A method for improving the sensitivity of a dual luciferase reporter gene system based on transcription level detection

Abstract

The application provides a method for improving the sensitivity of a dual-luciferase reporter gene system based on transcription level detection, and the method directly detects the mRNA transcript level of the dual-luciferase reporter gene by adopting RT-qPCR, replaces traditional enzyme activity detection, significantly improves the sensitivity and signal-to-noise ratio of transcription factor and promoter interaction detection, realizes efficient and stable detection of the transcription factor regulation effect, and solves the technical defects of insufficient sensitivity and poor recognition ability of weak regulation signals caused by the traditional dual-luciferase experiment which depends on enzyme activity detection.

Description

Technical Field

[0001] This invention relates to the field of molecular biology and gene function research technology, and in particular to a method for improving the sensitivity of a dual-luciferase reporter gene system based on transcriptional level detection. Background Technology

[0002] The dual-luciferase reporter gene system is one of the core technologies in the current biological field for verifying the interaction between transcription factors and promoters. This system typically uses firefly luciferase (Fluc) as the reporter gene and renal luciferase (Rluc) as the internal control gene. The regulatory role of transcription factors is indirectly reflected by detecting the ratio of their enzyme activities, i.e., Fluc / Rluc.

[0003] However, existing technologies have several significant problems: 1. Limited sensitivity: Enzyme activity detection relies on the post-translational activity state of proteins, which is easily affected by cell state, translation efficiency, protein stability, and detection conditions, leading to large signal fluctuations and insensitivity to weak regulatory signals. 2. Limited applicability: When transcription factor regulation is weak, changes in enzyme activity ratios are unlikely to show statistical significance. For weak interactions or weak promoter activity, traditional enzyme activity detection may affect the reliability of the results, especially for plants. 3. Protein expression is a multi-level, precisely regulated process, and the relationship between transcriptional level and final protein functional activity is not a simple linear one. Therefore, even if transcription factors cause changes in promoter transcription efficiency, these changes may be affected and masked by many downstream variables in the final enzyme activity reading. This makes traditional methods relying on enzyme activity detection inherently have a low signal-to-noise ratio when detecting weak, transient regulatory effects, making it difficult to reveal the true intensity of transcriptional regulation. Summary of the Invention

[0004] In view of this, the present invention provides a method for improving the sensitivity of a dual-luciferase reporter gene system based on transcriptional level detection. By using RT-qPCR to directly detect the mRNA transcript level of the dual-luciferase reporter gene, replacing the traditional enzyme activity detection, the sensitivity and signal-to-noise ratio of transcription factor and promoter interaction detection are significantly improved, achieving efficient and stable detection of transcription factor regulatory effects. This solves the technical defects of traditional dual-luciferase experiments that rely on enzyme activity detection, resulting in insufficient sensitivity and poor ability to recognize weak regulatory signals.

[0005] The technical solution of this invention is implemented as follows: In a first aspect, the present invention provides a method for improving the sensitivity of a dual-luciferase reporter gene system based on transcriptional level detection, comprising the following steps: S1. The reporter vectors carrying the target promoter and firefly luciferase and Renilla luciferase, and the expression vector carrying the transcription factor to be tested, were transfected into cells, and then the total RNA of the cells was extracted and reverse transcribed into cDNA. S2. The mRNA transcript levels of firefly luciferase and Renilla luciferase were detected separately. The mRNA transcript of the Renilla luciferase gene was used as an internal reference to calculate the relative expression level of firefly luciferase mRNA, thereby detecting the fold change in the regulation of the target promoter activity by the test transcription factor.

[0006] Based on the above technical solutions, the cells further include plant cells or animal cells.

[0007] Based on the above technical solutions, the transfection method further includes any one of Agrobacterium-mediated transformation, liposome transfection, or electroporation.

[0008] Based on the above technical solution, step S2 further includes: using quantitative real-time PCR to detect the mRNA transcript levels of firefly luciferase and kidney luciferase, respectively; The primer pairs for quantitative real-time PCR detection of the firefly luciferase include SEQ ID NO.1 and SEQ ID NO.2; the primer pairs for quantitative real-time PCR detection of the kidney luciferase include SEQ ID NO.3 and SEQ ID NO.4.

