Wheat chromosome 3A specific cytological probe and its application

Abstract

This invention belongs to the field of molecular genetics, specifically relating to a wheat chromosome 3A-specific cytological probe and its applications. The specific technical solution comprises a probe having a nucleic acid sequence as shown in SEQ ID NO. 1, with fluorescent groups attached to the thymine residues at the 3' and 5' ends of the probe and in the middle of the sequence. This invention proposes a novel wheat chromosome-specific DNA probe with high targeting and specificity, and excellent probe signal, providing a solid theoretical foundation and technical support for subsequent high-purity flow cytometry sorting of wheat chromosomes.

Description

Technical Field

[0001] The invention belongs to the field of molecular genetics, and in particular relates to a wheat 3A chromosome-specific cytological probe and an application thereof. Background Art

[0002] Wheat is the world's largest grain crop and the second largest grain crop in my country after rice. Wheat is an allohexaploid crop (genome is AABBDD), with a huge genome (about 17G) and many repetitive sequences and a complex structure. Therefore, the cost of genome sequencing of wheat is very high, and regions rich in repetitive sequences often cannot be assembled correctly. Compared with the entire genome, the amount of data for a single chromosome is small (often hundreds of megabytes), so the sequencing cost is low. Moreover, sequencing a single chromosome can effectively avoid interference from other chromosomes, and the assembly accuracy is higher. Therefore, single chromosome sequencing is of great significance for wheat.

[0003] However, accurate sorting of the target chromosomes to be sequenced is a prerequisite for single chromosome sequencing. Single chromosome sorting can be performed based on chromosome size and fluorescent labeling intensity. For wheat, due to the small size differences among the chromosomes in groups A, B, and D, it is difficult to obtain high-purity target chromosomes by directly sorting them by chromosome size, making subsequent single chromosome sequencing impossible. If single chromosome-specific probes are developed, the target chromosomes can carry specific fluorescent signals, thereby achieving the purpose of high-purity sorting. The copy number of specific repetitive sequences of some wheat chromosomes is low, resulting in extremely weak probe signals that are difficult to detect with the naked eye. Such probes are generally considered to be probes that have failed in development and cannot be used, which greatly limits the range of wheat chromosome-specific probes. Summary of the Invention

[0004] The purpose of the present invention is to provide a wheat 3A chromosome-specific cytological probe and application thereof.

[0005] In order to achieve the above-mentioned object of the invention, the technical solution adopted by the present invention is:

[0006] A probe, the nucleic acid sequence of the probe is shown in SEQ IN NO. 1, and fluorescent groups are connected to the 3' end, the 5' end and the thymine in the middle of the sequence of the probe.

[0007] Preferably, the middle position of the sequence is thymine at sequence number 23.

[0008] Correspondingly, the probe is used in wheat gene sequencing.

[0009] Accordingly, the probe is used in high-purity sorting of wheat.

[0010] Preferably, wheat cells are hybridized using the probe.

[0011] Preferably, the hybridization temperature is 42° C. and the hybridization time is 2 h.

[0012] Preferably, the hybridization buffer used in the hybridization is obtained by diluting the probe powder with sodium citrate buffer and TE buffer.

[0013] Preferably, the volume ratio of sodium citrate buffer to TE buffer is 1:1.

[0014] Correspondingly, test papers, reagents and kits are prepared using the probe.

[0015] Correspondingly, the invention also includes test papers, reagents and kits prepared using the probe.

