CN103160612A - Improved method for detecting and identifying transgenic wheat with high sensitivity and high efficiency - Google Patents

Abstract

The invention discloses an improved method for detecting and identifying wheat transgenes with high sensitivity and high efficiency. The method comprises the following steps: 1) pretreating with N ₂ O gas and enzymolyzing the cells in the root tip meristematic zone of wheat Obtain metaphase specimens with clear images and well-dispersed chromosomes; 2) use the nick translation method to label the fluorescent probes for the target fragment (for example, the transgene fragment carried by the transgene vector); 3) use the transgene constructed in step 2) The fluorescent probe pair of the target fragment of the carrier is carried out by using step 1) fluorescence in situ hybridization of the metaphase cleavage phase chromosome of transgenic wheat; and 4) analyzing the result of fluorescence in situ hybridization obtained in step 3), and comparing with the control material to determine the transgenic wheat to be detected The distribution of fluorescent signals in . The method provided by the invention can not only be applied to the chromosome location of exogenous transgenes in wheat, and the sensitivity can reach 2-3 kb, but also can be applied to the identification of transgene signals of other crops.

Description

An improved highly sensitive and efficient method for detection and identification of transgenic wheat

technical field

The invention relates to a highly sensitive and efficient method for detecting transgenic wheat chromosomes, and also relates to a fast and efficient method for obtaining metaphase plant chromosomes in good shape, belonging to the research fields of cell biology and molecular biology. the

Background technique

Wheat is one of the most important food crops in the world, which is closely related to social and economic development, food security supply and human nutrition and health. The narrow genetic base has become a key factor restricting the further improvement of important agronomic traits such as yield level, resistance and adaptability of crop varieties (Ye Xingguo et al. 2011). Genetic engineering breeding, as the main supplementary method of hybrid breeding, has achieved great success in crops such as soybeans, corn, cotton, and rapeseed. Genetic engineering has endowed these crops with some excellent traits such as herbicide resistance and insect resistance that they do not have. . Under the support of the national "863" plan and the "Special Project of Transgenic Plant Research and Industrialization" and other scientific and technological projects, my country's transgenic wheat research has made great progress, especially theoretically. With the continuous development of molecular biology and plant genetic engineering, more and more breeders have begun to use transgenic technology to obtain new germplasm and new varieties that are difficult to obtain by conventional breeding techniques. the

The commonly used methods to detect whether the transgene occurs are: PCR method, Southern hybridization and fluorescence in situ hybridization (FISH). PCR can detect whether the target gene is integrated on the chromosome of the recipient cell. The biggest feature of the PCR reaction is its large amplification ability and high sensitivity. cause false positives. Simultaneous detection of multiple insertions is difficult. The most reliable methods to detect exogenous gene integration in plant chromosomes are Southern hybridization and in situ hybridization. Southern hybridization can detect the copy number and insertion mode of exogenous gene insertion. It is a relatively accurate analysis. Southern method has high accuracy and strong specificity, but it has the disadvantage of time-consuming and labor-intensive. In addition, because the Southern method does not undergo target fragment amplification (PCR), generally 10-30 μg of DNA is required for each electrophoresis channel, and a relatively large amount of plant material is required to extract DNA in actual operation, and the callus of transgenic plants Tissues are generally weak after being screened and redifferentiated under sterile conditions, so it is not suitable to take a large number of samples. If the gene recombination occurs when the foreign gene is inserted, resulting in the loss of the restriction enzyme cutting site, the Southern method cannot detect it. These factors restrict the application of Southern method in detecting the copy number of exogenous gene in transgenic plants of T0 generation. the

Fluorescence in situ hybridization (FISH) is a new technology in the field of biology in the 20th century. It is a technology that uses labeled DNA probes to directly locate specific target DNA sequences on chromosomes, interphase nuclei and DNA fibers. It has a high Sensitivity and specificity have become an important means of cell biology and molecular biology research, and are widely used in various aspects of botany research, such as chromosome physical mapping of plant genes (Miller et al.1983; Mukai et al .1990), the identification of parental chromosomes in hybrids, the detection of foreign chromosomes or chromosome fragments (Schwarzacher et al.1989), the discussion of species evolution and kinship (Mukai et al.1993), the positioning of foreign genes in transgenic materials ( Kenton et al.1995) and so on. It has obvious advantages especially in detecting the transgene and its expression product. It can not only detect whether the exogenous gene exists but also locate the exogenous gene directly on the corresponding position of the chromosome. However, because the transformed gene is generally a small single copy Therefore, the technical requirements for FISH analysis of transgenic plants are relatively high and the difficulty is the greatest. Good chromosome preparation and highly sensitive FISH procedures are required (Harwood et al. 2004). the