[0009] Compared with the prior art, the present invention has the following beneficial effects: (1) Significantly improved detection sensitivity: The present invention can directly detect the relative changes in mRNA levels more significantly and directly than changes in enzyme activity, thereby improving the discrimination and reliability of the experiment; (2) Increased signal-to-noise ratio: This invention reduces errors introduced by factors such as differences in protein translation efficiency and protein stability, resulting in higher repeatability of the results; (3) Greater applicability: This invention has higher adaptability for scenarios such as low abundance transcription factors, weak promoters, and weak regulation; (4) Operation and cost advantages: This invention does not require the purchase of expensive dual-luciferase detection kits, and can be completed using conventional RNA extraction and qPCR equipment in the laboratory, thus reducing experimental costs. Attached Figure Description

[0010] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0011] Figure 1 This is a comparison of the effects of transcription factor / promoter interaction between Comparative Example 1 and Example 1. Detailed Implementation

[0012] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0013] This invention provides a method for improving the sensitivity of a dual-luciferase reporter gene system based on transcriptional level detection, comprising the following steps: (1) Vector construction: including reporter vectors carrying the target promoters of firefly luciferase (Fluc) and Renilla luciferase (Rluc) as well as expression vectors of transcription factors; (2) Transfection and culture: The above vectors were co-transfected into suitable cell materials according to the experimental requirements, and cultured under suitable conditions for a period of time; (3) RNA extraction and reverse transcription: high-quality total RNA was obtained using an RNA extraction kit and reverse transcribed into cDNA; (4) RT-qPCR detection: Specific primers for Fluc and Rluc mRNA were designed and synthesized. The relative expression levels of Fluc and Rluc were quantitatively detected by RT-qPCR, and the changes in the relative expression level of Fluc mRNA were calculated.

[0014] (5) Data analysis: The data were processed using a relative quantification algorithm to obtain the fold of regulation of promoter activity by transcription factors.

[0015] Example 1 1. Carrier Construction Using pGreenII-62sk as the backbone, AtMYB75 and AhZNF expression vectors were constructed to obtain pGreenII-62sk-AtMYB75 and pGreenII-62sk-AhZNF transcription factor expression vectors. The promoters AtDRFpro and AhKASpro were constructed into pGreenII 0800 vectors containing firefly luciferase (Fluc) and Renilla luciferase (Rluc), respectively, to initiate the expression of FLuc, resulting in reporter vectors pGreenII 0800-AtDFRpro-Fluc and pGreenII 0800-AhKASpro-Fluc. Among them, Rluc, driven by the 35S promoter, is used as an internal reference gene.

[0016] 2. Agrobacterium transformation and culture The recombinant plasmids were transferred into Agrobacterium GV3101 (pSoup-p19) by electroporation, plated on YEB solid medium containing the corresponding antibiotics, and cultured at 28°C for 3 days.

[0017] Single colonies were picked and inoculated into 10 mL of YEB liquid medium (yeast extract 4 g / L, mannitol 10 g / L, NaCl 0.1 g / L, MgSO4·7H2O 0.2 g / L, K2HPO4 0.5 g / L, pH 7.0), and cultured at 28℃ with shaking at 200 rpm until OD reached. 600 ≈0.6. Collect bacterial cells by centrifugation at 4000 rpm for 5 min, resuspend in 10 mM MgCl2 (containing 120 μM acetylsylcholine), and adjust OD. 600 Set to 1.0 and let stand at 4℃ for 3 hours.

[0018] 3. Leaf injection and culture Agrobacterium bacterial suspensions carrying different plasmids were mixed at a volume ratio of 1:1 (transcription factor expression bacterial suspension: reporter vector bacterial suspension), and the specific groupings are shown in Table 1 below: Table 1 Grouping of bacterial suspensions

[0019] Select healthy tobacco plants and inject them into the lower epidermis of the leaves using a 1mL syringe (without the needle). Inject three leaves per plant, and inject three areas on each leaf, marking each area clearly. After injection, place the plants in low light (light intensity approximately 50 μmol·m²). -2 ·s -1 Incubate at 25℃ for 3 days.

[0020] 4. Leaf collection Leaves from each injection region were collected separately, with three biological replicates per sample. Each leaf was divided into two parts: one part was used for RNA extraction in this embodiment, and the other part was used for luciferase activity detection in Comparative Example 1.

[0021] 5. RNA extraction and reverse transcription Total RNA was extracted from the tobacco leaves using a plant RNA extraction kit (NanoMagbio, NMR0311) and reverse transcribed into cDNA (Evo M-MLV Reverse Transcription Premix Kit, AG11728). The cDNA was then diluted to 200 ng / μL.

[0022] 6. qPCR detection The expression levels of Fluc and Rluc were detected by qPCR, with three replicates for each sample. Two [representations / methods] were used. -ΔΔCt The relative expression level was calculated using the method with Rluc mRNA as an internal reference and the control group (empty vector) as a calibration sample.

[0023] The qPCR detection primers for firefly luciferase are designated as Fluc-F and Fluc-R, respectively; the qPCR detection primers for kidney luciferase are designated as Rluc-F and Rluc-R, respectively. The nucleotide sequences of Fluc-F, Fluc-R, Rluc-F, and Rluc-R are shown in SEQ ID NO.1-4, as shown in Table 2 below.

