Chloroplast transit peptide

Abstract

The present invention relates to a chloroplast transit peptide, wherein the isolated polypeptide has an amino acid sequence shown in SEQ ID NO: 1. The isolated polypeptide of the present invention has chloroplast transit activity and can target a linked portion (such as a target protein) to the chloroplast.

Description

Technical Field

[0001] The present invention relates to the field of protein targeting, and in particular to a polypeptide that guides a linked portion (such as a target protein) to be targeted to chloroplasts. Background Art

[0002] Chloroplasts are the representative plastids in plant cells. Their most essential function is photosynthesis, but they also carry out many other biosynthetic processes that are important to plant cells. Furthermore, chloroplasts are a key target for the pesticide industry, as many herbicides are known to work by blocking functions performed within chloroplasts.

[0003] Most chloroplast proteins are encoded in the plant cell nucleus and synthesized as precursor proteins in the cytoplasm before being transported to the chloroplasts. Precursor proteins for chloroplast targeting include an N-terminal extension called a chloroplast transit peptide (CTP), which targets the protein of interest to the chloroplast.

[0004] While many proteins that can produce beneficial traits in crops, as well as chloroplast transit peptides for localizing proteins within cells, have been developed, the extent of effective subcellular localization and processing of any CTP and target protein combination is difficult to predict. Localization and processing determine the expression level and function of the target protein and affect the corresponding phenotype of transgenic cells, plants, or seeds containing the target protein. Therefore, more novel CTPs that can effectively localize and process target proteins are needed. Summary of the Invention

[0005] The object of the present invention is to provide a novel chloroplast transit peptide having chloroplast transport activity, which can target the linked portion (such as a target protein) to the chloroplast.

[0006] To achieve the above object, the present invention provides an isolated polypeptide having an amino acid sequence shown in SEQ ID NO: 1.

[0007] To achieve the above objectives, the present invention also provides a nucleic acid molecule comprising: (1) a nucleotide sequence encoding the isolated polypeptide or its complementary sequence; or (2) a nucleotide sequence as shown in SEQ ID NO: 2 or its complementary sequence.

[0008] To achieve the above objectives, the present invention also provides a fusion protein comprising the isolated polypeptide fused to a target protein.

[0009] Preferably, the isolated polypeptide is located at the N-terminus of the target protein in the fusion protein.

[0010] To achieve the above object, the present invention also provides an expression cassette comprising the coding sequence of the fusion protein.

[0011] To achieve the above object, the present invention also provides a DNA construct or recombinant vector comprising the expression cassette.

[0012] To achieve the above objectives, the present invention also provides a method for targeting a target protein to plant chloroplasts, comprising introducing the coding sequence of the fusion protein into a plant so that the target protein is localized in the plant chloroplasts.

[0013] Preferably, the target protein is a protein that modifies a physiological process present in chloroplasts, an abiotic stress resistance protein, or a biotic stress resistance protein.

[0014] Furthermore, the physiological process is photosynthesis, fatty acid synthesis, amino acid synthesis, oil synthesis, carotenoid synthesis, terpenoid synthesis or starch synthesis.

[0015] Furthermore, the abiotic stress resistance protein is a herbicide tolerance protein; and the biotic stress resistance protein is an insect resistance protein.

[0016] The following descriptions and definitions are provided to better define the present invention and to guide those skilled in the art to practice the present invention. Unless otherwise specified, terms should be understood according to conventional usage by those skilled in the art.

[0017] The articles "a" and "an" used in the present invention refer to one or more than one (ie, at least one). For example, "an element" means one or more elements.

[0018] In the present invention, the term "comprise" or its variations, such as "comprising", "containing" or "including", "comprising", means including the stated elements, integers or steps, or groups of elements, integers or steps, but does not exclude any other elements, integers or steps, or groups of elements, integers or steps.

[0019] As used herein, the term "chloroplast" refers to a specialized subunit of an organelle found in the cells of eukaryotic autotrophic organisms such as green plants and algae. Its primary function is photosynthesis. The photosynthetic pigment chlorophyll contained within it captures energy from sunlight and stores it in the energy-storage molecules ATP and NADPH, while simultaneously releasing oxygen from water. Chloroplasts then use ATP and NADPH to produce organic molecules from carbon dioxide in a process known as the Calvin cycle. Chloroplasts also perform many other functions, including fatty acid synthesis in plants, the synthesis of many amino acids, and immune responses.

