Vector composition for detecting protein transcriptional activity as well as preparation method and application thereof

Abstract

The invention provides a carrier composition for detecting protein transcriptional activity as well as a preparation method and application thereof, and belongs to the field of biology. The vector combination for detecting protein transcriptional activity comprises a first vector and a second vector, the sequence of the first vector is shown as SEQ ID NO: 1 in a sequence table, and the sequence of the second vector is shown as SEQ ID NO: 2 in the sequence table. The vector combination for detecting the transcriptional activity of the protein can be connected with the protein to be detected, the transcriptional activation activity of the protein to be detected in the rice protoplast is detected, and the detection is accurate.

Description

Technical Field

[0001] The present disclosure relates to the biological field, and in particular to a vector combination for detecting protein transcription activity, a preparation method thereof, and an application thereof. Background Art

[0002] Transcriptional activity assays can help scientists understand gene expression under specific conditions, thereby revealing the regulatory mechanisms of gene expression. By measuring transcriptional activity, it is possible to determine which genes are activated or inhibited under specific conditions, and thus understand how these regulatory mechanisms affect plant growth, development, and physiological functions.

[0003] Current transcriptional activity assays require constructing a vector, along with the construction of effector and reporter plasmids for the transcription factor and target gene promoter, respectively. These two plasmids are co-transfected into the same cell, where the transcription factor binds to the promoter and regulates FLuc expression. The ratio of FLuc to RLuc expression indicates whether the transcription factor has transcriptional activation or repression activity on the promoter. The reporter plasmid is pGreenⅡ0800-LUC, and the effector plasmid is pGreenⅡ62-SK.

[0004] However, the above plasmid combination requires obtaining a promoter sequence that specifically binds to the test protein (Effector) in order to detect the transcriptional activity of the test protein. This promoter sequence must be connected to pGreenⅡ0800-LUC before the transcriptional activity of the test protein can be detected. This makes it less versatile and is not conducive to revealing the transcriptional activation or inhibition characteristics of the test protein.

[0005] Public content

[0006] To address the problems of the prior art, the present disclosure provides a protein transcription activity detection vector combination, a preparation method, and an application thereof. The technical solution is as follows:

[0007] On the one hand, the present disclosure provides a combination of vectors for detecting protein transcriptional activity, which comprises: a first vector and a second vector, the sequence of the first vector being shown in SEQ ID NO: 1 in the sequence listing, and the sequence of the second vector being shown in SEQ ID NO: 2 in the sequence listing.

[0008] On the other hand, the present disclosure provides a method for preparing the above-mentioned vector combination for detecting protein transcription activity, the preparation method comprising: a method for preparing the first vector,

[0009] The backbone vector pGreen II-62-SK was double-digested with NotI and KpnI to obtain the first digestion product;

[0010] The coding sequence of the Gal4 DNA-binding domain, the MYC tag, and the multiple cloning site sequence are connected to the first enzyme digestion product to obtain the first vector, wherein the coding sequence of the Gal4 DNA-binding domain, the MYC tag, and the multiple cloning site sequence are shown in SEQ ID NO: 3 in the sequence listing;

[0011] The ligation product is connected to the first enzyme digestion product to obtain the first vector.

[0012] Specifically, the preparation method also includes a preparation method of the second carrier,

[0013] The backbone vector pGreen+II-0800-Luc was double-digested with KpnI and BamHI to obtain the second enzyme digestion product.

[0014] The 5×USA sequence shown in SEQ ID NO: 4 in the sequence listing was connected to the second enzyme digestion product to obtain the second vector.

[0015] In another aspect, the present disclosure provides an application of the above-mentioned vector combination for detecting protein transcriptional activity, the application comprising:

[0016] Connecting the coding region of the protein to be tested to the first vector to obtain a vector to be tested;

[0017] The test vector and the second vector are co-transformed into rice protoplasts and cultured as an experimental group;

[0018] Transforming the first vector and the second vector together into the protoplasts of the rice and culturing them as a control group;

[0019] The experimental group and the control group were tested respectively using an enzyme-labeled instrument;

[0020] The ratio of Luc to Ren is used to determine whether the protein to be tested has transcriptional activation activity.

