Method for enriching soybean root specific tissue cell nucleus
By expressing specific promoter-driven nuclear localization proteins and biotin ligases in soybean hairo roots, the use of streptavidin magnetic beads to enrich the nucleus of soybean roots, solving the problems of difficulty in cell nuclear enrichment and complex processing in the prior art, and achieving an efficient and simplified nuclear enrichment process.
Patent Information
- Application Number
- CN202510144276.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-02-10
- Publication Date
- 2025-05-13
Smart Images
Figure CN119979593A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the technical field of genetic engineering, in particular to a method for enriching soybean root-specific tissue cell nuclei. Background Art
[0002] Existing plant nucleus isolation techniques are widely used in the study of plant molecular biology, mainly for analyzing gene expression and epigenetic characteristics of specific plant tissues. However, traditional nucleus isolation techniques have shortcomings in tissue-specific separation, especially in the difficulty of enriching specific nuclei in root tissues. In addition, existing methods usually involve many steps, the processing process is relatively complicated, and it is difficult to obtain target tissue nuclei with high purity. Therefore, in response to the enrichment needs of soybean root-specific tissue nuclei, an efficient nucleus enrichment method is proposed to improve the efficiency of the experiment and the reliability of the results. Summary of the invention
[0003] The present invention aims to provide a method for enriching soybean root-specific tissue cell nuclei, so as to solve the technical problems of difficulty in enriching specific cell nuclei in root tissue and complex processing process.
[0004] To achieve the above object, the present invention provides a method for enriching soybean root-specific tissue cell nuclei, and the specific steps are as follows:
[0005] S1. construct a vector containing soybean root tissue-specific promoter, nuclear localization protein, biotin ligase recognition peptide and biotinidase;
[0006] S2. Expressing the vector by soybean hairy root transformation technique;
[0007] S3. Extract cell nuclei and use streptavidin magnetic beads to specifically enrich and separate biotin-labeled cell nuclei.
[0008] As one of the preferred technical solutions, in step S1, the soybean root tissue-specific promoter is a soybean nodule early promoter, and its nucleotide sequence is shown in SEQ ID NO.1.
[0009] As one of the preferred technical solutions, the specific method of step S1 is:
[0010] S1-1. Cloning of soybean root tissue-specific promoter;
[0011] S1-2. HindIII single restriction enzyme digestion of pCa-mCherry-KINTACT vector, the nucleotide sequence of which is shown in SEQ ID NO.2;
[0012] S1-3. The soybean root tissue-specific promoter was constructed into the pCa-mCherry-KINTACT vector by homologous recombinase;
[0014] S1-4. Verify the successful construction of the vector by Sanger sequencing and ensure that the promoter sequence is completely inserted into the vector.
[0015] As one of the preferred technical solutions, the specific method of step S2 is:
[0016] S2-1. The vector constructed in step S1 was transformed into Agrobacterium rhizogenes K599 by electroporation, and positive clones were screened;
[0017] S2-2. The single clone was transferred to LB liquid medium supplemented with kanamycin and streptomycin antibiotics for culture;
[0018] S2-3. The bacterial solution obtained in step S2-2 was transferred to LB liquid medium supplemented with kanamycin and streptomycin antibiotics at a volume ratio of 1:100, shaken to OD600 = 1.0, centrifuged; resuspended to OD600 = 0.6 using 10mM MgCl2, and cultured;
[0019] S2-4. Use a 1 ml syringe to inject the bacterial solution obtained in step S2-3 into the hypocotyls of 5-day-old soybean seedlings, and culture them at high humidity for about 14 days. When callus tissue is formed at the hypocotyl wound and roots extend out, transfer them to water and culture them until the roots are long enough. Use a handheld fluorescent ultraviolet lamp to screen positive roots for mCherry signals, and collect the positive roots for extracting cell nuclei.
[0020] As one of the further preferred technical solutions, in step S2-1, colony PCR technology is used to ensure that the screened monoclonal colonies are positive.
[0021] As one of the further preferred technical solutions, in step S2-2, the kanamycin content in the LB liquid culture medium is 50 mg / L, and the streptomycin content is 50 mg / L; the culture conditions are: 37°C, 200 rpm overnight.
[0022] As one of the further preferred technical solutions, in step S2-3, the kanamycin content in the LB liquid culture medium is 50 mg / L, and the streptomycin content is 50 mg / L.
[0023] As one of the further preferred technical solutions, in step S2-3, the centrifugation condition is: centrifugation at 8000 rpm for 5 minutes.
[0024] As one of the further preferred technical solutions, in step S2-3, the culture conditions are: 28°C, 200 rpm, culturing for 2 hours.
