Variable cleavage polypeptides and uses thereof

Abstract

The invention provides a variable cleavage polypeptide and application thereof, the variable cleavage polypeptide is a polypeptide composed of 23 amino acids at the N terminal of calendula spica HBFD, and can realize specific cleavage in tobacco and multi-protein tandem expression of a single expression cassette. The cleavage specificity of the variable cleavage polypeptide in plants is superior to that of a previously reported small RNA virus 2A self-cleavage polypeptide, and reduction of non-specific cleavage is facilitated. The variable cleavage polypeptide is a protein cleavage element derived from a plant, and the application value of a multi-protein tandem expression technology based on a single expression cassette in a plant bioreactor is improved.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to variable splicing polypeptides and applications thereof. Background Art

[0002] In the process of developing plant bioreactors, it is usually necessary to co-express multiple proteins. However, in plants as eukaryotic organisms, their protein translation is restricted by the "monocistron" principle. Multiple independent protein expression cassettes are required to express multiple proteins, which greatly increases the difficulty of constructing plant bioreactors. In order to achieve plant co-expression of multiple proteins, people have tried a variety of methods. Many studies use multiple vectors containing single expression cassettes or vectors containing multiple expression cassettes for multi-protein expression, but such methods are often affected by unbalanced gene expression, and the probability of achieving efficient expression of multiple proteins is relatively low, and it often requires a large amount of work to achieve. Some studies use plastid transformation methods, using prokaryotic polycistronic expression cassettes to express multiple proteins in plant plastids, but this method is only meaningful when expressing proteins that function in plastids, and has great limitations. Another part of the study uses a single expression cassette to express multiple proteins connected in series by variable splicing protein elements as linkers. After expression in the plant, the co-expression of multiple proteins is achieved by shearing of the protein linker. This method can achieve balanced expression of multiple proteins, but it will be affected by the shearing efficiency of the shearing element. At present, commonly used variable splicing protein elements include the 2A self-splicing polypeptide of small RNA viruses and tobacco etch virus protease. The splicing efficiency of these splicing peptides in plants is not high, and they are all protein elements of viral origin, so they are not the best choice for developing plant bioreactors.

[0003] Therefore, identifying efficient variable splicing protein elements of non-viral origin is of great significance to improving the application value of multi-protein tandem expression technology based on a single expression frame in plant bioreactors. Summary of the invention

[0004] The purpose of the present invention is to provide a variable splicing polypeptide and its application. The variable splicing polypeptide of the present invention is a polypeptide composed of 23 amino acids at the N-terminus of HBFD of Marigold, which can achieve specific splicing in tobacco and realize multi-protein tandem expression of a single expression frame. The splicing specificity of the variable splicing polypeptide of the present invention in plants is better than that of the small RNA virus 2A self-splicing polypeptide reported by previous researchers, which is conducive to reducing the occurrence of non-specific splicing.

[0005] To achieve the above object, the present invention adopts the following technical solution: In a first aspect of the present invention, a variable splicing polypeptide is provided, characterized in that the amino acid sequence of the variable splicing polypeptide is as shown in SEQ ID NO.1: MRTLHGACLPWIETNSLHWKLVK.

[0006] Furthermore, the variable splicing polypeptide also includes a polypeptide with the same function obtained by replacing, deleting and / or adding one or more amino acids in the amino acid sequence shown in SEQ ID NO.1.

[0007] In the second aspect of the present invention, the use of a variable splicing polypeptide in the tandem expression of plant multi-proteins is provided.

[0008] Furthermore, the plant includes at least one of Arabidopsis, tobacco, tomato, soybean, wheat and rice.

[0009] Furthermore, the application includes: achieving specific shearing in plants and realizing the tandem expression of multiple proteins in a single expression frame.

[0010] In a third aspect of the present invention, a method for tandem expression of multiple proteins is provided, the method comprising: Obtaining a first gene fragment and a second gene fragment to be expressed in tandem; The variable splicing polypeptide is used as a linker to express the first gene fragment and the second gene fragment in series.

[0011] Furthermore, the first gene fragment or the second gene fragment is a yFP gene with a nucleotide sequence as shown in SEQ ID NO.37 and a complete YFP gene with a nucleotide sequence as shown in SEQ ID NO.38.

