Method for inhibiting expression of target gene according to gene translation level and application of method

Abstract

The invention discloses a method for inhibiting expression of a target gene according to a gene translation level and application of the method, and relates to the technical field of gene engineering. The method comprises the following steps: constructing a recombinant expression vector for expressing a target protein; a translation inhibition element ATG-rich, ATGc02 or ATGc03 is inserted into the front end of an initiation codon of a target gene expression cassette of the recombinant expression vector. According to the invention, different translation inhibition elements are inserted at the translation level, so that different degrees of inhibition effects on target gene expression are realized, and the blank of accurate regulation and control of protein expression level at the translation level in the prior art is filled. The method provided by the invention has good universality and reliability in species such as different animals, plants or microorganisms, and provides powerful support for cross-species research and application.

Description

Technical Field

[0001] The present invention relates to the technical field of genetic engineering, and particularly relates to a method for inhibiting the expression of a target gene at the gene translation level and its application. Background Art

[0002] In the fields of life science and genetic engineering, the regulation of gene expression is a core link in understanding the physiological and pathological processes of organisms. Traditional methods for regulating gene expression mainly focus on the transcriptional level, such as regulating the promoter activity or intervening with transcription factors to adjust the transcriptional efficiency of genes. However, these methods often can only simply turn gene expression "on" or "off", and it is difficult to achieve precise gradient regulation of protein expression levels. In recent years, regulation at the translation level has gradually attracted attention because the translation process directly determines the conversion efficiency of gene transcripts into functional proteins.

[0003] Nevertheless, there is currently a lack of an efficient and general method to achieve gradient inhibition of target gene expression. Although some translation inhibitory elements have been discovered in the prior art, most of them can only achieve single-level inhibition and cannot achieve gradient regulation. In addition, the universality of these elements in different species has not been fully verified. Therefore, developing a method that can achieve gradient inhibition of target gene expression at the translation level has important scientific significance and practical value for molecular biology research, exploration of disease mechanisms, and biotechnological applications. Summary of the Invention

[0004] The present invention provides a method for inhibiting the expression of a target gene at the gene translation level and its application. This method realizes precise regulation of the expression of the target gene by inserting different translation inhibitory elements upstream of the CDS of the target gene. It can be used for studying gene functions, exploring disease mechanisms, and developing biotechnological applications, especially suitable for scenarios where precise regulation of protein expression levels is required. Specifically, it is achieved through the following techniques.

[0005] In the first aspect of the present invention, a method for inhibiting the expression of a target gene at the gene translation level is provided. A recombinant expression vector for expressing a target protein is constructed; a translation inhibitory element ATG-rich, ATGc02, or ATGc03 is inserted in front of the start codon (such as ATG) of the target gene expression frame (i.e., the gene sequence for expressing the target protein) in the recombinant expression vector; the nucleotide sequence of ATG-rich is as shown in SEQ ID NO.1, the nucleotide sequence of ATGc02 is atgcatgc, and the nucleotide sequence of ATGc03 is atgc.

[0006] In a second aspect of the present invention, there is provided a translational repression element for inhibiting the expression of a target gene, and the translational repression element is ATG-rich, ATGc02 or ATGc03; the nucleotide sequence of the ATG-rich is as shown in SEQ ID NO.1, the nucleotide sequence of the ATGc02 is atgcatgc, and the nucleotide sequence of the ATGc03 is atgc; in a recombinant expression vector for expressing a target protein, the translational repression element is inserted in front of the start codon of the target gene expression cassette.

[0007] In a third aspect of the present invention, there is provided a polynucleotide containing the above-mentioned translational repression element and a target gene expression cassette; the translational repression element is connected in front of the start codon of the target gene expression cassette.

[0008] In a fourth aspect of the present invention, there is provided a recombinant expression vector containing the above-mentioned polynucleotide.

[0009] In a fifth aspect of the present invention, there is provided a transfection system for transfecting the above-mentioned polynucleotide or the above-mentioned recombinant expression vector into a eukaryotic host cell.