[0020] The term "chloroplast transit peptide" (CTP) in the present invention refers to an amino acid sequence that mediates the targeting or localization of an amino acid sequence linked thereto (eg, as a fusion protein) to plant chloroplasts.

[0021] The present invention includes fusion proteins, i.e., a first nucleic acid molecule encoding a chloroplast transit peptide of the present invention is recombinantly linked to a second nucleic acid molecule encoding a protein of interest, such that translation of the nucleic acid molecules produces the fusion protein. The chloroplast transit peptide is generally fused to the N-terminus of the protein of interest. A fusion protein can consist of or contain the chloroplast transit peptide and the protein of interest. In certain embodiments, the chloroplast transit peptide is preferably located at the N-terminus of the fusion protein, for example, the chloroplast transit peptide is located within the N-terminal half, N-terminal third, or N-terminal quarter of the fusion protein. However, additional amino acid residues may be located at the N-terminus of the chloroplast transit peptide, provided that the fusion protein is at least partially targeted to the chloroplast. Generally, most or all of the chloroplast transit peptide is cleaved from the fusion protein upon insertion into the chloroplast. Chloroplast transit peptide cleavage can be uniform, such that the cleavage site is identical across a population of fusion proteins, or heterogeneous, such that the cleavage site varies by 1-10 amino acids across a population of fusion proteins. The chloroplast transit peptide can be recombinantly fused to a second protein in a variety of ways. For example, a restriction endonuclease recognition site can be introduced into the chloroplast transit peptide nucleotide sequence at a position corresponding to its C-terminus, and the same or a compatible site can be introduced into the N-terminus of the target protein nucleotide sequence. These sites should be designed to ensure that the coding sequence of the chloroplast transit peptide and the second protein remain in frame, allowing synthesis of the fusion protein. In some cases, it may be preferable to remove the initiator methionine codon of the second protein when introducing the new restriction site. Introduction of restriction endonuclease recognition sites into both parent molecules and subsequent ligation via recombinant DNA technology can result in the addition of one or more additional amino acids between the chloroplast transit peptide and the second protein. This generally does not affect targeting activity, as long as the chloroplast transit peptide cleavage site remains accessible and the addition of these additional amino acids to the N-terminus of the second protein does not alter its function. Alternatively, one skilled in the art can create a precise fusion between the chloroplast transit peptide and the second protein (with or without its initiator methionine) using gene synthesis or similar methods. In addition, the fusion protein can include amino acids downstream of the cleavage site. The amino acids at the N-terminus of the mature protein can affect the ability of the transit peptide to target the target protein to the chloroplast and / or the cleavage efficiency after the target protein enters. The target protein can be any polypeptide that needs to be located in the chloroplast. The target protein can be a full-length protein (for example, in its naturally occurring form), or it can be a modified form of the protein (for example, a part or fragment, variant or other non-natural form of protein). The target protein can come from any organism, including but not limited to bacteria, algae, yeast, plants, animals and synthetic proteins. For example, the target protein that can be included in the fusion protein includes but is not limited to proteins that modify physiological processes present in the chloroplast, abiotic stress resistance proteins and biotic stress resistance proteins.The proteins that modify physiological processes present in chloroplasts (such as photosynthesis or fatty acid, amino acid, oil, carotenoid, terpenoid, starch composition / synthesis) include but are not limited to ribulose 1,5-bisphosphate carboxylation / oxygenase, ribulose 1,5-bisphosphate carboxylation / oxygenase activating enzyme, fatty acid synthase, fatty acid desaturase, phytoene synthase, phytoene desaturase, starch synthase and ADP-glucose pyrophosphorylase; the abiotic stresses (such as drought, temperature, salinity, ozone and Proteins resistant to herbicides include, but are not limited to, herbicide tolerance proteins, such as 5-enolpyruvyl-3-phosphoshikimate synthase (EPSP synthase), glyphosate N-acetyltransferase (GAT), acetolactate synthase (ALS), protoporphyrinogen oxidase (PPO), and hydroxyphenylpyruvate dioxygenase (HPPD); proteins resistant to biotic stress (e.g., pathogen attack, including insects, viruses, bacteria, fungi, and nematodes) include, but are not limited to, insect resistance proteins, such as Bt toxin proteins. When different chloroplast transit peptides are used in combination with different proteins of interest, they exhibit varying degrees of efficacy (e.g., a higher ratio of targeted to non-targeted proteins of interest).

