A chloroplast localization signal peptide, its product, method and application

Abstract

This invention, entitled "A Chloroplast Localization Signal Peptide, Its Products, Methods, and Applications," belongs to the field of plant genetic engineering technology. The technical problem this invention aims to solve is the low targeting efficiency, unclear functional elements, and poor species adaptability of existing chloroplast localization signal peptides. The chloroplast localization signal peptide provided by this invention consists of an SP2 homologous peptide backbone, an N21 intermediate linker region, and a BS cleavage site region, sequentially linked from the N-terminus to the C-terminus. The SP2 homologous peptide backbone is constructed based on the tobacco RBCS homologous sequence. The N21 intermediate linker region is a flexible linker peptide rich in proline and glycine. The BS cleavage site region contains a specific recognition site for chloroplast matrix processing peptidases. This invention achieves the technical effects of highly efficient targeted transport of the signal peptide, clear functional elements, and broad species adaptability, and can be used in plant chloroplast genetic engineering.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering technology, specifically relating to a chloroplast localization signal peptide, its products, methods, and applications. Background Technology

[0002] Chloroplasts, as semi-autonomous organelles unique to plant cells, are not only the core site of photosynthesis but also participate in various key physiological processes such as amino acid synthesis, fatty acid metabolism, and the synthesis of secondary metabolites. Their maternally inherited genome avoids the risk of transgenic drift and possesses natural advantages such as multi-gene synergistic expression and efficient accumulation of recombinant proteins. Therefore, chloroplasts have become a highly promising targeted expression system in plant genetic engineering, possessing irreplaceable value in areas such as stress-resistant crop cultivation and plant bioreactors (e.g., pharmaceutical protein and industrial enzyme production).

[0003] The precise targeted transport of recombinant proteins into chloroplasts relies entirely on the mediation of the N-terminal chloroplast targeting peptide (CTP). This signal peptide acts as a "molecular navigation tag," which needs to be recognized by the cellular transport system to guide the precursor protein across the chloroplast membrane into the matrix, where it is then cleaved and removed by the signal peptidase, ensuring that the target protein folds correctly and functions properly.

[0004] Among the prior art represented by relevant patent and non-patent literature retrieved, the research and application of chloroplast localization signal peptides have been studied to some extent. However, many unresolved technical problems or defects still exist in the relevant technical solutions, which seriously limit the industrial application of chloroplast conversion technology. Specifically, these problems are reflected in the following aspects: 1. Low targeting efficiency: The structural design of natural signal peptides or existing artificially modified signal peptides lacks precision. The sequence combination of the N-terminal positive charge region, the central hydrophobic domain (H-region), and the C-terminal cleavage site is unreasonable, resulting in the efficiency of target protein transport to chloroplasts being less than 30%. A large amount of protein remains in the cytoplasm and is degraded by proteases, significantly reducing the yield of recombinant proteins.

[0005] 2. Ambiguous functional elements: Existing technologies have not clearly defined the key functional sites of each domain of the signal peptide—the number of basic amino acids at the N-terminus, the composition and length of hydrophobic residues in the H-region, and the mechanism of action of the C-terminal signal peptidase recognition motif are not thoroughly analyzed, making it impossible to achieve targeted efficiency improvement through structural optimization.

[0006] 3. Limited species adaptability: Most reported chloroplast signal peptides are designed only for specific crops (such as Arabidopsis thaliana and rice). They suffer from target activity decay in model plants such as tobacco and economic crops. Furthermore, no modular functional element library has been formed, making it difficult to meet the needs of different chloroplast metabolic engineering scenarios.

[0007] Furthermore, in plant recombinant protein expression systems, issues such as high intracytoplasmic protease activity, abnormal protein folding, and non-specific modifications further highlight the necessity of chloroplast-targeted transport—directing the target protein into chloroplasts via highly efficient signal peptides can significantly reduce degradation risk, optimize the modification environment, and increase the accumulation of recombinant proteins by several to tens of times. (Nicotiana benthamiana) Nicotiana benthamiana As a model host for plant genetic engineering, nuclear transformation has advantages such as short growth cycle, high transformation efficiency, and suitability for large-scale cultivation. Its nuclear transformation system has become the preferred platform for verifying the function of target signal peptides.

