzed by comparison with an internal reference gene of known copy number within the species.

1. Primer design

(1) The length of the PCR product should be between 80-250bp, preferably 100-150 bp.

(2) The length of the primer is usually 18-27bp, and the annealing temperature is consistent.

(3) The GC content of the primer is 40-60% (preferably 45-55%). The sequence should avoid the occurrence of consecutive repeats of bases, and individual portions avoid GC rich or AT rich (especially 3' end).

(4) Complementary sequences of more than 3 bases are avoided inside the primers or between two primers.

(5) Using a BLAST search, primer specificity was confirmed, avoiding the presence of primer dimers or non-specific amplification.

2. Confirmation of reaction Performance

The amplification efficiency E value of the primer is between 0.8 and 1.2, and the coefficient R is determined²Between 0.999 and 1. The fluorescent quantitative PCR can obtain good quantitative results within 30 cycles, and no non-specific product amplification and primer dimer generation exist within 40 cycles.

(1) Standard curve analysis of amplification efficiency of target gene and standardized gene

Determining PCR amplification efficiency by a concentration gradient dilution method, and diluting the standard substance DNA by 5-6 gradients. Ideal standard amplification curve: the base line is flat, the negative is a horizontal line, the index area is obvious, the gradient is large, the platform areas are converged together, and the linear range is wide. The amplification efficiency of the target gene is consistent with that of the standardized gene. Efficiency detection of rice internal reference gene Actin7 and exogenous gene Epsps-CP4 is shown in figure 1, Efficiency reaches 0.999, namely the Efficiency is 99.9%, and the quantitative primer is proved to meet the detection requirement and have consistent amplification Efficiency of the internal reference gene and the exogenous gene.

(2) Analysis of melting curves

The single dissolution curve of the product appears only as a single peak.

Third, quantitative determination of copy number

1. Preparation of quantitative reaction System

In this example, a fluorescent quantitative PCR reaction was carried out using FastStartUniversal SYBR Green Master [ ROX ] (Cat. No.04913914001) produced by Roche. The product requires that the initial genome content in a single quantitative reaction is not higher than 250ng, and the final system concentration of the single quantitative primer is 400nM at 100. The reaction was performed in 384-well PCR plates.

The reaction system was prepared as shown in Table 1.

TABLE 1 fluorescent quantitative PCR reaction System

Composition (I)	Volume of	Final concentration
			FastStart Universal SYBR Green Master[ROX]	5μl	1×
Forward and reverse primers	1.25. mu.l each	300nM
			Template DNA	2.5μl
Total volume	10μl

2. On-machine operation of quantitative reaction

The quantitative analyzer used in this example was a 7900HT Fastreal-Time PCR System from Applied Biosystems. And selecting a 384-hole quantitative plate and an RQ detection mode, and inputting reference gene information and gene information to be detected. The fluorescent quantitative PCR reaction was run according to the procedure in table 2.

TABLE 2 fluorescent quantitative PCR reaction procedure

3. Data analysis of quantitative results

After the fluorescent quantitative PCR reaction is finished, the instrument generates related data, downloads the average Ct value of the reference gene and the target gene, and calculates the RQ detection value of the corresponding sample according to a formula. If the RQ value is 1, the relative copy number of the target gene is consistent with that of the internal reference gene, if the RQ value is more than 1, the relative copy number of the target gene is more than that of the internal reference gene, otherwise, if the RQ value is less than 1, the relative copy number of the target gene is less than that of the internal reference gene. At this time, the relative copy number of the sample to be tested can be determined based on the relative copy number of the selected reference gene.

In the embodiment, the quantitative PCR adopts a 384-well plate module and a 10-microliter amplification system, each gene is doubly repeated, the amplification cycle number is selected according to needs, the amplification is generally completed within 28-32 cycles, and the method is accurate and efficient.

The results of detecting the copy number of the rice Epsps-CP4 gene are shown in Table 3.