[0021] Specifically, the pGreen II-62-SK-GAL4 vector was double-digested with EcoRI and BamHI to obtain a third digestion product; the third digestion product was connected to the coding region of the protein to be tested by a one-step cloning method to obtain the vector to be tested.

[0022] Specifically, the protoplast preparation method includes: cutting off the roots of rice seedlings and retaining the stems;

[0023] soaking the cut ends of the rice seedlings in mannitol;

[0024] The rice seedlings were cut into segments, immersed in a 0.6 M mannitol solution, and cultured in the dark at 28° C. for 10 min;

[0025] removing mannitol from the rice seedlings, and placing the rice seedlings in an enzymatic hydrolysis solution for enzymatic hydrolysis to obtain an enzymatic hydrolysis product;

[0026] Add an equal volume of W5 solution to the enzymatic hydrolysate, mix well, and filter to obtain a filtrate;

[0027] The filtrate is placed in a centrifuge tube and centrifuged to obtain a precipitate, which is the protoplast.

[0028] The beneficial effects of the technical solution provided by the embodiments of the present disclosure are as follows: the embodiments of the present invention provide a protein transcription activity detection vector combination and its preparation method and application, the protein transcription activity detection vector combination can be connected to the protein to be tested, and the transcription activation activity of the protein to be tested in rice protoplasts can be detected accurately. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] In order to more clearly illustrate the technical solutions in the embodiments of the present disclosure, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are only some embodiments of the present disclosure. For ordinary technicians in this field, other drawings can be obtained based on these drawings without any creative work.

[0030] Figure 1 is a diagram of the first carrier provided in Example 2 of the present disclosure;

[0031] Figure 2 is a diagram of the second carrier provided in Example 2 of the present disclosure;

[0032] Figure 3 This is a comparison chart of OsSND2 transcriptional activation activity detection provided in Example 3 of the present disclosure. DETAILED DESCRIPTION

[0033] In order to make the objectives, technical solutions and advantages of the present disclosure more clear, the embodiments of the present disclosure will be further described in detail below with reference to the accompanying drawings.

[0034] Example 1

[0035] The present disclosure provides a vector combination for detecting protein transcriptional activity, which comprises: a first vector and a second vector, wherein the sequence of the first vector is shown in SEQ ID NO: 1 in the sequence listing, and the sequence of the second vector is shown in SEQ ID NO: 2 in the sequence listing.

[0036] Example 2

[0037] The present disclosure provides a method for preparing a vector combination for detecting protein transcriptional activity provided in Example 1, the preparation method comprising: a method for preparing a first vector, specifically comprising: a coding sequence of a DNA-binding domain (Gal4 DNA-binding domain) in artificially synthesized yeast, a MYC tag, and a multiple cloning site sequence (MCS), wherein the coding sequence of the DNA-binding domain (Gal4 DNA-binding domain) in artificially synthesized yeast, the MYC tag, and the multiple cloning site sequence (MCS) are as shown in SEQ ID NO: 3 in the sequence listing; double-digesting the backbone vector pGreen II-62-SK (Novopro, V012539) with NotI and KpnI to obtain a first enzyme digestion product;

[0038] The synthetic sequence was connected to the first enzyme digestion product to obtain the first vector (pGreen II-62-SK-GAL4). The map of the first vector is as follows Figure 1 shown.

[0039] The preparation method also includes a preparation method for the second vector, specifically: the backbone vector pGreen+II-0800-Luc (Novopro, V010545) is double-digested with KpnI and BamHI to obtain a second enzyme digestion product;

[0040] The 5×USA sequence shown in SEQ ID NO: 4 in the sequence list was connected to the second enzyme digestion product by gene synthesis to obtain a second vector (pGreen+II-0800-Luc-5×USA). The map of the second vector is shown in FIG. Figure 2 shown.

