WO2020001547A1 - Harp1类多肽介导的细胞内转运及其在调节生物防御机制中的应用 - Google Patents

Harp1类多肽介导的细胞内转运及其在调节生物防御机制中的应用 Download PDF

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WO2020001547A1
WO2020001547A1 PCT/CN2019/093307 CN2019093307W WO2020001547A1 WO 2020001547 A1 WO2020001547 A1 WO 2020001547A1 CN 2019093307 W CN2019093307 W CN 2019093307W WO 2020001547 A1 WO2020001547 A1 WO 2020001547A1
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polypeptide
harp1
seq
amino acid
positions
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French (fr)
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毛颖波
陈春雨
陈芳艳
刘尧倩
王牧阳
王凌健
陈晓亚
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中国科学院上海生命科学研究院
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
    • C07K14/43563—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from insects
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
    • Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
    • Y02A40/146—Genetically Modified [GMO] plants, e.g. transgenic plants

Definitions

  • the invention belongs to the field of biotechnology and botany, and more specifically, the invention relates to the use of insect HARP1 polypeptide-mediated intracellular transport and its application in regulating biological defense mechanisms.
  • Phytophagous insects are insects that feed on living plants. Depending on the structure and function of their mouthparts and digestive tracts, they can be divided into two categories: feeding on intact plant organs and tissues and absorbing juice. Herbivorous insects are more common in insects such as Lepidoptera, Collembola, Isoptera, Coleoptera, Diptera. According to the different structures and functions of their mouth organs and digestive tracts, they can be divided into two categories: feeding on intact plant organs and tissues and absorbing juice. Many plant-eating insects only feed on live plants, and many agricultural pests have this kind of feeding habits, causing great harm to agriculture.
  • jasmonic acid signal pathway is one of the main defense signaling pathways in plants. Linolenic acid rapidly produces the signal substance jasmonic acid through the octadecane metabolism pathway. Jasmonic acid activates the gene encoding a protease inhibitor to produce a protease inhibitor. Proteasome inhibitors induced by insects in plants require the participation of jasmonic acid and ethylene.
  • protease inhibitor genes After the plants are fed by insects, the expression of protease inhibitor genes is positively regulated at the transcriptional level via the jasmonic acid pathway through a phylogenetic or cell membrane signal cascade, which causes the rapid increase of protease inhibitors to resist insect feeding.
  • Some peptides with cell transmembrane function include: 1 protein derived peptides (such as penetratin, TAT and pVEC); 2 model peptides (MAP and (Arg) 7); 3 designed peptides (designed CPPs) Such as MPG and Transportan.
  • amphipathic CPPs such as MPG, transportan, TP10, Pep-1
  • SaCPPs medium amphiphilic CPPs
  • NaCPPs Non-amphiphilic CPPs
  • the purpose of the present invention is to provide the application of HARP1 polypeptide-mediated intracellular transport; the object of the present invention is also to provide the application of HARP1 polypeptide in regulating plant defense mechanism.
  • a method for introducing an exogenous active molecule into a cell or a tissue comprising: (1) linking the exogenous active molecule with HARP1 or a conservative variant polypeptide thereof to obtain a connection Product; (2) contacting the ligation product of step (1) with a cell or tissue, so that an exogenous active molecule is introduced into the cell or tissue.
  • the exogenous active molecule is introduced into the cytoplasm or nucleus of a cell.
  • the exogenous active molecule includes: a polypeptide, a nucleic acid, a toxin, a compound, or a combination thereof.
  • the polypeptide or nucleic acid may be a therapeutic polypeptide or nucleic acid.
  • the exogenous active molecule includes a polypeptide, including: a functional polypeptide (such as an enzyme) or a structural polypeptide.
  • the exogenous active molecule is a polypeptide capable of changing the traits or characteristics of animals, plants or microorganisms.
  • the exogenous active molecule includes (but is not limited to): transcription factors, plant defense proteins, signal molecules, RNA binding proteins, drug molecules (such as molecules of peptides or nucleic acids), and the like.
  • connection includes: fusion, coupling, adsorption, coupling or complex.
  • the cells or tissues include: animal cells or tissues, plant cells or tissues, and microbial cells (including cell cultures).
  • the animals include, but are not limited to, humans, mammals, and insects.