TABLE 3 copy number detection results of rice Epsps-CP4 gene

Fourth, Southern hybridization detection of rice

In high throughput detection and calculation, there is often a deviation between the actual calculated RQ value and its theoretical value. To more accurately determine the copy number of the test sample, the actual measured RQ value needs to be compartmentalized.

Several samples of different size RQ were selected for Southern hybridization in the present invention. Southern hybridization Probe Labeling and hybridization and development Using the DIG High Primer DNA Labeling and Detection Starterkit I from Roche. For diploid material with a single copy internal reference in this example, the target gene with RQ in the interval 0.1-0.6 is single copy. The specific experimental method is as follows:

1. CTAB method for extracting total genome DNA of transformed plant

(1) Taking 0.5-1g of leaves from each plant, putting the leaves into a precooled mortar, adding liquid nitrogen to quickly grind the leaves into powder, and pouring the powder into a 2mL centrifuge tube. Adding 700 μ l of 1.5% CTAB extractive solution preheated to 65 deg.C, shaking, and keeping in 65 deg.C water bath for 30-60min, while shaking for several times;

(2) cooling at room temperature, adding 700 μ l chloroform, shaking, reversing, shaking gently for 10min, and centrifuging at 8000rpm at room temperature for 10 min;

(3) transferring the supernatant to another centrifuge tube, adding equal volume of precipitation solution (isopropanol), precipitating at-20 deg.C for 30min, and centrifuging at room temperature at 8000rpm for 10 min;

(4) rinsed 2-3 times with 700. mu.l of 75% ethanol, air-dried, and dissolved in 50. mu.l of TE, and stored at-20 ℃ for further use.

2. Enzyme digestion of genomic DNA

Selecting proper restriction enzyme to perform enzyme digestion on the total genome DNA of the transformed plant, wherein the enzyme digestion reaction system is as follows:

and (3) uniformly mixing, carrying out enzyme digestion at 37 ℃ for about 10-18h, carrying out pre-electrophoresis on 5 mu l of enzyme digestion product after enzyme digestion, and detecting the enzyme digestion effect. Finally, the total DNA after digestion is subjected to low voltage (30-40V) electrophoresis overnight in a 1% agarose gel.

3. Rotary film

Trimming the gel, cutting off the lower right corner as a mark, soaking in 0.25mol/L HCl until bromophenol blue turns yellow, and washing with distilled water twice; denaturing in alkali denaturing solution (1.5M NaCl,0.5M NaOH) for 45min, and rinsing with deionized water; rinsing in neutralization solution (1M Tris-HCl (pH7.4),1.5M NaCl) for 30min, replacing the neutralization solution, and rinsing for 15 min; placing on a well-built membrane transferring platform, using 10 XSSC as a membrane transferring solution, rinsing a Hybond-N + nylon membrane on the liquid surface of deionized water until the membrane is completely wet, and immersing the membrane in a transfer buffer solution; the DNA on the gel is transferred to a nylon membrane by capillary transfer for 16-20h using a 10 XSSC solution. After the transfer is finished, the nylon film is simply rinsed by 2 XSSC solution, crosslinked for 1min on an ultraviolet crosslinking instrument, dried at room temperature, wrapped by preservative film and stored at 4 ℃ for later use.

4. Probe synthesis and hybridization and development

Probe Labeling and hybridization and development were performed using the DIG High Primer DNA Labeling and detection Starter Kit I from Roche, according to the Kit instructions.

5. Hybridization of

(1) Heating the hybridization solution DIG Easy Hyb (10ml/100cm2), and prehybridizing for 30 minutes at 42 ℃ in a hybridization oven;

(2) probe (25ng/ml) was denatured at 95 ℃ and placed on ice after 5 minutes;

(3) the denatured probe was added to a previously heated DIG Easy Hyb (3.5ml/100 cm)²) Mixing uniformly;

(4) pouring out the pre-hybridization solution, and adding a denatured probe;

(5) hybridizing at 42 deg.C for 4 h-O/N.

