Alternative Splicing Polypeptides and Their Applications

Abstract

The present invention provides alternative splicing polypeptides and their applications. The alternative splicing polypeptide of the present invention is a polypeptide composed of 23 amino acids at the N-terminus of HBFD from Adonis amurensis, which can achieve specific splicing in tobacco and realize the tandem expression of multiple proteins in a single expression cassette. The alternative splicing polypeptide of the present invention has better splicing specificity in plants than the 2A self-cleaving polypeptide of picornavirus reported by predecessors, which is beneficial to reducing the occurrence of non-specific splicing. The alternative splicing polypeptide of the present invention is a protein splicing element derived from plants, which is beneficial to improving the application value of the tandem expression technology of multiple proteins based on a single expression cassette in plant bioreactors.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly to alternatively spliced polypeptides and their applications. Background Art

[0002] In the process of developing plant bioreactors, co-expression of multiple proteins is usually required. However, in plants, which are eukaryotes, protein translation is restricted by the "monocistronic" principle. To express multiple proteins, multiple independent protein expression cassettes are needed, which greatly increases the difficulty of constructing plant bioreactors. To achieve co-expression of multiple proteins in plants, various methods have been tried. Many studies have used multiple vectors containing single expression cassettes or vectors containing multiple expression cassettes for multi-protein expression. However, such methods are often affected by gene expression imbalance, and the probability of achieving high-efficiency expression of multiple proteins is relatively low, often requiring a large amount of work to achieve. Some studies have used plastid transformation methods, using polycistronic expression cassettes of prokaryotes to express multiple proteins in plant plastids. However, this method is only meaningful for proteins that function in plastids, and has great limitations. Another part of the studies uses a single expression cassette to express multiple proteins concatenated by alternatively spliced protein elements as linkers. After expression in plants, co-expression of multiple proteins is achieved through cleavage of the protein linker. This method can achieve balanced expression of multiple proteins, but is affected by the cleavage efficiency of the splicing element. Currently, commonly used alternatively spliced protein elements are the 2A self-cleaving polypeptide of picornaviruses, tobacco etch virus protease, etc. These cleavage peptides have low cleavage efficiency in plants and are all protein elements derived from viruses, not the best choice for developing plant bioreactors.

[0003] Therefore, identifying highly efficient alternatively spliced protein elements of non-viral origin is of great significance for improving the application value of multi-protein tandem expression technology based on single expression cassettes in plant bioreactors. Summary of the Invention

[0004] The object of the present invention is to provide alternatively spliced polypeptides and their applications. The alternatively spliced polypeptide of the present invention is a polypeptide consisting of 23 amino acids at the N-terminus of Adonis aestivalis HBFD, which can achieve specific cleavage in tobacco and realize multi-protein tandem expression of a single expression cassette. The alternatively spliced polypeptide of the present invention has better cleavage specificity in plants than the previously reported 2A self-cleaving polypeptide of picornaviruses, which is beneficial to reducing the occurrence of non-specific cleavage.

[0005] To achieve the above object, the present invention adopts the following technical solutions:

[0006] In the first aspect of the present invention, there is provided an alternatively spliced polypeptide, characterized in that the amino acid sequence of the alternatively spliced polypeptide is as shown in SEQ ID NO.1: MRTLHGACLPWIETNSLHWKLVK.

[0007] Furthermore, the alternative splicing polypeptide further includes a polypeptide with the same function obtained by substituting, deleting, and / or adding one or more amino acids to the amino acid sequence shown in SEQ ID NO.1.

[0008] In the second aspect of the present invention, an application of the alternative splicing polypeptide in tandem expression of plant multi-proteins is provided.

[0009] Furthermore, the plant includes at least one of Arabidopsis thaliana, tobacco, tomato, soybean, wheat, and rice.

[0010] Furthermore, the application includes: achieving specific splicing in plants to achieve tandem expression of multi-proteins in a single expression cassette.

