Cascade amplification gene expression regulation and control device or system based on GAL4 / UAS system and application of cascade amplification gene expression regulation and control device or system

Abstract

The invention belongs to the technical field of environmental detection and biology, and particularly discloses a cascade amplification gene expression regulation device or system based on a GAL4 / UAS system and application of the cascade amplification gene expression regulation device or system based on the GAL4 / UAS system. An estrogen receptor reaction element ERE responds to an estrogen signal, GAL4 fusion protein expression is driven, an upstream activation sequence UAS is combined to form a transcription complex, and the GAL4 / UAS cascade amplification gene expression regulation device or system based on the GAL4 / UAS system is obtained. And the expression of a downstream reporter gene (such as mScarlet3) or a target gene is amplified in a cascade manner. The system realizes high-sensitivity estrogen detection (the detection limit reaches 10 <-1 > pM) in mammalian cells, and an environmental estrogen biosensor can be constructed. The kit comprises a regulation and control system and a transfection reagent, is suitable for quantitatively monitoring fluorescence intensity change so as to evaluate estrogen activity, and has the technical advantages of high efficiency, sensitivity and modularization.

Description

Technical Field

[0001] The present invention belongs to the field of environmental detection and biotechnology, and in particular relates to a cascade amplification gene expression control device or system based on a GAL4 / UAS system and applications thereof. Background Art

[0002] As environmental pollution becomes increasingly severe, monitoring and early warning of persistent organic pollutants, such as environmental estrogens, have become crucial. Environmental estrogens are a class of exogenous chemicals with estrogen-like activity that are widely present in water, soil, and the atmosphere, posing a potential threat to ecosystems and human health. Therefore, effective monitoring of the presence and changes in environmental estrogens is essential to prevent their harmful effects.

[0003] However, existing monitoring technologies have many limitations in practical applications. On the one hand, traditional chemical analysis methods require the use of various chemical reagents and expensive precision instruments, such as high-performance liquid chromatography and gas chromatography-mass spectrometry, which not only increases the cost of detection but also limits its widespread application in resource-limited areas. On the other hand, these technologies usually require professional technicians to operate, and the detection process is complicated and time-consuming, making it difficult to achieve real-time and rapid monitoring of environmental estrogens, and unable to meet the high-frequency and large-area requirements of environmental monitoring.

[0004] In recent years, biomonitoring technologies have gained increasing attention. Xu et al. described a bioreporter system based on human cells that can autonomously generate bioluminescence. Using a human embryonic kidney cell line (HEK293), they enabled the cells to autonomously generate signals and bioluminescence under the control of a gene based on an estrogen response element (ERE), thereby enabling the detection of endocrine disruptors such as estrogen. This method reduces the number, steps, and cost of sample preparation required for analysis, providing new insights into environmental estrogen monitoring.

[0005] However, Xu et al.'s method still has some shortcomings. First, in terms of visualization, the signal output of this method is not intuitive and obvious, which is not conducive to rapid on-site judgment and data reading. Second, its sensitivity is moderate, which may have certain limitations for the detection of low concentrations of environmental estrogens and cannot meet the demand for high-sensitivity detection of trace environmental estrogens.

[0006] In summary, there is an urgent need for an efficient, economical, practical and highly sensitive persistent organic pollutant monitoring technology to overcome the shortcomings of existing technologies, achieve rapid, accurate and real-time monitoring of pollutants such as environmental estrogens, and provide strong technical support for environmental protection and pollution prevention and control. Summary of the Invention

[0007] The present invention aims to address the above-mentioned problems by providing a cascade amplification gene expression control device or system based on the GAL4 / UAS system and its applications. Specifically, by constructing a transgenic red fluorescent protein mammalian cell line, effective monitoring of persistent environmental organic pollutants such as E2 and BPA is achieved in the environment. This study establishes a red fluorescent cell line that is more sensitive to environmental estrogens by tandemly analyzing the specific binding sequence ERE of the estrogen receptor ER and enhancing the red fluorescence signal using the Gal4 / UAS amplification system. Furthermore, the constructed transgenic red fluorescent cell line was exposed to extracts of different Chinese herbal medicines, analyzing the agonistic effects of estrogens in Chinese herbal medicines on estrogen receptors and their relationship to cell fluorescence intensity. These studies have substantially promoted the practical application of transgenic fluorescent cell lines in environmental monitoring.