[0025] As one of the preferred technical solutions, the specific method of step S3 is:
[0026] S3-1. Grind 1 g of tissue sample into powder using a pre-cooled mortar and pestle, then resuspend in 10 mL of 4°C pre-cooled NPB buffer and gently homogenize;
[0027] S3-2. The extract was filtered through a 40-micron nylon mesh placed on top of a 15 mL tube on ice, centrifuged at 1000 g for 15 minutes at 4°C, and the precipitate, i.e., the nucleus, was taken;
[0028] S3-3. Gently resuspend the nuclei in 1 mL of ice-cold NPB; separate 25 μl of sample for nuclear quantification and carefully transfer the remaining sample from the centrifuge tube to a pre-labeled 1.5 ml centrifuge tube;
[0029] S3-4. Each sample requires 25 μl of Invitrogen M280 Streptavidin Dynabeads. Dilute the Dynabeads with 1 ml of NPB in advance, mix thoroughly, and place on a magnetic stand for 5 minutes. Discard the supernatant. Resuspend the Dynabeads with 25 μl of NPB. Add 25 μl of the magnetic bead suspension to the nuclear sample and incubate at 4°C for 30 minutes.
[0030] S3-5. Dilute 1 mL of the magnetic bead-core mixture to 14 mL with NPB (NPB-T) containing 0.1% triton X-100 by volume; gently mix and rotate for 30 seconds; place the tube in a 15 mL magnetic separator at 4°C for 8 minutes;
[0031] S3-6. Carefully remove the supernatant using a pipette, first aspirating out the bubbles, then the buffer; gently resuspend the beads in 14 mL of fresh NPB-T; gently mix and spin for 30 seconds; place the tube on a 15 mL magnetic stand and place at 4°C for 8 minutes;
[0032] S3-7. Repeat the washing and collecting steps;
[0033] S3-8. Gently use a pipette to remove the supernatant and suspend the magnetic beads in 1 mL of NPB-T; take 25 μl of the sample and count the captured nuclei on a hemocytometer;
[0034] S3-9. Transfer the suspended magnetic beads to a 1.5 mL tube and capture them on a 1.5 mL tube magnetic stand;
[0035] S3-10. Remove the supernatant, suspend the beads in 20 μl of NPB, and place on ice;
[0036] S3-11. Observe the purified cell nuclei under a microscope, add 1 μl of 0.2 μg / μl propidium iodide to each 25 μl sample of steps S3-3 and S3-8, and place on ice for 5 minutes; count the cell nuclei using a hemocytometer.
[0037] As one of the further preferred technical solutions, in step S3-3, the specific method for quantifying nuclei is: add 1 μl of 0.2 μg / μl propidium iodide aqueous solution to each 25 μl sample, and place on ice for 5 minutes away from light; use a hemocytometer to count cell nuclei.
[0038] The present invention has the following beneficial effects:
[0039] The present invention achieves the enrichment of soybean root specific tissue cell nuclei through soybean hairy root transformation technology. Specifically, a vector containing a specific tissue promoter is constructed, and the vector is introduced into soybean hairy roots for expression. The vector contains a nuclear localization protein, a biotin ligase recognition peptide and a biotinidase. The nuclear localization protein and the biotin ligase recognition peptide are driven by a specific promoter, and the biotinidase is expressed at the same time, thereby marking biotin in specific root tissues. After the cell nuclei are extracted, the biotin-labeled cell nuclei are specifically separated by streptavidin magnetic beads to achieve the enrichment of soybean root specific tissue cell nuclei, thereby meeting the needs of subsequent experiments.
[0040] In addition to the above-described purposes, features and advantages, the present invention has other purposes, features and advantages. The present invention will be further described in detail with reference to the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS
[0041] The drawings constituting a part of this application are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings:
[0042] Figure 1 It is the vector map;
[0043] Figure 2 It is the production of young roots after soybeans are injected with Agrobacterium rhizogenes;
[0044] Figure 3 It is to screen soybean positive roots through mCherry;
[0045] Figure 4 This is a slice of Promoter No. 6 GUS;
[0046] Figure 5 It is the fluorescence signal before and after the extraction of cell nuclei enrichment;
[0047] Figure 6 It is the Western blot to detect the expression of nuclear membrane original in soybean positive roots;
[0048] Figure 7 It is the statistics of differentially expressed genes in RNA-seq samples before and after Promoter6 enrichment;
[0049] Figure 8 It is IGV that shows that the nuclear membrane localization component is enriched in the sample after beads enrichment. DETAILED DESCRIPTION
[0050] The embodiments of the present invention are described in detail below with reference to the accompanying drawings, but the present invention can be implemented in many different ways as defined and covered by the claims.