[0008] Therefore, developing a chloroplast localization signal peptide with a well-defined structure, high targeting efficiency, and broad adaptability, and clarifying the key roles of its various functional elements, is of great significance for breaking through the bottlenecks of existing chloroplast transformation technology and promoting the application of plant genetic engineering in agriculture and biopharmaceutical fields. The related technology demand is urgent and the market prospects are broad. Summary of the Invention

[0009] The purpose of this invention is to provide: This invention provides a chloroplast localization signal peptide with a well-defined structure and high targeting efficiency, and its application. This signal peptide can efficiently mediate the directional transport of target proteins to the chloroplasts of Nicotiana benthamiana, and the functional elements can be modularly combined for use in different plant genetic engineering scenarios.

[0010] Terminology Explanation: Unless otherwise defined, all technical terms in this document have the same meanings as commonly understood by one of ordinary skill in the art to which the subject matter of the claims pertains. Unless otherwise stated, all patents, patent inventions, and publications cited in this document are incorporated herein by reference in their entirety. If multiple definitions exist for terms in this document, the definitions in this chapter shall prevail.

[0011] It should be understood that the above brief description and the following detailed description are exemplary and for illustrative purposes only, and do not limit the subject matter of the invention in any way. In this invention, the singular is used in conjunction with the plural unless otherwise specifically stated. It should also be noted that, unless otherwise stated, the use of “or” or “or” means “and / or”. Furthermore, the use of the term “comprising” and other forms such as “including,” “containing,” and “contains” are not limiting.

[0012] Unless otherwise stated, conventional methods within the scope of the art shall be used.

[0013] Unless specifically defined herein, the use of all commercially available products herein employs standard techniques. For example, it may be carried out using the manufacturer's instructions for use with the kit, or in accordance with methods known in the art or the description of this invention. The techniques and methods described herein can generally be implemented according to conventional methods well known in the art, based on the descriptions in the various summary and more specific documents cited and discussed in this specification.

[0014] The terms “optional / arbitrary” or “optionally / arbitrarily” mean that the event or situation described below may or may not occur, including both the occurrence and non-occurrence of the event or situation.

[0015] Chloroplast localization signal peptide: refers to a specific amino acid sequence located at the N-terminus of a protein. It can act as a molecular navigation tag to mediate the entry of precursor proteins across the chloroplast membrane into the chloroplast matrix. It can also be specifically cleaved and removed by signal peptidase. It is the core functional element for achieving targeted transport of proteins to chloroplasts. Its structure includes an N-terminal positively charged region, a central hydrophobic region, and a C-terminal cleavage site.

[0016] Basic amino acids: These are natural amino acids whose side chains have basic groups such as amino, guanidine, or imidazole groups, and which carry a positive charge under physiological pH conditions. They include lysine, arginine, and histidine.

[0017] Hydrophobic amino acid residues: These are natural amino acid residues whose side chains consist of nonpolar groups, are uncharged under physiological pH conditions, and are hydrophobic. They include leucine, isoleucine, valine, phenylalanine, methionine, and alanine.

[0018] Nucleic acid molecules: refer to polymers formed by deoxyribonucleotides linked by phosphodiester bonds. In this invention, the nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO:1, encoding the chloroplast localization signal peptide described in this invention.

[0019] Recombinant expression vectors: These are artificial vectors constructed by modifying natural plasmids through genetic engineering. They contain exogenous target nucleic acid molecules and expression regulatory elements such as promoters and terminators, which can introduce exogenous genes into host cells and achieve stable transcription and translation.

[0020] Operable ligation: refers to the in vitro ligation of nucleic acid molecules with gene expression regulatory elements such as promoters and terminators, in accordance with the natural logical order of gene expression, so that the regulatory elements can effectively recognize and regulate the transcription initiation and termination of target nucleic acid molecules.