In the plant FISH experiment, the traditional method is to take the vigorously dividing plant root tips, use low temperature treatment or chemical agents for pretreatment, so that the chromosomes can be properly condensed and dispersed, and then pressed into pieces. The commonly used method is to fix the pretreated material in Carnot's fixative solution, dissociate it with HCl or a mixture of vitaminase and pectinase, remove part of the cell wall and cytoplasm, and then press it with 45% acetic acid , liquid nitrogen freezing to remove the cover slip, but the success rate of this method is low, mainly because the pressing force is not easy to master, the chromosomes are difficult to completely disperse, and it is easy to overlap, deform, break, etc. The coverage of chromosomes by cytoplasm and cell wall increases the signal-to-noise ratio of in situ hybridization. At the same time, it may be easy to remove the chromosomes due to the cleanliness of the slides. The whole process is time-consuming, laborious, and technically difficult, making it difficult to obtain good chromosome preparations. Due to the high degree of chromatin condensation and thick cytoplasm, the resolution is generally 5-10Mb (Pedersen and Linde-Laursen 1995), and the sensitivity is about 10kb. For most laboratories, the detection of genes less than 10kb The rate is very low (Rong Hongying et al.2007). Jin Weiwei et al. (Jin Weiwei et al.2001) utilized meiotic metaphase chromosome production to locate the foreign gene of about 4.8 kb transferred into rice, and the detection rate was 11.7-30.4%. The degree of condensation of plant meiotic pachytene chromosomes is much lower than that of metaphase chromosomes, and its length is 7 to 50 times larger than that of the corresponding metaphase chromosomes, which makes the probe DNA easy to enter and thus improves the detection sensitivity. However, it is very difficult to select a suitable pollen mother cell for the preparation of pachytene chromosomes, and the pachytene chromosomes are very long, making it difficult to trace and identify individual chromosomes. These unfavorable factors limit the application of pachytene FISH. the

Wheat is an allohexaploid, the number of chromosomes is 2n=42, the genome is huge, and the repetitive sequences are many and complex. It is difficult to track and identify foreign chromosomes. We refer to the method of Akio Kato et al. (Kato et al.2004) to modify , according to different materials, process different time, get the chromosome in the best state, analyze and precisely locate the transgene. the

Contents of the invention

One object of the present invention is to provide a highly sensitive method for detecting chromosomes of transgenic wheat, wherein the sensitivity can reach 2-3kb. the

In a first aspect, the present invention provides a highly sensitive method for detecting the chromosome of transgenic wheat, said method comprising the steps of:

1) Pretreatment with N ₂ O gas and enzymatic hydrolysis of the tissues with vigorous cell division in the root apical meristem of transgenic wheat and control materials to obtain metaphase specimens with clear images and well-dispersed chromosomes;

2) Use the nick translation method to label the fluorescent probe of the target fragment to be detected;

3) using the fluorescent probe of the target fragment to be detected in step 2) to perform fluorescence in situ hybridization on the metaphase chromosome of transgenic wheat using step 1);

4) Analyzing the fluorescence in situ hybridization results obtained in step 3), and comparing them with the control materials to determine the distribution of fluorescent signals in the transgenic wheat to be detected. the

The most critical step in the above steps is step 1) Whether a metaphase sample with clear images and well-dispersed chromosomes can be obtained will affect the sensitivity of the detection of transgenes. As mentioned above, step 1) pretreatment with N ₂ O (commonly known as laughing gas) and enzymatic treatment of transgenic wheat and control material root apical meristem cells, including the following steps: (a) N ₂ O gas pretreatment: take Appropriate root tips are placed in a wetted centrifuge tube and placed in a N ₂ O gas chamber for 0.5-5 hours. The pressure of N ₂ O is 10ATM (1.01Mpa); (b) pre-treated with N ₂ O gas Root tip fixation; (c) enzymatic treatment: wash the fixed root tip with water, cut off the root tip meristem, and treat with pectinase and cellulase for 30min-2h.