[0008] In order to achieve the above object, the technical solution adopted by the present invention is as follows:

[0009] A cascade amplification gene expression control device or system based on the GAL4 / UAS system, comprising:

[0010] (a) a promoter regulatory unit comprising at least one estrogen receptor response element (ERE);

[0011] (b) a GAL4 expression unit operably linked to the promoter regulatory unit, the GAL4 expression unit encoding a fusion protein of a GAL4 binding protein comprising a nuclear localization signal NLS and a GAL4 activator protein;

[0012] (c) an upstream activation sequence UAS element located downstream of the GAL4 expression unit;

[0013] (d) a target gene expression cassette operably linked to the UAS element, comprising a reporter gene and / or a target gene to be enhanced;

[0014] The GAL4 fusion protein forms a transcription complex after binding to UAS, and enhances the expression of downstream genes through cascade amplification.

[0015] It is further described that the promoter regulatory unit comprises more than three ERE elements connected in series and a minimal promoter.

[0016] It is further described that the GAL4 activator protein comprises a VP16 or p65 activation domain.

[0017] It is further specified that the reporter gene and / or the target gene to be enhanced is the mScarlet3 red fluorescent protein gene.

[0018] It is further specified that the nuclear localization signal is selected from SV40 large T antigen NLS or nucleoplasmin NLS.

[0019] Further explanation, it is applicable to mammalian cell lines, including but not limited to MCF7 and 293T cells.

[0020] The present invention also provides a method for detecting environmental estrogens, using the above-mentioned device or system to quantify estrogen activity by monitoring changes in the fluorescence intensity of mScarlet3.

[0021] The method further illustrates that the detection sensitivity is 10 -4 nM-10nM.

[0022] The present invention also provides an application of the above-mentioned device or system in constructing an environmental estrogen biosensor.

[0023] The present invention also provides a kit comprising the above-mentioned gene expression regulation device or system and a cell transfection reagent.

[0024] The cell transfection reagent is a common reagent used by those skilled in the art, such as Lipofectamine 2000 / 3000 (Thermo Fisher), EZ transfection reagent (Invitrogen), Opti-MEM (Invitrogen), etc.

[0025] In summary, due to the adoption of the above technical solution, the beneficial effects of the present invention are:

[0026] The cascade amplification gene expression control device or system based on the GAL4 / UAS system of the present invention specifically uses the strong estrogen E2 and the weak estrogen BPA to achieve the desired effect in 10 -4 The fluorescence of 293T and MCF7 cells transfected with the Gal4 / UAS system was enhanced in the range of nM-10nM, and the fluorescence intensity gradually increased with the extension of the induction time of environmental estrogen, reaching a peak at 24-48h. In the real environment, the transgenic fluorescent cell line of this application can still sensitively and effectively monitor environmental estrogen, with a detection limit of 10 -4 This application successfully detected estrogens in the environment for the first time using Gal4-UAS-mScarlet3. This is a sensitive, efficient, low-cost, visual and high-throughput detection method, providing a new direction for the detection of persistent organic pollutants in the environment. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 Schematic diagram of the construction of the system of the present invention (ERE-UAS-GAL4-mScarlet3).

[0028] Figure 2 Flowchart of the signal transduction mechanism of the present invention.