[0022] As used herein, the term "herbicide" refers to an active ingredient used to kill, control, or adversely regulate the growth of plants. The preferred amount or concentration of the herbicide is an "effective dose" or "effective concentration," which is sufficient to kill similar wild-type plants, plant tissues, plant cells, or host cells, or to inhibit their growth, but does not kill, or severely inhibit the growth of, the herbicide-tolerant / resistant plants, plant tissues, plant cells, or host cells of the present invention. Generally, an "effective dose" or "effective concentration" of a herbicide is an amount routinely used to kill the desired weeds in agricultural production systems, and such amounts are known to those skilled in the art.

[0023] As used herein, the term "herbicide tolerance" or "herbicide resistance" is heritable and allows a plant to grow and reproduce despite treatment with a herbicide that is generally effective against a given plant. As will be recognized by those skilled in the art, even if a given plant experiences some degree of damage from herbicide treatment, such as minimal necrosis, dissolution, chlorosis, or other damage, but at least without a significant impact on yield, the plant can still be considered to possess an enhanced ability to resist various degrees of herbicide-induced damage that would normally result in damage to wild-type plants of the same genotype at the same herbicide dose. As used herein, "conferring or enhancing herbicide tolerance / resistance to a plant" refers to imparting tolerance / resistance to a plant that does not possess herbicide tolerance / resistance and / or enhancing the tolerance / resistance of a plant that possesses herbicide tolerance / resistance. Herbicide tolerance can be complete or partial insensitivity to a particular herbicide and can be expressed as a percentage (%) of tolerance or insensitivity to a particular herbicide.

[0024] The terms "control" and / or "prevent and control" in the present invention refer to at least applying an effective dose of herbicide directly (for example, by spraying) to the plant growth environment to minimize the development of weeds and / or stop their growth. At the same time, the cultivated transgenic plants should be normal in morphology and can be cultivated under conventional methods for consumption and / or production of products; preferably, they have reduced plant damage and / or increased plant yield compared to wild-type plants. The reduced plant damage specifically includes but is not limited to improved stem resistance and / or increased grain weight. Among them, "weeds" refer to plants that compete with cultivated transgenic plants in the plant growth environment.

[0025] As used herein, the term "insect resistance protein" can inhibit (inhibit growth, feeding, fertility, or viability), curb (inhibit growth, feeding, fertility, or viability), control (control pest infestation, control pest feeding activity on a specific crop containing an effective dose of the insecticidal protein), or kill (cause disease, mortality, or reduced fertility) pests. An effective dose of the insecticidal protein is provided to the pest, exposing the pest to the insecticidal protein to cause disease, mortality, reduced fertility, or stunted growth. "Insect resistance protein" also includes the expulsion of pests from plants, plant tissues, plant parts, seeds, plant cells, or specific geographical locations where plants may grow due to the provision of an effective dose of the insecticidal protein in or on the plant. The insecticidal protein can be produced by the plant or can be applied to the plant or the environment within the location where the plant is located.

[0026] The terms "nucleic acid," "polynucleotide," "nucleic acid molecule," or "nucleic acid sequence" are used interchangeably herein to include reference to deoxyribonucleotide or ribonucleotide polymers in single-stranded or double-stranded form, and unless otherwise limited, include known analogs (e.g., peptide nucleic acids) that have the essential properties of natural nucleotides in that they hybridize to single-stranded nucleic acids in a manner similar to naturally occurring nucleotides.

[0027] The terms "polypeptide," "peptide," "protein," or "proteinaceous" are used interchangeably herein to refer to a polymer of amino acid residues. One or more of the amino acid residues in the polymer is an artificial chemical analog of a corresponding naturally occurring amino acid, as well as naturally occurring amino acid polymers. The polypeptides of the present invention can be produced recombinantly or by chemical synthesis.

[0028] As used herein, the term "amino acid" refers to naturally occurring amino acids, synthetic amino acids, and amino acid analogs and mimetics that function in a manner similar to naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code. Amino acid analogs include, but are not limited to, naturally occurring amino acids that have been subsequently modified, such as hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acids may be referred to herein by their commonly known three-letter symbols or by the single-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission.

[0029] As used herein, the term "sequence" refers to the sequential arrangement of nucleotides or amino acids. The terms "transgene expression" and "protein expression" refer to the production of protein by transcribing DNA into messenger RNA (mRNA) and translating mRNA into a polypeptide chain (which ultimately folds into protein).