[0010] Furthermore, the transfection system can select various method routes such as plasmid transfection of protoplasts, virus transfection method, etc., specifically including plasmid transfection reagents, liposome transfection reagents, lentiviruses, and retroviruses.

[0011] In a sixth aspect of the present invention, there is provided the use of the above-mentioned translational repression element in the preparation of a preparation for inhibiting the expression of a target gene.

[0012] In the present invention, Arabidopsis thaliana and tobacco are used as experimental research objects. Expression cassettes of two luciferases FLUC and RLUC are connected on the same recombinant vector, and three different translational repression elements are respectively connected in front of the FLUC expression cassette, while no translational repression element is connected in front of the RLUC expression cassette. By detecting and calculating the ratio of the luminescence signal intensities of the two luciferases, it is found that the three different translational repression elements ATG-rich, ATGc02 and ATGc03 provided by the present invention can effectively inhibit the expression of luciferase FLUC.

[0013] In the above experiments of the present invention, Arabidopsis thaliana and tobacco selected as model plants are commonly used test plants in current industrial experimental research. These two plants can be used as representative test objects for all plants and even all eukaryotes. Compared with the recombinant vector without inserting the 3 translational repression elements, selecting other plants will not affect the relative repression effect of the 3 translational repression elements on the translation of the target gene.

[0014] The luciferase selected in the above experiments of the present invention is also a commonly used luciferase under test in experimental research in the industry. This protein can be used as a representative object under test for all target proteins / target genes. Similarly, the selected basic vector pSYB is only used as an example and does not have a special limiting effect on the effect of the three translation inhibition elements on inhibiting the expression of the target gene. Compared with the recombinant vector without the insertion of the three translation inhibition elements, selecting different basic vectors and / or target proteins will not affect the relative inhibition effect of the three translation inhibition elements on the target protein / target gene.

[0015] Compared with the prior art, the advantages of the present invention are as follows:

[0016] 1. By inserting different translation inhibition elements at the translation level, the present invention realizes different degrees of inhibition effects on the expression of target genes, filling the gap in accurately regulating the protein expression level at the translation level in the prior art.

[0017] 2. Through experimental verification in Arabidopsis and tobacco protoplasts, it is proved that the method of the present invention has good universality and reliability in different species such as animals, plants or microorganisms, providing strong support for cross-species research and application.

[0018] 3. Using the dual-luciferase reporter vector, through the luminescence signal ratio of FLUC and RLUC, the effect of translation inhibition elements on the expression of target genes can be quantitatively analyzed, providing an efficient and accurate tool for studying the gene expression regulation mechanism.

[0019] 4. The method of the present invention has broad application prospects in molecular biology research, disease mechanism exploration and biotechnology applications. For example, in drug screening, it can be used to evaluate the regulatory effect of drugs on protein expression; in gene therapy, it can be used to accurately regulate the expression level of therapeutic genes; in genetic engineering, it can be used to optimize the expression efficiency of recombinant proteins. This method can also be used to study the translation regulation mechanism, develop new gene expression regulation elements and explore the fine regulation network of gene expression.

[0020] All in all, from the perspective of the translation level, the present invention provides a novel method for regulating the translation and expression of proteins, with broad application prospects and significant technical advantages. By using specific translation inhibition elements, precise control of the expression level of target genes can be achieved, thus playing an important role in the fields of molecular biology, genetics, synthetic biology, etc. Brief Description of the Drawings

[0021] Figure 1 It is a dual-luciferase reporter system containing three translation inhibition elements.

[0022] Figure 2Test results for detecting the inhibitory effects of different translation inhibitors using Arabidopsis protoplasts.

[0023] Figure 3 Test results for detecting the inhibitory effects of different translation inhibitors using tobacco protoplasts. Detailed implementation manners

[0024] The technical solutions of the present invention will be described clearly and completely below. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts belong to the scope of protection of the present invention.