[0029] Figure 3 This is a comparison diagram of the dose-effect curve of the present invention. Figure 3 A is the fluorescence enhancement factor of cells after transfection of ERE-UAS-GAL4-mScarlet3 system in 293T cells and induction with different concentrations of E2. Figure 3 B is the fluorescence enhancement factor of cells after transfection of ERE-UAS-GAL4-mScarlet3 system in 293T cells and induction with different concentrations of BPA. Figure 3 C is the red fluorescence image of cells after transfection of ERE-UAS-GAL4-mScarlet3 system in 293T cells and induction with different concentrations of E2. Figure 3 D is the red fluorescence image of cells after being transfected with ERE-UAS-GAL4-mScarlet3 system and induced with different concentrations of BPA. Figure 3 E is the fluorescence enhancement factor of cells after transfection of ERE-UAS-GAL4-mScarlet3 system with different concentrations of E2. Figure 3 F is the fluorescence enhancement factor of MCF7 cells after transfection with the ERE-UAS-GAL4-mScarlet3 system and induction with different concentrations of BPA.

[0030] Figure 4 This is the time course response curve diagram of the present invention (dynamic changes from 0 to 72 hours). Figure 4 A is the fluorescence enhancement factor in 293T cells after transfection of the ERE-UAS-GAL4-mScarlet3 system and induction with E2 (0.1 nM) for different time periods. Figure 4 B is the fluorescence enhancement factor of cells after transfection of ERE-UAS-GAL4-mScarlet3 system in MCF7 cells and induction with E2 (0.1nM) for different time periods. Figure 4 C is the red fluorescence image of cells after being transfected with ERE-UAS-GAL4-mScarlet3 system in 293T cells and induced with E2 (0.1nM) for different time periods. Figure 4 D is the red fluorescence image of cells after being transfected with the ERE-UAS-GAL4-mScarlet3 system in MCF7 cells and induced with E2 (0.1 nM) for different time periods.

[0031] Figure 5 This is an application diagram of the present invention in a real environment. Figure 5 A is a schematic diagram of the extraction process of Chinese herbal medicines in four environments. Figure 5 B is the experiment on the agonistic effect of these four extracts on estrogen receptor ER. Figure 5C is the fluorescence enhancement factor after 293T cells were transfected with ERE-UAS-GAL4-mScarlet3 and induced with Psoralen at different drug concentrations. Figure 5 D is the fluorescence enhancement factor after MCF7 cells were transfected with ERE-UAS-GAL4-mScarlet3 and induced with Psoralen at different drug concentrations. Figure 5 E is a red fluorescence image of cells after being transfected with ERE-UAS-GAL4-mScarlet3 in 293T cells and induced with Psoralen at different concentrations. Figure 5 F is a red fluorescence image of cells after being transfected with ERE-UAS-GAL4-mScarlet3 in MCF7 and induced with Psoralen at different concentrations. DETAILED DESCRIPTION

[0032] To make the above-mentioned objects, features, and advantages of the present invention more readily apparent, specific embodiments of the present invention are described in detail below with reference to the accompanying drawings. The following description sets forth numerous specific details to facilitate a full understanding of the present invention. However, the present invention can be implemented in many other ways than those described herein, and those skilled in the art may make similar modifications without departing from the scope of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0033] Example 1:

[0034] A cascade amplification gene expression control device or system based on the GAL4 / UAS system, comprising:

[0035] (a) a promoter regulatory unit comprising at least one estrogen receptor response element (ERE); wherein the promoter regulatory unit comprises three or more tandem ERE elements and a minimal promoter;

[0036] (b) a GAL4 expression unit operably linked to the promoter regulatory unit, the GAL4 expression unit encoding a fusion protein of a GAL4 binding protein comprising a nuclear localization signal NLS and a GAL4 activator protein; the GAL4 activator protein comprises a VP16 or p65 activation domain; the nuclear localization signal is selected from SV40 large T antigen NLS or nucleoplasmin NLS;

[0037] (c) an upstream activation sequence UAS element located downstream of the GAL4 expression unit;

[0038] (d) a target gene expression cassette operably linked to the UAS element, comprising a reporter gene and / or a target gene to be enhanced; wherein the reporter gene and / or the target gene to be enhanced is the mScarlet3 red fluorescent protein gene;

[0039] The GAL4 fusion protein forms a transcription complex after binding to UAS, and enhances the expression of downstream genes through cascade amplification.

