Gene of RMR1 recombinant protein, recombinant expression vector and application thereof

Abstract

The invention relates to the technical field of biotechnology and genetic engineering, and discloses a gene of RMR1 recombinant protein, a recombinant expression vector and application thereof. The gene of the RMR1 recombinant protein provided by the invention is used for coding the RMR1 recombinant protein, and the nucleotide sequence of the gene is shown as SEQ ID NO: 1. The gene of the RMR1 recombinant protein is obtained by optimizing an RMR1 gene and comprises a section of specific CT signal sequence, so that the RMR1 recombinant protein can be expressed in a plant cell endoplasmic reticulum in a targeted manner; meanwhile, through optimization design of the gene, the N end of the gene of the RMR1 recombinant protein can be fused with genes of other functional proteins to form fusion protein. The fusion protein has the capability of targeting endoplasmic reticulum, and the fused functional protein can still perform the original function after targeting the endoplasmic reticulum, so that a new thought and a new method are provided for research and application in the field of biomedicine.

Description

Technical Field

[0001] The present invention relates to the technical fields of biotechnology and genetic engineering, and in particular to a gene of an RMR1 recombinant protein, a recombinant expression vector and applications thereof. Background Art

[0002] In today's biotechnology and genetic engineering fields, protein fusion technology has become a powerful tool for creating new proteins with multiple biological activities. By combining the functions of different proteins, fusion proteins have shown great potential in drug development, disease diagnosis and treatment. Among them, RMR1 protein, as a protein with endoplasmic reticulum targeting, has attracted much attention in cell biology and medical research due to its unique properties. In recent years, with the rapid development of gene editing and recombinant technology, protein fusion technology has also made significant progress. By precisely designing the structure and function of fusion proteins, researchers have successfully created a variety of fusion proteins with specific functions, providing strong support for research and application in the biomedical field. However, its application in complex biological research is limited. First, the stability and activity of fusion proteins are often difficult to maintain, which may lead to reduced or lost functions. Secondly, the targeting of fusion proteins is also an important issue. How to ensure that fusion proteins can accurately target the endoplasmic reticulum is a difficult problem that needs to be solved urgently. Summary of the invention

[0003] The purpose of the present invention is to overcome the problems of the prior art that the stability and activity of the fusion protein are difficult to maintain and the targeting is insufficient, and a gene of a RMR1 recombinant protein, a recombinant expression vector and the application thereof are provided.

[0004] In order to achieve the above object, the present invention provides a gene of RMR1 recombinant protein on the one hand, the gene is used to encode RMR1 recombinant protein, and the nucleotide sequence of the gene is shown in SEQ ID NO: 1. The second aspect of the present invention provides a RMR1 recombinant protein encoded by the gene as described above, and the amino acid sequence of the RMR1 recombinant protein is shown in SEQ ID NO: 2.

[0005] The third aspect of the present invention provides a fusion protein, which comprises a functional protein and the RMR1 recombinant protein as described above.

[0006] Preferably, the functional protein is a fluorescent protein.

[0007] The fourth aspect of the present invention provides a recombinant expression vector, which comprises the RMR1 recombinant protein as described above or a gene encoding the fusion protein as described above.

[0008] A fifth aspect of the present invention provides a method for expressing a recombinant protein, the method comprising the following steps:

[0009] (1) constructing the recombinant expression vector as described above;

[0010] (2) transfecting the recombinant expression vector into plant cells, and culturing the plant cells to express the recombinant protein.

[0011] Preferably, the plant is Arabidopsis thaliana, tobacco or tomato.

[0012] The sixth aspect of the present invention provides the use of the above-mentioned gene, RMR1 recombinant protein, the above-mentioned recombinant expression vector, and the above-mentioned fusion protein in the field of targeting the endoplasmic reticulum.