[0011] In the third aspect of the present invention, a method for tandem expression of multi-proteins is provided, and the method includes:

[0012] Obtaining a first gene fragment and a second gene fragment to be tandemly expressed;

[0013] Using the alternative splicing polypeptide as a linker to tandemly express the first gene fragment and the second gene fragment.

[0014] Furthermore, the first gene fragment or the second gene fragment is the yFP gene with the nucleotide sequence shown in SEQ ID NO.37 and the complete YFP gene with the nucleotide sequence shown in SEQ ID NO.38.

[0015] One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages:

[0016] 1. The alternative splicing polypeptide and its application provided by the present invention are based on an alternative splicing polypeptide found at the N-terminus of HBFD (carotenoid β-ring 4-hydroxylase dehydrogenase) in Adonis amurensis. A method for tandem expression of plant multi-proteins using this polypeptide as a linker is provided. The alternative splicing polypeptide of the present invention is a polypeptide consisting of 23 amino acids at the N-terminus of HBFD in Adonis amurensis, which can achieve specific splicing in tobacco and achieve tandem expression of multi-proteins in a single expression cassette.

[0017] 2. The splicing specificity of the alternative splicing polypeptide of the present invention in plants is better than that of the previously reported picornavirus 2A self-splicing polypeptide, which is beneficial to reducing the occurrence of non-specific splicing. The alternative splicing polypeptide of the present invention is used for tandem expression of multi-proteins in a single expression cassette of other proteins in plants, and co-expression of multi-proteins in plants can be achieved through splicing.

[0018] 3. The alternative splicing polypeptide of the present invention is a protein splicing element derived from plants, which is beneficial to improving the application value of the multi-protein tandem expression technology based on a single expression cassette in plant bioreactors. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following will briefly introduce the drawings required for the description of the embodiments. Obviously, the drawings in the following description are some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0020] Figure 1 : Construction of astaxanthin-producing tobacco expressing CBFD-2A-HBFD. A. Astaxanthin-producing tobacco expressing CBFD-2A-HBFD. Plants transformed with the empty vector were used as blank controls. B. Analysis of the expression of CBFD and HBFD in transgenic tobacco by semi-quantitative RT-PCR. C. Detection of HA-tagged CBFD-2A-HBFD (stable expression) and HBFD (transient expression) proteins by Western Blotting. The red arrow indicates unspliced CBFD-2A-HBFD, and the black arrow indicates spliced HBFD and transiently expressed HBFD. 1-2 are two independent transgenic single plants.

[0021] Figure 2 : Construction of astaxanthin-producing tobacco expressing CBFD-HBFD. A. Astaxanthin-producing tobacco expressing CBFD-HBFD, and plants transformed with the empty vector were used as blank controls. B. Semi-quantitative RT-PCR analysis of the expression of CBFD and HBFD in transgenic tobacco. C. Detection of Flag-tagged CBFD-HBFD protein expressed in tobacco by Western Blotting. The black arrow indicates unspliced CBFD-HBFD, and the red arrow indicates spliced HBFD. 1-2 represent two independent transgenic tobaccos.

[0022] Figure 3 : Identification of the HBFD splicing peptide segment. A. Co-immunoprecipitation (Co-IP) analysis of transgenic tobacco expressing HA-tagged CBFD-2A-HBFD protein and Flag-tagged CBFD-HBFD protein. The black arrow indicates the protein band corresponding to HBFD, and the larger band is the antibody band. 1-2 are two replicate samples. B. Localization map of the detected protein fragments on the full-length fusion protein. The blue box indicates the fragment detected only in one transgenic tobacco sample; the orange box indicates the overlapping fragment detected in both transgenic tobacco samples; the green box indicates the same fragment detected in both transgenic tobacco samples. C. Results of the 5'-terminal RT-PCR amplification experiment. 1-4 are the PCR amplification products of 4 rounds.