[0051] 1. Vector Construction ( Figure 1 )
[0052] The vector of the present invention is constructed by genetic engineering means, and comprises a specific tissue promoter, a nuclear localization protein, a biotin ligase recognition peptide and a biotinidase. The promoter is a soybean root tissue specific promoter, and can effectively drive the expression of exogenous genes in specific root tissues. The vector construction process includes inserting a specific promoter sequence, a nuclear localization protein coding sequence, a biotin ligase recognition peptide sequence and a biotinidase coding sequence, and the successfully constructed vector can be used for the subsequent hairy root transformation step.
[0053] Detailed steps:
[0054] 1) Cloning of soybean root tissue-specific promoter;
[0055] 2) Hind III single restriction digestion of pCa-mCherry-KINTACT vector;
[0056] 3) The soybean root tissue-specific promoter was constructed into the pCa-mCherry-KINTACT vector using homologous recombinase;
[0057] 4) Verify the success of vector construction by Sanger sequencing and ensure that the promoter sequence is completely inserted into the vector.
[0058] 2. Hairy Root Transformation
[0059] The constructed vector is introduced into soybean hairy roots through Agrobacterium-mediated transformation to ensure the stable expression of the vector in soybean hairy roots. This process uses soybean hairy root transformation technology to express the vector with a specific promoter and biotinidase in soybean hairy roots.
[0060] Detailed steps:
[0061] 1) The vector plasmid constructed in 1 is transformed into Agrobacterium rhizogenes K599 by electroporation, positive clones are screened, and colony PCR technology is used to ensure that the screened monoclonal colonies are positive;
[0062] 2) Transfer the single clone to LB liquid medium supplemented with kanamycin (50 mg / L) and streptomycin (50 mg / L) antibiotics and culture at 37°C, 200 rpm overnight;
[0063] 3) Transfer the bacterial solution containing the target vector to a medium containing kanamycin (50 mg / L) and streptomycin (50 mg / L) at a dilution of 1:100.
[0064] LB liquid medium containing antibiotics, shake to OD600 = 1.0, centrifuge at 8000 rpm for 5 min; resuspend with 10 mM MgCl2 to OD600 = 0.6, culture at 28°C, 200 rpm for 2 h;
[0065] 4) Use a 1 ml syringe to inject the bacterial solution into the hypocotyl of 5-day-old soybean seedlings, and culture them at high humidity for about 14 days. When callus tissue forms at the wound of the hypocotyl and roots extend out, transfer them to water and culture them until the roots are long enough ( Figure 2 ), using a handheld fluorescent UV lamp, mCherry signal screening positive roots ( Figure 3 ), where positive roots were collected for extraction of nuclei.
[0066] 3. Cell Nucleus Extraction and Isolation
[0067] After obtaining transgenic soybean hairy roots containing the target vector, the root tissue is treated with a nuclear extraction reagent to extract the nuclei. Then, the biotin-labeled nuclei are specifically enriched and separated using streptavidin magnetic beads to ensure that high-purity root-specific tissue nuclei are obtained.
[0068] Detailed steps:
[0069] 1) Grind tissue (approximately 1 g) to powder using a pre-chilled mortar and pestle, then resuspend in 10 mL of cold NPB buffer and gently homogenize.
[0070] 2) Filter the extract through a 40 μm nylon mesh placed on top of a 15 mL tube on ice. Centrifuge at 1000 g for 15 minutes at 4°C.
[0071] 3) Gently resuspend the nuclei in 1 mL of ice-cold NPB. Separate a 25 μl sample for nuclei quantification. Carefully transfer the remaining sample from the centrifuge tube to a pre-labeled 1.5 ml centrifuge tube.
[0072] 4) Wash 25 μl of Invitrogen M280 Streptavidin Dynabeads suspension with 1 mL NPB per sample and resuspend to original volume with NPB. Add the magnetic bead suspension to the nuclear sample and incubate at 4°C with rotation for 30 minutes.
[0073] 5) Dilute 1 mL of the magnetic bead-core mixture to 14 mL with NPB containing 0.1% triton X-100 (NPB-T). Mix gently and rotate for 30 seconds. Place the tube in a 15 mL magnetic separator at 4°C for 8 minutes.
[0074] 6) Carefully remove the supernatant using a pipette, first removing the bubbles, then the buffer. Gently resuspend the beads in 14 mL
[0075] Add fresh NPB-T. Mix gently and spin for 30 seconds. Place the tube on a 15 mL magnetic stand and place at 4 °C for 8
[0076] minute.