[0041] Example 3

[0042] The present disclosure provides an application of the above-mentioned protein transcription activity detection vector combination, the application including:

[0043] The coding region of the protein to be tested is ligated into a first vector to produce a test vector. Specifically, in this embodiment, the test protein is the (OsSND2)LOC_Os05g48850 protein, known to have transcriptional activation activity in rice cells. This protein is ligated into the pGreen II-62-SK-GAL4 vector and, after correct sequencing, is constructed into the test vector (pGreen II-62-SK-GAL4-SND2). In other embodiments, OsSND2 can be replaced with another protein whose transcriptional activity needs to be tested.

[0044] The vector to be tested and the second vector are co-transfected into rice protoplasts and cultured as the experimental group;

[0045] The first vector and another second vector were co-transformed into rice protoplasts and cultured as a control group;

[0046] The experimental group and the control group were tested using an enzyme-labeled instrument.

[0047] The ratio of Luc to Ren was used to determine whether the protein under test had transcriptional activation activity.

[0048] Specifically, the pGreen II-62-SK-GAL4 vector was double-digested with EcoRI and BamHI to obtain a third digestion product; the third digestion product was connected to the coding region of the protein to be tested by a one-step cloning method to obtain the vector to be tested.

[0049] RNA extraction

[0050] Total RNA was extracted from the rice plant Nipponbare. In this example, whole rice plants grown for 15 days were used. In other examples, the rice plant may be other varieties, and the procedures described in this example apply. Total RNA was extracted from the rice plant using the RNAsimple Total RNA Extraction Kit (TIANGEN Code: DP419). Specific steps are described in the kit's manual.

[0051] cDNA synthesis

[0052] Total RNA was reverse transcribed to obtain cDNA; in this example, PrimeScript TM Reverse transcription was performed using the RT reagent Kit with gDNA Eraser (Code: RR047Q). For specific operation procedures, please refer to the instructions of the kit.

[0053] Amplification of the OsSND2 coding region

[0054] Using cDNA as a template, amplification is performed using a forward primer with a linker and a reverse primer with a linker to obtain an amplified product, i.e., the coding region of the gene. The sequence of the forward primer with a linker is shown in SEQ ID NO: 5 in the sequence listing, the reverse primer with a linker is shown in SEQ ID NO: 6 in the sequence listing, and the sequence of the amplified product with a linker is shown in SEQ ID NO: 7 in the sequence listing.

[0055] In this embodiment, the coding sequence of OsSND2 was amplified using KOD-Plus-Neo high-fidelity DNA polymerase (Takara Code: KOD-401), and the specific operation was as follows:

[0056] The amplification system is 50 μL, and the specific amplification system is shown in Table 1.

[0057] Table 1 shows the amplification system

[0058] Reagents Usage 10×buffer 5.0μL 2mM dNTP 5.0μL Forward primer (10 μM) 1.5 μL Reverse primer (10 μM) 1.5 μL cDNA 1.0μL KOD-Plus-Neopolymerase 1.0 μL <![CDATA[ddH2O]]> 35μL

[0059] The amplification procedure is shown in Table 2.

[0060] Table 2 shows the amplification procedure.

[0061]

[0062] After the amplified product was purified and recovered, an OsSND2 coding sequence fragment with a linker was obtained, which was 987 bp in length.

[0063] Linearization of pGreen II-62-SK-GAL4 vector

[0064] The pGreen II-62-SK-GAL4 vector was double-digested with EcoRI and BamHI to linearize it. The enzyme digestion system of 50 μL is shown in Table 3.

[0065] Table 3 shows the enzyme digestion system

[0066] Reagents Usage 10×FastDigestbuffer 5.0μL FastDigestEcoRI (Thermo, FD0274) 2.0 μL FastDigestBamHI (Thermo, FD0054) 2.0 μL GreenII-62-SK-GAL4 vector 4 μg <![CDATA[ddH2O]]> 37μL

[0067] The enzyme digestion procedure was: 37°C water bath for 3 h.

