Visual reporter plasmid for plant cell transcriptional regulation and related applications thereof

Abstract

The application belongs to the technical field of bioengineering, and particularly relates to a visual report plasmid for plant cell transcription regulation and related application thereof. The visual report plasmid for plant cell transcription regulation is obtained by taking cis-acting elements and mini35S as core promoter components to drive DODA gene expression. The visual report plasmid is co-transformed with a plasmid expressing a transcription activation or inhibition transcription factor combined with cis-acting elements into plant protoplast cells. If the transcription factor plays an activation or inhibition function, the expression of DODA protein is affected, and then the accumulation of betalain is affected, so that the cells become yellow or the accumulation of yellow slows down. Therefore, the change of transcription regulation can be compared with a control group and visually recognized by naked eyes. The technology is simple and intuitive, and is free from dependence on high-cost instruments and equipment in the prior art, and has great application potential in scientific research and popularization.

Description

Technical Field

[0001] This application belongs to the field of bioengineering technology, specifically relating to a visual reporter plasmid for plant cell transcriptional regulation and its related applications. Background Technology

[0002] Transcriptional regulation of genes refers to the precise regulation of the initiation, elongation, termination, and spatiotemporal specificity of gene transcription by various intracellular molecular mechanisms, ultimately determining whether a gene is expressed and at what level. In addition to various omics research methods, research on transcriptional regulation of genes typically requires one-to-one validation of specific transcription factors (trans-acting factors) and DNA sequences (cis-acting elements) to clarify the activation or repression relationship between them.

[0003] Studies on transcriptional regulation of plant genes mainly employ two methods: heterologous expression in yeast and in vivo expression in plants. Heterologous expression in yeast is typically validated through auxotrophic growth and lacZ-based colorimetric reactions. While yeast systems allow for direct observation of results, they risk failing to accurately reflect endogenous regulatory relationships within the plant. In vivo expression in plants is usually validated using a dual-luciferase reporter system. The main principle is that a constitutive promoter drives REN gene expression, and the target cis-acting element is placed in the LUC gene promoter region. If the transcription factor binds to the cis-acting element, it activates or inhibits LUC expression. The activation or inhibition of the target gene promoter can then be quantitatively detected by the ratio of the activities of the two luciferases. However, dual-luciferase reporter systems require microplate readers, are expensive, and do not allow for visual observation of results. Therefore, a simpler and more intuitive method for studying plant cell transcriptional regulation is urgently needed. Summary of the Invention

[0004] Based on this need, this invention proposes a visual reporter plasmid for plant cell transcriptional regulation, a detection method, and its application. This reporter system is intuitive and low-cost. This invention utilizes betaine accumulation to cause cell discoloration and designs two novel visual reporter plasmids for plant cell transcriptional regulation (hereinafter referred to as pDODA-Tr1 and pDODA-Tr3).

[0005] To achieve the above objectives, the present invention provides a visual reporter plasmid for transcriptional regulation in plant cells, wherein the visual reporter plasmid is composed of a cis-acting element, a mini35S promoter, and a DODA gene in sequence.

[0006] In the actual operation of this invention, the core promoter of the visualization reporter plasmid consists of a cis-acting element and a mini35S;

[0007] The vector backbone of the report plasmid is: pVSr (CN 117363644 A).

[0008] The reporter gene of the visualization reporter plasmid is the DODA gene;

[0009] For information on the use of the DODA reporting system, please refer to patent CN 117230109 A.

[0010] In this invention, the upstream of the cis-acting element also includes a Ubi promoter;

[0011] In the actual operation of this invention, the visualization report plasmids include pDODA-Tr1 and pDODA-Tr3;

[0012] The pDODA-Tr1 has a Ubi promoter at the 5' end of its cis-acting element, thus exhibiting a higher DODA basal expression level and being more suitable for transcriptional repression studies.

[0013] The pDODA-Tr3 has a low DODA basal expression level, making it more suitable for research on transcriptional activation.

[0014] This invention also provides a visual detection method for transcriptional regulation in plant cells, the detection method comprising the following steps:

[0015] (1) The above-mentioned visualization reporter plasmid and the plasmid expressing transcriptional activation protein / transcriptional repressor protein were co-transformed into plant protoplasts;

[0016] (2) Twelve hours after transformation, the change in the proportion of yellow cells was observed to determine the result of transcriptional regulation;

[0017] In the actual operation of this invention, when co-transforming the above-mentioned plasmids into protoplasts, due to the large differences between different experimental batches, it is necessary to prepare a control group for comparative observation.