[0030] As used herein, the term "operably linked" refers to the connection of nucleic acid sequences such that one sequence provides a function required of the linked sequence. As used herein, "operably linked" includes a functional connection between a chloroplast transit peptide coding region and a target protein coding region, wherein the CTP sequence directs the localization of the target protein to plant chloroplasts. Typically, the two protein coding regions "operably linked" are contiguous and in the same reading frame. As used herein, "operably linked" also includes the connection of a regulatory element to the target protein coding sequence such that transcription of the target protein coding sequence is controlled and regulated by the regulatory element. Nucleic acid sequences that can be "operably linked" include, but are not limited to, sequences that provide gene expression function (i.e., gene expression elements, such as promoters, 5' untranslated regions, introns, protein coding regions, 3' untranslated regions, polyadenylation sites and / or transcription terminators), sequences that provide DNA transfer and / or integration function (i.e., T-DNA border sequences, site-specific recombinase recognition sites, integrase recognition sites), sequences that provide selection function (i.e., antibiotic resistance markers, biosynthetic genes), sequences that provide scorable marker function, sequences that assist in sequence manipulation in vitro or in vivo (i.e., polylinker sequences, site-specific recombination sequences), and sequences that provide replication function (i.e., bacterial replication origins, autonomous replication sequences, centromere sequences).

[0031] Among the present invention, term " promoter " refers to the region or sequence that is positioned at the upstream and / or downstream of the transcription start site, and it participates in identifying and combining RNA polymerase and other albumen, to start transcription.Promoter comprises the necessary nucleic acid sequence near the transcription start site, for example, comprises TATA element with respect to polymerase II type promoter.Promoter also comprises distal enhancer or repressor element optionally, and it can be positioned at the position of up to thousands of base pairs apart from the transcription start site. " constitutive " promoter is the promoter that is active under most environments and development conditions. " inducible " promoter is the promoter that is active under environment or development regulation.

[0032] As used herein, the term "DNA construct" refers to a recombinant DNA molecule comprising two or more heterologous DNA sequences. DNA constructs can be used for transgenic expression and can be contained in vectors and plasmids. DNA constructs can be used in vectors for transformation purposes, i.e., to introduce heterologous DNA into host cells to produce transgenic cells and plants. They can also be contained in plasmid DNA or genomic DNA of transgenic cells, plants, parts thereof, or seeds. A "plant transformation vector" typically contains a plasmid vector containing cis-acting sequences required for T-DNA transfer (e.g., left and right borders), a selectable marker engineered to be expressed in plant cells, and a target gene. This plasmid vector also contains sequences required for bacterial replication. The cis-acting sequences are arranged in a manner that allows for efficient transfer and expression in plant cells. For example, the selectable marker gene and the pesticidal gene are located between the left and right borders. As understood in the art, the "plant transformation vector" contains virulence functions (Vir genes) that allow infection of plant cells by Agrobacterium and transfer of DNA by cleavage at border sequences and vir-mediated DNA transfer (Hellens and Mullineaux, Trends in Plant Science, (2000) 5:446-451).

[0033] Suitable methods for transforming host plant cells include any method for introducing DNA into cells (e.g., stably integrating a recombinant DNA construct into a plant chromosome), which are well known in the art. An exemplary and widely used method for introducing recombinant DNA constructs into plants is the Agrobacterium transformation system, well known to those skilled in the art. Another exemplary method for introducing recombinant DNA constructs into plants is by inserting the recombinant DNA construct into the plant genome at a predetermined site via site-directed integration. Site-directed integration can be achieved by any method known in the art, such as using zinc finger nucleases, engineered or natural meganucleases, TALE endonucleases, or RNA-guided endonucleases (such as the CRISPR / Cas9 system). Transgenic plants can be regenerated from transformed plant cells using plant cell culture methods. Methods for regenerating plants are also well known in the art. For example, Ti plasmid vectors have been used to deliver exogenous DNA, as well as direct DNA uptake, liposomes, electroporation, microinjection, and microprojectiles.

[0034] In the present invention, the term "recombinant" refers to a non-natural DNA, protein, cell, seed or organism that is caused by genetic engineering and is not usually present in nature. A "recombinant DNA molecule" refers to a DNA molecule that is contained in nature and is not naturally occurring and is produced by human intervention, such as a DNA molecule consisting of at least two DNA molecules that are heterologous to each other. Wherein, "genetic engineering" refers to the creation of non-natural DNA, protein or organism that is not usually present in nature and requires human intervention. Genetic engineering can be used to conceive and create engineered DNA, protein or organisms using one or more techniques in biotechnology, such as molecular biology, protein biochemistry, bacterial transformation, and plant transformation.