[0023] Figure 4 : Application of HBFD cleavage peptide in other multi-protein tandem expressions. A. Schematic diagram of the construction of the transient expression vector. B. Detection of the specific cleavage effect of the cleavage peptide segment in tobacco by Western Blotting. "-" represents the tandem protein without HBFD cleavage peptide, and "+" represents the tandem protein with HBFD cleavage peptide. Specific embodiments

[0024] The present invention will be specifically described below in combination with specific embodiments and examples, and the advantages and various effects of the present invention will be presented more clearly therefrom. Those skilled in the art should understand that these specific embodiments and examples are used to illustrate the present invention, rather than to limit the present invention.

[0025] Throughout the specification, unless otherwise specifically stated, the terms used herein should be understood as having the meanings commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which the present invention pertains. In case of contradiction, this specification shall prevail.

[0026] Unless otherwise specifically stated, various raw materials, reagents, instruments, and equipment used in the present invention can be obtained through market purchases or can be obtained by existing methods.

[0027] The alternative splicing polypeptides and their applications of the present application will be described in detail below in combination with examples and experimental data.

[0028] Example 1: Novel alternative splicing exists in the fusion protein expression of HBFD

[0029] 1. Gene expression design: When co-expressing the astaxanthin synthase CBFD (GenBank accession number: AY644757.1) and HBFD (GenBank accession number: DQ902555.1) of Adonis amurensis in tobacco, the picornavirus 2A self-cleaving polypeptide was used as a linker and added between CBFD and HBFD. A single expression cassette was used for the sequential tandem expression of the two genes CBFD and HBFD. The gene sequences used were codon-optimized for tobacco expression. The optimized nucleotide sequence of CBFD is shown in SEQ ID NO.2, the optimized nucleotide sequence of HBFD is shown in SEQ ID NO.3, and the optimized nucleotide sequence of the 2A cleavage peptide is shown in SEQ ID NO.4.

[0030] 2. Construction of the expression vector pBin-CBFD-2A-HBFD-HA:

[0031] In the first round of PCR, specific primers in Table 1 (CBFD-5’ / CBFD-FUS3’, HBFD-FUS5’ / HBFD-3’, 2A-5 / 2A-3’) were used to amplify the CBFD gene, HBFD gene, and peptide fragments of the 2A polypeptide coding sequence respectively. The PCR reaction conditions were as follows: pre-denaturation at 95°C for 30 s; 35 cycles of denaturation at 95°C for 15 s, annealing at 58°C for 15 s, and extension at 72°C for 30 s; incubation at 72°C for 5 min. The PCR amplification products were subjected to gel cutting and recovery purification to serve as the amplification template for the second round of PCR.

[0032] In the second round of PCR, the above-obtained amplified fragments were used as templates, and the specific primers in Table 1 (CBFD-5’ / HBFD-3’) were used to amplify the fusion gene fragment CBFD-2A-HBFD. The PCR reaction conditions were as follows: pre-denaturation at 95°C for 30 s; 35 cycles of denaturation at 95°C for 15 s, annealing at 58°C for 15 s, and extension at 72°C for 1.5 min; incubation at 72°C for 5 min.

[0033] The PCR amplification products were subjected to gel cutting and recovery purification, and the fusion fragment CBFD-2A-HBFD was cloned into the pENTR-D-TOPO vector (purchased from Invitrogen, catalog number K240020) using restriction endonuclease Sac II and Asc I.

[0034] Then, the fusion fragment CBFD-2A-HBFD was recombined into the pBin19-attR-HA vector containing the 2×CaMV35S promoter (Zhuangmeng Biotech, catalog number ZK1941) through the LR reaction mediated by Gateway® LR Clonase® II Enzyme mix (Invitrogen) to obtain the expression vector pBin-CBFD-2A-HBFD-HA, where HA is the tag protein.

[0035] After sequencing verification, the expression vector pBin-CBFD-2A-HBFD-HA was transferred into Agrobacterium tumefaciens LBA4404 by the freeze-thaw method. Then, tobacco TN90 ( Nicotiana tabacum cv. TN90) was transformed using the leaf disc method to obtain transgenic tobacco of pBin-CBFD-2A-HBFD-HA. After antibiotic screening, the regenerated seedlings differentiated from the callus were transferred to the rooting medium for further cultivation, and then transferred to the nutrient soil for further cultivation when they grew to an appropriate size.