[0025] In the following specific implementation manners provided by the present invention, in order to verify the inhibitory effects of three translation inhibitory elements on the expression of the target gene, protoplasts of Arabidopsis and tobacco are respectively selected for corresponding experiments. The steps of the experiment include the construction of a dual-luciferase reporter vector, the transformation of protoplasts, and the verification of the gradient inhibitory effect.

[0026] It should be noted that in the following specific implementation manners provided by the present invention, the selected luciferases FLUC and RLUC, the basic vector pSYB, and the plant-derived protoplasts are only examples for verifying the inhibitory effects of the three translation inhibitory elements on the expression of the target gene, and do not have any limiting effect on the above effects of the three translation inhibitory elements. That is to say, the selection of protoplasts of different plants / animals or other eukaryotes, the selection of different basic vectors for constructing recombinant expression vectors, and the selection of target proteins have no direct influence on the inhibitory effects of the three translation inhibitory elements on the expression of the target gene. As long as any one of the three translation inhibitory elements is connected to the front of the start codon of the target gene expression cassette in the recombinant expression vector, a significant inhibitory effect can be produced on the target protein.

[0027] 1. Construction of the dual-luciferase reporter vector

[0028] Construct a dual-luciferase reporter vector containing two independent luciferase expression cassettes; the first expression cassette encodes Firefly Luciferase (FLUC), and the second expression cassette encodes Renilla Luciferase (RLUC). The structural schematic diagram of the dual-luciferase reporter vector is as Figure 1 shown.

[0029] It can be seen that the FLUC gene expression cassette already contains the start codon ATG itself. By directly inserting three different translation inhibitory elements upstream of the start codon ATG in the FLUC gene expression cassette (i.e., the start codon ATG is directly connected to the translation inhibitory element without any other bases or linker sequences), the luminescence signal ratio of FLUC and RLuc is used to quantify the effect of the translation inhibitory element on the expression level of the target gene. Four vectors were constructed in the present invention, named pSYB-Dual-LUC (blank control), pSYB-Dual-LUC-ATG-rich, pSYB-Dual-LUC-ATGc02, and pSYB-Dual-LUC-ATGc03, respectively.

[0030] 2. Transformation of protoplasts

[0031] A transformation method based on Arabidopsis or tobacco protoplasts is provided for detecting the inhibitory effect of translation inhibitory elements. This method includes the preparation, transformation, and fluorescence value detection of protoplasts.

[0032] (1) Preparation of Arabidopsis protoplasts. After culturing Arabidopsis seedlings under specific conditions, protoplasts are isolated by enzymatic digestion and adjusted to a concentration of 2×10 5 cells / mL through steps such as filtration, centrifugation, resuspension, and washing.

[0033] (2) Transformation reaction. The constructed dual-luciferase reporter vector is transformed into Arabidopsis protoplasts by the PEG-mediated method and cultured under specific conditions.

[0034] (3) Fluorescence value detection. The activities of FLUC and RLuc are detected through cell lysis and luciferase reaction, and the luminescence signal ratio is calculated to evaluate the inhibitory effect of the translation inhibitory element.

[0035] 3. Verification of gradient inhibitory effect

[0036] The experimental results show that three different translation inhibitory elements (ATGc02, ATG-rich, and ATGc03) all exhibit significant gradient inhibitory effects in Arabidopsis and tobacco protoplasts. In Arabidopsis, the FLUC activities of the vectors pSYB-Dual-LUC-ATGc02, pSYB-Dual-LUC-ATG-rich, and pSYB-Dual-LUC-ATGc03 are 1 / 3.16, 1 / 9.58, and 1 / 32.74 times that of the control vector pSYB-Dual-LUC, respectively; in tobacco, they are 1 / 6.81, 1 / 8.22, and 1 / 9.77 times, respectively. This indicates that the translation inhibitory elements ATGc02, ATG-rich, and ATGc03 can achieve gradient inhibition of target gene expression and have good universality in different species.