[0021] A promoter is a specific DNA sequence located upstream of the 5' end of a gene. It is the site where RNA polymerase recognizes and binds, and it initiates the transcription process of downstream target genes. In this invention, the promoter can be selected from constitutive promoters, tissue-specific promoters, chloroplast-specific promoters, or inducible promoters.

[0022] Terminator: A specific DNA sequence located downstream of the 3' end of a gene that can be recognized by the transcription system and terminate the transcription process of RNA polymerase, causing the transcription product mRNA to be released from the template DNA.

[0023] Host cell: refers to a cell that can receive and accommodate exogenous nucleic acid molecules or recombinant expression vectors, and can provide the necessary cellular environment and raw materials for the transcription and translation of exogenous genes, thereby realizing the expression of exogenous genes. In this invention, the host cell includes Escherichia coli or Agrobacterium.

[0024] Fusion expression: refers to the expression of a fusion protein by linking the coding sequences of two or more genes in vitro through genetic engineering, which is then transcribed into an mRNA in the host cell and translated into a fusion protein containing multiple protein functional regions.

[0025] Targeted transport: refers to the process of directionally transporting a target protein from its site of synthesis to a specific organelle within the cell, mediated by specific functional elements. In this invention, it specifically refers to the targeted transport of a target protein from the plant cytoplasm to the chloroplast.

[0026] Reagent kit: refers to a complete set of products prepared by combining the required biological materials, reagents, enzyme tools and supporting consumables in a certain proportion to achieve a specific biological experiment or technical operation.

[0027] Subcellular localization: refers to experimental methods that use specific detection techniques to determine the specific distribution location of target proteins within plant cells. It is a core detection method for verifying the targeting function of chloroplast localization signal peptides.

[0028] Fluorescence overlap rate: refers to the proportion of the overlapping area between the fluorescence signal of the target protein and the autofluorescence signal of chloroplast in the microscopic observation field. It is the core indicator for quantifying the targeted transport efficiency of chloroplast localization signal peptides. The higher the overlap rate, the stronger the targeted transport efficiency.

[0029] In a first aspect, the present invention provides a chloroplast localization signal peptide.

[0030] The chloroplast localization signal peptide is composed of an SP2 homologous peptide backbone, an N21 intermediate linker region, and a BS cleavage site region connected sequentially from the N-terminus to the C-terminus. The SP2 homologous peptide backbone is constructed based on the tobacco RBCS homologous sequence. The N21 intermediate linker region is a flexible linker peptide rich in proline and glycine. The BS cleavage site region contains specific recognition sites for chloroplast matrix processing peptidases.

[0031] Based on a further solution to the technical problem of the present invention, a preferred embodiment of the technical solution provided in the first aspect of the present invention includes: The first preferred embodiment is a chloroplast localization signal peptide, wherein the amino acid sequence of the SP2 homologous peptide backbone is shown in SEQ ID NO:1, the amino acid sequence of the N21 intermediate linker region is shown in SEQ ID NO:2, and the amino acid sequence of the BS cleavage site region is shown in SEQ ID NO:3. This technical solution, based on solving the technical problems of low targeting efficiency and ambiguous functional elements of chloroplast localization signal peptides in the prior art, further provides specific functional module sequences, achieving highly efficient targeted transport of the target protein to the chloroplast.

[0032] Secondly, the present invention provides a nucleic acid molecule.

[0033] The nucleic acid molecule encodes the chloroplast localization signal peptide described in the first aspect.

[0034] Based on a further solution to the technical problem of the present invention, in the technical solution provided in the second aspect of the present invention, a preferred solution includes: The first preferred embodiment is a nucleic acid molecule, the nucleotide sequence of which is shown in SEQ ID NO:5. This technical solution, while addressing the technical problem of unclear signal peptide coding sequences, further provides a specific nucleotide sequence, facilitating genetic engineering operations.

[0035] Thirdly, the present invention provides a recombinant expression vector.