Specifically, the operation method of laughing gas pretreatment is as follows:

Cut off suitable wheat root tips, put them in a wet centrifuge tube, treat with nitrous oxide (N ₂ O) for 30 minutes to 5 hours, and the pressure of N ₂ O is 10ATM (1.01Mpa), and the N ₂ O pretreated The root tips were fixed for the next step of enzymatic digestion.

Depending on the plant species, the genome size is different, and the treatment time of nitrous oxide is different. For example, the root tips or shoot tips of plants such as peanuts and soybeans are treated for 30-40 minutes, and the root tips or shoot tips of plants such as wheat and corn are treated for 2 hours. The root tip or shoot tip treatment time of such a large genome plant can reach 4-5 hours. If the processing time is too long or too short, it will affect the preparation of chromosomes. For example, if the root tip or shoot tip of wheat is treated for 1 hour, the chromosomes will be too long, and the chromosomes in one cell will overlap, which will affect the subsequent transgenic identification and other tests. Highly condensed, even some chromosomes will appear filamentous at the edge, which will also affect the later experimental part. Those skilled in the art may need to select an appropriate N ₂ O treatment time according to factors such as the size of the plant genome and the pressure of N ₂ O.

Among them, the operation method of enzymatically hydrolyzing the root tip or shoot tip is as follows:

Wash the root tip or shoot tip that has been pretreated and fixed with water 3 times to remove residual acetic acid and _alcohol , use filter paper to dry the water on the root tip or shoot tip slightly, and remove the root tip or shoot tip The meristem (approximately 1-2 mm) was excised and placed in 20 μL of a mixture of pectinase and cellulase. Depending on the size of the plant genome, the enzymatic hydrolysis time is also different, such as 20-35 minutes for the root or shoot tips of plants such as peanuts and soybeans, 48 minutes for the enzymatic hydrolysis of the root or shoot tips of plants such as wheat and corn, and The enzymatic hydrolysis time of the root tip or shoot tip of a plant with a large genome like lily can reach 1-2 hours. The grasp of the enzymatic hydrolysis time will also directly affect the preparation of chromosomes and the identification of later chromosomes. For example, the enzymatic hydrolysis of wheat chromosomes takes 40 minutes. If the enzymatic hydrolysis time is short, the chromosomes will appear elongated and irregular. If it takes 55 minutes or even longer, the chromosomes in the cells will have different shapes, and most of the chromosomes cannot be completely separated, so no good chromosomes can be obtained, which will bring a lot of inconvenience to the later identification work. Those skilled in the art may need to select an appropriate enzymatic hydrolysis time according to factors such as the size of the plant genome.

In the second aspect, the present invention also provides a method for quickly and efficiently obtaining metaphase plant chromosomes in good shape, comprising the following steps: (a) N ₂ O gas pretreatment: taking a suitable Root tip or shoot tip, placed in a wet centrifuge tube, placed in N ₂ O gas chamber for 0.5-5 hours, the pressure of N ₂ O is 10ATM (1.01Mpa); (b) Pretreatment with N ₂ O gas (c) enzymatic treatment: wash the fixed root tip or shoot tip with water, cut off the root tip or shoot tip meristem, and treat with pectinase and cellulase for 30min-2h .

Specifically, the method for quickly and efficiently obtaining the plant chromosomes of the metaphase cleavage phase with good shape comprises the following steps:

1) N ₂ O treatment of the root tip or shoot tip meristematic zone of the plant

Put the appropriate root tip or shoot tip in a wet centrifuge tube, and treat it in an N2O air chamber for 1-3 hours, with a pressure of 10ATM (1.01Mpa);

2) Root tip or shoot tip fixation

Fix with 90% acetic acid for 5-10 minutes (on ice, no more than 1 hour), and wash with distilled water twice. the

3) Enzymolysis

Cut off the root tip or shoot tip meristem, put it in the medium of pectinase and cellulase, and put it in a 37°C water bath. the

4) Drop tablets

Wash off the enzymatic solution with 70% alcohol, mash the root tip with a dissecting needle, centrifuge to drain the alcohol, and add glacial acetic acid to suspend. the