[0037] Example 1: Detecting the inhibitory effect of translation inhibitory elements in Arabidopsis protoplasts

[0038] 1. Construction of a dual-luciferase vector

[0039] In the present invention, a dual-luciferase reporter vector was constructed, which contains two independent luciferase expression cassettes. The first expression cassette encodes Firefly Luciferase (FLUC), and the second expression cassette encodes Renilla Luciferase (RLUC). As Figure 1 shown, three different translation inhibitory elements in Table 1 were inserted in front of FLUC in the present invention.

[0040] Table 1 Information of three translation inhibitory elements

[0041]

[0042] By measuring the luminescence signal ratio of FLUC and RLUC, the influence of the translation inhibitory element on the expression level of the target gene can be quantified and precisely analyzed. The constructed vectors were named: pSYB-Dual-LUC, pSYB-Dual-LUC-ATG-rich, pSYB-Dual-LUC-ATGc02, and pSYB-Dual-LUC-ATGc03, respectively.

[0043] 2. Preparation and transformation of Arabidopsis protoplasts

[0044] (1) Culturing Arabidopsis seedlings: The Arabidopsis seedlings were cultured under the conditions of a photoperiod of 12 hours of light / 12 hours of darkness and a temperature of about 22°C for 25 - 30 days.

[0045] (2) Enzymatic digestion treatment: Take several leaves, add 5 - 10 mL of enzymatic digestion solution to ensure that the leaves are completely immersed. Incubate at 24°C for 3 hours for enzymatic digestion. The formula of the enzymatic digestion solution is shown in Table 2 below.

[0046] Table 2

[0047]

[0048] Adjust the pH to 5.8, heat at 55°C for 10 min, invert and mix three times, and then cool to room temperature and continue to add the solution shown in Table 3 below.

[0049] Table 3

[0050]

[0051] Add ddH 20 to 10 mL, filter-sterilize with a 0.22 μm filter membrane and use immediately after preparation.

[0052] (3) Filtration and centrifugation: Filter the enzymatically digested tissue using a 40 μm filter mesh, and then centrifuge at 300 rpm for 3 min to remove the supernatant.

[0053] (4) Resuspension and washing: Add 5 - 10 mL of W5 solution to resuspend the precipitate, and let it stand on ice for 30 min. Subsequently, centrifuge at 300 rpm for 4 min to remove as much supernatant as possible. The formula of the W5 solution is shown in Table 4 below.

[0054] Table 4

[0055]

[0056] Adjust the pH to 5.7 - 5.8, and finally add ddH 2 O to 100 mL; filter-sterilize with a 0.22 μm filter membrane and store at 4°C.

[0057] (5) Adjust the protoplast concentration: According to the amount of protoplasts, add an appropriate amount (3 - 10 mL) of MMG solution to suspend the protoplasts (100 μL of protoplasts are used for each sample), and adjust the concentration to 2×10 5 cells / mL. Check under a microscope to ensure that the protoplasts have a round shape and few ruptures. The formula of the MMG solution is shown in Table 5 below.

[0058] Table 5

[0059]

[0060] Adjust the pH to 5.7 - 5.8, and finally add ddH 2 O to 10 mL; filter-sterilize with a 0.22 μm filter membrane and store at 4°C.

[0061] (6) Transformation reaction: Take 100 μL of the protoplast suspension, and add 10 μL of each of the 4 dual-luciferase reporter vectors in (1) (the plasmid mass is 8 - 10 μg). Then take a PEG4000 solution (110 μL) equal to the total volume of the dual-luciferase reporter vector and the protoplasts, gently mix well, and let it stand at room temperature for 10 min. The formula of the PEG4000 solution is shown in Table 6 below.

[0062] Table 6

[0063]

[0064] Adjust the pH to 5.8, and finally add ddH 2 O to 10 mL, use immediately after preparation.