[0012] One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages: 1. The variable splicing polypeptide and its application provided by the present invention are based on a variable splicing polypeptide found at the N-terminus of HBFD (carotene β-ring 4-hydroxydehydrogenase) of Asteraceae, and provide a plant multi-protein tandem expression method using the polypeptide as a linker. The variable splicing polypeptide of the present invention is a polypeptide composed of 23 amino acids at the N-terminus of HBFD of Asteraceae, which can achieve specific splicing in tobacco and realize multi-protein tandem expression of a single expression frame.

[0013] 2. The shearing specificity of the variable splicing polypeptide of the present invention in plants is better than that of the previously reported small RNA virus 2A self-splicing polypeptide, which is beneficial to reduce the occurrence of non-specific shearing. The variable splicing polypeptide of the present invention is used for the single expression frame multi-protein tandem expression of other proteins in plants, and the co-expression of multi-proteins in plants can be achieved through shearing.

[0014] 3. The variable splicing polypeptide of the present invention is a protein splicing element derived from plants, which is beneficial to improving the application value of multi-protein tandem expression technology based on a single expression frame in plant bioreactors. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0016] Figure 1 :Construction of astaxanthin-producing tobacco expressing CBFD-2A-HBFD. A. Astaxanthin-producing tobacco expressing CBFD-2A-HBFD. Plants transformed with empty vector were used as blank controls. B. The expression of CBFD and HBFD in transgenic tobacco was analyzed by semi-quantitative RT-PCR. C. HA-tagged CBFD-2A-HBFD (stable expression) and HBFD (transient expression) proteins were detected by Western Blotting. The red arrow indicates the unspliced ​​CBFD-2A-HBFD, the black arrow indicates the spliced ​​HBFD and the transiently expressed HBFD, and 1-2 are two independent transgenic plants.

[0017] Figure 2 :Construction of astaxanthin-producing tobacco expressing CBFD-HBFD. A. Astaxanthin-producing tobacco expressing CBFD-HBFD, and plants transformed with an 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 the unspliced ​​CBFD-HBFD, the red arrow indicates the spliced ​​HBFD, and 1-2 indicate two independent transgenic tobaccos.

[0018] Figure 3 :Identification of HBFD cleavage peptides. 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 two transgenic tobacco samples; the green box indicates the same fragment detected in two transgenic tobacco samples. C. 5'-end RT-PCR amplification experiment results, 1-4 are 4 rounds of PCR amplification products.

[0019] Figure 4:Application of HBFD cleavage peptide in other multi-protein tandem expression. A. Schematic diagram of transient expression vector construction. B. Western Blotting to detect the specific cleavage effect of cleavage peptide fragments in tobacco. “—” indicates a tandem protein without HBFD cleavage peptide, and “+” indicates a tandem protein with HBFD cleavage peptide. DETAILED DESCRIPTION

[0020] The present invention will be described in detail below in conjunction with specific implementations and examples, and the advantages and various effects of the present invention will be more clearly presented. It should be understood by those skilled in the art that these specific implementations and examples are used to illustrate the present invention, rather than to limit the present invention.

[0021] Throughout the specification, unless otherwise specifically stated, the terms used herein should be understood as 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 belongs. In the event of a conflict, the present specification takes precedence.

[0022] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or obtained by existing methods.

[0023] The variable splicing polypeptide and its application of the present application will be described in detail below with reference to the examples and experimental data.

[0024] Example 1: Fusion protein expression of HBFD has novel variable splicing 1. Gene expression design: When the astaxanthin synthase CBFD (GenBank accession number: AY644757.1) and HBFD (GenBank accession number: DQ902555.1) of Marigold acuminata were co-expressed in tobacco, the picornavirus 2A self-cleavage polypeptide was used as a linker and added between CBFD and HBFD, and a single expression frame was used to express the two genes CBFD and HBFD in series. 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 2A cleavage peptide is shown in SEQ ID NO.4.

[0025] 2. Construction of expression vector pBin-CBFD-2A-HBFD-HA: The first round of PCR used the specific primers in Table 1 (CBFD-5' / CBFD-FUS3', HBFD-FUS5' / HBFD-3', 2A-5 / 2A-3') to amplify the peptide fragments of the CBFD gene, HBFD gene, and 2A polypeptide coding sequence, respectively. The PCR reaction conditions were: 95°C pre-denaturation for 30s; 35 cycles of 95°C denaturation for 15s, 58°C annealing for 15s, and 72°C extension for 30s; and 72°C incubation for 5min. The PCR amplification product was excised and purified and used as the amplification template for the second round of PCR.