[0013] In the present invention, the existing gene encoding the RMR1 protein is optimized so that the gene of the obtained RMR1 recombinant protein contains a specific CT signal sequence, thereby ensuring that the RMR1 recombinant protein can be targeted and expressed in the endoplasmic reticulum of plant cells; after the above-mentioned optimization design, the N-terminus of the gene of the RMR1 recombinant protein can be fused with the genes of other functional proteins to form a fusion protein.

[0014] The fusion protein provided by the present invention not only has the ability of RMR1 recombinant protein to target the endoplasmic reticulum, but also the fused functional protein can still perform its original function after targeting the endoplasmic reticulum, providing new ideas and methods for research and application in the biomedical field. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 is the PCR amplification band of RMR1;

[0016] Figure 2 is the plasmid construction map NbG-RFP-RMR1 (red fluorescent protein-tagged fusion protein);

[0017] Figure 3 The secretory protein NbG-RFP-RMR1 is expressed in tobacco leaf protoplasts, and the expressed secretory protein is collected;

[0018] Figure 4 The endoplasmic reticulum marker protein GFP-CNX labeled with green fluorescent protein was expressed in tobacco leaf protoplasts. The transformed cells were incubated in Figure 3 Secretory proteins collected in the culture medium;

[0019] Figure 5 The signals in tobacco cells were detected under laser confocal microscope (Nikon A1R laser confocal microscope). For GFP and RFP excitation, 488 and 561 nm laser excitation were used, and the acceptance wavelengths were 500-550 and 570-620 nm, respectively;

[0020] Figure 6The signals in Arabidopsis cells were detected under laser confocal (Nikon A1R laser confocal microscope), and for GFP and RFP excitation, 488 and 561 nm laser excitation were used, respectively, with acceptance wavelengths of 500-550 and 570-620 nm;

[0021] Figure 7 The signals in tomato cells were detected under laser confocal microscope (Nikon A1R laser confocal microscope). For GFP and RFP excitation, 488 and 561 nm laser excitation were used, respectively, and the acceptance wavelengths were 500-550 and 570-620 nm, respectively. DETAILED DESCRIPTION

[0022] The specific implementation of the present invention is described in detail below in conjunction with the accompanying drawings. It should be understood that the specific implementation described here is only used to illustrate and explain the present invention, and is not used to limit the present invention.

[0023] The endpoints and any values ​​of the ranges disclosed in this article are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

[0024] The present invention also provides a gene of RMR1 recombinant protein, which is used to encode the RMR1 recombinant protein. The nucleotide sequence of the gene is shown in SEQ ID NO:1.

[0025] SEQ ID NO: 1:

[0026] atgactgttgtcctcaattccatctccgcctcttttgccgatctcccagccaaatttgacggctccgtgaccaaaaacggaatctgtggagctctatacgtcgcagatcctctcgacggttgctcaccgcttctccacgccgccgcatccaactggacgcaacacagaactactaagttcgctttgataatcagaggcgaatgttcttttgaggataagctgctcaatgcccagaactcaggttttcaagctgtgattgtctatgacaacattgacaacgaagatctcatcgtcatgaaggtgaaccctcaggacattacagttgatgcagtcttcgtttcaaatgtcgccggtgagattttgagaaagtacgcgagaggccgagatggtgaatgctgccttaatccgccagacagagggagcgcttggactgtgttggccatctccttcttctctctccttcttatagtcactttcctgttgattgccttctttgcacccagacactggacccaatggcgagggaggcacaccaggaccatcaggttagatgcaaagctcgtccacacactcccctgcttcaccttcactgattctgctcaccacaaggccggggaaacatgtgctatatgtctcgaggattacagatttggagaaagcctcagacttctcccctgccaacatgcttttcacttgaattgcatcgactcttggttgacaaaatggggtacatcttgccctgtgtgcaagcatgacataagaaccgagactatgtcttctgaggtacataaacgagagagtccgagaacagatacaagtacgagtagatttgcctttgcccaatccagtcaaagccgt。

[0027] In the present invention, the gene encoding the RMR1 recombinant protein is designed so that the N-terminus of the encoded RMR1 recombinant protein can be fused with other genes to form a fusion protein; the fusion protein has good stability and activity and is targeted, so that it can not only be targeted for expression in the endoplasmic reticulum of plant cells, but also enable the fused functional protein to still perform its original function after being targeted to the endoplasmic reticulum.