[0036] 3. Construction of the transient expression vector pBin-HBFD-HA: In the experiment, the transiently expressed HBFD was used as a control. When constructing the transient expression vector of HBFD, the coding sequence of HBFD was amplified using the specific primers (HBFD-5’ / HBFD-3’) in Table 1. The PCR reaction conditions were: pre-denaturation at 95°C for 30 s; 35 cycles of denaturation at 95°C for 15 s, annealing at 58°C for 15 s, and extension at 72°C for 30 s; incubation at 72°C for 5 min. Subsequently, using the same method as described above, the PCR product HBFD was recombined into the pBin19-attR-HA vector (Zhuangmeng Biotech, product number ZK1941) containing the 2×CaMV35S promoter to construct the expression vector pBin-HBFD-HA, and sequencing verification was performed. The correctly sequenced vector pBin-HBFD-HA was transformed into Agrobacterium tumefaciens GV3101 and injected into tobacco leaves for transient expression of HBFD.

[0037] Table 1 Primers for constructing vectors used in the experiment

[0038]

[0039] Gene expression analysis: To determine the expression of CBFD and HBFD in the above transgenic tobacco, total RNA of transgenic tobacco was extracted using TRIzol ® reagent, and the RNA was reverse-transcribed into cDNA according to the instructions of the PrimeScript™ II 1st Strand cDNA Synthesis Kit (Takara). The expression levels of CBFD and HBFD were analyzed by semi-quantitative RT-PCR. The semi-quantitative RT-PCR reaction used the tobacco Actin gene as an internal reference gene and specific primers (Table 2) for PCR amplification. The PCR reaction amplification conditions were: pre-denaturation at 95°C for 3 min; 25 cycles of denaturation at 95°C for 15 s, annealing at 58°C for 15 s, and extension at 72°C for 15 s; incubation at 72°C for 5 min. The PCR products were separated on a 1% (w / v) agarose gel and stained with ethidium bromide for observation. The images were taken by the JUNYI JY04S-3E gel imaging analysis system.

[0040] Table 2 Primers for semi-quantitative RT-PCR

[0041]

[0042] Protein expression detection by Western Blotting: Take 0.5 g of fresh leaves of pBin-CBFD-2A-HBFD-HA transgenic tobacco and leaves of pBin-HBFD-HA transient expression, grind them into powder with liquid nitrogen, and boil in 2× protein extraction buffer (100 mM Tris-Cl, pH = 6.8; 200 mM DTT; 4% SDS; 20% glycerol) for 10 min to extract total protein. After centrifugation of the extract, take the supernatant protein solution and separate it by SDS-PAGE gel electrophoresis. Transfer the separated protein sample to a PVDF membrane by wet transfer method, and perform Western Blotting analysis using the monoclonal antibody Anti-HA (12CA5) (1:4000) (Roche) antibody produced in mice. Then generate antibody binding signals through anti-mouse IgG (H+L) HRP (1:4000) (Affinity Biosciences) and Pierce™ ECL Western Blotting Substrate (Thermo Fisher Scientific), and take images with Tanon 5200 fully automatic chemiluminescence image analysis system.

[0043] The test results showed that the leaves, stems, flowers and seed coats of positive transgenic tobacco plants of pBin-CBFD-2A-HBFD-HA all showed red due to the synthesis of astaxanthin ( Figure 1 in A), indicating that the CBFD and HBFD proteins linked by the picornavirus 2A cleavage polypeptide were successfully expressed and cleaved. The gene expression results of transgenic tobacco also showed that the CBFD and HBFD genes in pBin-CBFD-2A-HBFD-HA transgenic tobacco were successfully expressed, and the expression levels were relatively close ( Figure 1 in B).