[0077] 7) Repeat the washing and collecting steps.
[0078] 8) Gently remove the supernatant using a pipette and resuspend the beads in 1 mL of NPB-T. Take a 25 μl sample and count the captured nuclei on a hemocytometer.
[0079] 9) Transfer the suspended magnetic beads to a 1.5 mL tube and capture them on a 1.5 mL tube magnet.
[0080] 10) Remove the supernatant, suspend the beads in 20 μl of NPB, and place on ice.
[0081] 11) Observe the purified nuclei under a microscope, add 1 μl of 0.2 μg / μl propidium iodide to each 25 μl sample from steps 3 and 8 and place on ice for 5 minutes. Count the nuclei using a hemocytometer.
[0082] Nuclei Purification Buffer (NPB):
[0083] -20 mM 3-morpholinepropanesulfonic acid (MOPS, pH=7)
[0084] -40mM sodium chloride (NaCl)
[0085] -90mM Potassium Chloride (KCl)
[0086] -2mM EDTA
[0087] -0.5 mM ethylene glycol diethyl ether diamine tetraacetic acid (EGTA)
[0088] -0.5mM Spermidine
[0089] -0.2mM Spermine
[0090] -1× protease inhibitor cocktail
[0091] 4. Verification Method
[0092] In order to verify the effectiveness of the technology of the present invention, RNA-seq experiments were carried out using soybean nodule early promoters, and the feasibility of the technology in soybean roots was demonstrated through gene expression data.
[0093] Detailed steps:
[0094] The cell nuclei extracted above were used to conduct relevant experiments to verify the feasibility;
[0095] 1) Promoter 6 is an early promoter of soybean nodule, and its expression site can be seen in the GUS section diagram ( Figure 4 ); (This promoter is expressed in the nodule primordium in the early stage of nodule formation, so the applicant inoculated the hairy roots with rhizobia to promote the formation of nodule tissue)
[0096] 2) Observe fluorescence; Figure 5 You can see that the cell nuclei purified by beads have fluorescent signals)
[0097] 3) Detect the expression of target protein by western blot technology ( Figure 6 , a specific promoter drives an element localized in the nuclear membrane, as can be seen in the map; the size of its expressed protein is about 42 kDa, so the western blot technology can be used to detect whether the protein is expressed through GFP antibody to determine whether the target tissue exists in the positive root);
[0098] Specifically: take 0.5g of positive roots, grind into powder, add 100 microliters of 5% SDS (sodium dodecyl sulfate), boil at 95℃ for 10 minutes; centrifuge and aspirate the supernatant into a new centrifuge tube, add 5× protein loading buffer, load 20 microliters for SDS-Page; run at 150V, 340mA for 1.5h, wet transfer membrane at 200V, 340mA, 2h, block with 5% skimmed milk powder, incubate with primary antibody at 4℃ overnight, wash 3 times with TBST, incubate with secondary antibody for 2h, wash 3 times with TBST, 15min each time; expose and develop.
[0099] 4) Use RNA-seq to observe whether the nuclear membrane localization components are enriched in the nucleus after beads pulling;
[0100] Specifically: RNA was extracted from the cell nuclei extracted in 3 (before and after beads enrichment, 20 μl was aspirated from 1 ml of cell nuclei resuspension before enrichment and retained for RNA extraction). After RNA was extracted, RNA-seq library was constructed.
[0101] After the construction is completed, it will be sent to the company for quality inspection and sequencing. Figure 7 , 8 )
[0102] The composition of 5× protein loading buffer is as follows:
[0103]
[0104] The composition of TBST buffer is as follows:
[0105] 20mM Tris
[0106] 150mM Sodium Chloride (NaCl)
[0107] Mass concentration 0.1% Polysorbate 20 (Tween-20)
[0108] Adjust to about pH 7.4.
[0109] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.
Claims
1. A method for enriching soybean root-specific tissue cell nuclei, characterized in that: The specific steps are as follows: S1. construct a vector containing soybean root tissue-specific promoter, nuclear localization protein, biotin ligase recognition peptide and biotinidase; S2. Expressing the vector by soybean hairy root transformation technique; S3. Extract cell nuclei and use streptavidin magnetic beads to specifically enrich and separate biotin-labeled cell nuclei.
2. The method according to claim 1, characterized in that: In step S1, the soybean root tissue-specific promoter is a soybean nodule early promoter, and its nucleotide sequence is shown in SEQ ID NO.
1.