[0024] Reaction system: 2× SYBR qPCR Master Mix 10 μl, primer F 1 μl, primer R 1 μl, cDNA template 1 μl, RNase Free water 7 μl, total 20 μl; Reaction conditions: 95℃ for 30s; 95℃ for 5s, 60℃ for 30s, 40 cycles; 95℃ for 15s, 60℃ for 60s, 95℃ for 0.05℃ / s.

[0025] Table 2 qRCR primer sequences

[0026] Comparative Example 1 This comparative example uses traditional luciferase activity detection, including the following steps: The procedure was performed according to the instructions of the Dual Luciferase Reporter Assay Kit (Vazyme DL101-01). 100 mg of leaf samples were taken, and 1×Cell Lysis Buffer was added. The samples were ground by shaking at 60 Hz for 90 sec, followed by shaking lysis for 5 min. The samples were then centrifuged at 12,000 g at room temperature for 5 min, and the supernatant was collected. 20 μL of the supernatant was added to 100 μL of Luciferase Substrate, and Fluc activity was immediately detected using a fluorescence detector (PerkinElmer VICTOR Nivo™).

[0027] Then, 100 μL of freshly prepared Renilla substrate working solution was added, and Rluc activity was immediately measured. The Fluc / Rluc ratio was calculated, and the fold change was calculated using the control group as a baseline.

[0028] Performance testing The detection results in Example 1 and the conventional luciferase activity detection results in Comparative Example 1 are as follows: Figure 1 As shown.

[0029] Depend on Figure 1 As shown in Comparative Example 1, the Fluc / Rluc ratio in the experimental group of AtMYB75 + AtDFRpro was 14.87 times higher than that in the control group (P<0.01), indicating that AtMYB75 significantly activated AtDFRpro. In the case of AhZNF + AhKASpro, there was no statistically significant difference in the Fluc / Rluc ratio between the experimental group and the control group (P>0.05), indicating that no regulatory effect of AhZNF on AhKASpro was detected at the enzyme activity level.

[0030] In Example 1, the relative expression level of Fluc mRNA in AtMYB75 + AtDFRpro was 22.16 times higher than that in the control group (P<0.001), significantly higher than the 14.87 times (P<0.01) observed by enzyme activity detection, indicating higher sensitivity in transcriptional level detection. The relative expression level of Fluc mRNA in AhZNF + AhKASpro was 1.46 times lower than that in the control group (P<0.05), a significant difference. However, enzyme activity detection did not show a significant difference, indicating that transcriptional level detection can identify weak regulatory signals.

[0031] In summary, this invention provides a method for improving the sensitivity of a dual-luciferase reporter gene system based on transcriptional level detection. By using RT-qPCR to directly detect the mRNA transcript level of the dual-luciferase reporter gene, replacing the traditional enzyme activity detection, it significantly improves the sensitivity and signal-to-noise ratio of transcription factor and promoter interaction detection, achieving efficient and stable detection of transcription factor regulatory effects. This solves the technical defects of traditional dual-luciferase experiments that rely on enzyme activity detection, resulting in insufficient sensitivity and poor ability to recognize weak regulatory signals.

[0032] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for improving the sensitivity of a dual-luciferase reporter gene system based on transcriptional level detection, characterized in that, Includes the following steps: S1. The reporter vectors carrying the target promoter and firefly luciferase and Renilla luciferase, and the expression vector carrying the transcription factor to be tested, were transfected into cells, and then the total RNA of the cells was extracted and reverse transcribed into cDNA. S2. The mRNA transcript levels of firefly luciferase and Renilla luciferase were detected separately. The mRNA transcript of the Renilla luciferase gene was used as an internal reference to calculate the relative expression level of firefly luciferase mRNA, thereby detecting the fold change in the regulation of the target promoter activity by the test transcription factor.

2. The method for enhancing the sensitivity of a dual-luciferase reporter gene system based on transcriptional level detection as described in claim 1, characterized in that, The cells include plant cells or animal cells.

3. The method for enhancing the sensitivity of a dual-luciferase reporter gene system based on transcriptional level detection as described in claim 1, characterized in that, The transfection method includes any one of Agrobacterium-mediated transformation, liposome transfection, or electroporation.

4. The method for improving the sensitivity of a dual-luciferase reporter gene system based on transcriptional level detection as described in claim 1, characterized in that, Step S2 includes: using quantitative real-time PCR to detect the mRNA transcript levels of firefly luciferase and kidney luciferase, respectively; The primer pair for quantitative real-time PCR detection of the firefly luciferase includes SEQ ID NO.1 and SEQ ID NO.2; the primer pair for quantitative real-time PCR detection of the kidney luciferase includes SEQ ID NO.3 and SEQ ID NO.4.

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