[0036] The recombinant expression vector comprises the nucleic acid molecule described in the second aspect.

[0037] Based on a further solution to the technical problem of the present invention, a preferred embodiment of the technical solution provided in the third aspect of the present invention includes: The first preferred embodiment is a recombinant expression vector, which further comprises a promoter and a terminator operatively linked to the nucleic acid molecule. This technical solution ensures efficient transcription and translation of the signal peptide in the host cell.

[0038] The second preferred embodiment is a recombinant expression vector, wherein the promoter is selected from constitutive promoters, tissue-specific promoters, chloroplast-specific promoters, or inducible promoters. This technical solution provides a variety of promoter options to meet different expression needs.

[0039] The third preferred embodiment is a recombinant expression vector, wherein the promoter is a CaMV 35S promoter and the terminator is a NOS terminator. This technical solution provides a promoter and terminator combination that is widely used in plant genetic engineering and has high expression efficiency.

[0040] Fourthly, the present invention provides a host cell.

[0041] The host cell contains either the nucleic acid molecule described in the second aspect or the recombinant expression vector described in the third aspect.

[0042] Based on a further solution to the technical problem of the present invention, in the technical solution provided in the fourth aspect of the present invention, a preferred embodiment includes: The first preferred embodiment is a host cell, wherein the host cell is *Escherichia coli* or *Agrobacterium*. This technical solution provides a host cell suitable for vector cloning and plant genetic transformation, facilitating the standardized implementation and promotion of the technology.

[0043] Fifthly, the present invention provides a method for targeted transport of a target protein to plant chloroplasts.

[0044] The method includes fusing the target protein with the chloroplast localization signal peptide described in the first aspect for expression.

[0045] Based on a further solution to the technical problem of the present invention, a preferred embodiment of the technical solution provided in the fifth aspect of the present invention includes: The first preferred embodiment is a method for targeted transport of a target protein to plant chloroplasts, wherein the plant is selected from tobacco benthamiana, Arabidopsis thaliana, tomato, rice, corn, cotton, soybean, or spinach. This technical solution illustrates the versatility of the signal peptide of the present invention in a variety of dicotyledonous and monocotyledonous plants.

[0046] In a sixth aspect, the present invention provides a reagent kit.

[0047] The kit contains at least one of the following: the chloroplast localization signal peptide described in the first aspect, the nucleic acid molecule described in the second aspect, the recombinant expression vector described in the third aspect, or the host cell described in the fourth aspect.

[0048] Based on a further solution to the technical problem of the present invention, a preferred embodiment of the technical solution provided in the sixth aspect of the present invention includes: The first preferred option is a kit that further comprises at least one of a restriction endonuclease, a T4 DNA ligase, an infiltration buffer, or an instruction manual. This technical solution provides the necessary reagents and instructions for signal peptide application, facilitating direct experimentation for users.

[0049] In a seventh aspect, the present invention provides the use of the above-mentioned chloroplast localization signal peptide, nucleic acid molecule, recombinant expression vector, host cell or kit in the preparation of products for targeted delivery of target proteins to plant chloroplasts.

[0050] The present invention has at least the following beneficial effects: 1. High targeting efficiency: The chloroplast localization signal peptide provided by this invention consists of a three-segment structure: the SP2 homologous peptide backbone, the N21 intermediate linker region, and the BS cleavage site region. Experimental results show that the overlap rate between the GFP fluorescence mediated by this signal peptide and the autofluorescence of chloroplasts is over 85%, and the accumulation of the target protein in chloroplasts is higher compared to existing signal peptides.

[0051] 2. Clear functional components: This invention clarifies for the first time the independent roles and synergistic mechanisms of three functional modules: SP2 (chloroplast targeting recognition), N21 (flexible connection, conformation maintenance), and BS (SPP enzyme cleavage recognition), realizing the modular design of signal peptides, which can be directionally modified according to different needs.