[0044] Protein Western Blotting detection of pBin-CBFD-2A-HBFD-HA transgenic tobacco showed that in addition to a 100 KDa protein band corresponding to the unspliced CBFD-2A-HBFD, there were also multiple protein bands inconsistent with the protein bands of transiently expressed HBFD ( Figure 1 in C), indicating that there may be unknown novel protein variable splicing sites in the CBFD-2A-HBFD fusion protein in pBin-CBFD-2A-HBFD-HA transgenic tobacco.

[0045] Example 2: There is a protein variable splicing element at the N-terminus of HBFD

[0046] Experimental protocol: The above results indicate that there is a new protein alternative splicing in the CBFD and HBFD proteins linked by the picornavirus 2A cleavage polypeptide. When identifying the position where this splicing occurs, we detected whether specific protein cleavage could occur after the expression of the direct fusion protein of CBFD and HBFD in tobacco and whether astaxanthin synthesis could be achieved.

[0047] Construction of pMDC-CBFD-HBFD-Flag vector: In the first round of PCR, specific primers in Table 3 (CBFD-5’ / CBFD-FusH3’, HBFD-FusC5’ / HBFD-3’) were used to amplify the gene fragments of CBFD and HBFD respectively. The PCR reaction conditions were: pre-denaturation at 95°C for 30 s; 35 cycles of denaturation at 95°C for 15 s, annealing at 58°C for 15 s, and extension at 72°C for 30 s; incubation at 72°C for 5 min. After the PCR products were recovered and purified by gel cutting, they were used as templates for the second round of PCR. In the second round of PCR, using the specific primers in Table 3 (CBFD-5’ / HBFD-3’) and the template fragments obtained by the above amplification, the directly tandem fragment CBFD-HBFD of CBFD and HBFD was obtained by amplification. The PCR reaction conditions were: pre-denaturation at 95°C for 30 s; 35 cycles of denaturation at 95°C for 15 s, annealing at 58°C for 15 s, and extension at 72°C for 1.5 min; incubation at 72°C for 5 min. Subsequently, according to the method described above, the CBFD-HBFD fragment was cloned into the pENTR-D-TOPO vector (the same as above), and a recombination reaction mediated by Gateway® LR Clonase® II Enzyme mix (Invitrogen) was used to recombine it into the pMDC-attR-Flag vector (Zhuangmeng Biology, product number ZK29707) to construct the pMDC-CBFD-HBFD-Flag vector, and tobacco transformation was carried out to obtain pMDC-CBFD-HBFD-Flag transgenic tobacco, where Flag is a tag protein.

[0048] Table 3 Primers for vector construction

[0049]

[0050] Gene expression analysis: RNA was extracted from pMDC-CBFD-HBFD-Flag transgenic tobacco and reverse transcribed into cDNA, and the expression levels of CBFD and HBFD were analyzed by semi-quantitative RT-PCR. The semi-quantitative RT-PCR method was the same as that described above.

[0051] Protein expression detection by Western Blotting: Total protein was extracted from 0.5 g of fresh leaves of pMDC-CBFD-HBFD-Flag transgenic tobacco. The protein samples transferred to the PVDF membrane were detected by the monoclonal antibody Anti-FLAG produced in mice® Western blotting analysis was performed using M2 (1:4000) (Sigma Aldrich). Subsequently, antibody binding signals were generated using anti-mouse IgG (H+L) HRP (1:4000) (Affinity Biosciences) and Pierce™ ECL Western Blotting Substrate (Thermo Fisher Scientific), and images were captured by a Tanon 5200 fully automatic chemiluminescence image analysis system.

[0052] Research results: The test results showed that the leaves, stems, flowers, and seed coats of the positive transgenic tobacco plants of pMDC-CBFD-HBFD-Flag were all red due to the synthesis of astaxanthin ( Figure 2 in A), indicating that the directly tandem CBFD and HBFD proteins were successfully expressed and cleaved. The gene expression results of the transgenic tobacco also showed that the CBFD and HBFD genes in the pMDC-CBFD-HBFD-Flag transgenic tobacco were successfully expressed, and the expression levels were relatively close ( Figure 2 in B).