[0028] The present invention optimizes the gene encoding the RMR1 protein in Arabidopsis thaliana so that the optimized encoding gene contains a specific CT signal sequence, thereby ensuring that the RMR1 recombinant protein can be targeted and expressed in the endoplasmic reticulum of plant cells. The specific optimization idea of ​​the present invention is: taking the RMR1 gene in Arabidopsis thaliana as the starting point for improvement, after the prediction of the functional domain and the functional analysis of RMR1 in cells, the signal peptide sequence of RMR1 is removed, and the start codon is added, and then the remaining sequence is connected to the CT signal sequence, and the sequence after the CT sequence is deleted, thereby obtaining the improved and optimized RMR1 recombinant protein sequence. During the specific preparation, the gene sequence of the new RMR1 recombinant protein is obtained by gene PCR amplification and enzyme cutting and connecting multiple fragments.

[0029] The present invention provides a RMR1 recombinant protein encoded by the gene as described above, and the amino acid sequence of the RMR1 recombinant protein is shown in SEQ ID NO:2.

[0030] SEQ ID NO:2:

[0031] mvaapflssllrvslatvvlnsisasfadlpakfdgsvtkngicgalyvadpldgcspllhaaasnwtqhrttkfaliirgecsfedkllnaqnsgfqavivydnidnedlivmkvnpqditvdavfvsnvageilrkyargrdgecclnp pdrgsawtvlaisffslllivtflliaffaprhwtqwrgrhtrtirldaklvhtlpcftftdsahhkagetcaicledyrfgeslrllpcqhafhlncidswltkwgtscpvckhdirtetmssevhkresprtdtstsrfafaqssqsr.

[0032] In the present invention, the recombinant protein is encoded by a gene sequence from a plant source or other biological source, and is prepared by genetic engineering technology.

[0033] The present invention also provides a fusion protein, which includes a functional protein or the RMR1 recombinant protein as described above.

[0034] In some embodiments, two or more exogenous functional proteins can be fused to the N-terminus of the RMR1 recombinant protein, so that the fused functional proteins still have functional activity after entering the cell.

[0035] In some embodiments, two or more exogenous functional proteins are fused to the N-terminus of the RMR1 recombinant protein, so that the resulting fusion protein can have biological activity in different species.

[0036] The present invention does not limit the specific type of the functional protein, which can be a functional protein commonly used in the art. By fusing different functional proteins, a delivery system or tool with multiple biological functions can be created to achieve a more precise and efficient effect. For example, in the field of gene therapy, RMR1 recombinant protein can be used to deliver therapeutic genes to the endoplasmic reticulum to achieve precise gene therapy; in the field of cell imaging, RMR1 recombinant protein can be used to deliver markers such as fluorescent proteins to the endoplasmic reticulum to achieve real-time imaging and monitoring in cells. Therefore, the RMR1 recombinant protein described in the present invention has important scientific significance and practical applications.

[0037] In a specific embodiment, the functional protein is a fluorescent protein, and by selecting the fluorescent protein and fusing it with the RMR1 recombinant protein, the resulting fusion protein has a wide range of application prospects in the fields of targeting the endoplasmic reticulum of cells. By fusing different functional proteins, a delivery system or tool with multiple biological functions can be created to achieve a more precise and efficient effect.

[0038] The present invention also provides a recombinant expression vector, which comprises the gene of the RMR1 recombinant protein as described above or the gene encoding the fusion protein as described above.

[0039] The present invention also provides a method for expressing a recombinant protein, which comprises the following steps:

[0040] (1) constructing the recombinant expression vector as described above;

[0041] (2) transfecting the recombinant expression vector into plant cells, and culturing the plant cells to express the recombinant protein.