[0052] 3. Wide species adaptability: The signal peptide of this invention has been verified to have a highly efficient targeting function in Nicotiana benthamiana and can be applied to a variety of dicotyledonous and monocotyledonous plants such as Arabidopsis thaliana, tomato, rice, corn, cotton, and soybean, and has a wide range of species applicability.

[0053] 4. Standardized technical process: This invention establishes a standardized technical process from vector construction and Agrobacterium-mediated transformation to transient expression in tobacco. All materials used are commercially available, the operation is simple and highly reproducible, and it is easy to promote and apply.

[0054] In summary, this invention provides a chloroplast localization signal peptide with high targeting efficiency, well-defined function, and broad adaptability, offering an efficient tool for plant genetic engineering and chloroplast transformation technology. Attached Figure Description

[0055] Figure 1 For the experimental group p1300 - GFP fluorescence expression 48 h after injection of SP2-N21-BS-GCS-GFP (containing a complete signal peptide backbone) into Agrobacterium, including GFP (GFP fluorescence), chlorophyll (chloroplast autofluorescence), BF (bright field image), and Merge (superposition of GFP, chlorophyll, and bright field signals), scale bar: 20 μm.

[0056] Figure 2 p1300 was the control group. -GFP fluorescence expression 48 h after injection of SP2-DNB-GCS-GFP (signal peptide backbone does not contain N21 and BS sequences) into Agrobacterium, including GFP (GFP fluorescence), chlorophyll (chloroplast autofluorescence), BF (bright field image), and Merge (superposition of GFP, chlorophyll, and bright field signals), scale bar: 20 μm. Detailed Implementation

[0057] Unless otherwise specified, all raw materials and reagents used in this invention were purchased from commercial suppliers, and experiments were conducted in accordance with the operating instructions. Unless otherwise specified, all instruments, equipment, and apparatus used in this invention are conventional instruments, equipment, and apparatus, and experiments were conducted in accordance with the operating instructions and the accompanying reagents.

[0058] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. Unless otherwise specified in the embodiments, conditions are performed under conventional conditions or conditions recommended by the manufacturer. All reagents or instruments without specified manufacturers are commercially available conventional products. Numerous specific details are provided in the following detailed embodiments to better illustrate the invention. The specific embodiments described herein are for illustrative purposes only and are not intended to constitute any limitation on the invention.

[0059] Example 1 1.1 Experimental Materials (1) Target signal peptide sequence Artificially designed and synthesized (synthetic company: Qingke Biotechnology). It consists of the following functional modules connected sequentially from the N-terminus to the C-terminus: SP2 (Homologous Peptide Backbone): Constructed based on the tobacco RBCS homologous sequence, the amino acid sequence is shown in SEQ ID NO:1, located at the N-terminus, specifically the sequence: MASSVLSSAAVATRTNVAQANMVAPFTGLKSAASFPVSRKQNLDITSIASNGG, which is responsible for recognizing the transport complex (TOC / TIC complex) on the chloroplast membrane and initiating protein transmembrane transport.

[0060] N21 (intermediate linker region): A flexible linker peptide rich in proline and glycine, with an amino acid sequence as shown in SEQ ID NO:2, located in the middle segment, specifically RVQCMQVWPPYGKKKYETLSYLPDL. It is used to link the SP2 guide peptide with the BS cleavage site, maintain the spatial conformation of the guide peptide, and ensure cleavage efficiency.

[0061] BS (cleavage site region): Contains the specific recognition site of chloroplast matrix processing peptidase (SPP). The amino acid sequence is shown in SEQ ID NO:3. It is located at the C-terminus, and the specific sequence is: GNSGFGNVSNGGRIRC. After the protein enters the chloroplast matrix, it is cleaved at this site, the signal peptide is removed, and the mature functional protein is released.

[0062] The complete amino acid sequence of the chloroplast localization signal peptide is shown in SEQ ID NO:4 (i.e., the tandem sequence of SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3), and the specific sequence is: MASSVLSSAAVATRTNVAQANMVAPFTGLKSAASFPVSRKQNLDITSIASNGGRVQCMQVWPPYGKKKYETLSYLPDLGNSGFGNVSNGGRIRC.