[0035] The term "transgene" as used herein refers to a DNA molecule that has been incorporated into the genome of an organism as a result of human intervention (eg, plant transformation procedures).

[0036] The term "native" refers to a naturally occurring ("wild-type") nucleic acid sequence.

[0037] The term "heterologous" sequence refers to a sequence that originates from a foreign source or species, or when derived from the same source, a sequence that has been modified from its original form.

[0038] The present invention includes variant polypeptides related to SEQ ID NO:1 but altered in some way (e.g., any fragment thereof, or substitution, deletion, or addition of one or more residues). Specifically, the variant polypeptides possess at least 50%, 60%, 70%, 75%, 85%, 90%, 95%, 97%, 98%, or 99% of at least a portion of the chloroplast transport functional activity (e.g., the ability to import the attached moiety into the chloroplast) compared to the unaltered polypeptide; alternatively, the variant polypeptides possess the same or greater chloroplast transport functional activity compared to the unaltered polypeptide. Variant polypeptides are generally constructed to enhance desirable characteristics of a chloroplast transit peptide or a polypeptide containing a chloroplast transit peptide (e.g., increased targeting efficacy, conferring chloroplast specificity, or enhancing transcription and / or translation efficiency) or to mitigate undesirable characteristics (e.g., susceptibility to degradation). Various variant polypeptides can be obtained by methods known in the art.

[0039] As used herein, the term "functional activity" or "activity" refers to the ability of a chloroplast transit peptide of the present invention to import the attached moiety (e.g., a protein of interest) into chloroplasts. When attached to the chloroplast transit peptide, the attached moiety is enriched in the chloroplast (e.g., at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% more than the moiety not attached to the chloroplast transit peptide). Typically, the activity of the chloroplast transit peptide is compared to a positive control (i.e., a chloroplast transit peptide known to target a specific protein of interest) and / or a negative control (i.e., a polypeptide lacking a chloroplast transit peptide or containing a nonfunctional chloroplast transit peptide). Assays for analyzing chloroplast transit peptide activity may include, but are not limited to, creating a recombinant fusion between a candidate chloroplast transit peptide and the protein of interest and expressing the fusion protein in plants or plant cells. In one embodiment, the fusion protein functions exclusively or substantially exclusively in chloroplasts; thus, the functionality of the protein of interest is used to determine its localization, for example, by measuring its enzymatic activity. In another embodiment, the fusion protein fluoresces; thus, the localization of the protein of interest can be determined by monitoring the accumulation of fluorescence in chloroplasts, for example, using a fluorescence microscope. In another embodiment, the localization of the protein of interest is determined by measuring its size. When the fusion protein is inserted into the chloroplast, the chloroplast transit peptide is typically cleaved in whole or in part. If the chloroplast transit peptide contains an accessible cleavage site as part of the fusion protein, the chloroplast transit peptide will be cleaved, reducing the length of the fusion protein and, therefore, the molecular weight of the fusion protein. If the sequence of the cleavage site is not readily accessible (for example, if surrounding sequences prevent proper recognition of the cleavage site or if the fusion protein folds in a way that prevents matrix proteases from accessing the cleavage site), cleavage will be ineffective and may occur at one or more alternative positions. Although the length of the processed fusion protein may vary slightly in this case, it will still be reduced in length and molecular weight compared to the unprocessed fusion protein. In another embodiment, chloroplasts are isolated from plant tissue and then analyzed for the presence of the protein of interest. The presence of the protein of interest can be tested using any polypeptide detection method known in the art, including immunoblotting, immunoprecipitation, ELISA, or by measuring a property of the protein of interest (e.g., fluorescence or enzymatic activity).

[0040] The chloroplast transit peptide of the present invention can be applied to a variety of plants, including dicotyledonous plants such as alfalfa, beans, cauliflower, cabbage, carrots, celery, cotton, cucumber, eggplant, lettuce, melon, peas, peppers, zucchini, radish, rapeseed, spinach, soybean, pumpkin, tomato, Arabidopsis, peanut or watermelon; preferably, the dicotyledonous plant is cucumber, soybean, Arabidopsis, tobacco, cotton, peanut or rapeseed. The monocotyledonous plant includes but is not limited to corn, rice, sorghum, wheat, barley, rye, millet, sugarcane, oats or lawn grass; preferably, the monocotyledonous plant is corn, rice, sorghum, wheat, barley, millet, sugarcane or oats.