[0042] In a specific embodiment, step (1) is specifically: constructing a recombinant expression vector containing the gene of the RMR1 recombinant protein. The present invention uses genetic engineering technology to screen out gene sequences with specific functions from plants, construct a recombinant expression vector containing the gene encoding the RMR1 recombinant protein, and transform it into plant cells for expression, thereby obtaining a plant recombinant protein that can target the endoplasmic reticulum of plant cells. During the preparation process, the purity and activity of the recombinant protein can be improved by optimizing the expression conditions, purification process and other steps. Experiments have shown that the fusion protein not only has the ability of the RMR1 recombinant protein to target the endoplasmic reticulum, but also the fused functional protein can still perform its original function after targeting the endoplasmic reticulum.

[0043] The RMR1 recombinant protein provided by the present invention can be successfully targeted among different plant species and has wide applicability. In some embodiments, the plant is Arabidopsis, tobacco or tomato.

[0044] The present invention also proposes the use of the gene of the RMR1 recombinant protein, the RMR1 recombinant protein, the fusion protein, and the recombinant expression vector described above in the field of endoplasmic reticulum targeting.

[0045] The RMR1 recombinant protein of the present invention can be used as a research tool to explore the function and regulatory mechanism of the plant endoplasmic reticulum. In the field of cell imaging, the RMR1 recombinant protein can be used to deliver markers such as fluorescent proteins to the endoplasmic reticulum to achieve real-time imaging and monitoring in cells.

[0046] The present invention will be described in detail below by way of examples, but the protection scope of the present invention is not limited thereto. The experimental methods described in the present invention can be found in Molecular Cloning Experiment Guide (3rd Edition) (Science Press, 2002). The experimental reagents used in the present examples, unless otherwise specified, can be purchased through commercial channels.

[0047] Example 1

[0048] This example is used to illustrate the construction of a recombinant expression vector containing a gene encoding a recombinant protein.

[0049] (1) Extraction of plant total RNA using a total RNA kit: Use a total RNA kit (Thermo Fisher Scientific Plant RNA Extraction Kit, 12183018A) to extract RNA from Arabidopsis thaliana. This can yield RNA with good reproducibility and high purity. For detailed instructions, please refer to the kit manual.

[0050] (2) Reverse transcription to cDNA: using a reverse transcription kit (Roche)

[0051] (3) PCR amplification system: 10X Taq PCR Buffer 3μl, 2mM dNTP Mix 3μl, 10pmol upstream and downstream primers 1μl each, 5U / μl Taq DNA polymerase 0.5μl, 50ng total genomic DNA, ddH20 to 30μl. The PCR amplification conditions of the primer pair were 94℃3min; 94℃30sec, 55℃30sec, 72℃1min, 30 cycles; 72℃3min.

[0052] The primers used were:

[0053] Upstream primer: 5′-CAGTACCATATGTACTGTTGTCCTCAATTCCATC-3′;

[0054] Downstream primer: 5′-AGTCTAGGATCCCTAACGGCTTTGACTGGATTGGG-3′.

[0055] Figure 1 The PCR amplification band of RMR1.

[0056] (4) The double restriction enzyme cutting sites are: NdeI and BamHI.

[0057] (5) Connect the enzyme-digested products.

[0058] (6) constructing a plasmid, wherein the constructed plasmid map is as follows Figure 2 ( Figure 2 In the middle, antiGFP is Nb G ): RMR1 with red fluorescent protein (RFP) labeled at the N-terminus.

[0059] Example 2

[0060] This example is used to illustrate the large-scale extraction of plasmid (obtaining approximately 500 mL of plasmid with a concentration of 3-10 ug / mL).

[0061] (1) The above plasmids were prepared into a solution of 500 mL / plasmid using TB medium (11.8 g / L tryptone, 23.6 g / L yeast powder, 9.4 g / L dimethyl phosphate, 2.2 g / L potassium dihydrogen phosphate) and sterilized by high pressure steam at 121°C for 16 minutes.