[0016] The present invention has the following beneficial effects: It proposes two new wheat chromosome-specific DNA probes with extremely high targeting and specificity. Furthermore, the present invention proposes a fluorescent group connection method that significantly amplifies the signal of extremely weak signal probes, providing a solid theoretical foundation and technical guarantee for subsequent high-purity flow cytometry sorting of wheat chromosomes. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Figure 1 is a fluorescence in situ hybridization image using Oligo-3A-5' as a probe;

[0018] Figure 2 To use Oligo-pSc119.2 and Oligo-pTa535 as probes in Figure 1 Fluorescence in situ hybridization images of hybridization performed on the same chromosome division phase;

[0019] Figure 3 This is a fluorescence in situ hybridization image using Oligo-3A-3'5' as the probe and an exposure time of 2 seconds;

[0020] Figure 4 To use Oligo-pSc119.2 and Oligo-pTa535 as probes in Figure 3 Fluorescence in situ hybridization images of hybridization performed on the same chromosome division phase;

[0021] Figure 5 Oligo-3A plus The fluorescence in situ hybridization image with the probe and exposure time of 2 seconds;

[0022] Figure 6 To use Oligo-pSc119.2 and Oligo-pTa535 as probes in Figure 5 Fluorescence in situ hybridization images of hybridization performed on the same chromosome division phase;

[0023] Figure 7 This is a fluorescence in situ hybridization image using Oligo-3A-3'5'3T as a probe;

[0024] Figure 8 To use Oligo-pSc119.2 and Oligo-pTa535 as probes in Figure 7 Fluorescence in situ hybridization images of hybridization performed on the same chromosome division phase;

[0025] Figure 9 This is a fluorescence in situ hybridization image using Oligo-3A-3'5'43T as a probe;

[0026] Figure 10 To use Oligo-pSc119.2 and Oligo-pTa535 as probes in Figure 9 Fluorescence in situ hybridization images of hybridization performed on the same chromosome division phase DETAILED DESCRIPTION

[0027] The present invention provides a method for amplifying the signal of extremely weak signal probes. These are synthetic probes whose signal, after being linked to a fluorescent group at one end, is so weak that it is undetectable to the naked eye. In research, synthesized probes of this type are generally considered failures and unsuitable for practical use.

[0028] The present invention simultaneously attaches fluorescent groups to both ends of the very weak probe, effectively amplifying the probe signal. A more preferred approach is to simultaneously attach fluorescent groups to both ends of the probe and to the middle or near the middle. Even more preferred is to simultaneously attach fluorescent groups to both ends of the probe and to the thymine (T) residue in the middle or near the middle, to reduce attachment costs.

[0029] According to the above method, the present invention provides a wheat 3A chromosome-specific cytological probe, the nucleic acid sequence of the probe is shown in SEQ IN NO. 1, and the probe sequence is from 5' end to 3' end from left to right.

[0030] Oligo-3A: AATTAACAGAAAAGGATTTTAGTTAGATTAATGAACAGTGTATGA.

[0031] The technical solutions in the embodiments of the present invention will be described clearly and completely below. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art. The data obtained are the average values ​​obtained after at least 3 repetitions, and all the data obtained in each repetition are valid data.

[0032] Example: Demonstration of the effect of the probe anchoring chromosome 3A in hexaploid wheat

[0033] Probes for wheat chromosome 3A have not been reported to date, likely due to the low copy number of specific repetitive sequences on wheat chromosome 3A, resulting in extremely weak probe signals and the difficulty in obtaining effective probes using conventional methods. To demonstrate the effectiveness of the method provided by the present invention, a 3A chromosome probe was selected for demonstration in this example.

[0034] 1. Synthesize the probe. According to SEQ IN NO.1, the probe Oligo-3A was synthesized by a nucleic acid synthesis company. The fluorescent group FAM was attached to the 3' end, 5' end, and the 23rd thymine (T) from the 3' end to the 5' end of the sequence SEQ IN NO.1 to synthesize the probe Oligo-3A. plus At the same time, the probe Oligo-3A-5', in which the fluorescent group FAM is linked only at one end (5' end) of the sequence, and the probe Oligo-3A-3'5', in which the fluorescent group FAM is linked only at both ends of the sequence, were synthesized.