[0041] The term "plant" in the present invention includes the entire plant, aboveground vegetative organs / structures (e.g., leaves, stems, and tubers), roots, flowers, and floral organs / structures (e.g., bracts, sepals, petals, stamens, carpels, anthers, and ovules), seeds (including embryos, endosperms, and seed coats), and fruits (mature ovaries), plant tissues (e.g., vascular tissues, ground tissues, etc.), and cells (e.g., guard cells, egg cells, trichomes, etc.), and their progeny. The plant classes that can be used in the methods of the present invention generally encompass a wide range of higher and lower plant classes that can withstand genetic transformation techniques, including angiosperms (monocots and dicots), gymnosperms, ferns, and multicellular algae. This includes plants of various ploidy levels, including aneuploids, polyploids, diploids, haploids, and hemizygotes.

[0042] The present invention provides a novel chloroplast transit peptide having the following beneficial effects:

[0043] 1. Chloroplast transport activity: The chloroplast transit peptide of the present invention can target the part to which it is connected (eg, target protein) to the chloroplast.

[0044] 2. Promote the enrichment of target proteins in chloroplasts. The chloroplast transit peptide of the present invention can target one or more target proteins to chloroplasts to enrich them in chloroplasts.

[0045] The technical solution of the present invention is further described in detail below through examples. DETAILED DESCRIPTION

[0046] The technical solution of the chloroplast transit peptide of the present invention is further illustrated by specific examples below. The methods and operations described in the following examples are exemplary and should not be construed as limiting.

[0047] Example 1: Obtaining the Chloroplast Transit Peptide (CTP) of the Present Invention

[0048] The amino acid sequence of the chloroplast transit peptide HCP1 of the present invention is shown in SEQ ID NO: 1 in the sequence listing; the HCP1 nucleotide sequence encoding the HCP1 is shown in SEQ ID NO: 2 in the sequence listing.

[0049] It is well known to those skilled in the art that due to the degeneracy of the genetic code, different codons can encode the same amino acid. Therefore, SEQ ID NO: 2 in the present invention is only an exemplary nucleotide sequence encoding the chloroplast transit peptide HCP1.

[0050] Second Example: Targeted Activity Analysis of Chloroplast Transit Peptides in Tobacco

[0051] A DNA construct GR01 comprising the nucleotide sequence of the chloroplast transit peptide HCP1 of the present invention was constructed. The DNA construct GR01 comprises an expression cassette consisting of, in sequence, the Arabidopsis thaliana ubiquitin 10 promoter (SEQ ID NO: 3), operably linked to the HCP1 (SEQ ID NO: 2), the HCP1 fused to the green fluorescent protein (GFP) gene (SEQ ID NO: 4), and the GFP gene operably linked to the terminator of the nopaline synthase gene (SEQ ID NO: 5).

[0052] A control DNA construct, GR02, was constructed. The DNA construct GR02 comprises an expression cassette consisting of the Arabidopsis thaliana ubiquitin 10 promoter (SEQ ID NO: 3), operably linked to the green fluorescent protein (GFP) gene (SEQ ID NO: 4), and operably linked to the nopaline synthase gene terminator (SEQ ID NO: 5).

[0053] The DNA constructs GR01 and GR02 were connected to plant transformation vectors V01 and V02, respectively. The plant transformation vectors V01 and V02 were transiently expressed in tobacco leaves using the Agrobacterium infection method known in the art and subjected to the following targeting analysis.

[0054] Experiment 1: Targeted Activity Assay of Transiently Expressed Genes in Tobacco

[0055] The infected tobacco was placed under conditions of 23-26°C, 70-80% relative humidity, and a photoperiod (light / dark) of 16h:8h for 3 days. Infected tobacco leaves and uninfected tobacco leaf sections were cut from the plants and observed for green fluorescence using a laser confocal microscope. 50 cells transformed with each plant transformation vector were taken, and the proportion of GFP protein localized in chloroplasts in cells transformed with each plant transformation vector in the microscope field was counted to evaluate CTP targeting activity. The proportion of CTP targeting chloroplasts = the number of cells with GFP protein localized in chloroplasts / the total number of cells (50) × 100%. The experimental results of CTP targeting activity of each plant transformation vector are shown in Table 1.

[0056] Table 1. Experimental results of CTP targeting activity of various plant transformation vectors

[0057] CTP DNA constructs Plant transformation vectors Number of cells localized in chloroplasts Proportion of CTP targeting chloroplasts HCP1 GR01 V01 43 86% none GR02 V02 0 0%

[0058] The results in Table 1 indicate that the chloroplast transit peptide HCP1 of the present invention has chloroplast transport activity and can effectively target the linked GFP protein (target protein) to the chloroplast.