[0068] After the enzyme digestion product is purified and recovered, the linearized first vector pGreen II-62-SK-GAL4 is obtained.

[0069] The OsSND2 coding region was ligated to the first vector pGreen II-62-SK-GAL4

[0070] The OsSND2 coding sequence fragment with a linker was ligated to the linearized first vector pGreen II-62-SK-GAL4 using a one-step cloning kit (Vazyme, C112) to construct the test vector pGreen II-62-SK-GAL4-SND2. The ligation system is shown in Table 4.

[0071] Table 4 shows the one-step cloning connection system

[0072] Reagents Usage 5×CEIIBuffer 2.0 μL ExnaseII 1.0 μL Linearized first vector pGreenII-62-SK-GAL4 1.0μL OsSND2 coding sequence fragment 1.5 μL <![CDATA[ddH2O]]> 4.5 μL

[0073] The ligation reaction conditions were: constant temperature reaction at 37°C for 30 min.

[0074] After the sequence was sequenced correctly, the pGreen II-62-SK-GAL4-SND2 vector was obtained. During sequencing, the forward primer shown in SEQ ID NO: 8 and the reverse primer shown in SEQ ID NO: 9 in the sequence listing were used for sequencing.

[0075] Rice protoplasts co-transformed with pGreen II-62-SK-GAL4-SND2 and pGreen+II-0800-Luc-5×USA were used as the experimental group, and rice protoplasts co-transformed with pGreen II-62-SK-GAL4 and pGreen+II-0800-Luc-5×USA were used as the control group. After the rice protoplasts in the control and experimental groups were cultured in the dark for 18 hours, the Luc / Ren value was measured to detect the transcriptional activation activity of OsSND2.

[0076] Preparation of rice seedlings

[0077] Mature Nipponbare rice seeds were hulled and 200 hulled seeds were placed in a 50 mL centrifuge tube. The seeds were washed with deionized water to remove impurities and then treated with 75% ethanol for 2 minutes for initial sterilization. The seeds were then rinsed several times with deionized water and finally treated with 3% sodium hypochlorite solution for 12 minutes to complete the sterilization. After sterilization, the seeds were washed three times with sterile water on a laminar flow hood to remove any residual sodium hypochlorite solution. Using sterile forceps, the seeds were placed in 50 mL Erlenmeyer flasks containing solid 1 / 2 MS culture medium, with 20 seeds per flask. The tubes were incubated at 28°C for 12 days.

[0078] Specifically, the method for preparing protoplasts includes: cutting off the roots of rice seedlings and retaining the stems;

[0079] Soak the cut ends of rice seedlings in mannitol;

[0080] Rice seedlings were cut into segments, immersed in 0.6 M mannitol solution, and incubated in the dark at 28°C for 10 min;

[0081] removing mannitol from the rice seedlings, and placing the rice seedlings in an enzymatic hydrolysis solution for enzymatic hydrolysis to obtain an enzymatic hydrolysis product;

[0082] Add an equal volume of W5 solution to the enzymatic hydrolysate, mix well, and filter to obtain a filtrate;

[0083] The filtrate is placed in a centrifuge tube and centrifuged to obtain a precipitate, which is the protoplast.

[0084] Furthermore, healthy rice seedlings were pulled out of the culture medium, and the roots of the seedlings and the culture medium were cut off with scissors. A portion of a 0.6 M mannitol solution (0.6 M mannitol solution comprises: 5.465 g mannitol, 50 mL sterile water) was poured into a glass culture dish, and the cut end of the rice seedling was immersed in the 0.6 M mannitol solution.

[0085] Pour the remaining 0.6M mannitol solution into another glass Petri dish. Place the rice seedling stems on a glass dish and soak them in the 0.6M mannitol solution. Use a razor blade to cut the stems into as small segments as possible; smaller segments facilitate enzymatic hydrolysis. Submerge the cut segments in another glass Petri dish containing the 0.6M mannitol solution. Equilibrate in a dark incubator at 28°C for 10 minutes.