[0053] Protein Western blotting detection of the pMDC-CBFD-HBFD-Flag transgenic tobacco showed that in addition to a 100KDa protein band corresponding to the unspliced CBFD-2A-HBFD, there were also multiple bands formed by protein cleavage, and two of the main bands were close to the theoretical sizes of the CBFD and HBFD proteins ( Figure 2 in C), indicating that the directly tandemly expressed CBFD-HBFD fusion protein in the pMDC-CBFD-HBFD-Flag transgenic tobacco was cleaved and correctly performed the function of astaxanthin synthesis.

[0054] Example 3: The 23 amino acids at the N-terminus of HBFD are protein alternative splicing elements

[0055] Experimental protocol: To identify the sequence of the cleavage peptide segment in CBFD-HBFD, immunoprecipitation (Co-IP) experiments were performed on pBin-CBFD-2A-HBFD-HA transgenic tobacco and pMDC-CBFD-HBFD-flag transgenic tobacco using HA antibody and Flag antibody respectively to obtain the cleaved protein samples, and mass spectrometry analysis was carried out on them.

[0056] Protein extraction: Take 5 g of fresh leaves of the above transgenic tobacco, grind them in liquid nitrogen, and add 6 mL of Co-IP binding buffer (50 mM Tris-Cl, pH = 7.5; 100 mM NaCl; 0.2% Triton X-100; 0.5 mM phenylmethylsulfonyl fluoride; protease inhibitor) for total protein extraction. Add 12 μL of mouse monoclonal antibody Anti-HA (12CA5) (Roche) or Anti-FLAG ® M2 (Sigma Aldrich) to the extraction mixture and incubate at 4°C for 3 hours. Then add Protein G-Agarose (Roche) beads and continue to incubate at 4°C for 3 hours. Collect Protein G-Agarose (Roche) and boil it in 2× protein extraction buffer for 10 min. Centrifuge to collect the supernatant protein sample for SDS-PAGE electrophoresis.

[0057] Protein mass spectrometry detection: Cut the gel strips into pieces and decolorize them with decolorizing solution. Then treat the decolorized gel particles with acetonitrile until they become completely white and air-dry naturally. Digest the decolorized and dehydrated gel particles, centrifuge, and collect the supernatant for freeze-drying. Dissolve the freeze-dried powder in 0.1% formic acid aqueous solution, centrifuge, and take the supernatant to slowly pass through a C18 desalting column. After eluting the column with an appropriate amount of eluent, collect the filtrate and freeze-dry it. After redissolving the freeze-dried powder in 0.1% formic acid aqueous solution, centrifuge to collect the supernatant for high-performance liquid chromatography-mass spectrometry (LC-MS / MS) analysis. Use an EASY-nLC™ 1200 nano ultra-high performance liquid chromatography system (Thermo Fisher Scientific) for chromatographic analysis. The pre-column and analytical column are both self-made by Novogene. The liquid chromatography elution conditions are shown in Table 4. The separated peptides are analyzed by Q Exactive TM HF-X mass spectrometer (Thermo Fisher Scientific). The ion source is Nanospray Flex™ (ESI) (Thermo Fisher Scientific), the spray voltage is 2.1 kV, the ion transfer tube temperature is 320°C, and the mass spectrometry uses data-dependent acquisition mode. Analyze the obtained spectra for the target protein sequence through Proteome Discover 2.2.

[0058] Table 4 Gradient elution system for UPLC analysis of protein gel strips

[0059]

[0060] Terminal amplification detection of the expressed gene: Four gene-specific primers were designed at approximately 800 bp, 700 bp, 600 bp, and 500 bp downstream of the start codon ATG of CBFD, respectively, and were used in combination with universal primers (Table 5). Using Smarter ® RACE 5’ / 3’ Kit (Takara) to perform the rapid amplification experiment of the cDNA 5’-end.