[0063] The nucleotide sequence encoding the complete chloroplast localization signal peptide is shown in SEQ ID NO:5. The specific sequence is: ATGGCAAGTTCAGTTCTCTCTTCAGCTGCAGTTGCTACAAGAACCAATGTTGCACAGGCTAATATGGTGGCACCATTTACTGGTTTGAAATCAGCTGCTTCATTTCCAGTTAGTAGAAAGCAGAATCTCGACATTACCTCTATTGCAAGCAATGGTGGAAGAGTGCAATGTATGCAAGTTTGGCCTCCGTATGGAAAGAAGAAGTACGAAACCTTGTCTTATCTGCCTGATCTTGGAAATTCTGGATTTGGAAATGTGTCTAATGGTGGCAGAATTAGGTGC.

[0064] (2) Reporter genes GFP coding sequence (GenBank accession number: U55762.1).

[0065] (3) Plant expression vectors pCAMBIA1300 (including CaMV 35S promoter, NOS terminator, Kan) ).

[0066] (4) Escherichia coli competent cells TOP10 competent cells (purchased from Coolaber, catalog number CC504-100×100μL).

[0067] (5) Agrobacterium competent cells GV3101 (pSoup-p19) competent cells (purchased from Coolaber, catalog number CC407-100×100μL).

[0068] (6) Benedict's tobacco Soil-grown seedlings, 3-4 weeks, greenhouse cultivation (25℃, 16h light / 8h darkness). (7) Tool enzymes BsaI-HF®v2 restriction endonuclease (NEB, catalog number R3733S) and T4 DNA Ligase (NEB, catalog number M0202S). (8) Resistance culture medium LB medium (containing 50 μg / mL Kan) and LB medium (containing 50 μg / mL Kan + 25 μg / mL Rif). (9) Other reagents Rapid plasmid miniprep kit (centrifuge column type, purchased from TIANGEN, catalog number DP105-02), Gel Extraction Kit D2500 (purchased from omega BIO-TEK, catalog number D2500-02), Agrobacterium infection liquid (Coolaber, catalog number SL0911-500 mL), 2 × Phanta Flash Master Mix (Dye Plus) (Vazyme, catalog number P520-03), 2 × Rapid Taq Master Mix (Vazyme, catalog number P222-03).

[0069] (10) Instruments and equipment Rotary laser super-resolution microscope system (Olympus, SpinSR10), PCR instrument, constant temperature shaker, and clean bench.

[0070] 1.2 Specific Implementation Steps Step 1: Design and synthesis of target chloroplast localization signal peptides Based on the functional element design principles of this invention, the nucleotide sequence of the target signal peptide (SEQ ID NO:5) was designed.

[0071] Step 2: Construction of the recombinant expression vector Target fragment amplification: First, log in to the official website ( https: / / goldengate.neb.com / #! / The fragment was input into the website for primer design. After synthesis by the company (Qingke Biotechnology), PCR amplification was performed. The synthesized genes SP, GCS, and GFP were amplified according to the system shown in Table 1. The PCR reverse procedure was performed according to Table 2. After PCR, the fragment was recovered using a DNA gel recovery kit (Tiangen Biotech). Golden Gate method for vector recombination: The recovered fragments were mixed according to Table 3, and the mixed system was digested and ligated according to Table 4.

[0072] Table 1 PCR reaction system

[0073] Table 2 PCR amplification program

[0074] Table 3 Golden Gate reaction system

[0075] Table 4 Golden Gate Reaction Procedure

[0076] The resulting carrier: Experimental group (A): p1300-SP2-N21-BS-GCS-GFP (containing the complete signal peptide backbone); Control group (B): p1300-SP2-DNB-GCS-GFP (signal peptide backbone does not contain N21 and BS sequences).