[0040] Example 2:

[0041] A method for detecting environmental estrogens, using the device or system described in Example 1, quantifying estrogen activity by monitoring changes in mScarlet3 fluorescence intensity, with a detection sensitivity of 10 -4 nM-10nM.

[0042] Example 3:

[0043] A kit comprising the gene expression control device or system described in Example 1 and a cell transfection reagent. The cell transfection reagent is Lipofectamine 2000 / 3000 (Thermo Fisher), EZ transfection reagent (Invitrogen), Opti-MEM (Invitrogen), etc.

[0044] Example 4:

[0045] test:

[0046] 1. Materials and Methods

[0047] 1.1 Reagents, antibodies, and chemicals

[0048] Transfection reagents, including Lipofectamine 3000 and Opti-MEM, were purchased from Invitrogen. EZ Cell Transfection Reagent II was purchased from Life-iLab Biotechnology (Shanghai, China). A plasmid extraction kit was purchased from Tiangen Biotechnology (Beijing, China). 10× RIPA lysis buffer was purchased from Merck Millipore. The dual-luciferase reporter assay system was purchased from Promega. ER primary and secondary antibodies were purchased from Santa Cruz Biotechnology Inc. 17β-estradiol (E2) and bisphenol A were purchased from MCE (Shanghai, China).

[0049] 1.2 Development of estrogen-responsive MCF7-ERE / Gal4-mScarlet3 and HEK293-ERE / Gal4-mScarlet3 bioreporters

[0050] The Gal4-UAS system was first established in the model organism Drosophila melanogaster. Since then, it has been widely used by researchers, successfully establishing a large number of different Gal4 expression lines in Drosophila.

[0051] Three tandem repeats of ERE, the Gal4 DNA binding domain, and five tandem repeats of the upstream activation sequence (UAS) were inserted into the mammalian vector pPB to amplify the expression of luciferase or mScarlet3. These dual-module amplification circuits, named 3XERE-Gal4 / UAS-mScarlet3, were transfected into HEK293T or MCF7 cell lines to verify the amplification effect of the UAS system.

[0052] The ERE sequence is 5′-GTCAGGTCACTGTGACCTGAT-3′,

[0053] The TATA sequence is 5′-TAGGGTATATAATGGAAGCTCGACTTCCAG-3′,

[0054] The UAS sequence is 5′-CGGAGTACTGTCCTCCG-3′.

[0055] 1.3 Cell culture and plasmid transfection

[0056] MCF7 and 293T cells were cultured in Dulbecco's modified Eagle's medium (DMEM, Invitrogen, Carlsbad, USA) containing 10% (v / v) fetal bovine serum (FBS, Gemini BioProducts, Woodland, USA) and 1% streptomycin-penicillin mixture (Bio-U Biotech, Shanghai, China) at 37°C and 5% CO2. 6 × 10 4 Cells were seeded into 96-well plates for luciferase reporter gene activity detection and fluorescence value measurement; 8×10 5 Cells were cultured in 3.5 cm dishes and analyzed by Western blot. Plasmids were transfected into MCF7 or 293T cells using Lipofectamine 3000 or EZ Cell Transfection Reagent II. Cells were further analyzed 48 hours after transfection.

[0057] 1.4 Fluorescence imaging and fluorescence value measurement

[0058] 48 hours after plasmid transfection, cells were treated with E2 or BPA for 24 hours. Changes in mScarlet3 fluorescence were observed under a fluorescence microscope and fluorescence intensity was measured using a fluorescence detector. Cells were washed with 100 μl of PBS. Red fluorescence intensity was measured using excitation and emission wavelengths of 560 nm and 610 nm, respectively. Images were simultaneously captured using a fluorescence microscope with a 10x objective.