3. The method according to claim 1, characterized in that The specific method of step S1 is: S1-1. Cloning of soybean root tissue-specific promoter; S1-2. Hind III single enzyme digestion of pCa-mCherry-KINTACT vector, the nucleotide sequence of which is shown in SEQ ID NO.2; S1-3. The soybean root tissue-specific promoter was constructed into the pCa-mCherry-KINTACT vector by homologous recombinase; S1-4. Verify the successful construction of the vector by Sanger sequencing and ensure that the promoter sequence is completely inserted into the vector.
4. The method according to claim 1, characterized in that: The specific method of step S2 is: S2-1. The vector constructed in step S1 was transformed into Agrobacterium rhizogenes K599 by electroporation, and positive clones were screened; S2-2. The single clone was transferred to LB liquid medium supplemented with kanamycin and streptomycin antibiotics for culture; S2-3. The bacterial solution obtained in step S2-2 was transferred to LB liquid medium supplemented with kanamycin and streptomycin antibiotics at a volume ratio of 1:100, shaken to OD600 = 1.0, centrifuged; resuspended to OD600 = 0.6 using 10mM MgCl2, and cultured; S2-4. Use a 1 ml syringe to inject the bacterial solution obtained in step S2-3 into the hypocotyls of 5-day-old soybean seedlings, and culture them at high humidity for about 14 days. When callus tissue is formed at the hypocotyl wound and roots extend out, transfer them to water and culture them until the roots are long enough. Use a handheld fluorescent ultraviolet lamp to screen positive roots for mCherry signals, and collect the positive roots for extracting cell nuclei.
5. The method according to claim 4, characterized in that In step S2-1, colony PCR technology is used to ensure that the screened monoclonal colonies are positive.
6. The method according to claim 4, characterized in that In step S2-2, the kanamycin content in the LB liquid culture medium is 50 mg / L, and the streptomycin content is 50 mg / L; the culture conditions are: 37°C, 200 rpm overnight.
7. The method according to claim 4, characterized in that In step S2-3, the kanamycin content in the LB liquid culture medium is 50 mg / L, and the streptomycin content is 50 mg / L.
8. The method according to claim 4, characterized in that In step S2-3, the centrifugation condition is: 8000 rpm for 5 min.
9. The method according to claim 4, characterized in that In step S2-3, the culture conditions are: 28°C, 200 rpm, and 2 h.
10. The method according to claim 1, characterized in that The specific method of step S3 is: S3-1. Grind 1 g of tissue sample into powder using a pre-cooled mortar and pestle, then resuspend in 10 mL of 4°C pre-cooled NPB buffer and gently homogenize; S3-2. The extract was filtered through a 40-micron nylon mesh placed on top of a 15 mL tube on ice, centrifuged at 1000 g for 15 min at 4°C, and the precipitate, i.e., the nucleus, was taken; S3-3. Gently resuspend the nuclei in 1 mL of ice-cold NPB; separate 25 μl of sample for nuclear quantification and carefully transfer the remaining sample from the centrifuge tube to a pre-labeled 1.5 ml centrifuge tube; S3-4. Each sample requires 25 μl of Invitrogen M280 Streptavidin Dynabeads. Dilute the Dynabeads with 1 ml of NPB in advance, mix thoroughly, and place on a magnetic rack for 5 minutes. Discard the supernatant. Resuspend the Dynabeads with 25 μl of NPB. Add 25 μl of the magnetic bead suspension to the nuclear sample and incubate at 4°C for 30 minutes. S3-5. Dilute 1 mL of the magnetic bead-core mixture to 14 mL with NPB containing 0.1% triton X-100 by volume; gently mix and rotate for 30 seconds; place the tube in a 15 mL magnetic separator at 4°C for 8 minutes; S3-6. Carefully remove the supernatant using a pipette, first aspirating out the bubbles, then the buffer; gently resuspend the beads in 14 mL of fresh NPB-T; gently mix and spin for 30 seconds; place the tube on a 15 mL magnetic stand and place at 4°C for 8 minutes; S3-7. Repeat the washing and collecting steps; S3-8. Gently use a pipette to remove the supernatant and suspend the magnetic beads in 1 mL of NPB-T; take 25 μl of the sample and count the captured nuclei on a hemocytometer; S3-9. Transfer the suspended magnetic beads to a 1.5 mL tube and capture them on a 1.5 mL tube magnetic stand; S3-10. Remove the supernatant, suspend the beads in 20 μl of NPB, and place on ice; S3-11. Observe the purified cell nuclei under a microscope, add 1 μl of 0.2 μg / μl propidium iodide to each 25 μl sample of steps S3-3 and S3-8, and place on ice for 5 minutes; count the cell nuclei using a hemocytometer.