[0086] Filter through two layers of 300-mesh nylon membrane to remove mannitol. Transfer the cut pieces to the prepared enzymatic hydrolysis solution and hydrolyze in a light-proof shaker at 28°C for 4-5 hours at a speed of 80 rpm.

[0087] The enzymatic hydrolysis solution includes: Cellulase R-100.15g, Macerozyme R-100.075g, mannitol 1.093g, BSA 0.01g, 100mM MES 1mL and sterile water 8mL.

[0088] Add an equal volume of W5 solution to the enzymatic hydrolysate and mix to obtain a mixed solution. 1L of W5 solution contains: 9g NaCl, 13.87g CaCl2, 50mL 100mM KCl, 0.9g glucose, 20mL 100mM MES, and ddH2O. Bring the volume to 1L with ddH2O, adjust the pH to 5.8, and filter sterilize. Filter the mixture through a filter membrane. During filtration, squeeze the enzymatic hydrolysate to release the protoplasts as much as possible. Transfer the filtrate to a 10mL round-bottom centrifuge tube and centrifuge horizontally at 905rcf for 3 minutes at room temperature. Discard the supernatant and collect the precipitate (protoplasts). Add 10mL of W5 solution to the centrifuge tube, gently invert to wash the protoplasts, and centrifuge horizontally at 905rcf for 3 minutes at room temperature.

[0089] Discard the supernatant and retain the precipitate. Add MMG solution to the precipitate. The MMG solution consists of 30 mL of 100 mM MgCl2, 8 mL of 100 mM MES, 21.86 g of mannitol, and ddH2O. Bring the volume up to 200 mL with ddH2O, adjust the pH to 5.6, and filter sterilize. The amount of MMG solution to be added can be calculated based on the desired conversion amount. After mixing, remove a small amount of the solution for microscopic examination to check the condition of the protoplasts. If the protoplasts are relatively round, the protoplasts are in good condition.

[0090] Protoplast transformation

[0091] The plasmid (protein) to be transformed is added to a 2mL centrifuge tube. If it is a single plasmid, 10 μg is generally added. If it is a multiple plasmid, the amount of each plasmid added can be adjusted according to actual needs. In the present embodiment, 100 μL of protoplasts are added to each centrifuge tube, and then an equal volume (the sum of the protoplast and plasmid volumes) of PEG solution is added and flicked to mix. Protect from light, 28°C, and transform for 18 minutes. The PEG solution includes: 2.5mL of 0.8M mannitol, 1mL of 1M CaCl2, 4g of PEG 4000, and 3mL of sterile water.

[0092] After transformation is complete, add 4 volumes of W5 solution to terminate the reaction. Centrifuge horizontally at 905 rcf for 3 minutes at room temperature, discard the supernatant, and retain the precipitate. Add 1 mL of W5 solution to the precipitate, gently invert and mix, and incubate in the dark at 28°C for 12–18 hours.

[0093] Fluorescence value detection

[0094] Using the dual-luciferase reporter gene assay system ( Reporter Assay System, Promega, E1910) to measure fluorescence values. Specific steps are as follows:

[0095] (1) Cell lysis: Protoplasts were collected by centrifugation, and 100 μL of 1× Cell Lysis Buffer was added. The cells were shaken for 5 min and the lysate was transferred to a 1.5 mL centrifuge tube. The cells were centrifuged at 12,000 g for 2 min at room temperature and the supernatant was used for subsequent detection.

[0096] (2) Firefly luciferase reaction detection: Take 100 μL of Luciferase Substrate and add it to the ELISA plate. Pipette 20 μL of cell lysis supernatant into the wells of the ELISA plate. Mix quickly and immediately detect the Firefly luciferase reporter gene activity in an ELISA reader.

[0097] (3) Renilla luciferase reaction detection: Add 100 μL of freshly prepared Renilla working solution to the above reaction solution, mix quickly and immediately detect the Renilla luciferase reporter gene activity in a microplate reader. Perform three replicates for each test.