  • the plants include, but are not limited to, monocotyledons and dicotyledons; or the plants include, but are not limited to, seed plants, ferns, algae, and moss plants.
  • a method for reducing a plant's defense response to damage comprising: treating a plant with HARP1 or a conservative variant polypeptide thereof; or encoding a gene encoding HARP1 or a conservative variant polypeptide thereof Transformed plants.
  • the HARP1 reduces the defense response ability of a plant to damage by inhibiting the expression or activity of a jasmonate signaling pathway gene or polypeptide.
  • the jasmonate signal pathway includes: a Jasmonate (JA) response gene, a protease inhibitor gene, a secondary metabolic synthesis-related transcription factor, and a synthase.
  • JA Jasmonate
  • the Jasmonate response genes include: TAT1, VSP2, and MYC2.
  • the protease inhibitor genes include: Gh_Sca005135G01, Gh_A10G2353, and Gh_D11G1335.
  • the ability to reduce the plant's defense response to damage includes: reducing the production of poisonous defense substances.
  • HARP1 or its conservative variant polypeptide includes: (a) a polypeptide having the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4; (b) starting from SEQ ID NO: 2 A polypeptide that terminates at any amino acid at positions 21 to 39 and ends at the amino acid sequence shown at positions 119 to 122 in SEQ ID NO: 2; or, starts at any amino acid at positions 21 to 38 in SEQ ID NO: 4 , A polypeptide terminating in the amino acid sequence shown at positions 118 to 121 in SEQ ID NO: 4; (c) passing one or more (such as 1-20, preferably 1-) from the (a) or (b) polypeptide 10; more preferably 1-5; more preferably 1-3) polypeptides formed by substitution, deletion or addition of amino acid residues and having the function of a polypeptide (a) or (b); (d) amino acids
  • the sequence and the amino acid sequence of the (a) or (b) polypeptide are 40% or more (preferably 50% or more,
  • the sequence of the polypeptide starts at positions 22 to 38, 23 to 37, 24 to 38, 25 to 37 in SEQ ID NO: 2, Any of amino acids 26 to 36, 27 to 35, 28 to 34, 29 to 33, 30 to 31, or 30 to 32, which terminates at 120 in SEQ ID NO: 2 Or 121; or, starting at 22 to 37, 23 to 36, 24 to 35, 25 to 34, 26 to 33, and 27 to 32 in SEQ ID NO: 4 Or any of the amino acids in positions 28 to 31 or 29 to 30, which ends at positions 119 and 120 in SEQ ID NO: 4.
  • HARP1 or its conservative variant polypeptide is derived from: Lepidoptera insects; Preferably, the Lepidoptera insects include Noctuidae insects.
  • the Lepidoptera insect includes diamondback moth, S. exigua, H. virescens, S. frugiperda, and small tiger (A.ipsilon), M. configurata, T.ni.
  • the plants include: plants that can be eaten by Lepidoptera insects, and plants that have a damage defense response mechanism.
  • the plants include (but are not limited to): cruciferous plants, mallow plants, gramineous plants, and solanaceous plants.
  • HARP1 or a conservative variant polypeptide thereof for introducing an exogenously active molecule into a plant cell or tissue.
  • the exogenous active molecule is introduced into the cytoplasm or nucleus of a plant cell.
  • the exogenous active molecule is introduced into the plant cell by HARP1 or a conservative variant polypeptide thereof by linking the exogenous active molecule with HARP1 or a conservative variant polypeptide thereof.
  • HARP1 or a conservative variant polypeptide thereof is provided for reducing the defense response ability of a plant to damage.