[0026] The second round of PCR used the obtained amplified fragment as a template to amplify the fusion gene fragment CBFD-2A-HBFD with the specific primers (CBFD-5' / HBFD-3') in Table 1. The PCR reaction conditions were: 95°C pre-denaturation for 30 s; 35 cycles of 95°C denaturation for 15 s, 58°C annealing for 15 s, and 72°C extension for 1.5 min; and 72°C incubation for 5 min.

[0027] The PCR amplification product was purified by gel excision and then the restriction endonuclease sites were used to Sac II and Asc I. Clone the fusion fragment CBFD-2A-HBFD into the pENTR-D-TOPO vector (purchased from Invitrogen, catalog number K240020).

[0028] Then, the fusion fragment CBFD-2A-HBFD was recombined into the pBin19-attR-HA vector (Zhuangmeng Bio, Catalog No. ZK1941) containing the 2×CaMV35S promoter through an LR reaction mediated by Gateway® LR Clonase® II Enzyme mix (Invitrogen) to obtain the expression vector pBin-CBFD-2A-HBFD-HA, in which HA is the tag protein.

[0029] After sequencing verification, the expression vector pBin-CBFD-2A-HBFD-HA was transferred into Agrobacterium LBA4404 by freeze-thaw method, and then transformed into tobacco TN90 by leaf disc method ( Nicotiana tabacum cv. TN90), and obtained 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 after growing to a suitable size.

[0030] 3. Construction of transient expression vector pBin-HBFD-HA: The transiently expressed HBFD was used as a control in the experiment. 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: 95°C pre-denaturation for 30s; 35 cycles of 95°C denaturation for 15s, 58°C annealing for 15s, and 72°C extension for 30s; incubation at 72°C for 5min. Subsequently, the PCR product HBFD was recombined into the pBin19-attR-HA vector (Zhuangmeng Biology, Product No. ZK1941) containing the 2×CaMV35S promoter using the same method as above, and the expression vector pBin-HBFD-HA was constructed and sequenced for verification. The correctly sequenced vector pBin-HBFD-HA was transformed into Agrobacterium GV3101 and injected into tobacco leaves for transient expression of HBFD.

[0031] Table 1 Primers for constructing vectors used in the experiment

[0032] Gene expression analysis: To determine the expression of CBFD and HBFD in the above transgenic tobacco, TRIzol ® Total RNA from transgenic tobacco was extracted using a reagent and converted into cDNA according to the instructions of PrimeScript™ II 1st Strand cDNA Synthesis Kit (Takara). The expression levels of CBFD and HBFD were analyzed by semi-quantitative RT-PCR. Actin The gene was used as an internal reference gene and PCR amplified using specific primers (Table 2). The PCR reaction amplification conditions were: 95℃ pre-denaturation for 3min; 25 cycles of 95℃ denaturation for 15s, 58℃ annealing for 15s, and 72℃ extension for 15s; incubation at 72℃ for 5min. The PCR products were separated in 1% (w / v) agarose gel and observed by ethidium bromide staining. The images were taken by the JUNYI JY04S-3E gel imaging analysis system.

[0033] Table 2 Semi-quantitative RT-PCR primers

[0034] Protein expression Western Blotting detection: 0.5 g of fresh pBin-CBFD-2A-HBFD-HA transgenic tobacco leaves and pBin-HBFD-HA transient expression leaves were ground into powder using liquid nitrogen and boiled for 10 min in 2× protein extraction buffer (100 mM Tris-Cl, pH = 6.8; 200 mM DTT; 4% SDS; 20% glycerol) to extract total protein. After centrifugation of the extract, the supernatant protein solution was separated by SDS-PAGE gel electrophoresis. The separated protein samples were transferred to PVDF membranes by wet transfer and analyzed by Western Blotting using the monoclonal antibody Anti-HA (12CA5) (1:4000) (Roche) produced in mice. Antibody binding signals were then generated by 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 automated chemiluminescence image analysis system.

[0035] The test results showed that the leaves, stems, flowers and seed coats of the positive transgenic tobacco plants of pBin-CBFD-2A-HBFD-HA all showed red color due to the synthesis of astaxanthin ( Figure 1 (A), indicating that the CBFD and HBFD proteins connected 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 the pBin-CBFD-2A-HBFD-HA transgenic tobacco were successfully expressed, and the expression levels were relatively close ( Figure 1 Middle B).