[0065] (7) Termination of reaction: Dilute to terminate the reaction with 1 mL of W5 solution. Centrifuge at 300 rpm for 3 min, collect the protoplasts, and remove the supernatant.

[0066] (8) Washing and culturing: Add 1 mL of W5 solution and wash 1 - 2 times. Finally, add 100 μL of W5 solution, gently mix well, and transfer the protoplasts to a six-well cell culture plate (previously added with 1 mL of WI solution), and incubate in the dark at 23 °C for 18 - 24 hours. The formula of the WI solution is shown in Table 7 below.

[0067] Table 7

[0068]

[0069] Adjust the pH to 5.7 - 5.8, add 100 μl of Kana (50 mg / ml), and finally add ddH 2 O and make up the volume to 100 mL; filter and sterilize with a 0.22 μm filter membrane and store at 4 °C.

[0070] 3. Fluorescence value detection

[0071] (1) Cell lysis: Centrifuge to collect the protoplasts, add 100 μL of 1×Cell Lysis Buffer, and let it stand or shake at room temperature for 5 min to lyse. Transfer the cell lysis product to a 1.5 mL centrifuge tube, centrifuge at 12000 g at room temperature for 2 min, and take the supernatant for subsequent detection.

[0072] (2) Detection of Firefly luciferase reaction: Add 100 μL of Luciferase Substrate equilibrated at room temperature to the detection tube or microplate, carefully add 20 μL of the cell lysis supernatant, quickly mix well, and immediately detect the activity of the Firefly luciferase reporter gene in a microplate reader. Each detection is repeated 6 times to ensure the reliability of the results.

[0073] (3) Detection of Renilla luciferase reaction: Add 100 μL of freshly prepared Renilla substrate working solution to the above reaction solution, quickly mix well, and immediately detect the activity of the Renilla luciferase reporter gene in a microplate reader. Each detection is repeated 6 times to ensure the reliability of the results.

[0074] 4. Result analysis

[0075] As Figure 2As shown in the figure, all three different translation inhibitory elements significantly inhibited the expression of FLUC. The vectors pSYB-Dual-LUC-ATGc02, pSYB-Dual-LUC-ATG-rich, and pSYB-Dual-LUC-ATGc03 showed FLUC activities of 1 / 3.16, 1 / 9.58, and 1 / 32.74 of the control vector pSYB-Dual-LUC, respectively. That is, the inhibitory degrees of the translation inhibitory elements ATGc02, ATG-rich, and ATGc03 on the target gene were 3.16, 9.58, and 32.74 times, respectively.

[0076] Example 2: Detection of the inhibitory effect of translation inhibitory elements in tobacco protoplasts

[0077] Using Figure 1 the four vectors constructed in Example 1, tobacco protoplasts were transformed, and the specific method was the same as that in Example 1.

[0078] The results are as Figure 3 shown. It can be seen that all three different translation inhibitory elements significantly inhibited the expression of FLUC. The vectors pSYB-Dual-LUC-ATGc02, pSYB-Dual-LUC-ATG-rich, and pSYB-Dual-LUC-ATGc03 showed FLUC activities of 1 / 6.81, 1 / 8.22, and 1 / 9.77 of the control vector pSYB-Dual-LUC, respectively. That is, the inhibitory degrees of the translation inhibitory elements ATGc02, ATG-rich, and ATGc03 on the target protein were 6.81, 8.22, and 9.77 times, respectively, also showing a gradient inhibition. The results were consistent with those in Example 1, indicating that the translation inhibitory elements ATGc02, ATG-rich, and ATGc03 have the effect of gradient inhibition of target gene expression in different species.

[0079] The present invention provides a method for gradient inhibition of target gene expression based on translation inhibitory elements. Through the construction of a dual-luciferase reporter vector and protoplast transformation technology, precise regulation of the target gene expression level is achieved. The experimental results show that three different translation inhibitory elements (ATGc02, ATG-rich, and ATGc03) all showed significant gradient inhibition effects in Arabidopsis thaliana and tobacco protoplasts, and the inhibitory degrees were 3.16 times, 9.58 times, and 32.74 times (Arabidopsis thaliana) and 6.81 times, 8.22 times, and 9.77 times (tobacco), respectively.