  • an isolated HARP1 polypeptide fragment which is: (i) starts at any amino acid at positions 21 to 39 in SEQ ID NO: 2 and terminates at SEQ ID NO: 2 Polypeptide having the amino acid sequence shown at positions 119 to 122; or, starting from any amino acid at positions 21 to 38 in SEQ ID NO: 4 and terminating at the amino acid sequence shown at positions 118 to 121 in SEQ ID NO: 4 (Ii) one or more (such as 1-20, preferably 1-10; more preferably 1-5; more preferably 1-3) amino acid residues from (i) the polypeptide A polypeptide formed by substitution, deletion, or addition, and having the function of the polypeptide (i); (iii) the amino acid sequence and (i) the amino acid sequence of the polypeptide are 40% or more (preferably 50% or more, 60% or more, 70%) More than 8% or more than 80%; more preferably more than 85%, more than 90%, more than 95% or more than 99%), and a polypeptide
  • the sequence of the polypeptide starts at positions 22 to 38, 23 to 37, 24 to 38, 25 to 37 in SEQ ID NO: 2, Any of amino acids 26 to 36, 27 to 35, 28 to 34, 29 to 33, 30 to 31, or 30 to 32, which terminates at 120 in SEQ ID NO: 2 Or 121; or, starting at 22 to 37, 23 to 36, 24 to 35, 25 to 34, 26 to 33, and 27 to 32 in SEQ ID NO: 4 Or any of the amino acids in positions 28 to 31 or 29 to 30, which ends at positions 119 and 120 in SEQ ID NO: 4.
  • a method for improving the efficiency of introducing a foreign active molecule into a cell or tissue by a HARP1 polypeptide comprising: truncating the N-terminus or C-terminus of the HARP1 polypeptide, thereby obtaining SEQ ID NO: 2, any amino acid at positions 21 to 39, a polypeptide that terminates at the amino acid sequence shown at positions 119 to 122 in SEQ ID NO: 2; or, starting at 21 to 39 in SEQ ID NO: 4 Any amino acid at position 38, which terminates in the polypeptide of the amino acid sequence shown at positions 118 to 121 in SEQ ID NO: 4.
  • FIG. 1 Sequence analysis of the protein revealed that HARP1 has a signal peptide at the N-terminus. The full-length sequence of the HARP1 protein is shown. The underlined font indicates the signal peptide sequence that the HARP1 protein has at the N-terminus.
  • HARP1 protein accumulates heavily in the cotton boll mouthparts.
  • B HARP1 protein levels in the midgut of cotton bollworm (Midgut); intestinal fluid (Gut fluid) and oral secretions (OS).
  • Gossypol gossypol, + means that the cotton bollworm eats artificial feed containing gossypol,-means that the cotton bollworm eats artificial feed that does not contain gossypol.
  • CBB Coomassie Brilliant Blue staining for determination of protein loading.
  • C The abundance of HARP1 protein in the mouth organ secretions of Helicoverpa armigera that ate different foods.
  • AD artificial feed; GL; phenol-free cotton; GD: phenol cotton; AT: Arabidopsis.
  • HARP1 was significantly distributed in the damaged parts of plant leaves after feeding by cotton bollworm.
  • Mechanical wounding indicates man-made mechanical damage
  • Instrument wounding indicates a wound caused by plant feeding.
  • the signal of HARP1 on the leaves is dark purple, the area pointed by the arrow is the area where the HARP1 signal is strong, and the scale indicates 100 ⁇ m.
  • Prokaryotic purified HARP1 protein can attenuate plant responses to mechanical damage.
  • Venus-HARP1 fusion protein can enter plant cells and is partially localized in the nucleus.
  • HARP1 protein transiently expressed in tobacco is localized in the nucleus of plant cells.
  • the 35S: GFP-HARP1 (GFP-HARP1) and 35S: GFP (GFP) vectors were transferred into tobacco leaves in succession and observed with a laser confocal microscope after 2 days.
  • the scale represents 100 ⁇ m.
  • HARP1 is widely present in Lepidoptera insects, and has a high similarity in Noctuidae.
  • HARP1 protein is widely distributed in Lepidoptera insects and more conserved in Spodoptera insects.
  • the phylogenetic tree of HARP1 protein in Lepidoptera insects was constructed using Mega software.
  • HARP1-like polypeptide REPAT38 has similar functions to HARP1. After treatment of plant wounds with prokaryotic expression of purified REPAT38 protein, compared with the control Venus protein, the induction level of JA response genes TAT1, VSP2, and MYC2 was significantly reduced after REPAT38 treatment.
  • the prokaryotic purified Venus-REPAT38 fusion protein and Venus protein were used to treat the damaged Arabidopsis.
  • the experiments showed that only the Venus-REPAT38 fusion protein can detect signals in Arabidopsis leaves and hypocotyls, and the Venus protein Hardly detectable.