5) Microscopic examination

Put the clean glass slides in a humid box, drop 6-7μl root tip or shoot tip cell suspension on each slide, cover the box lid, and check under the microscope after 5 minutes. the

The plants suitable for the chromosome preparation method include wheat (2n=42), brachypodium (2n=10, 20, 30), kelp (n=31), soybean (2n=40), tomato (2n=24), Cotton (2n=52), pear (2n=34), lily (2n=24, 36, 60), sorghum (2n=20) and other dicots and monocots. the

In a specific embodiment of the present invention, the present invention provides a kind of highly sensitive and efficient detection and the method for identification wheat transgene, it comprises the following steps:

1) Utilize the method for the second aspect of the present invention to prepare the mitosis metaphase of transgenic wheat and control material;

2) fluorescently labeling the target gene contained in the transgenic wheat to make a fluorescent probe;

3) using the fluorescent probe labeled in step 2) to carry out fluorescence in situ hybridization on metaphase chromosomes of transgenic wheat; and

4) Analyzing the fluorescence in situ hybridization result obtained in step 3) to determine the distribution of the transgenic fluorescent signal in the plant to be tested. the

In a specific embodiment of the present invention, the transgenic plants to be detected are TaDREB3 transgenic wheat and the control material Jimai 19. the

The method of the present invention is suitable for preparing metaphase cell chromosomes of different plants. In particular, the method of the present invention successfully obtains plant chromosomes quickly, efficiently, simply and conveniently for the first time, which overcomes the problem of unsatisfactory detection results of plant autosomes in the prior art, and is a technological breakthrough. Specifically, the method of the present invention has the following technical breakthroughs: 1) the improvement of chromosome preparation technology, the method of the present invention has adopted laughing gas (N ₂ O) to root tip or shoot tip pretreatment, rather than traditional The main advantages of chemical reagents such as colchicine, octahydroxyquinoline, and p-dichlorobenzene are that they are non-toxic and have good dispersion effects. At the same time, the influence of the treatment time of laughing gas (N ₂ O) on the subsequent chromosome preparation is also mastered. ; 2) Enzyme digestion treatment of root tip or shoot apex meristematic zone cells, mastered the influence of different enzyme digestion treatment time on subsequent chromosome preparation, which is mainly related to genome size; 3) Fluorescent probe construction and fluorescence in situ Compared with similar methods, hybridization technology also reflects the characteristics of fastness, high efficiency and strong signal;

Although one or several of the above technologies have been applied in related research, there are no relevant research reports on the comprehensive application of these technologies to form a complete experimental program and their application to highly sensitive and efficient detection of wheat transgenics. For the first time, the present invention successfully detects transgenic wheat with high sensitivity using this set of research schemes, which overcomes the problem of unsatisfactory detection of plant autosomes in the prior art. It is a technological breakthrough and reflects the advanced nature of the research method , innovation and practicality. the

In summary, the present invention provides the following items:

1. A method for highly sensitive and efficient detection and identification of transgenic wheat, comprising the steps of:

1) Pretreatment with N ₂ O gas and enzymatic hydrolysis of the tissues with vigorous cell division in the root apical meristem of transgenic wheat and control materials to obtain metaphase specimens with clear images and well-dispersed chromosomes;

2) Use the nick translation method to label the fluorescent probe of the target fragment to be detected;

3) using the fluorescent probe of the target fragment to be detected in step 2) to perform fluorescence in situ hybridization on the metaphase chromosome of transgenic wheat using step 1);

4) Analyzing the fluorescence in situ hybridization results obtained in step 3), and comparing them with the control materials to determine the distribution of fluorescent signals in the transgenic wheat to be detected. the

2. The method described in item 1, wherein step 1) comprises the following steps: (a) N ₂ O gas pretreatment: get a suitable wheat root tip, put it in a wetted centrifuge tube, put N ₂ O gas Treat in the chamber for 0.5-5 hours, the pressure of N ₂ O is 10ATM (1.01Mpa); (b) fix the root tip pretreated by N ₂ O gas; (c) enzymatic treatment: wash the fixed root tip with water , cut off the root tip meristem, and treated with pectinase and cellulase for 30min-2h.