[0077] Detailed Primer and Template Sequences Table 5: Table 5

[0078] GFP-LINKER (SEQ ID NO:18): GGTGGTGGTTCTGGTGGTGGTTCTGGTATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCTTCACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCACGGCATGGACGAGCTGTACAGATCTTAA。

[0079] GFP(SEQ ID NO:19): ATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCTTCACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCACGGCATGGACGAGCTGTACAGATCTTAA。

[0080] GCS(SEQ ID NO:20):

[0081] 1300 skeleton (SEQ ID NO:21):

[0082] Step 3: Transformation of E. coli TOP10 and Vector Validation (1) Transformation: Take 15 μL of ligation product and add it to 100 μL of TOP10 competent cells, incubate on ice for 10 min, heat shock at 42℃ for 90 s, and immediately incubate on ice for 10 min; add 700 μL of antibiotic-free LB medium, and culture at 37℃ and 270 rpm for 40 min with shaking. (2) Screening: Spread 200 μL of bacterial suspension onto LB agar plates containing 50 μg / mL Kan, incubate upside down at 37℃ for 12 h, and pick single colonies; (3) Sequencing verification: Single colonies were inoculated into 10 mL of Kan resistant LB liquid medium and cultured overnight at 37°C with shaking. Recombinant plasmids were extracted using a plasmid extraction kit. Using the recombinant plasmid as a template, colony PCR was performed with amplification primers. Single colonies with bands were selected and sent to Qingke Biotechnology for third-generation nanopore whole plasmid sequencing. The sequencing results were compared with the designed sequence to confirm that the signal peptide and GFP sequence were correctly inserted and that there was no frameshift in the reading frame. Step 4: Transformation with Agrobacterium GV3101 (1) Freeze-thaw transformation: Take 100 μL of GV3101 competent cells and thaw them in an ice bath; add 1 μL of the correctly sequenced recombinant plasmid, mix gently, and incubate in an ice bath for 10 min; freeze in liquid nitrogen for 5 min, revive in a 37℃ water bath for 5 min, and incubate in an ice bath for 10 min; add 800 μL of antibiotic-free LB medium and culture at 28℃ and 270 rpm for 2 h with shaking; (2) Screening: Spread 200 μL of bacterial suspension onto LB plates containing 50 μg / mL Kan + 50 μg / mL Rif, incubate upside down at 28℃ for 48 h, and pick single colonies; (3) Verification: A single colony was inoculated into 10 mL of Kan+Rif resistant LB liquid medium and cultured overnight at 28°C with shaking. The presence of the recombinant vector was verified by bacterial PCR, and engineered Agrobacterium GV3101 / pCAMBIA1300SP-GCS-GFP and GV3101 / pCAMBIA1300SP-GFP were obtained. Step 5: Transient transformation of Tobacco Benzoviae (Agrobacterium-mediated transformation) (1) Agrobacterium activation: Take 200 μL of engineered Agrobacterium culture and inoculate it into 10 mL of Kan+Rif resistant LB liquid medium. Incubate at 28℃ and 270 rpm for 16 h with shaking. (2) Preparation of bacterial suspension: Centrifuge the activated bacterial suspension at 5000 rpm for 7 min and discard the supernatant; resuspend the bacterial suspension with Agrobacterium infection solution, adjust OD600 = 0.8, and let stand at room temperature for 3 h; (3) Tobacco injection: Select healthy leaves of tobacco that have grown for 3-4 weeks, and slowly inject the bacterial solution from the back of the leaf with a 1 mL needleless syringe to ensure that the bacterial solution evenly infiltrates the leaf tissue; after injection, place the tobacco in a greenhouse (25℃, 16 h light / 8 h dark) for 48-72 h. Step 6: Subcellular localization detection and result analysis (1) Sample preparation: Take tobacco leaves 48 h after injection, cut a 1 cm × 1 cm piece of mesophyll tissue, place it on a glass slide, add a small amount of sterile water, and cover with a coverslip; (2) Laser confocal observation: Excitation wavelength: GFP (488 nm), emission wavelength (507-520 nm); Chloroplast autofluorescence: excitation wavelength (640 nm), emission wavelength (660-680 nm); Observation field: Select mesophyll cells and take images immediately; (3) Reference settings: Negative control: Injection of Agrobacterium empty spp. GV3101 (without recombinant vector) to exclude background fluorescence interference; (4) Result determination: The result is as follows Figure 1 As shown, the GFP fluorescence signal of the target signal peptide group highly overlaps with the autofluorescence of chloroplasts (overlap rate ≥ 85%), demonstrating that the target signal peptide can efficiently mediate the targeted transport of GFP to chloroplasts. Step 7: Functional verification of each functional element of the signal peptide (1) Construction of mutants: Construct the control group vector (p1300-SP2-DNB-GCS-GFP (signal peptide backbone does not contain N21 and BS sequences)). (2) Repeat steps 3-6: After transforming and injecting each mutant vector into tobacco, observe the GFP fluorescence localization; (3) Results analysis: The results are as follows Figure 2 After the cleavage site shown is deleted, GFP fluorescence appears randomly in the cell.