  • the scale represents 250 ⁇ m.
  • Venus-REPAT38 fusion protein was used to treat tobacco leaf wounds using prokaryotic purification.
  • the Venus-REPAT38 and Venus-HARP1 fusion protein can detect signals in tobacco leaf wounds and inside the leaf, but Venus protein is almost undetectable.
  • the scale represents 250 ⁇ m.
  • the internal cells were observed on the material for treating tobacco leaf wounds.
  • the Venus-REPAT38 and Venus-HARP1 fusion proteins could be detected in the leaf cells, while the Venus protein could hardly be detected.
  • the scale represents 50 ⁇ m.
  • a to G are Venus-HARP1; Venus-HARP1 ⁇ C5 (5aa removed from the C-terminal); Venus-HARP1 ⁇ N10 (10aa removed from the N-terminal after the signal peptide); Venus-HARP1 ⁇ N15 (15aa removed from the N-terminal after the signal peptide); Venus-HARP1 ⁇ N20 (removed 20aa at the N-terminus after the signal peptide); Venus-HARP1 ⁇ N25 (25aa at the N-terminus after removal of the signal peptide); Venus.
  • the HARP1 protein has the ability to enter animal cells and partially localizes the nucleus.
  • HARP1 protein has the ability to enter human cells, illustrating Venus-HARP1 enters A549 cells and partially enters the nucleus.
  • A549 cells were immersed in Venus-HARP1 and Venus for 4 hours to observe the results of fluorescence microscopy.
  • HARP1 and its similar peptides have the characteristics of entering the cell and the nucleus through complex structures such as cell walls and cell membranes, so that it can be used to establish efficient transportation systems, through protein fusion, etc. Means to help foreign active molecules enter cells or tissues of an organism, thereby changing the characteristics, characteristics or state of the organism.
  • the HARP1 and similar peptides also have effector activity, which can reduce the plant defense response, thereby reducing the production of poisonous defense substances.
  • HARP1 Jasmonate
  • HARP1 conservative variant polypeptides are derived from, for example, but not limited to, diamondback moth, S. exigua, H. virescens, S. frugiperda, and A. ipsilon.
  • the HARP1 polypeptide (protein) or REPAT38 polypeptide (protein) according to the present invention also includes their conservative variant polypeptides, fragments, derivatives and analogs.
  • conservative variant polypeptide refers to a polypeptide that substantially maintains the same biological function or activity of the HARP1 polypeptide or REPAT38 polypeptide of the invention.
  • polypeptide fragments, derivatives or analogs of the present invention may be (i) one or more (such as 1-50; preferably 1-20; more preferably 1-10; more preferably 1-8) (Such as 5, 3) proteins with conservative or non-conservative amino acid residues (preferably conservative amino acid residues) substituted, and such substituted amino acid residues may or may not be encoded by the genetic code, or (ii) a protein having a substituent group in one or more (eg, 1-30; preferably 1-20; more preferably 1-10; such as 5, 3) amino acid residues, or (iii) A protein formed by fusing an additional amino acid sequence to this protein sequence.
  • These fragments, derivatives and analogs are within the scope of those skilled in the art as defined herein.
  • the biologically active fragment of any HARP1 polypeptide or REPAT38 polypeptide can be used in the present invention.
  • the meaning of the biologically active fragment of HARP1 polypeptide or REPAT38 polypeptide means that as a polypeptide, it can still maintain all or part of the function of the full-length HARP1 polypeptide or REPAT38 polypeptide.
  • the biologically active fragment retains at least 50% of the activity of the full-length HARP1 polypeptide or REPAT38 polypeptide. Under more preferred conditions, the active fragment is capable of maintaining 60%, 70%, 80%, 90%, 95%, 99%, or 100% activity of the full-length HARP1 polypeptide or REPAT38 polypeptide.
  • the term "HARP1 polypeptide” refers to a polypeptide having a SEQ ID NO: 2 sequence having HARP1 polypeptide activity.
  • the term also includes variants of the SEQ ID NO: 2 sequence that have the same function as the HARP1 polypeptide. These variants include (but are not limited to): several (such as 1-50; preferably 1-20; more preferably 1-10; such as 5, 3) amino acid deletions, insertions and / Or substitutions, and the addition or deletion of one or several (usually within 20, preferably within 10, more preferably within 5) amino acids at the C-terminus and / or N-terminus.