[0059] 1.5 Actual samples in the testing environment

[0060] Four substances were randomly selected from the environment, including Psoralen, Licorice flavonoid A, Naringenin, Daidzein ( Figure 5 First, they were exposed to 293T-3XERE-luc at a concentration of 10 μM, and their relative luciferase activities were detected using a dual-luciferase reporter gene assay. Then, two Psoralen substances with higher estrogen receptor agonist effects were selected ( Figure 5 C and D), exposed them to our GAL4 cell sensors, and analyzed the sensitivity of the synthesized biosensor in detecting estrogen in a real environment using fluorescence values ​​and fluorescence images.

[0061] 1.6 Statistical analysis

[0062] Statistical analyses were performed using SPSS, ORIGIN, and GraphPad. Limits of detection and quantification could be reliably distinguished based on "analytical noise." Furthermore, a Z factor was introduced to assess the accuracy of the detection limit. All experiments were repeated at least three times. Data are presented as mean ± SD and compared using independent-sample t-tests and one-way analysis of variance. A P value of less than 0.05 was considered statistically significant.

[0063] 2. Results

[0064] Figure 1 Figure 1 is a schematic diagram of the plasmid construction of the present invention. The signal output is significantly enhanced by the cascade amplification mechanism of the ERE-UAS-GAL4-mScarlet3 system. Initially, three tandem ERE repeat sequences and a minimal TATA promoter were used to drive the expression of downstream target genes. In order to amplify the signal, a positive feedback circuit was introduced through self-responsive Gal4 expression. In addition, a biosensor was designed to express the bright and stable red fluorescent protein mScarlet3, making it very suitable for fluorescence detection. Next, this system was verified in human cell lines (HEK293T and MCF7) to enable it to serve as a platform for high-throughput screening of EDCs detection.

[0065] Figure 2 This is a flow chart of the signal transduction mechanism of the present invention. After entering the cell nucleus through the nuclear pore, estrogen binds to the ligand-binding domain (LBD) of the nuclear receptor (ER). This triggers the ER's DNA-binding domain (DBD) to bind to the estrogen response element (ERE) upstream of the target gene. Under the action of RNA polymerase and related cofactors, transcriptional activation of the target gene is initiated. Once the target gene is transcribed into protein, it alters cellular function or can serve as a cellular indicator for detecting estrogen in the environment.

[0066] Table 1-2 and Figure 3 AB shows that the fluorescence enhancement factor of 293T cells after induction with different concentrations of E2 and different concentrations of BPA first increases and then decreases. Figure 3 AD can be seen in 293T cells, E2 (0.01nM) and BPA (10 -5 After 24 hours of treatment with 500 nM, the fluorescence value of mScarlet3 reached a peak value ( Figure 3 AB), the sensitivity is about 1000 times higher than that of the traditional system. Table 3-4 and Figure 3 EF shows that after MCF7 cells were transfected with the ERE-UAS-GAL4-mScarlet3 system and induced with different concentrations of E2, the fluorescence enhancement factor of the cells first increased and then decreased. Figure 3 EF showed that the detection peaks in MCF7 cells were 0.1nM (E2) and 0.01nM (BPA), respectively.

[0067] Table 1: Fluorescence enhancement folds in 293T cells after transfection with the ERE-UAS-GAL4-mScarlet3 system and induction with different concentrations of E2

[0068] Figure 3A mean <![CDATA[10 -4 ]]> 1.574 <![CDATA[10 -3 ]]> 1.482 <![CDATA[10 -2 ]]> 1.968 <![CDATA[10 -1 ]]> 1.870 <![CDATA[10 0 ]]> 1.547 <![CDATA[10 1 ]]> 1.371

[0069] Table 2: Fluorescence enhancement folds in 293T cells after transfection with the ERE-UAS-GAL4-mScarlet3 system and induction with different concentrations of BPA

[0070] Figure 3B mean <![CDATA[10 -7 ]]> 1.007 <![CDATA[10 -6 ]]> 1.165 <![CDATA[10 -7 ]]> 1.707 <![CDATA[10 -4 ]]> 1.441 <![CDATA[10 -3 ]]> 1.443 <![CDATA[10 -2 ]]> 1.390 <![CDATA[10 -1 ]]> 1.300 <![CDATA[10 0 ]]> 1.264