[0062] (2) One day in advance, transform the plasmid to be extracted into an LB plate with the corresponding resistance (Kanamycin resistance) for activation to obtain a single clone. Add 2.5 mL of LB solution with the corresponding resistance to a 15 mL shaking tube, select a single colony for activation culture, and culture at 37°C with shaking.

[0063] (3) After shaking and culturing for about 3 hours, the bacterial solution in the test tube becomes slightly turbid. Pour the 2.5 mL activated bacterial solution into 500 mL TB medium (preheated at 37°C) for large-scale culture for at least 24 hours.

[0064] (4) At this point, the following steps will be performed on ice. Pour the cultured bacterial solution into a collection bottle (500 mL collection bottle, using a horizontal rotor), centrifuge at 4000 rpm at 4°C for 30 minutes, and discard the supernatant.

[0065] (5) Resuspend the bacteria in pre-cooled TE 50 / 1 solution to a final volume of 8 mL (4 mL + 4 mL). Transfer the bacteria in the collection bottle to a pre-cooled round-bottom SS34 test tube and place it on ice.

[0066] (6) Quickly add 5 mL of freshly prepared lysozyme solution (10 mg / mL), tighten the cap, and mix by inverting at least 10 times. The solution will become slightly viscous after sufficient contact with the bacteria, but do not shake the tube. Let it stand on ice for 5 min.

[0067] (7) Quickly add 2 mL of 0.5 M EDTA pH 8.0 solution, tighten the cap, and mix by inverting at least 10 times until the solution becomes more viscous. Do not shake the tube and place on ice for 5 min.

[0068] (8) Add 1 mL of mixed solution (150 μL 10% Triton X-100 solution plus 850 μL TE 50 / 1 solution), tighten the lid, and mix by inverting at least 10 times. The solution will become very viscous. Do not shake the tube and let it stand on ice for 30 to 60 minutes.

[0069] (9) Centrifuge at 18,000 rpm for 60 min using a Sorvall SS34 rotor (Sorvall RC-5B centrifuge) to obtain a precipitate and a supernatant. Sometimes, if the bacterial culture time is insufficient, the precipitate volume may be very large, leaving a very small supernatant volume. In this case, repeat the centrifugation process.

[0070] (10) Transfer the supernatant to a new 50 mL centrifuge tube. The procedure can be interrupted here by freezing the recovered supernatant at -20 °C.

[0071] (11) Add balanced phenol (pH 8.0, containing 0.1% 8-hydroxyquinoline) in a 1:1 ratio, shake vigorously for 1 min, and centrifuge at maximum speed for 20 min in a swing-out rotor (the centrifuge speed is set to 0).

[0072] (12) Slowly transfer the upper layer of solution into a new 50 mL centrifuge tube and add chloroform in a 1:1 ratio. Shake again to mix and centrifuge at maximum speed in a swing-out rotor for 20 min (set the centrifuge speed to 0).

[0073] (13) Slowly recover the upper solution and transfer it to a new 50 mL centrifuge tube. Add 200 μL of RNase (20 mg / mL) and place the centrifuge tube in a 37°C water bath for 2 hours (or place the centrifuge tube in a 37°C incubator for overnight reaction).

[0074] (14) After the reaction with the enzyme, balanced phenol (pH 8.0, containing 0.1% 8-hydroxyquinoline) was added in a 1:1 ratio, shaken vigorously for 1 min, and centrifuged at maximum speed for 20 min in a swing-out rotor (the centrifuge speed was set to 0).

[0075] (15) Slowly recover the upper layer of solution and transfer it to a new centrifuge tube. Add chloroform in a 1:1 ratio, shake again to mix, and centrifuge at maximum speed in a swing-out rotor for 20 min (set the centrifuge speed to 0).

[0076] (16) Slowly recover the upper solution and transfer it to a 30 mL COREX glass tube. The volume of the supernatant is generally between 10 and 15 mL. Add 1 mL of 5 M NaClO4 solution to the supernatant and mix it by blowing with a pipette. Then add 8 mL of isopropanol solution and seal it with a sealing film. Invert the glass tube to mix the solution.