3. The method described in item 1, wherein step 2) separates the target fragment in the transgenic vector by enzyme digestion, and carries out fluorescent probe labeling to the target gene amplification product by means of the nick translation method, and the target fragment is carried by the transgenic vector Gene fragment. the

4. A method for obtaining the plant chromosome of the metaphase cleavage phase with good shape, said method may further comprise the steps:

(a) N ₂ O gas pretreatment: take the suitable root tip or bud tip of the plant, put it in a wet centrifuge tube, put it into a N ₂ O gas chamber and process it for 0.5-5 hours, and the pressure of N ₂ O is 10ATM (1.01Mpa); (b) fix the root tip or shoot tip pretreated by N ₂ O gas; (c) enzymatic treatment: wash the fixed root tip or shoot tip with water, cut off the root tip or shoot tip Meristem, treated with pectinase and cellulase for 30min-2h.

5. The method of item 4, wherein the plant comprises a monocot or a dicot. the

6. The method of item 4, wherein the plant is selected from wheat, brachypodium, kelp, soybean, tomato, cotton, pear, lily or sorghum. the

Description of drawings

From the following detailed description in conjunction with the accompanying drawings, the above-mentioned features and advantages of the present invention will be more apparent, wherein:

Figure 1 shows the chromosomal location of the wheat transgenic signal:

Figure 1A, Metaphase of control material Jimai 19 cells, 42 wheat chromosomes in the cell, which is the negative control of this experiment; Figure 1B: Metaphase of Ta DREB3 transgenic wheat cells, the arrow indicates the transgene signal, located at the end of the homologous chromosome In the section, the length of the target fragment used by the probe is 1264bp, and its sensitivity can reach about 2kb, and the green fluorescent signal can be clearly seen under the microscope. the

Figure 2 shows the metaphase chromosomes of different plants, A, soybean (2n=40), B, cotton (2n=52), C, rice (2n=24), D, kelp sporophyte (2n=62), E, sorghum (2n=20), F, Brachypodium (2n=10), G, Lily (2n=36), H, Pear (2n=34), I, Tomato (2n=24). the

Figure 3 shows metaphase chromosomes at different times of nitrous oxide treatment:

Figure 3A, the chromosomes of metaphase cells prepared under the condition of common wheat root tip treatment for 1 hour, the chromosomes are too long, and the chromosomes in one cell will overlap. Figure 3B, the chromosomes of metaphase cells prepared under the condition of common wheat root tip treatment for 2 hours, the chromosomes are clearly visible and clearly identified. Figure 3C, pear root tip cells treated for 1:30h, the chromosomes will be highly condensed, and some chromosomes will even appear filamentous at the edge. the

Figure 4 shows metaphase chromosomes at different enzymatic hydrolysis times:

Figure 4A, the enzymatic hydrolysis of wheat chromosomes is 40m, and the enzymatic hydrolysis time is short, the chromosomes will appear elongated and irregular. Figure 4B, when the enzymatic hydrolysis time of wheat root tip cells reaches 55 m or even longer, the chromosomes in the cells will have different shapes, and most of the chromosomes overlap and cannot be fully identified. Figure 4C, pear root tip cells were enzymatically hydrolyzed for 30m, and the time was short, and the chromosomes would appear filamentous at the edge, which could not be fully identified. the

Detailed ways

The present invention is described in detail below with reference to Examples and drawings. Those of ordinary skill in the art can understand that the following examples are for the purpose of illustration and should not be construed as limiting the present invention in any way. The protection scope of the present invention is defined by the appended claims. the

In addition, those skilled in the art should also understand that unless otherwise specified, the reagents used in the following examples are commercially available reagents of analytical grade. the

Example 1. Identification of Ta DREB3 transgenic wheat and control material Jimai 19 fluorescence in situ hybridization

1. Preparation of transgenic wheat mitotic metaphase

The Ta DREB3 transgenic wheat and the reference material Jimai 19 were provided by Ma Youzhi Laboratory, Chinese Academy of Agricultural Sciences (XiaoYan H, Ming C, HuiJun X, ShiQing G, XianGuo C, LianCheng L, LiPu D, XinGuo Y, YouZhi M( 2005) Obtaining of transgenic wheats with GH-DREB gene and their physiological index analysis on drought tolerance. Southwest China Journal of Agricultural Sciences 18:616-620). the