[0036] Western Blotting of proteins from pBin-CBFD-2A-HBFD-HA transgenic tobacco showed that, in addition to a 100KDa protein band corresponding to the unspliced ​​CBFD-2A-HBFD, there were several other protein bands that were inconsistent with the transiently expressed HBFD protein bands ( Figure 1 (C) indicates that there may be unknown novel protein variable splicing sites in the CBFD-2A-HBFD fusion protein in the pBin-CBFD-2A-HBFD-HA transgenic tobacco.

[0037] Example 2: There is a protein variable splicing element at the N-terminus of HBFD Experimental plan: The above results indicate that there is a new protein variable splice in the CBFD and HBFD proteins connected by the picornavirus 2A splice polypeptide. When identifying the location of the splice, we tested whether the direct fusion protein of CBFD and HBFD can undergo specific protein splicing and achieve the synthesis of astaxanthin after being expressed in tobacco.

[0038] Construction of pMDC-CBFD-HBFD-Flag vector: In the first round of PCR, the 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: 95℃ pre-denaturation for 30s; 35 cycles of 95℃ denaturation for 15s, 58℃ annealing for 15s, and 72℃ extension for 30s; incubation at 72℃ for 5min. The PCR product was purified by gel excision and recovery and used as the template for the second round of PCR. In the second round of PCR, the specific primers in Table 3 (CBFD-5' / HBFD-3') and the template fragment obtained by the above amplification were used to amplify the direct tandem fragment of CBFD and HBFD, CBFD-HBFD. The PCR reaction conditions were: 95℃ pre-denaturation for 30s; 35 cycles of 95℃ denaturation for 15s, 58℃ annealing for 15s, and 72℃ extension for 1.5min; incubation at 72℃ for 5min. Subsequently, the CBFD-HBFD fragment was cloned into the pENTR-D-TOPO vector (same as above) according to the method described above, and recombined into the pMDC-attR-Flag vector (Zhuangmeng Bio, Catalog No. ZK29707) using a recombination reaction mediated by Gateway® LR Clonase® II Enzyme mix (Invitrogen) to construct the pMDC-CBFD-HBFD-Flag vector, and tobacco transformation was performed to obtain pMDC-CBFD-HBFD-Flag transgenic tobacco, in which Flag is a tag protein.

[0039] Table 3 Vector construction primers

[0040] Gene expression analysis: RNA from pMDC-CBFD-HBFD-Flag transgenic tobacco was extracted and converted into cDNA, and the expression levels of CBFD and HBFD were analyzed by semi-quantitative RT-PCR. The semi-quantitative RT-PCR method was consistent with that described above.

[0041] Protein expression Western Blotting detection: Total protein was extracted from 0.5 g fresh pMDC-CBFD-HBFD-Flag transgenic tobacco leaves. The protein sample transferred to the PVDF membrane was stained with the monoclonal antibody Anti-FLAG produced in mice. ®Western Blotting analysis was performed using M2 (1:4000) (Sigma Aldrich). Antibody binding signals were then 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 using a Tanon 5200 fully automated chemiluminescence image analysis system.

[0042] Research results: The test results show that the leaves, stems, flowers and seed coats of the positive transgenic tobacco plants of pMDC-CBFD-HBFD-Flag are red due to the synthesis of astaxanthin ( Figure 2 A in the middle), indicating that the directly tandem CBFD and HBFD proteins were successfully expressed and spliced. The gene expression results of 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 Middle B).

[0043] Western Blotting of proteins from pMDC-CBFD-HBFD-Flag transgenic tobacco showed that in addition to a 100KDa protein band corresponding to unspliced ​​CBFD-2A-HBFD, there were multiple bands formed by protein splicing, of which the two main bands were close to the theoretical sizes of CBFD and HBFD proteins ( Figure 2 (C) shows that the CBFD-HBFD fusion protein directly expressed in tandem in the pMDC-CBFD-HBFD-Flag transgenic tobacco was cleaved and correctly played the role of astaxanthin synthesis.

[0044] Example 3: The N-terminal 23 amino acids of HBFD are protein variable splicing elements Experimental plan: In order to identify the sequence of the cleaved peptide in CBFD-HBFD, HA antibody and Flag antibody were used to perform co-immunoprecipitation (Co-IP) experiments on pBin-CBFD-2A-HBFD-HA transgenic tobacco and pMDC-CBFD-HBFD-flag transgenic tobacco, respectively, to obtain the cleaved protein samples, which were then subjected to mass spectrometry analysis.