[0098] (4) The value obtained by the firefly luciferase (LUC) assay was divided by the value obtained by the Renilla luciferase (REN) assay, that is, the experimental result was Luc / Ren. Figure 3 As shown, Figure 3Data in the table = mean (three independent experiments repeated) ± standard error. Asterisks on the error bars indicate significant differences compared with the control group (p value calculated by one-way ANOVA, **p<0.01). pGreen II-62-SK-GAL4-SN D2+pGreen+II-0800-Luc-5×USA is the experimental group, and pGreen II-62-SK-GAL4+pGreen+II-0800-Luc-5×USA is the control group. Larger values indicate stronger transcriptional activity. Figure 3 It can be seen that the values of the experimental group are significantly higher than those of the control group, which indicates that OsSND2 has transcriptional activation activity in rice protoplasts, which is consistent with its actual situation. This further confirms that the protein transcriptional activity detection vector combination provided by the present invention can be used to detect the transcriptional activity of proteins in rice protoplasts.

[0099] The above description is merely an optional embodiment of the present disclosure and is not intended to limit the present disclosure. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present disclosure shall be included in the scope of protection of the present disclosure.

Claims

1. A vector combination for detecting protein transcription activity, characterized in that: The protein transcription activity detection vector combination includes: a first vector and a second vector, the sequence of the first vector is shown in SEQ ID NO: 1 in the sequence listing, and the sequence of the second vector is shown in SEQ ID NO: 2 in the sequence listing.

2. A method for preparing a vector combination for detecting protein transcriptional activity according to claim 1, characterized in that: The preparation method includes: a preparation method of the first carrier, The backbone vector pGreen II-62-SK was double-digested with NotI and KpnI to obtain the first digestion product; The coding sequence of the Gal4 DNA-binding domain, the MYC tag and the multiple cloning site sequence are connected to the first enzyme digestion product to obtain the first vector. The coding sequence of the Gal4 DNA-binding domain, the MYC tag and the multiple cloning site sequence are shown in SEQ ID NO: 3 in the sequence listing.

3. The preparation method according to claim 2, characterized in that The preparation method also includes a preparation method of the second carrier, The backbone vector pGreen+II-0800-Luc was double-digested with KpnI and BamHI to obtain the second enzyme digestion product. The 5×USA sequence shown in SEQ ID NO: 4 in the sequence listing was connected to the second enzyme digestion product to obtain the second vector.

4. A use of the vector combination for detecting protein transcriptional activity according to claim 1, characterized in that: The applications include: Connecting the coding region of the protein to be tested to the first vector to obtain a vector to be tested; The test vector and the second vector are co-transformed into rice protoplasts and cultured as an experimental group; Transforming the first vector and the second vector together into the protoplasts of the rice and culturing them as a control group; The experimental group and the control group were tested respectively using an enzyme-labeled instrument; The ratio of Luc to Ren is used to determine whether the protein to be tested has transcriptional activation activity.

5. The use according to claim 4, characterized in that The pGreen II-62-SK-GAL4 vector was double-digested with EcoRI and BamHI to obtain a third digestion product; the third digestion product was connected to the coding region of the protein to be tested by a one-step cloning method to obtain the vector to be tested.

6. The use according to claim 4, characterized in that The protoplast preparation method comprises: cutting off the root of the rice seedling and retaining the stem; soaking the cut ends of the rice seedlings in mannitol; The rice seedlings were cut into segments, immersed in a 0.6 M mannitol solution, and cultured in the dark at 28° C. for 10 min; removing mannitol from the rice seedlings, and placing the rice seedlings in an enzymatic hydrolysis solution for enzymatic hydrolysis to obtain an enzymatic hydrolysis product; Add an equal volume of W5 solution to the enzymatic hydrolysate, mix well, and filter to obtain a filtrate; The filtrate is placed in a centrifuge tube and centrifuged to obtain a precipitate, which is the protoplast.

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