1) N ₂ O treatment of root tips

When the root tip grows to 2-3cm, cut it off, put it in a wet centrifuge tube, cover it and put it in a N2O air chamber for 1-3h, the pressure is 10ATM (1.01Mpa);

2) root tip fixation

Fix with 90% acetic acid for 5-10 minutes (no more than 1 hour), and wash with distilled water twice. The fixed root tips can be transferred to 70% ethanol and stored at -20°C for many years;

3) Enzymolysis

Use filter paper to blot the water on the root tip slightly dry, cut off the root tip meristem, and put in 20 μL of a mixture of pectinase and cellulase (enzymolysis solution preparation: put a plastic petri dish on a balance, Add 9.7 g of 1 × citric acid buffer (10 mM sodium citrate + 10 mM EDTA, pH 5.5), take it out and put it on ice, weigh 0.1 g of pectinase Y-23 (purchased from Japan Yakult Company, 1% w/w) and 0.2g cellulase Onozuka R-10 (purchased from Japan Yakult Company, 2% w/w) were added to the buffer solution, blown and sucked with a pipette tip to aid dissolution, and the enzyme was dissolved and then packed with a dispenser into a 0.5ml thin-walled PCR tube, stored at -20°C), in a 37°C water bath for 40-60 minutes,

4) Drop tablets

Wash twice with 70% alcohol, leave a little alcohol for the second wash, put about 200ul in the centrifuge tube, mash the root tip with a dissecting needle, centrifuge at low speed (<2000 rpm) for about 10 seconds, drain the alcohol, add Glacial acetic acid (20-40 μl per root tip, depending on the size of the root tip of the plant) was used to suspend the root tip cells. the

5) Microscopic examination

Put the clean glass slides in a humid box, drop 6-7μl root tip cell suspension on each slide, cover the box lid, check under the microscope after 5 minutes, and keep the chromosome preparation specimens in the refrigerator for later use. the

2. Probe labeling of Ta DREB3 target gene

1) Add the following components to a centrifuge tube on ice

Ta DREB3 target gene DNA 2μg (200ng/μl) 10μl 10x Nick buffer 2.0μl Labeled-dNTP (1mM) 0.5μl Unlabeled dNTPs (2mM each, mixed) 2.0μl DNA polymerase I (10U/μl) 5.0μl DNase I (100mU/μl) 0.5μl

Among them, Ta DREB3 (SEQ ID No.1, GenBank: FJ560494.1) was excised from the transgenic vector by enzyme digestion, and the concentration was 200ng/μl. Vortex and incubate at 15°C for 2 hours. Place it in a metal bath (H2O3-PRO, China) at 15°C for 2h. (The labeled-dNTP is Alexa Fluor-488-dUTP (green light), purchased from Invitrogen; DNA polymerase I was purchased from Invitrogen, with a concentration of 10U/μL, which can be used directly, 10x Nick buffer: in 100ml buffer Contains Tris 6.05g, MgCl ₂ , 0.47g, adjusted to pH 7.8 with HCl);

2) Centrifuge at 13,000 rpm for 30-40 minutes, discard the supernatant, wash the precipitate once with 70% ethanol and absolute ethanol, and dry in the dark. the

3) Add 10 μl of 2×SSC, 1×TE buffer (pH 7.0) to make the final concentration of the probe 200ng/ul. the

3. Identification of Ta DREB3 transgenic wheat and control material Jimai 19 by fluorescence in situ hybridization using the probe of the labeled Ta DREB3 target gene

1) Prepare metaphase chromosome samples of Ta DREB3 transgenic wheat and control material Jimai 19 (from Ma Youzhi Laboratory, Chinese Academy of Agricultural Sciences) by the method of procedure 1. the

2) Put the specimen (prepared sheet) to be hybridized into an ultraviolet crosslinker (code-No. CL-1000, purchased from UVP Company, USA) for crosslinking at 0.125J/cm2. the

3) Dilute the probe with 2×SSC (0.3mol/L NaCl, 0.03mol/L sodium citrate), 1×TE buffer (10mmol/L Tris-HCl, 1mmol/L EDTA, pH 8.0), different probes The needle dilution ratio is different, generally 1:5-1:10 dilution. the