[0045] Protein extraction: 5 g of fresh transgenic tobacco leaves were ground in liquid nitrogen and 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 inhibitors) was added to extract total protein. 12 μL of mouse monoclonal antibody Anti-HA (12CA5) (Roche) or Anti-FLAG ® M2 (Sigma Aldrich) was incubated at 4°C for 3 hours. Protein G-Agarose (Roche) beads were then added and incubated at 4°C for another 3 hours. Protein G-Agarose (Roche) beads were collected and boiled in 2× protein extraction buffer for 10 minutes. The supernatant protein samples were collected by centrifugation and subjected to SDS-PAGE electrophoresis.

[0046] Protein spectrum detection: After the gel strips are chopped up, they are decolorized with a decolorizing solution, and then the decolorized gel particles are treated with acetonitrile until they turn completely white and air-dried naturally. The decolorized and dehydrated gel particles are enzymatically hydrolyzed, and the supernatant is collected after centrifugation and freeze-dried. The freeze-dried powder is dissolved in a 0.1% formic acid aqueous solution, and the supernatant is slowly passed through a C18 desalting column after centrifugation. After eluting the column with an appropriate amount of eluent, the filtrate is collected and freeze-dried. After the freeze-dried powder is redissolved in a 0.1% formic acid aqueous solution, the supernatant is collected by centrifugation for high-performance liquid chromatography-mass spectrometry (LC-MS / MS) analysis. Chromatographic analysis was performed using an EASY-nLC™ 1200 nanoliter ultra-high performance liquid chromatography system (Thermo Fisher Scientific). The pre-column and analytical column were both homemade by Novogene, and the liquid chromatography elution conditions are shown in Table 4. The separated peptides were prepared by Q Exactive TM Mass spectrometry analysis was performed using an HF-X mass spectrometer (Thermo Fisher Scientific), with an ion source of Nanospray Flex™ (ESI) (Thermo Fisher Scientific), a spray voltage of 2.1 kV, an ion transfer tube temperature of 320 °C, and a data-dependent acquisition mode. The obtained spectra were analyzed for target protein sequences using Proteome Discover 2.2.

[0047] Table 4 Gradient elution system for UPLC analysis of protein strips

[0048] Terminal amplification detection of expressed genes: 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 and matched with universal primers (Table 5). ® The cDNA 5'-end rapid amplification experiment was performed using the RACE 5' / 3' kit (Takara).

[0049] RNA of pBin-CBFD-2A-HBFD-HA and pMDC-CBFD-HBFD-flag transgenic tobacco was synthesized according to the kit instructions for 5'-end rapid amplification of cDNA templates. The first round of PCR amplification was performed using primers GSP-800bp with the cDNA of the two as templates; the second round of PCR amplification was performed using primers GSP-700bp with the first round of PCR products as templates; the third round of PCR amplification was performed using primers GSP-600bp with the second round of PCR products as templates; the fourth round of PCR amplification was performed using primers GSP-500bp with the third round of PCR products as templates. The PCR reaction conditions were: 95℃ pre-denaturation for 3min; 25 cycles of 94℃ denaturation for 30s, 58℃ annealing for 30s, and 72℃ extension for 3min; incubation at 72℃ for 5min. The obtained PCR products were separated by electrophoresis in 1% (w / v) agarose gel and observed by ethidium bromide staining.

[0050] Table 5 5'-terminal rapid amplification primers

[0051] Test results: By co-immunoprecipitation (Co-IP) experiments, proteins with HA tags and Flag tags were separated from pBin-CBFD-2A-HBFD-HA and pMDC-CBFD-HBFD-Flag transgenic tobacco protein extracts, respectively. After electrophoresis separation and gel silver staining, an antibody band of about 60 KDa and a protein band of about 40 KDa, as well as a small amount of lighter-stained protein bands ( Figure 3 A in the middle). The 40KDa protein bands were sent to Novogene for LC-MS / MS analysis and were found to be HBFD proteins. One peptide corresponding to the 2A shear peptide and six peptides of HBFD protein were detected in the pBin-CBFD-2A-HBFD-HA transgenic tobacco sample, and eight HBFD protein peptides were detected in the pMDC-CBFD-HBFD-Flag transgenic tobacco sample, one of which was only 23 amino acids away from the N-terminus of HBFD. Moreover, no peptide corresponding to CBFD was detected in samples of both materials. The above results show that there are variable shear sites at the N-terminus of the HBFD protein or the 5' end of its mRNA ( Figure 3 Then, RT-PCR was used to amplify the 5' end of the cDNA 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, the primers at both ends of the gene fusion junction could only amplify one band ( Figure 3 (C) shows that there is no shearing at the mRNA level, and the shearing of CBFD-HBFD occurs at the protein level.