[0018] In this invention, the observation method is to observe using an optical microscope or a stereofluorescence microscope and compare with a control group.

[0019] This invention provides the application of the above-mentioned visualization reporter plasmid and detection method in the regulation of cis-acting element function by transcription factors.

[0020] This invention provides the application of the above-described visualization reporter plasmid and the above-described detection method in screening transcriptional activation or repression domains.

[0021] This invention provides the application of the above-described visualization reporter plasmid and the above-described detection method in screening transcription factor binding elements.

[0022] The principle of the visual reporter plasmids (pDODA-Tr1 and pDODA-Tr3; the visually visible plant cell transcription regulation reporter system mentioned below also refers to the visual reporter plasmids for plant cell transcription regulation) provided by this invention is as follows: Figure 1 As shown, the carrier schematic diagram is as follows. Figure 2 As shown, the pDODA-Tr1 and pDODA-Tr3 plasmids use cis-acting elements and the mini35S promoter to drive DODA expression. When transcriptional activators or repressors bind to the cis-acting elements, they activate or inhibit DODA protein expression. Differences in DODA protein expression lead to differences in betalain accumulation, causing cells to turn yellow or slow down yellow accumulation. This allows for direct observation of transcriptional regulation in plant cells with the naked eye.

[0023] The beneficial effects of this invention are reflected in:

[0024] This invention utilizes the activation or inhibition of DODA gene expression to influence betalain accumulation in cells, thereby enabling transcriptional regulation to be observed with the naked eye under a regular optical microscope. This method can be used not only for transcriptional regulation research in basic life sciences but also for science education. Attached Figure Description

[0025] Figure 1 A schematic diagram illustrating the principle of a reporter system for transcriptional regulation in plant cells that is visible to the naked eye.

[0026] Figure 2 A schematic diagram of a vector for a plant cell transcriptional regulatory reporter system visible to the naked eye;

[0027] Figure 3 Bright-field and fluorescence observation results of protoplasts co-transformed with pDODA-Tr1 and pDBD;

[0028] Figure 4 Bright-field and fluorescence observation results of protoplast cotransformation of pDODA-Tr1 and pDBD-3×SRDX;

[0029] Figure 5 Bright-field and fluorescence observation results of protoplasts co-transformed with pDODA-Tr3 and pDBD;

[0030] Figure 6 The results are bright-field and fluorescence observations of protoplasts co-transformed with pDODA-Tr3 and pDBD-VP16. Detailed Implementation

[0031] The present application will be further explained below with reference to the embodiments. Before introducing the specific embodiments, the experimental background of some embodiments is briefly described below.

[0032] Example 1

[0033] Evaluation of the transcriptional repressive activity of 3×SRDX using a visual plant cell transcriptional regulation reporter system.

[0034] In this embodiment, the pVSr plasmid was first modified by deleting the LUC gene and adding the cis-acting elements 6×UAS, the mini35S promoter, and the DODA gene to obtain the transcriptional regulation reporter system pDODA-Tr1 plasmid. Subsequently, the negative control plasmid pDBD and the transcriptional repressor plasmid pDBD-3×SRDX were constructed. pDODA-Tr1 was co-transfected with pDBD and pDBD-3×SRDX into protoplasts, respectively. The transcriptional repressive activity of 3×SRDX was evaluated by counting the number of yellow cells using a conventional optical microscope.

[0035] The specific implementation process is briefly described below:

[0036] (I) Construction of pDODA-Tr1 plasmid

[0037] The pDODA-Tr1 plasmid was used by GenScript for gene synthesis. The synthesized 6×UAS, mini35S promoter, and DODA gene sequences replaced the LUC gene sequence between BamHI and EcoRI in the pVSr plasmid. The complete sequences of the 6×UAS, mini35S promoter, and DODA gene in the synthesized plasmid are shown in SEQ ID NO.1.

[0038] SEQ ID NO.1:

[0039]

[0040] (II) Construction of pDBD and pDBD-3×SRDX plasmids

[0041] Using the pGBKT7 BD plasmid (a commonly used plasmid, publicly available at https: / / www.snapgene.com / plasmids / yeast_plasmids / pGBKT7) as a DNA template, the GAL4 DNA binding domain gene fragment (this sequence can specifically bind the cis-acting element 6×UAS) was amplified. The following primer pair was used for the PCR reaction:

[0042] GAL4 F: 5'-cgctctagaactagtggatccatgaagctactgtcttctatcgaacaagc-3', SEQ ID NO.2;

[0043] GAL4 HindIII: 5'-gtcgacggtatcgataagcttttacgatacagtcaactgtctttgaccttt-3', SEQ ID NO. 3.