[0071] Table 3: Fluorescence enhancement folds in MCF7 cells after transfection with the ERE-UAS-GAL4-mScarlet3 system and induction with different concentrations of E2

[0072] Figure 3E mean <![CDATA[10 -7 ]]> 1.007 <![CDATA[10 -6 ]]> 1.165 <![CDATA[10 -5 ]]> 1.707 <![CDATA[10 -4 ]]> 1.441 <![CDATA[10 -3 ]]> 1.443 <![CDATA[10 -2 ]]> 1.390 <![CDATA[10 -1 ]]> 1.300 <![CDATA[10 0 ]]> 1.264

[0073] Table 4: Fluorescence enhancement folds in MCF7 cells after transfection with the ERE-UAS-GAL4-mScarlet3 system and induction with different concentrations of BPA

[0074] Figure 3F mean <![CDATA[10 -5 ]]> 1.033 <![CDATA[10 -4 ]]> 1.111 <![CDATA[10 -3 ]]> 1.163 <![CDATA[10 -2 ]]> 1.262 <![CDATA[10 -1 ]]> 1.210 <![CDATA[10 0 ]]> 1.199

[0075] Table 5 Figure 4 A shows that after the ERE-UAS-GAL4-mScarlet3 system was transfected into 293T cells and induced with E2 (0.1 nM) for different time periods, the fluorescence enhancement factor of the cells first increased and then decreased. Table 6 Figure 4B shows that after MCF7 was transfected with the ERE-UAS-GAL4-mScarlet3 system and induced with E2 (0.1nM) for different times, the fluorescence enhancement factor in the cells first increased and then decreased. Figure 4 AD found that as the treatment time of 0.1nM E2 increased, the fluorescence value of mScarlet3 in 293T and MCF7 gradually increased and reached a peak after 48h of treatment.

[0076] Table 5: Fluorescence enhancement folds in 293T cells after transfection of the ERE-UAS-GAL4-mScarlet3 system and induction with E2 (0.1 nM) for different times

[0077] Figure 4A mean 15min 0.624 30min 0.873 60min 0.760 3h 1.446 6h 1.712 12h 2.158 24h 3.173 48h 4.194 72h 3.404

[0078] Table 6: Fluorescence enhancement folds in MCF7 cells after transfection with the ERE-UAS-GAL4-mScarlet3 system and induction with E2 (0.1 nM) for different times

[0079] Figure 4B mean 15min 2.455 30min 2.474 60min 2.627 3h 2.838 6h 3.182 12h 3.229 24h 3.389 48h 3.614 72h 1.642

[0080] From Table 7, Figure 5 C shows the increasing trend of fluorescence enhancement after the cells were transfected with ERE-UAS-GAL4-mScarlet3 and induced with Psoralen at different concentrations. Figure 5 D shows the fluorescence enhancement trend of MCF7 cells after being transfected with ERE-UAS-GAL4-mScarlet3 and induced with Psoralen at different concentrations, which first increased and then decreased. Figure 5 The results of the detection of CF environmental herbal extracts (such as psoralen) showed that the Gal4-UAS system can effectively identify EDCs as low as 0.01nM.

[0081] Table 7: Fluorescence enhancement after cells were transfected with ERE-UAS-GAL4-mScarlet3 and induced with Psoralen at different drug concentrations in 293T cells

[0082] Figure 5C Psoralen <![CDATA[10 -7 ]]> 1.066 <![CDATA[10 -6 ]]> 1.041 <![CDATA[10 -5 ]]> 1.009 <![CDATA[10 -4 ]]> 1.101 <![CDATA[10 -3 ]]> 1.111 <![CDATA[10 -2 ]]> 1.138 <![CDATA[10 -1 ]]> 1.366 <![CDATA[10 0 ]]> 2.068 <![CDATA[10 1 ]]> 2.018 <![CDATA[10 2 ]]> 1.707