[0083] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. A chloroplast localization signal peptide, characterized in that, The chloroplast localization signal peptide is composed of an SP2 homologous peptide backbone, an N21 intermediate linker region, and a BS cleavage site region connected sequentially from the N-terminus to the C-terminus. The SP2 homologous peptide backbone is constructed based on the tobacco RBCS homologous sequence; The N21 intermediate linker region is a flexible linker peptide rich in proline and glycine. The BS cleavage site region contains specific recognition sites for chloroplast matrix processing peptidases.

2. The chloroplast localization signal peptide according to claim 1, characterized in that, The amino acid sequence of the SP2 homologous peptide backbone is shown in SEQ ID NO:1, the amino acid sequence of the N21 intermediate linker region is shown in SEQ ID NO:2, and the amino acid sequence of the BS cleavage site region is shown in SEQ ID NO:

3.

3. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the chloroplast localization signal peptide as described in claim 1 or 2.

4. The nucleic acid molecule according to claim 3, characterized in that, The nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO:

5.

5. A recombinant expression vector, characterized in that, It includes the nucleic acid molecule as described in claim 3 or 4.

6. The recombinant expression vector according to claim 5, characterized in that, The recombinant expression vector also includes a promoter and a terminator operatively linked to the nucleic acid molecule.

7. The recombinant expression vector according to claim 6, characterized in that, The promoters are selected from constitutive promoters, tissue-specific promoters, chloroplast-specific promoters, or inducible promoters.

8. The recombinant expression vector according to claim 7, characterized in that, The promoter is the CaMV 35S promoter; the terminator is the NOS terminator.

9. A host cell, characterized in that, It comprises the nucleic acid molecule as described in claim 3 or 4 or the recombinant expression vector as described in any one of claims 5-8.

10. The host cell according to claim 9, characterized in that, The host cell is either Escherichia coli or Agrobacterium.

11. A method for targeted transport of a target protein to plant chloroplasts, characterized in that, This includes expressing the target protein by fusing it with the chloroplast localization signal peptide as described in claim 1 or 2.

12. The method according to claim 11, characterized in that, The plants are selected from tobacco, Arabidopsis thaliana, tomato, rice, corn, cotton, soybean or spinach.

13. A reagent kit, characterized in that, The present invention comprises the chloroplast localization signal peptide of claim 1 or 2, or the nucleic acid molecule of claim 3 or 4, or the recombinant expression vector of any one of claims 5-8, or the host cell of claim 9 or 10.

14. The kit according to claim 13, characterized in that, It also contains at least one of the following: restriction endonuclease, T4 DNA ligase, infection buffer, or instructions for use.

15. The use of the chloroplast localization signal peptide of claim 1 or 2, or the nucleic acid molecule of claim 3 or 4, or the recombinant expression vector of any one of claims 5-8, or the host cell of claim 9 or 10, or the kit of claim 13 or 14 in the preparation of a product for targeted delivery of a target protein to plant chloroplasts.

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