[0044] Table 1 PCR reaction system

[0045] Components volume Phanta Max Super-Fidelity DNA Polymerase 1μL 2 × Phanta Max Buffer 10μL dNTP Mix 0.5μL upstream primer 0.8μL Downstream primer 0.8μL Template DNA 1μL Add double-distilled water to make up to the required level. 20μL

[0046] Table 2 PCR amplification program

[0047]

[0048] The amplified PCR products were recovered using an enhanced agarose gel DNA recovery kit.

[0049] The vector's backbone plasmid was pGreenII 62-SK (SlMYB75, an MYB-type transcription factor, which promotes anthocyanin accumulation and enhances volatile aroma production in tomato fruits). The vector was linearized using restriction endonucleases BamHI and HindIII.

[0050] Table 3 Enzyme digestion system

[0051] Components volume 10x Cut smart buffer 10μL BamHI 1μL HindIII 1μL carrier 2000ng Add double-distilled water to make up to the required level. 100μL

[0052] The reaction system was incubated overnight at 37°C for enzyme digestion.

[0053] Furthermore, the linearized vector was recovered using an ultrathin DNA product purification kit;

[0054] Furthermore, the recombinant PCR products and linearized vectors were obtained using the ClonExpress® II One Step Cloning Kit.

[0055] Furthermore, the recombinant product was transformed into Escherichia coli strain DH5α, and after overnight growth on plates, single clones were selected for colony PCR identification to screen for positive transformants.

[0056] Furthermore, Sanger sequencing was used to confirm whether the GAL4 DNA binding domain gene fragment was correctly constructed into the plasmid.

[0057] The pDBD-3×SRDX plasmid was synthesized by GenScript, replacing the GAL4 DNA binding domain gene fragment between BamHI and HindIII in the pDBD plasmid. The complete sequences of the GAL4 DNA binding domain and the 3×SRDX fusion gene in the synthesized plasmid are shown in SEQ ID NO.4.

[0058] SEQ ID NO.4:

[0059] .

[0060] (III) Protoplast Transformation

[0061] pDODA-Tr1, pDBD, and pDBD-3×SRDX plasmids were extracted using an endotoxin-free plasmid large-scale extraction kit. The ratio of pDBD / pDBD-3×SRDX plasmid to pDODA-Tr1 reporter plasmid was adjusted to 1:1 and added to 100 μL of extracted and isolated cotton protoplasts (approximately 10,000 cells). The mixture was gently tapped and incubated in the dark for 10 minutes. Protoplast transformation solution was then added, gently tapped, and incubated in the dark for 30 minutes. The reaction was terminated by adding W5 solution, and the transformation solution was discarded by centrifugation. The transformed protoplasts were resuspended in W5 solution.

[0062] (iv) Cell observation

[0063] After incubation at room temperature for 12 h, the transformed protoplasts were observed under a stereofluorescence microscope. Figure 3 and Figure 4 As shown, yellow cells were observed in the control group protoplasts co-transformed with pDODA-Tr1 and pDBD under bright-field microscopy, while no yellow cells were observed in the experimental group protoplasts co-transformed with pDODA-Tr1 and pDBD-3×SRDX under bright-field microscopy. Correspondingly, the control group protoplasts emitted autofluorescence of betaine under a fluorescence light source of 465-495 nm, while no fluorescence was observed in the experimental group. As shown in Table 4, the two groups showed statistically significant differences under bright-field microscopy, indicating that 3×SRDX inhibited the expression of DODA in the transcriptional regulatory reporter system pDODA-Tr1.

[0064] Table 4. Proportion of yellow cells 12 hours after transformation

[0065] Percentage of yellow cells (%) Standard deviation pDODA-Tr1+pDBD 8.73 1.7 pDODA-Tr1+pDBD-3×SRDX 0 0

[0066] Example 2

[0067] Evaluation of VP16 transcriptional activation activity using a visual plant cell transcriptional regulation reporter system.