4) Put the preparation on ice, add 5-6 μL of hybridization solution, cover with a plastic cover glass, put it into a small iron basin (the iron basin is lined with absorbent paper, spray it with water), cover it with a plastic box, Put into boiling water for 5min, and then put into a preheated humid box at 55°C for overnight hybridization. the

5) Take the slide out of the wet box, put it in 2×SSC to make the cover glass slide off, dry the back of the slide with absorbent paper, and drop a drop of antifade agent containing DAPI (H-1200, purchased from the United States Vector Labs), cover with a 24×50mm cover glass, and use a fluorescent microscope (Olympus BX61) for microscopic examination and photography (Magnafier CCD camera) after a few minutes. the

4. Results and Discussion

The results of the study are shown in Figure 1. the

As shown in Figure 1A, the control material Jimai 19 cell metaphase, 42 wheat chromosomes in the cell can be clearly identified, the green channel is the probe signal, even if the exposure is strong, the green probe has no fluorescent signal, for this experiment negative control; as shown in Figure 1B: Ta DREB3 transgenic wheat cell metaphase, the arrow shows the transgene signal, located at the end of the homologous chromosome, the length of the target fragment used by the probe is 1264bp, and its sensitivity can reach about 2kb, green fluorescence The signal is clearly visible under the microscope and intuitively clear on the chromosome, confirming that it is a transgene-positive plant, which provides a basis for subsequent biological experiments. The biological experiment was repeated three times with consistent results. the

For the first time in this study, the target gene PCR amplification product of about 2 kb was used as a fluorescent probe to identify transgenic wheat, and the results showed that the signal was very clear, indicating that the positive plants of transgenic wheat were successfully detected and identified by the method adopted in the present invention. It only takes 3 days from sending seeds to the end of identification, which is not only efficient but also sensitive, and the sensitivity can reach 2-3kb. the

Embodiment 2. The metaphase chromosome preparation of different plants

1. Germination of seeds

(1) The seeds of various plants are respectively placed in petri dishes lined with moist filter paper, and germinated in an incubator at 21-23°C. the

2. Plant chromosome specimen preparation

1) With embodiment 1.1. When the root tip grows to 2-3cm, cut it off, put it in a humid centrifuge tube, and treat it with nitrous oxide for 30 minutes to 5 hours. Depending on the plant species, the genome size is different, and the time of nitrous oxide treatment is different, such as peanuts, soybeans, etc. Plant root tips are treated for 30-40 minutes, wheat, corn and other plant root tips are treated for 2 hours, and for plants with large genomes such as lilies, the treatment time can reach 4-5 hours. the

2) With embodiment 1.2. the

3) With embodiment 1.3. the

4) With embodiment 1.4. Blot the water on the root tip with filter paper, cut off the root tip meristem, put it in a mixture of pectinase and cellulase, and put it in a water bath at 37°C for 40-80 minutes. Depending on the size of the plant genome, The enzymatic hydrolysis time is also different, such as 20-35 minutes for the root tips of plants such as peanuts and soybeans, and 48 minutes for the root tips of plants such as wheat and corn, and the enzymatic hydrolysis time for the root tips of plants with large genomes such as lilies can reach 1-2 hours. the

5) With embodiment 1.5. the

3. Results and Discussion

The results of the study are shown in Figure 2, Figure 3 and Figure 4. the

As shown in Figure 2, soybean (2n=40), cotton (2n=52), rice (2n=24), kelp sporophyte (2n=62), sorghum (2n=20), Brachypodium (2n=10 ), lily (2n=36), pear (2n=34), tomato (2n=24) and other monocotyledonous and dicotyledonous plant metaphase cell chromosomes, the chromosome shape is rod-shaped structure, showing obvious centromere, clearly visible , which is convenient for subsequent experiments. the