[0052] Example 4: The N-terminal splicing element of HBFD can be used for tandem expression of other proteins Experimental plan: To verify whether the N-terminal splicing element of HBFD can be used for the tandem expression and specific splicing of other proteins, the 84 amino acids at the C-terminus of yellow fluorescent protein (YFP) (yFP) (SEQ ID NO.37) and complete YFP (SEQ ID NO.38) were expressed in tandem using the splicing element (HB23) as a linker, and Flag protein tags were added to the C-termini of yFP and YFP, respectively. After expression in tobacco, their variable splicing was detected by Western Blotting assay.

[0053] Expression vector construction: The schematic diagram of transient expression vector construction is shown in Figure 4 In Figure A, the first round of PCR amplification used the 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') to amplify the Flag tag, yFP fragment, HB23 peptide and YFP coding fragment respectively. The PCR reaction conditions were: 95℃ pre-denaturation for 3min; 35 cycles of 95℃ denaturation for 15s, 58℃ annealing for 30s, 72℃ extension for 3min; 72℃ incubation for 5min. The PCR product was excised, recovered and purified and used as the template for the second round of PCR.

[0054] The second round of PCR amplification uses the specific primers in Table 6 (yFP-flag-HB23-5' / yFP-flag-HB23-3') and the template fragments obtained above to amplify the yFP-Flag-YFP-flag and yFP-Flag-HB23-YFP-Flag fusion fragments. Specifically: The mixture of PCR products of the Flag tag, yFP fragment and YFP coding fragment was used as a template and a primer pair (yFP-flag-HB23-5' / yFP-flag-HB23-3') was used for PCR amplification to obtain the yFP-Flag-YFP-flag fusion fragment.

[0055] A mixture of PCR products of the Flag tag, yFP fragment, HB23 peptide and YFP coding fragment was used as a template and a primer pair (yFP-flag-HB23-5' / yFP-flag-HB23-3') was used for PCR amplification to obtain the yFP-Flag-HB23-YFP-Flag fusion fragment.

[0056] The PCR reaction conditions were as follows: 95°C pre-denaturation for 30 s; 35 cycles of 95°C denaturation for 15 s, 58°C annealing for 15 s, 72°C extension for 1.5 min; and 72°C incubation for 5 min. The pMDC-yFP-Flag-YFP-flag and pMDC-yFP-Flag-HB23-YFP-Flag vectors were constructed using the methods described above.

[0057] Table 6 Vector construction primers

[0058] Transient expression in tobacco leaves: The above vectors were transferred into Agrobacterium GV3101 and injected into tobacco leaves. After 5 days of cultivation in the greenhouse, samples were taken, ground into powder with liquid nitrogen, and the total protein of the leaves was extracted by the method described above. ® Western Blotting analysis was performed using M2 (Sigma Aldrich) antibody. Antibody binding signals were then 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 using a Tanon 5200 fully automated chemiluminescence image analysis system.

[0059] Experimental results: Taking the tobacco transient expression sample of pMDC-yFP-flag-YFP-flag as the control, the protein shearing of yFP-flag-HB23-YFP-flag after expression in tobacco was analyzed. The results showed that in the protein sample without HBFD shearing peptide, there was only one uncut protein band of about 40KDa, while in the protein containing HBFD shearing peptide, the tandem protein was sheared, and two protein bands appeared, one about 30KDa and the other about 10KDa ( Figure 4 (B) shows that the 23-amino acid peptide at the N-terminus of the HBFD protein can undergo specific cleavage at the protein level in tobacco and can be used for the single expression frame multi-protein tandem expression of other proteins.

[0060] Finally, it should be noted that the terms "comprises," "includes," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that includes a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements that are inherent to such process, method, article, or apparatus.

[0061] Although the preferred embodiments of the present invention have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0062] 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 equivalents, the present invention is also intended 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. A variable splicing polypeptide according to claim 1, characterized in that The variable splicing polypeptide also includes a polypeptide with the same function obtained by replacing, deleting and / or adding one or more amino acids in the amino acid sequence shown in SEQ ID NO.1 of HBFD of Aristolochia serrata.

3. Use of the variable splicing polypeptide according to claim 1 as a linker in plant multi-protein tandem expression.

4. The use according to claim 3, characterized in that: The plant comprises at least one of Arabidopsis, tobacco, tomato, soybean, wheat and rice.

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

6. 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 fragment and the second gene fragment in tandem.

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