[0068] In this embodiment, the pVSr plasmid was first modified by deleting the Ubi promoter and LUC gene, and adding the cis-acting elements 6×UAS, mini35S promoter, and DODA gene to obtain the transcriptional regulatory reporter system pDODA-Tr3 plasmid. Subsequently, the plasmid pDBD-VP16, containing transcriptional activation function, was constructed. pDODA-Tr3 was co-transformed with pDBD and pDBD-VP16 into protoplasts, respectively. The transcriptional activation activity of VP16 was evaluated by counting the number of yellow cells using a conventional optical microscope.

[0069] The specific implementation process is briefly described below:

[0070] (I) Construction of pDODA-Tr3 plasmid

[0071] The pDODA-Tr3 plasmid was synthesized by GenScript, and the synthesized sequence replaced the Ubi promoter and LUC gene sequences between XhoI and EcoRI in the pVSr plasmid. The complete sequences of the 6×UAS, mini35S promoter, and DODA gene in the synthesized plasmid are shown in SEQ ID NO. 5.

[0072] SEQ ID NO.5:

[0073]

[0074] (II) Construction of pDBD-VP16 plasmid

[0075] The pDBD-VP16 plasmid was synthesized by GenScript, and the GAL4 DNA binding domain and VP16 transcriptional activation domain sequences were synthesized to replace the GAL4 DNA binding domain gene fragment between BamHI and HindIII in the pDBD plasmid. The complete sequences of the GAL4 DNA binding domain and VP16 transcriptional activation domain in the synthesized plasmid are shown in SEQ ID NO. 6.

[0076] SEQ ID NO.6:

[0077] .

[0078] (III) Protoplast Transformation

[0079] pDODA-Tr3, pDBD, and pDBD-VP16 plasmids were extracted using an endotoxin-free plasmid large-scale extraction kit. The ratio of pDBD / pDBD-VP16 plasmid to pDODA-Tr3 reporter plasmid was adjusted to 1:1 and added to 100 μL of extracted and isolated cotton protoplasts (approximately 10,000 cells). The mixture was gently tapped and incubated in the dark for 10 minutes. Protoplast transformation solution was then added, gently tapped and mixed, and incubated in the dark for 30 minutes. The reaction was terminated by adding W5 solution, and the transformation solution was discarded by centrifugation. The transformed protoplasts were resuspended in W5 solution.

[0080] (iv) Cell observation

[0081] After incubation at room temperature for 12 h, the transformed protoplasts were observed under a stereofluorescence microscope. Figure 5 and Figure 6 As shown, no yellow cells were observed in the control group protoplasts co-transformed with pDODA-Tr3 and pDBD under bright-field microscopy, while yellow cells were observed in the experimental group protoplasts co-transformed with pDODA-Tr3 and pDBD-VP16 under bright-field microscopy. Correspondingly, no autofluorescence of betaine was observed in the control group protoplasts under a fluorescence light source of 465–495 nm, while fluorescence was observed in the experimental group. As shown in Table 5, the two groups showed statistically significant differences under bright-field microscopy, indicating that VP16 activates the expression of DODA in the transcriptional regulatory reporter system pDODA-Tr3.

[0082] Table 5. Proportion of yellow cells 12 hours after transformation

[0083] Percentage of yellow cells (%) Standard deviation pDODA-Tr3+pDBD 0 0 pDODA-Tr3+pDBD-VP16 13.39 4.62

[0084] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A visual reporter plasmid for plant cell transcriptional regulation, characterized in that, The visualization reporter plasmid is composed of cis-acting elements, a mini35S promoter, and a DODA genome.

2. The visualization report plasmid according to claim 1, characterized in that, The upstream of the cis-acting element also includes a Ubi promoter.

3. A visual detection method for transcriptional regulation in plant cells, characterized in that, The detection method includes the following steps: (1) The visual reporter plasmid described in claim 1 is co-transformed with a plasmid expressing a transcriptional activating protein / a plasmid expressing a transcriptional repressor protein into plant protoplasts; (2) Twelve hours after transformation, the change in the proportion of yellow cells was observed to determine the result of transcriptional regulation.

4. The detection method according to claim 3, characterized in that, The observation method involved using an optical microscope and comparing the results with a control group.

5. The application of the visualization reporter plasmid as described in claim 1 or 2 and the detection method as described in claim 3 or 4 in the regulation of cis-acting element function by transcription factors.

6. The application of the visualization reporter plasmid of claim 1 or 2 and the detection method of claim 3 or 4 in screening transcriptional activation or repression domains.

7. The application of the visualization reporter plasmid of claim 1 or 2 and the detection method of claim 3 or 4 in screening transcription factor binding elements.

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