[0077] (17) Centrifuge at 10,000 rpm for 15 min at room temperature, discard the supernatant, wash once with 70% ethanol, and dry the glass tube.

[0078] (18) Add 500 μL TE buffer to dissolve the DNA in the glass tube, transfer the dissolved solution to a new centrifuge tube, and store it at 4°C.

[0079] Example 3

[0080] This example is used to illustrate the application of the gene of the RMR1 recombinant protein of the present invention in targeting the endoplasmic reticulum.

[0081] 1. Preparation and transient transformation of plant protoplasts

[0082] (1) Prepare the culture buffer (3.05 g / L B5 salt, 0.5 g / L MES, 0.75 g / L calcium chloride dihydrate, 0.25 g / L ammonium nitrate, 139.3 g / L sucrose, and adjust the pH to 5.7 with KOH) in advance, filter and sterilize using a 0.22 μM filter in a clean bench, and store at 4 °C.

[0083] (2) Prepare an enzymatic hydrolysis solution (2% cleavage enzyme, 4% cellulase, dissolved in culture buffer) and filter the sterilized supernatant using a 0.22 μM filter membrane.

[0084] (3) Select fresh green leaves of tobacco. Gently cut the leaves from the jar, place the leaves on a square petri dish, and use a Derma-Roll to punch holes on the lower leaf surface (gently roll the leaves). Use a knife to remove the middle vein of the leaf, and transfer the two halves of the leaf to a petri dish containing 7 mL of enzymatic solution, with the side rolled by the roller facing down. Each plate should provide 5 × 10 6 A protoplast.

[0085] (4) The digestion mixture is filtered through a 100 μM nylon filter, and the filter can be washed with electroporation buffer. This will release more protoplasts from the tissue residue. The protoplasts are then centrifuged at 700 rpm for 15 minutes at room temperature in a 50 mL centrifuge tube. The living protoplasts will move to the surface of the solution, while the cell debris will form a pellet or remain in the solution.

[0086] (5) Insert a Pasteur pipette into the floating cell layer and discard the solution.

[0087] (6) Add 25 mL of electroporation buffer (2.4 g / L HEPES, 0.6 g / L calcium chloride, 6 g / L potassium chloride, 139.3 g / L sucrose, pH adjusted to 7.2 with KOH) and centrifuge again at 700 rpm for 10 min.

[0088] (7) Remove the bottom solution as above and repeat this process twice. The solution below the protoplasts should become clear with almost no visible particles. Finally, resuspend the protoplasts in an appropriate volume to obtain 2 to 5 × 10 6 protoplasts / mL.

[0089] (8) Gently transfer 500 μL of protoplasts into a disposable 1 mL plastic cuvette, add 100 μL of electroporation buffer containing plasmid, and gently shake to mix; wherein, 100 μL of electroporation buffer is obtained by diluting 10 to 50 μg of the plasmid extracted in Example 2 above to a final solution of 100 μL.

[0090] (9) The cell mixture was incubated at room temperature for 5 minutes, and then electroporation was performed. The electroporation device used here has a pair of stainless steel plate electrodes embedded in a polytetrafluoroethylene insulator, which will be directly inserted into the cuvette. During electroporation, no metal parts are exposed on the surface. Electroporation was performed at a voltage of 160V.

[0091] (10) After electroporation, incubate the cells at room temperature for 15 to 30 minutes. The live cells will float to the surface, and 300 to 400 μL of the bottom electroporation buffer and cell debris can be removed.

[0092] (11) The obtained cell suspension was cultured in a small culture dish in the dark for 48 hours to express the secretory protein.

[0093] 2. Collect the secretory protein solution expressed by transcription and translation, enter the cell through the endocytosis pathway and target the endoplasmic reticulum

[0094] (1) After the cells expressed secreted protein for 48 hours, the cell culture mixture was collected, the culture medium was aspirated with a syringe, filtered through a 0.22 μm filter membrane, and the filtrate was collected (eg Figure 3 ).