  • REPAT38 polypeptide refers to a polypeptide of SEQ ID NO: 4 sequence having REPAT38 polypeptide activity.
  • the term also includes variants of the SEQ ID NO: 4 sequence that have the same function as the REPAT38 polypeptide. These variants include (but are not limited to): several (such as 1-50; preferably 1-20; more preferably 1-10; such as 5, 3) amino acid deletions, insertions and / Or substitutions, and the addition or deletion of one or several (usually within 20, preferably within 10, more preferably within 5) amino acids at the C-terminus and / or N-terminus.
  • Polynucleotide sequences encoding HARP1 polypeptide or REPAT38 polypeptide or a conservative variant polypeptide thereof can also be used in the present invention.
  • the coding region sequence encoding the mature HARP1 polypeptide or REPAT38 polypeptide may be substantially the same as the sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3 or a degenerate variant.
  • a "degenerate variant" in the present invention refers to a protein encoding SEQ ID NO: 2 or SEQ ID NO: 4, but the encoding shown in SEQ ID NO: 1 or SEQ ID NO: 3 The sequence of a region has a different nucleic acid sequence.
  • coding gene may include a polynucleotide that encodes the polypeptide, or a polynucleotide that also includes additional coding and / or non-coding sequences.
  • variants of the above-mentioned polynucleotides are also available, which encode polypeptides or fragments, analogs, and derivatives of polypeptides having the same amino acid sequence as the present invention.
  • Variants of this polynucleotide may be naturally occurring allelic variants or non-naturally occurring variants. These nucleotide variants include substitution variants, deletion variants, and insertion variants.
  • an allelic variant is an alternative form of a polynucleotide that may be a substitution, deletion, or insertion of one or more nucleotides without substantially altering the function of the polypeptide it encodes .
  • the present invention provides a truncated form of a HARP1 polypeptide or a REPAT38 polypeptide, which is a polypeptide obtained by removing the N-terminal or C-terminal partial sequence on the basis of the HARP1 polypeptide or the REPAT38 polypeptide.
  • the truncated body is a polypeptide obtained by removing 1 to 19, 2 to 18, and 3 to 17 amino acids of the N-terminus (excluding the signal peptide). More specifically, for example, a polypeptide obtained by removing 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 amino acids at the N-terminus (excluding the signal peptide).
  • HARP1 polypeptide or REPAT38 polypeptide For the C-terminus of HARP1 polypeptide or REPAT38 polypeptide, generally less than 5, less than 4, less than 3, or less than 2 amino acids are removed.
  • the inventors have unexpectedly found that the truncated body can more efficiently introduce foreign active molecules into cells or tissues.
  • the HARP1 gene or REPAT38 gene of the present invention is obtained from Spodoptera exigua cotton bollworm or Spodoptera exigua, respectively, it is highly homologous to the HARP1 gene or REPAT38 gene obtained from other insects, such as having more than 40%; Preferably, more than 50%, more than 60%, more than 70%, or more than 80%; more preferably, more than 85%, more than 90%, more than 95%, or more than 99% of other genes are also within the scope of the present invention. Based on the homology comparison, the inventors found that HARP1 like polypeptide (HARP1like, HL) is ubiquitous in Lepidoptera insects, but highly conserved in Noctuidae.
  • a vector comprising the coding sequence, and a host cell genetically engineered using the vector or the HARP1 polypeptide or the REPAT38 polypeptide coding sequence are also included in the present invention.
  • Methods well known to those skilled in the art can be used to construct expression vectors containing the HARP1 polypeptide REPAT38 polypeptide coding sequence and appropriate transcription / translation control signals. These methods include in vitro recombinant DNA technology, DNA synthesis technology, and in vivo recombinant technology.
  • the sequence can be operably linked to an appropriate promoter in an expression vector to guide mRNA synthesis.
  • a vector containing the above-mentioned appropriate coding sequence and appropriate promoter or control sequence can be used to transform an appropriate host cell so that it can express a protein.
  • the host cell may be a plant cell or an animal cell.