[0059] Experiment 2: Western blotting

[0060] GFP protein was extracted from tobacco leaves infected with each plant transformation vector and from uninfected tobacco leaves for western blot hybridization. The experimental results showed that: (1) GFP protein was expressed in infected tobacco leaves, but not in uninfected tobacco leaves. (2) The size of the GFP band in tobacco leaves expressing the plant transformation vector V01 was comparable to that in tobacco leaves expressing the plant transformation vector V02, indicating that the chloroplast transit peptide HCP1 of the present invention can be cleaved from the mature GFP protein.

[0061] Example 3: Evaluation of the targeting efficiency of chloroplast transit peptides in corn

[0062] A DNA construct GR03 comprising the chloroplast transit peptide HCP1 nucleotide sequence of the present invention was constructed. The DNA construct GR03 comprises two tandem expression cassettes: the first expression cassette is composed of the cauliflower mosaic virus 35S promoter (SEQ ID NO: 6) operably linked to the HCP1 (SEQ ID NO: 2), the HCP1 being fused to the protoporphyrinogen oxidase (PPO) gene (SEQ ID NO: 7), which is operably linked to the cauliflower mosaic virus 35S terminator (SEQ ID NO: 8); and the second expression cassette is composed of the maize ubiquitin 1 promoter (SEQ ID NO: 9) operably linked to the phosphinothricin acetyltransferase (PAT) gene (SEQ ID NO: 10), and operably linked to the terminator of the maize In2-1 gene (SEQ ID NO: 11).

[0063] A control DNA construct, GR04, was constructed. The DNA construct GR04 comprises two expression cassettes in series: the first expression cassette consists of the cauliflower mosaic virus 35S promoter (SEQ ID NO: 6), operably linked to the protoporphyrinogen oxidase (PPO) gene (SEQ ID NO: 7), and operably linked to the cauliflower mosaic virus 35S terminator (SEQ ID NO: 8); and the second expression cassette consists of the maize ubiquitin 1 promoter (SEQ ID NO: 9), operably linked to the phosphinothricin acetyltransferase (PAT) gene (SEQ ID NO: 10), and operably linked to the maize In2-1 gene terminator (SEQ ID NO: 11).

[0064] The DNA constructs GR03 and GR04 were ligated to the plant transformation vectors V03 and V04, respectively. Wild-type maize immature embryos were transformed with the plant transformation vectors V03 and V04 using Agrobacterium infection methods known in the art to generate transgenic maize plants. Single-copy transgenic maize plants were obtained using Taqman assays for testing.

[0065] Fifteen single-copy transgenic maize plants harboring DNA construct GR03 and fifteen single-copy transgenic maize plants harboring DNA construct GR04 were sprayed with fluazifop-butyl (240 g ai / ha) at a concentration four times the field standard. Seven days after spraying, the CTP targeting efficiency was evaluated by the percentage of leaves showing no damage (tolerance to the phytotoxicity) (percentage of leaves showing no damage = area of ​​leaves showing no damage / total leaf area × 100%). The experimental results for the CTP targeting efficiency of each plant transformation vector are shown in Table 2.

[0066] Given that fluazifop-butyl causes plant leaf damage by blocking the function of the PPO enzyme in the chloroplast, the results in Table 2 indicate that the chloroplast transit peptide HCP1 of the present invention can effectively target the linked PPO enzyme to the chloroplast and enrich it in the chloroplast, thereby conferring good tolerance to fluazifop-butyl on corn plants.

[0067] Table 2. Experimental results of CTP targeting efficiency of various plant transformation vectors

[0068] CTP DNA constructs Plant transformation vectors Number of genetically modified corn plants Percentage of leaves without damage HCP1 GR03 V03 15 90% none GR04 V04 15 0%

[0069] Example 4: Evaluation of the targeting efficiency of chloroplast transit peptides in soybeans

[0070] A DNA construct GR05 containing the chloroplast transit peptide HCP1 sequence of the present invention was constructed. The DNA construct GR05 comprises two tandem expression cassettes: the first expression cassette consists of the Cauliflower Mosaic Virus 35S promoter (SEQ ID NO: 6) operably linked to the HCP1 (SEQ ID NO: 2), which is fused to the protoporphyrinogen oxidase (PPO) gene (SEQ ID NO: 7), which is operably linked to the Cauliflower Mosaic Virus 35S terminator (SEQ ID NO: 8); and the second expression cassette consists of the Arabidopsis thaliana ubiquitin 10 promoter (SEQ ID NO: 3) operably linked to the Arabidopsis thaliana EPSPS chloroplast transit peptide AtCTP2 gene (SEQ ID NO: 12), which is fused to the 5-enolpyruvyl-3-phosphoshikimate synthase (EPSPS) gene (SEQ ID NO: 13), which is operably linked to the nopaline synthase gene terminator (SEQ ID NO: 5).