[0061] The RNA of pBin-CBFD-2A-HBFD-HA and pMDC-CBFD-HBFD-flag transgenic tobacco was synthesized into cDNA templates for rapid amplification of the 5’-end according to the kit instructions. Using the cDNA of the two as templates respectively, the first round of PCR amplification was carried out using the primer GSP-800bp; using the product of the first round of PCR as the template, the second round of PCR amplification was carried out using the primer GSP-700bp; using the product of the second round of PCR as the template, the third round of PCR amplification was carried out using the primer GSP-600bp; using the product of the third round of PCR as the template, the fourth round of PCR amplification was carried out using the primer GSP-500bp. The PCR reaction conditions were: pre-denaturation at 95°C for 3 min; 25 cycles of denaturation at 94°C for 30 s, annealing at 58°C for 30 s, and extension at 72°C for 3 min; incubation at 72°C for 5 min. The obtained PCR products were electrophoretically separated in 1% (w / v) agarose gel and stained with ethidium bromide for observation.

[0062] Table 5 Primers for rapid amplification of the 5’-end

[0063]

[0064] Test results:

[0065] Through the immunoprecipitation (Co-IP) experiment, the proteins with HA tags and Flag tags were separated from the protein extracts of pBin-CBFD-2A-HBFD-HA and pMDC-CBFD-HBFD-Flag transgenic tobacco respectively. After electrophoretic separation and silver staining of the gel, an antibody band of approximately 60 kDa, a protein band of approximately 40 kDa, and a small number of lightly stained protein bands could be seen ( Figure 3In A), the 40KDa protein bands were sent to Novogene for LC-MS / MS analysis and found to be all HBFD proteins. One peptide segment corresponding to the 2A cleavage peptide and six peptide segments of the HBFD protein were detected in the pBin-CBFD-2A-HBFD-HA transgenic tobacco samples. Eight peptide segments of the HBFD protein were detected in the pMDC-CBFD-HBFD-Flag transgenic tobacco samples, and one of the peptide segments was only 23 amino acids away from the N-terminus of HBFD. Moreover, no peptide segments corresponding to CBFD were detected in the samples of both materials. The above results show that there are alternative splicing sites at the N-terminus of the HBFD protein or the 5' end of its mRNA ( Figure 3 In B), subsequently, RT-PCR was used to amplify the 5' ends of the cDNAs corresponding to CBFD-2A-HBFD-HA and CBFD-HBFD-Flag in the two transgenic tobaccos. It was found that after the second round of amplification, only one band could be amplified by the primers at both ends of the gene fusion junction ( Figure 3 In C), indicating that there is no splicing at the mRNA level, and the splicing of CBFD-HBFD occurs at the protein level.

[0066] Example 4: The N-terminal splicing element of HBFD can be used for tandem expression of other proteins

[0067] Experimental scheme: To verify whether the N-terminal splicing element of HBFD can be used for tandem expression and specific splicing of other proteins, 84 amino acids (yFP) (SEQ ID NO.37) at the C-terminus of the yellow fluorescent protein (YFP) and the complete YFP (SEQ ID NO.38) were tandemly expressed using this splicing element (HB23) as a linker, and Flag protein tags were added to the C-terminus of yFP and YFP respectively. After expression in tobacco, their alternative splicing was detected by Western Blotting.

[0068] Construction of expression vector: The schematic diagram of the transient expression vector construction is shown in Figure 4 In A, for the first round of PCR amplification, specific primers in Table 6 (yFP-Flag-HB23-5’ / yFP-Flag-fus1-3’ or yFP-Flag-HB23-FUS1-3’, yFP-Flag-fus2-5’ or yFP-Flag-HB23-FUS2-5’ / yFP-Flag-HB23-3’) were used to amplify the Flag tag, yFP fragment, HB23 peptide segment and the coding fragment of YFP respectively. The PCR reaction conditions were: pre-denaturation at 95°C for 3 min; 35 cycles of denaturation at 95°C for 15 s, annealing at 58°C for 30 s, extension at 72°C for 3 min; incubation at 72°C for 5 min. After the PCR products were recovered and purified by gel cutting, they were used as templates for the second round of PCR.