[0083] Table 8: Fluorescence enhancement after cells were transfected with ERE-UAS-GAL4-mScarlet3 and induced with Psoralen at different drug concentrations in MCF7 cells

[0084] Figure5D Psoralen <![CDATA[10 -7 ]]> 1.029 <![CDATA[10 -6 ]]> 1.043 <![CDATA[10 -5 ]]> 1.040 <![CDATA[10 -4 ]]> 1.175 <![CDATA[10 -3 ]]> 1.181 <![CDATA[10 -2 ]]> 1.216 <![CDATA[10 -1 ]]> 1.359 <![CDATA[10 0 ]]> 1.166 <![CDATA[10 1 ]]> 1.029 <![CDATA[10 2 ]]> 1.043

[0085] In summary, the present application uses strong estrogen E2 and weak estrogen BPA ranging from 0.0001nM to 10nM to sensitively induce the process of fluorescence from slow enhancement to continuous decay in the GAL4 system of 293T and MCF7 cells, reaching a peak value between 0.01 and 0.1nM. The fluorescence also showed a trend of first increasing and then decreasing after the pollutants acted on the cells at 0 minutes, 15 minutes, 30 minutes, 60 minutes, 3 hours, 6 hours, 12 hours, 24 hours, 48 ​​hours and 72 hours, reaching a peak value at 24 to 48 hours. The method of the present application is more sensitive, efficient, simple, and visual than the traditional method, and can be used for high-throughput screening in 96-well plates, providing efficient and reliable technical support for toxicity research and risk assessment of environmental pollutants.

[0086] The above-described embodiments merely illustrate several implementations of the present invention. While the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that variations and modifications are possible within the scope of the present invention, as would be apparent to those skilled in the art. These variations and modifications fall within the scope of the present invention. Therefore, the scope of the present invention shall be determined by the appended claims.

Claims

1. A cascade amplification gene expression control device or system based on the GAL4 / UAS system, characterized by: include: (a) a promoter regulatory unit comprising at least one estrogen receptor response element (ERE); (b) a GAL4 expression unit operably linked to the promoter regulatory unit, the GAL4 expression unit encoding a fusion protein of a GAL4 binding protein comprising a nuclear localization signal NLS and a GAL4 activator protein; (c) an upstream activation sequence UAS element located downstream of the GAL4 expression unit; (d) a target gene expression cassette operably linked to the UAS element, comprising a reporter gene and / or a target gene to be enhanced; The GAL4 fusion protein forms a transcription complex after binding to UAS, and enhances the expression of downstream genes through cascade amplification.

2. The cascade amplification gene expression control device or system based on the GAL4 / UAS system according to claim 1, characterized in that: The promoter regulatory unit comprises more than three ERE elements connected in series and a minimal promoter.

3. The cascade amplification gene expression control device or system based on the GAL4 / UAS system according to claim 1, characterized in that: The GAL4 activator protein comprises a VP16 or p65 activation domain.

4. The cascade amplification gene expression control device or system based on the GAL4 / UAS system according to claim 1, characterized in that: The reporter gene and / or target gene to be enhanced is the mScarlet3 red fluorescent protein gene.

5. The cascade amplification gene expression control device or system based on the GAL4 / UAS system according to claim 1, characterized in that: The nuclear localization signal is selected from SV40 large T antigen NLS or nucleoplasmin NLS.

6. The cascade amplification gene expression control device or system based on the GAL4 / UAS system according to claim 1, characterized in that: Suitable for mammalian cell lines, including but not limited to MCF7 and 293T cells.

7. A method for detecting environmental estrogens, characterized in that: The device or system according to any one of claims 1 to 6 is used to quantify estrogen activity by monitoring changes in the fluorescence intensity of mScarlet3.

8. The method according to claim 7, wherein the detection sensitivity reaches 10 -4 nM-10nM.

9. Use of the device or system according to any one of claims 1 to 6 in constructing an environmental estrogen biosensor.

10. A kit comprising the gene expression regulation device or system according to any one of claims 1 to 6 and a cell transfection reagent.

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