The results of the study are shown in Figures 3 and 4. the

Figure 3A, the chromosomes of metaphase cells prepared under the condition of common wheat root tip treatment for 1 hour, the chromosomes are too long, and the chromosomes in one cell will overlap. Figure 3B, the chromosomes of metaphase cells prepared under the condition of common wheat root tip treatment for 2 hours, the chromosomes are clearly visible and clearly identified. Figure 3C, pear root tip cells treated for 1:30h, the chromosomes will be highly condensed, and some chromosomes will even appear filamentous at the edge. Improper treatment time that is too long or too short will affect the preparation of chromosomes. Depending on the plant species, the genome size is different, and the treatment time with nitrous oxide is different. For example, the root tips of plants such as peanuts and soybeans are treated for 30-40 minutes, and the root tips of plants such as wheat and corn are treated for 30-40 minutes. The treatment time is 2 hours, and the treatment time can reach 4-5 hours for the root tips of plants with large genomes like lilies. the

Figure 4A, wheat chromosomes were enzymatically hydrolyzed for 40 minutes. If the enzymatic hydrolysis time is short, the chromosomes will appear elongated and irregular. Figure 4B, when the enzymatic hydrolysis time of wheat root tip cells reaches 55 minutes or longer, the chromosomes in the cells will have different shapes, and most of the chromosomes overlap and cannot be fully identified. Figure 4C, pear root tip cells were enzymatically hydrolyzed for 30 minutes. If the time is short, the chromosomes will appear filamentous at the edge and cannot be fully identified. The grasp of the enzymatic hydrolysis time directly affects the preparation of chromosomes and the identification of later chromosomes. Depending on the size of the plant genome, the enzymatic hydrolysis time is also different. For example, the root tip of plants such as peanuts and soybeans is enzymatically hydrolyzed for 20-35 minutes, and the root tip of plants such as wheat and corn is enzymatically hydrolyzed for 48 minutes. For plants with large genomes such as lily The root tip enzymatic hydrolysis time can reach 1-2 hours. the

It should be understood that while the invention has been particularly shown and described with reference to exemplary embodiments thereof, those skilled in the art will appreciate that, without departing from the spirit and scope of the invention as defined by the appended claims, Various changes in form and details can be made therein, and any combination of various embodiments can be made. the

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Claims (6)

1. A method for highly sensitive and efficient detection and identification of transgenic wheat, comprising the steps of: 1) Pretreatment with N ₂ O gas and enzymatic hydrolysis of the tissues with vigorous cell division in the root apical meristem of transgenic wheat and control materials to obtain metaphase specimens with clear images and well-dispersed chromosomes; 2) Using the nick translation method to label the fluorescent probe of the target fragment to be detected; 3) using the fluorescent probe of the target fragment to be detected in step 2) to perform fluorescence in situ hybridization on the transgenic wheat metaphase chromosomes in step 1); 4) Analyzing the fluorescence in situ hybridization results obtained in step 3), and comparing them with the control materials to determine the distribution of fluorescent signals in the transgenic wheat to be detected. 2. The method according to claim 1, wherein step 1) comprises the following steps: (a) N ₂ O gas pretreatment: get a suitable root tip, put it in a wetted centrifuge tube, put it into N ₂ O gas chamber 0.5-5 hours, the pressure of N ₂ O is 10ATM (1.01Mpa); (b) fix the root tip pretreated by N ₂ O gas; (c) enzymatic treatment: wash the fixed root tip with water, Cut off the root tip meristem and treat it with pectinase and cellulase for 30min-2h. 3. The method according to claim 1, wherein step 2) separates the target segment in the transgenic carrier by enzyme digestion, and carries out fluorescent probe labeling to the target gene amplification product by means of the nick translation method, and the target segment is carried by the transgenic carrier Gene fragment. 4. A method for obtaining the plant chromosomes of a good metaphase division phase, said method comprising the steps of: (a) N ₂ O gas pretreatment: take the suitable root tip or bud tip of the plant, put it in a wet centrifuge tube, put it into a N ₂ O gas chamber and process it for 0.5-5 hours, and the pressure of N ₂ O is 10ATM (1.01Mpa); (b) fix the root tip or shoot tip pretreated by N ₂ O gas; (c) enzymatic treatment: wash the fixed root tip or shoot tip with water, cut off the root tip or shoot tip Meristem, treated with pectinase and cellulase for 30min-2h. 5. The method of claim 4, wherein the plant comprises a monocot or a dicot. 6. The method of claim 4, wherein the plant is selected from wheat, brachypodium, kelp, soybean, tomato, cotton, pear, lily or sorghum.

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