[0035] 2. Obtain wheat raw material. Place hexaploid wheat seeds on moistened filter paper and incubate at 23°C for 24 hours. Then, transfer the seeds to 4°C for another 24 hours. Then, transfer the seeds to 23°C until roots develop.

[0036] When wheat roots reach 1–2 cm in length, remove them and place them in a moistened 0.5 mL EP tube on ice. A small hole approximately 3 mm in diameter is pre-punctured in the cap of the EP tube to allow for nitrous oxide (N2O) treatment of the root tip. The EP tube containing the roots is placed in a nitrous oxide tank, which is then filled with nitrous oxide (0.9 MPa) for 2 hours.

[0037] Remove the EP tube and place it on ice. Add approximately 0.4 mL of 90% acetic acid (enough to completely submerge the root) and fix the root tip for 8–10 minutes. Rinse the roots twice with ddH₂O. Add 70% ethanol to the EP tube and store at -20°C for long-term storage.

[0038] 3. Preparation of slides. Wash the roots in the EP tube with ddH2O twice to remove the ethanol on the roots. Then cut off the root tip and place it in enzyme solution (1% pectinase, mass volume ratio; 2% cellulase, mass volume ratio, prepared with 0.01M, pH = 5.5 citric acid buffer), and enzymatically hydrolyze it at 37°C for 1 hour. Rinse the root tip with 70% ethanol twice to wash out the enzyme solution. Pour out most of the ethanol in the EP tube, leaving a small amount of ethanol, and crush the root tip with a sterilized needle tip. After centrifugation (4000rmp, 2min), turn the EP tube upside down and control the ethanol. Add 15-50μL of acetic acid to the EP tube and vortex to obtain a root tip cell suspension. Place the slide on a moistened paper box, use a pipette to aspirate 10μL of cell suspension, and drop it on the slide.

[0039] 4. Hybridization. Prepare the working solution: Dilute each probe powder synthesized in step 1 with hybridization buffer to a final concentration of 1 nmol / 100 μL (probe powder / hybridization buffer). For use, pipette 2 μL of working solution per slide, then add 8 μL of hybridization buffer per slide and mix thoroughly to prepare the hybridization solution. Apply 10 μL of hybridization solution to each slide. Hybridization buffer formula: 2×SSC (sodium citrate buffer): 1×TE (10 mM Tris-HCl, 1 mM EDTA) = 1:1 (V:V), pH = 7.0. Hybridize at 42°C in the dark for 2 h. After hybridization, stain with DAPI, cover with a coverslip, and examine under a Leica DM2500 fluorescence microscope. Images were taken using a black and white cooled CCD camera, and the fluorescence signal was pseudo-colored. It should be noted that the inventors tried various hybridization conditions, including the conventional 37°C. The results showed that even after 5 h of hybridization at 37°C, the signal was still weak, demonstrating that hybridization conditions can influence the results.

[0040] The results of the Oligo-3A series probes are as follows Figures 1 to 6 As shown, the arrow position in the figure is the position where the signal should be detected. Figure 1 This is a fluorescence in situ hybridization image using Oligo-3A-5' (the fluorescent group FAM is linked only to the 5' end) as the probe, with an exposure time of 2 seconds; the signal is not visible to the naked eye. Figure 2 Oligo-pSc119.2 ( Figure 2 The brighter white spot in the figure) and Oligo-pTa535 as probes ( Figure 2 Fluorescence in situ hybridization image of the light gray dot in the middle (tested with the same materials and conditions as Oligo-3A-5'). Figure 3 This is a fluorescence in situ hybridization image using Oligo-3A-3'5' (fluorescent group FAM is connected only at the head and tail ends) as the probe with an exposure time of 2 seconds. Figure 4 Oligo-pSc119.2 ( Figure 4The brighter white spot in the figure) and Oligo-pTa535 as probes ( Figure 4 Fluorescence in situ hybridization image of the light gray dot in the middle (tested with the same materials and conditions as Oligo-3A-3'5'). Figure 5 Oligo-3A plus The fluorescence in situ hybridization image with a probe and an exposure time of 2 seconds. The white spots indicated by the arrows are visible signals (circled in the circle). Figure 6 Oligo-pSc119.2 ( Figure 6 The brighter white spot in the figure) and Oligo-pTa535 as probes ( Figure 6 Fluorescence in situ hybridization image of the lighter gray dot in the middle) (with Oligo-3A plus Same materials and same conditions tested).