[0071] A control DNA construct, GR06, was constructed. The DNA construct GR06 comprises two tandem expression cassettes: the first expression cassette consists of the Cauliflower Mosaic Virus 35S promoter (SEQ ID NO: 6), operably linked to the protoporphyrinogen oxidase (PPO) gene (SEQ ID NO: 7), and operably linked to the Cauliflower Mosaic Virus 35S terminator (SEQ ID NO: 8); and the second expression cassette consists of the Arabidopsis thaliana ubiquitin 10 promoter (SEQ ID NO: 3), operably linked to the Arabidopsis thaliana EPSPS chloroplast transit peptide AtCTP2 gene (SEQ ID NO: 12), fused to the 5-enolpyruvyl-3-phosphoshikimate synthase (EPSPS) gene (SEQ ID NO: 13), which is operably linked to the nopaline synthase gene terminator (SEQ ID NO: 5).

[0072] The DNA constructs GR05 and GR06 were ligated to the plant transformation vectors V05 and V06, respectively. Wild-type soybean seeds were transformed with the plant transformation vectors V05 and V06 using Agrobacterium infection, a method known in the art, to generate transgenic soybean plants. Single-copy transgenic soybean plants were obtained using Taqman assays for testing.

[0073] Twelve single-copy transgenic soybean plants containing DNA construct GR05 and twelve single-copy transgenic soybean plants containing DNA construct GR06 were used to evaluate the targeting efficiency of CTP according to the method described in Example 3. The experimental results of the CTP targeting efficiency of each plant transformation vector are shown in Table 3.

[0074] Table 3. Experimental results of CTP targeting efficiency of various plant transformation vectors

[0075] CTP DNA constructs Plant transformation vectors Number of genetically modified soybean plants Percentage of leaves without damage HCP1 GR05 V05 12 89% none GR06 V06 12 0%

[0076] The results in Table 3 indicate that the chloroplast transit peptide HCP1 of the present invention can effectively target the linked PPO enzyme to the chloroplast and enrich it in the chloroplast, thereby imparting good tolerance to fluazifop-butyl to soybean plants.

[0077] In summary, the present invention discloses for the first time a new chloroplast transit peptide. The chloroplast transit peptide of the present invention has chloroplast transport activity and can effectively target the part to which it is connected (such as the target protein) to the plant chloroplast to promote the enrichment of the target protein in the chloroplast.

[0078] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not limiting. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present invention.

Claims

1. An isolated polypeptide, characterized in that The amino acid sequence is shown in SEQ ID NO:

1.

2. A nucleic acid molecule, characterized in that include: (1) a nucleotide sequence encoding the isolated polypeptide of claim 1 or its complementary sequence; or (2) The nucleotide sequence shown in SEQ ID NO: 2 or its complementary sequence.

3. A fusion protein, characterized in that The invention also comprises the isolated polypeptide of claim 1 fused to the N-terminus of a protein of interest.

4. An expression cassette, characterized in that Contains the coding sequence of the fusion protein according to claim 3.

5. A DNA construct or recombinant vector, characterized in that: Comprising the expression cassette of claim 4.

6. A method for targeting a target protein to plant chloroplasts, characterized in that: The method comprises introducing the coding sequence of the fusion protein according to claim 3 into a plant, so that the target protein is localized in the chloroplast of the plant.

7. The method for targeting a target protein to plant chloroplasts according to claim 6, characterized in that: The target protein is a protein that modifies a physiological process present in chloroplasts, an abiotic stress resistance protein, or a biotic stress resistance protein.

8. The method for targeting a target protein to plant chloroplasts according to claim 7, characterized in that: The physiological process is photosynthesis, fatty acid synthesis, amino acid synthesis, oil synthesis, carotenoid synthesis, terpenoid synthesis or starch synthesis.

9. The method for targeting a target protein to plant chloroplasts according to claim 7, characterized in that: The abiotic stress resistance protein is a herbicide tolerance protein; the biotic stress resistance protein is an insect resistance protein.