6. Washing membrane

The hybridization solution was decanted and then washed twice with 2 XSSC, 0.1% SDS at room temperature for 5 minutes each; finally, the column was washed twice with 0.5 XSSC, 0.1% SDS at 65 ℃ for 15 minutes each.

7. Color development

And (5) detecting with the probe. The results are shown in FIG. 3.

The results of the rice Southern hybridization and Real-time PCR identification control are shown in Table 4.

TABLE 4 copy number detection results of rice Epsps-CP4 Gene control

When the RQ value is 0.5 +/-0.1, the copy number of the target gene of the transgenic rice is 1, namely, the copy number is single, and the accuracy of identifying the copy number by Real-time PCR is proved.

The transgenic rice with the RQ value of 0.1-1.5 is selected for breeding, the corresponding range of the copy number is 1-3, the transgenic rice belongs to a low-copy plant, and the transgenic rice is suitable for breeding application.

Example 2 identification of target Gene copy number in transgenic maize and screening for Low copy plants

The transgenic maize (containing the target gene GFP gene) in this example was obtained from China seed group, Inc.

The method comprises the following basic steps: high-flux extraction of rice genome DNA, screening of target gene and standardized gene primer, preparation of quantitative reaction system, operation of quantitative reaction on computer and data analysis, and Southern hybridization detection of transformed plant exogenous gene. In this example, the procedure was substantially the same as in example 1, except that the number of cycles of the reaction program on the quantitative reaction was 32.

Screening of corn target gene and standardized gene primer

The Efficiency detection of the corn internal reference gene Ivr1 and the exogenous gene Epsps-CP4 is shown in figure 2, the Efficiency reaches 0.999, namely the Efficiency is 99.9 percent, and the quantitative primer is proved to meet the detection requirement and have consistent amplification Efficiency of the internal reference gene and the exogenous gene.

Second, data analysis of quantitative results

In the embodiment, the fluorescence quantitative PCR adopts a 384-well plate module and a 10-microliter amplification system, each gene is subjected to double repetition, amplification is completed in 32 cycles, and the method is accurate and efficient.

The copy number assay results for the maize GFP gene are shown in table 5.

TABLE 5 copy number assay results for maize GFP gene

Third, Southern hybridization detection of maize

The Southern hybridization assay for maize is shown in FIG. 4.

The results of the Southern hybridization and Real-time PCR identification controls of maize are shown in Table 6.

TABLE 6 comparison of copy number detection results for maize GFP gene

Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Claims

1. A method for high-throughput identification of copy number of a target gene in a transgenic plant and screening for low-copy plants, comprising the steps of:

1) extracting the genome DNA of the transgenic plant to be detected;

4) screening low-copy transgenic plants;

5) correcting the obtained copy number by Southern hybridization;

wherein the transgenic plant is transgenic rice, the target gene is a glyphosate-resistant gene Epsps-CP4, and the reference gene is a rice Actin7 gene; the target gene fluorescent quantitative PCR primer comprises a forward primer and a reverse primer, the nucleotide sequences of the forward primer and the reverse primer are respectively shown as SEQ ID No.5 and SEQ ID No. 6, the reference gene fluorescent quantitative PCR primer comprises a forward primer and a reverse primer, and the nucleotide sequences of the forward primer and the reverse primer are respectively shown as SEQ ID No.9 and SEQ ID No. 10;

when the fluorescent quantitative PCR amplification is carried out on the transgenic rice, the reaction system is as follows: FastStart Universal SYBRGreen Master [ ROX ] 5. mu.l, forward and reverse primers of 300nM each 1.25. mu.l, template DNA 2.5. mu.l; the reaction procedure is as follows: 10min at 95 ℃; 28 cycles of 95 ℃ 10sec, 60 ℃ 30 sec;

the threshold value of the fluorescence signal in the step 3) is 0.2, and the RQ value is 0.1-1.5.

2. The method according to claim 1, wherein the DNA template used for screening the fluorescent quantitative PCR primers of the target gene and the fluorescent quantitative PCR primers of the reference gene in the step 2) is the genomic DNA of the transgenic plant to be tested.