[0069] The second round of PCR amplification was performed using the specific primers in Table 6 (yFP-flag-HB23-5’ / yFP-flag-HB23-3’) and the template fragment obtained above to amplify the yFP-Flag-YFP-flag and yFP-Flag-HB23-YFP-Flag fusion fragments. Specifically:

[0070] Using the mixture of PCR products of the coding fragments of the Flag tag, yFP fragment and YFP as a template, PCR amplification was carried out with the primer pair (yFP-flag-HB23-5’ / yFP-flag-HB23-3’) to obtain the yFP-Flag-YFP-flag fusion fragment.

[0071] Using the mixture of PCR products of the coding fragments of the Flag tag, yFP fragment, HB23 peptide segment and YFP as a template, PCR amplification was carried out with the primer pair (yFP-flag-HB23-5’ / yFP-flag-HB23-3’) to obtain the yFP-Flag-HB23-YFP-Flag fusion fragment.

[0072] The PCR reaction conditions were as follows: pre-denaturation at 95°C for 30 s; 35 cycles of denaturation at 95°C for 15 s, annealing at 58°C for 15 s, and extension at 72°C for 1.5 min; incubation at 72°C for 5 min. By the method described above, the pMDC-yFP-Flag-YFP-flag and pMDC-yFP-Flag-HB23-YFP-Flag vectors were constructed.

[0073] Table 6 Primers for vector construction

[0074]

[0075] Transient expression in tobacco leaves: The above vectors were separately transferred into Agrobacterium tumefaciens GV3101 and injected into tobacco leaves. Samples were taken after 5 days of cultivation in the greenhouse, ground into powder with liquid nitrogen, and total leaf proteins were extracted by the method described above. Monoclonal Anti-FLAG ® M2 (Sigma Aldrich) antibody was used for Western Blotting analysis. Subsequently, antibody-binding signals were generated by anti-mouse IgG (H+L) HRP (1:4000) (Affinity Biosciences) and Pierce™ ECL Western Blotting Substrate (Thermo Fisher Scientific), and images were taken by the Tanon 5200 fully automatic chemiluminescence image analysis system.

[0076] Test results: Using the tobacco transient expression sample of pMDC-yFP-flag-YFP-flag as a control, the protein cleavage of yFP-flag-HB23-YFP-flag after expression in tobacco was analyzed. The results showed that in the protein sample without the HBFD cleavage peptide segment, there was only one uncleaved protein band of approximately 40KDa, while in the protein containing the HBFD cleavage peptide segment, the tandem protein was cleaved, and two protein bands appeared, one approximately 30KDa and one approximately 10KDa ( Figure 4 in B), indicating that the 23-amino acid peptide segment at the N-terminus of the HBFD protein can undergo specific cleavage at the protein level in tobacco and can be used for the multi-protein tandem expression of a single expression cassette of other proteins.

[0077] Finally, it should also be noted that the term "comprising", "including" or any other variant thereof is intended to cover non-exclusive inclusion, so that a process, method, article or device comprising a series of elements not only includes those elements but also includes other elements not expressly listed, or further includes elements inherent to such process, method, article or device.

[0078] Although the preferred embodiments of the present invention have been described, those skilled in the art can make additional changes and modifications once they learn the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments as well as all changes and modifications falling within the scope of the present invention.

[0079] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalent technologies, the present invention also intends to include these modifications and variations.

Claims

1. A variable splicing polypeptide, characterized in that: The amino acid sequence of the variable splicing polypeptide is shown in SEQ ID NO.

1.

2. Use of the variable splicing polypeptide according to claim 1 as a linker in the tandem expression of tobacco polyproteins.

3. The use according to claim 2, characterized in that: The application includes: realizing specific shearing in tobacco and realizing multi-protein tandem expression in a single expression frame.

4. A method for tandem expression of multiple proteins, characterized in that: The method comprises: Obtaining a first gene fragment and a second gene fragment to be expressed in tandem; The variable splicing polypeptide according to claim 1 is used as a linker to express the first gene segment and the second gene segment in series in tobacco.

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