[0041] It should be noted that: because the patent requires the provision of black and white images, Figures 1 to 6 The Oligo-pSc119.2 probe and the Oligo-pTa535 probe are from the prior art of the inventor's research group, and the relevant patent publication number is: CN111118002A.

[0042] The results showed that the probes oligo-pSc119.2 and oligo-pTa535 could identify chromosome 3A, while the probe Oligo-3A provided by the present invention plus It hybridizes specifically to the centromere region in the middle of chromosome 3A, demonstrating strong specificity. Furthermore, under identical exposure conditions, the signal intensity of probes with fluorescent groups added to both ends and the middle of the probe is significantly higher than that of probes with fluorescent groups added to only one or both ends. Compared to probes with fluorescent groups added only to the ends, adding fluorescent groups to both the ends and the middle of the probe amplifies the signal more effectively. Adding fluorescent groups to the middle of the probe is more cost-effective when added to thymine.

[0043] Comparative Example

[0044] Under the same conditions as in the example, the Oligo-3A-3' probe (only connected to the fluorescent group FAM at the 3' end), the Oligo-3A-3'5'3T probe (connected to the fluorescent group FAM at both ends and the 3rd thymine from the 3' end to the 5' end), and the Oligo-3A-3'5'43T probe (connected to the fluorescent group FAM at both ends and the 43rd thymine from the 3' end to the 5' end) were set up, and the fluorescence in situ hybridization image with an exposure time of 2 seconds was obtained under the same conditions. Among them, the result of the Oligo-3A-3' probe is a signal that is not visible to the naked eye, and the picture is not shown. The observation results of the Oligo-3A-3'5'3T probe are as follows Figure 7As shown, Oligo-pSc119.2 and Oligo-pTa535 were used as probes in Figure 7 The results of fluorescence in situ hybridization on the same chromosome division phase are as follows Figure 8 The results of the Oligo-3A-3'5'43T probe are shown in Figure 9 As shown, Oligo-pSc119.2 and Oligo-pTa535 were used as probes in Figure 9 The results of fluorescence in situ hybridization on the same chromosome division phase are as follows Figure 10 The results show that the position of the central fluorescent group has a significant impact on the signal. If the central fluorescent group is placed near the two ends, the signal is very weak or even invisible.

[0045] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various deformations, modifications, and substitutions made to the technical solutions of the present invention by ordinary technicians in this field should fall within the scope of protection determined by the claims of the present invention.

Claims

1. A probe, characterized in that: The nucleic acid sequence of the probe is shown in SEQ IN NO.

1. Fluorescent groups are connected to the 3' end, 5' end and the thymine in the middle of the sequence of the probe; the middle position of the sequence is the thymine No. 23 in the sequence.

2. Use of the probe according to claim 1 in wheat chromosome 3A sequencing.

3. Use of the probe according to claim 1 in high-purity sorting of wheat chromosome 3A.

4. The use according to claim 2 or 3, characterized in that: The wheat cells are hybridized using the probe according to claim 1.

5. The application according to claim 4, characterized in that: The hybridization temperature was 42°C and the hybridization time was 2 h.

6. The application according to claim 4, characterized in that: The hybridization buffer used in the hybridization is obtained by diluting the probe powder with sodium citrate buffer and TE buffer.

7. The use according to claim 6, characterized in that: The volume ratio of sodium citrate buffer to TE buffer was 1:

1.

8. A test paper, reagent or kit prepared using the probe according to claim 1.

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