[0080] For verification and actual industrial application, the applicant of the present invention also adopted the method of the present invention for plants such as rice and corn, and connected ATGc02, ATG-rich or ATGc03 in front of the start codon of the target gene expression cassette, and found that the expression of the target gene could also be inhibited. This indicates that the method of the present invention can not only effectively inhibit the expression of the target gene, but also achieve gradient regulation of different intensities, and has good universality in different species.

[0081] The significance of the present invention lies in filling the gap in the prior art that lacks precise regulation of protein expression levels. Traditional gene expression regulation methods mainly focus on the transcriptional level, but these methods can often only achieve the "on" or "off" of gene expression, and it is difficult to achieve fine regulation of protein expression levels. By inserting different translation inhibition elements at the translation level, the present invention realizes gradient inhibition of the expression of the target gene, providing new ideas and tools for gene expression regulation.

[0082] In addition, the method of the present invention has broad application prospects in molecular biology research, disease mechanism exploration and biotechnology applications. In basic research, this method can be used to study the fine regulation mechanism of gene expression and explore the functions and action mechanisms of translation regulation elements. In disease research, this method can be used to simulate abnormal protein expression related to diseases and provide model support for disease mechanism research. In biotechnology applications, this method can be used to optimize the expression efficiency of recombinant proteins, develop new gene therapy vectors, and screen and evaluate the regulatory effects of drugs on protein expression.

[0083] In summary, the present invention not only enriches the mechanism research of gene expression regulation theoretically, but also provides strong technical support for biomedical research and biotechnology development in practical applications. By precisely regulating the protein expression level, the present invention is expected to promote the rapid development of related fields and provide new strategies and methods for solving key problems in life sciences.

[0084] The above specific embodiments have described the implementation of the present invention in detail. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the claims and technical concept of the present invention, various simple modifications and changes can be made to the technical solutions of the present invention, and these simple variations all belong to the protection scope of the present invention.

Claims

1. A method for inhibiting target gene expression at the gene translation level, characterized in that: A recombinant expression vector for expressing a target protein is constructed; a translation inhibition element ATG-rich, ATGc02 or ATGc03 is inserted into the front end of the start codon of the target gene expression frame of the recombinant expression vector; the nucleotide sequence of the ATG-rich is shown in SEQ ID NO.1, the nucleotide sequence of the ATGc02 is atgcatgc, and the nucleotide sequence of the ATGc03 is atgc.

2. A translation inhibitory element for inhibiting the expression of a target gene, characterized in that: The translation inhibition element is ATG-rich, ATGc02 or ATGc03; the nucleotide sequence of the ATG-rich is shown in SEQ ID NO.1, the nucleotide sequence of the ATGc02 is atgcatgc, and the nucleotide sequence of the ATGc03 is atgc; in the recombinant expression vector expressing the target protein, the translation inhibition element is inserted into the front end of the start codon of the target gene expression frame.

3. A polynucleotide, characterized in that Contains the translation inhibition element according to claim 2 and a target gene expression frame; the translation inhibition element is connected to the front end of the start codon of the target gene expression frame.

4. A recombinant expression vector, characterized in that: The recombinant expression vector contains the polynucleotide according to claim 3.

5. A transfection system, characterized in that: The polynucleotide according to claim 3 or the recombinant expression vector according to claim 4 is transfected into a eukaryotic host cell.

6. The viral transfection system according to claim 5, characterized in that The transfection system includes a plasmid transfection reagent, a liposome transfection reagent, a lentivirus, and a retrovirus.

7. Use of the translation inhibition element according to claim 2 in preparing a preparation for inhibiting the expression of a target gene.

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