[0095] (2) The protein filtrate collected in the above step is incubated in the culture medium to be introduced into the cells (such as Figure 4 ).

[0096] (3) Detect signals of exogenously expressed proteins entering cells and targeting the endoplasmic reticulum.

[0097] The results of detecting the signal in tobacco cells under laser confocal microscope (Nikon A1R laser confocal microscope) are as follows: Figure 5 shown.

[0098] Figure 5 The results showed that green endoplasmic reticulum marker protein signals and red signals indicating that the secretory protein NbG-RFP-RMR1 entered the cell and was targeted to the endoplasmic reticulum could be observed.

[0099] Example 4

[0100] The method described in Example 3 was followed, except that during the preparation of plant protoplasts and transient transformation, the fresh green leaves of Arabidopsis thaliana were selected in step (3).

[0101] The signal detection results in Arabidopsis cells were detected under laser confocal microscope (Nikon A1R laser confocal microscope). Figure 6 shown.

[0102] Figure 6 The results showed that green endoplasmic reticulum marker protein signals and red signals indicating that the secretory protein NbG-RFP-RMR1 entered the cell and was targeted to the endoplasmic reticulum could be observed.

[0103] Example 5

[0104] The method described in Example 3 was followed, except that during the preparation and transient transformation of plant protoplasts, fresh green young leaves of tomatoes were selected in step (3).

[0105] The signal detection results in tomato cells were detected under laser confocal microscope (Nikon A1R laser confocal microscope). Figure 7 shown.

[0106] Figure 7 The results showed that green endoplasmic reticulum marker protein signals and red signals indicating that the secretory protein NbG-RFP-RMR1 entered the cell and was targeted to the endoplasmic reticulum could be observed.

[0107] The above results show that the fusion protein is active for ≥48 hours after expression and can enter the cell and target the endoplasmic reticulum, indicating that the present invention, through the design of the gene of the RMR1 recombinant protein, makes the fusion protein containing the RMR1 recombinant protein not only have the ability to target the endoplasmic reticulum, but also the fused functional protein can still perform its original function after targeting the endoplasmic reticulum.

[0108] The preferred embodiments of the present invention are described in detail above, but the present invention is not limited thereto. Within the technical concept of the present invention, the technical solution of the present invention can be subjected to a variety of simple modifications, including the combination of various technical features in any other suitable manner, and these simple modifications and combinations should also be regarded as the contents disclosed by the present invention and belong to the protection scope of the present invention.

Claims

1. A gene for RMR1 recombinant protein, characterized in that: The gene is used to encode RMR1 recombinant protein, and the nucleotide sequence of the gene is shown in SEQ ID NO:

1.

2. The RMR1 recombinant protein encoded by the gene according to claim 1, wherein the amino acid sequence of the RMR1 recombinant protein is shown in SEQ ID NO:

2.

3. A fusion protein, characterized in that The fusion protein comprises a functional protein and the RMR1 recombinant protein according to claim 2.

4. The fusion protein according to claim 3, characterized in that The functional protein is a fluorescent protein.

5. A recombinant expression vector, characterized in that: The recombinant expression vector comprises the gene of the RMR1 recombinant protein according to claim 1 or the gene encoding the fusion protein according to any one of claims 3-4.

6. A method for expressing a recombinant protein, characterized in that: The method comprises the following steps: (1) constructing the recombinant expression vector according to claim 5; (2) transfecting the recombinant expression vector into plant cells, and culturing the plant cells to express the recombinant protein.

7. The method according to claim 6, characterized in that The plant was Arabidopsis thaliana, tobacco or tomato.

8. Use of the gene according to claim 1, the RMR1 recombinant protein according to claim 2, the fusion protein according to claim 3 or 4, or the recombinant expression vector according to claim 5 in the field of targeting the endoplasmic reticulum.

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