  • the present invention provides a method for introducing an exogenous active molecule into a cell or tissue, comprising: (1) linking the exogenous active molecule with a HARP1 type polypeptide to obtain a connection product; (2) connecting in step (1) The product contacts the cell or tissue so that an exogenous active molecule is introduced into the cell or tissue.
  • the exogenous active molecule is introduced into the nucleus of a cell.
  • the HARP1 type polypeptide includes a HARP1 conservative variant polypeptide such as REPAT38, and the conservative variant polypeptide includes a truncated form of a polypeptide such as HARP1, REPAT38.
  • exogenous active molecule is also referred to as a "functional molecule”, which refers to a group of molecules that have a specific function and can modify the traits, structures, characteristics, or states of organisms (including animals and plants).
  • Class-like molecules for example, they can make plants improve at least one aspect of their traits, and they can also improve the disease state of animals or humans; they can also be a class of molecules with the function of indicating or reporting, which can make certain organisms Some organs, tissues and cells are detected or localized, such as fluorescent proteins, tag proteins (such as myc, HA, His, etc.).
  • Exogenous active molecules that can be used include: polypeptides, nucleic acids, compounds (such as some hormones).
  • the HARP1 type polypeptide of the present invention can introduce exogenous active molecules into cells or tissues, especially can enter human cells, which will have great application prospects in the field of clinical disease treatment.
  • diseases that require the introduction of exogenous active molecules (such as drugs) for treatment.
  • tumors can carry inhibitory molecules or toxins to tumor cells as HARP1 polypeptides, thereby achieving the purpose of suppressing tumors.
  • some diseases related to low enzyme or protein function can use HARP1 polypeptides to carry active enzymes or proteins into cells or tissues to achieve the purpose of alleviating or treating such diseases.
  • the human cells may include, but are not limited to, tumor cells, somatic cells, and embryonic cells.
  • the somatic cells include, but are not limited to, fibroblasts, germ cells, bone marrow cells, blood cells, and the like.
  • human lung cancer cells, human embryonic kidney cells, and animal cells such as fly cells and plant cells are demonstrated, but it should be understood that the cells applicable to the technical scheme of the present invention are not limited to those listed in the embodiments.
  • connection mode of the HARP1 type polypeptide according to the present invention and the exogenous active molecule may be covalent connection or non-covalent connection.
  • the connection includes: fusion, coupling, adsorption, coupling or compounding.
  • the "connection” refers to "operative connection", that is, a functional spatial arrangement between two or more molecules. It should be understood that, as long as the functions of the HARP1 polypeptides and exogenous active molecules can be retained, and the effect of penetrating cell membranes and body tissue barriers is maintained, any connection manner can be included in the present invention.
  • Covalent linking usually connects two molecules in a manner that forms a covalent bond. And some non-covalent linkages (without forming covalent bonds) such as coupling, adsorption, binding, etc. can also be applied.
  • the exogenous active molecule is a polypeptide, including: a functional polypeptide (such as an enzyme) or a structural polypeptide.
  • a functional polypeptide such as an enzyme
  • a structural polypeptide When the exogenous active molecule is a polypeptide, it is fused with a HARP1 polypeptide to obtain a fusion protein.
  • the HARP1 type polypeptide may be directly connected to the exogenous active molecule, or may be connected through a polypeptide linker (linking peptide).
  • the linker includes, for example, 1-30 amino acids; preferably, 1-20 amino acids; for example, 15, 10, 8, 6, 5, 4, 3, 2, 1 amino acids.
  • the setting of the linker peptide basically does not affect the effect of HARP1 and exogenous active molecules to penetrate cell membranes and body tissue barriers, nor does it affect the function of exogenous active molecules.
  • the exogenous active molecule may be located at the amino terminal of the HARP1 polypeptide or the carboxyl terminal of the HARP1 polypeptide according to needs.
  • the exogenous active molecule may be RNA or DNA, for example, it is an RNA molecule capable of producing a homologous interference effect on a certain gene in an organism.
  • the HARP1 type polypeptide can be connected to an exogenous active molecule through a chemical reaction such as an amino group, a carboxyl group, or a thiol group, including but not limited to the connection between the polypeptide and a polymer.
  • a chemical reaction such as an amino group, a carboxyl group, or a thiol group, including but not limited to the connection between the polypeptide and a polymer.
  • the non-covalent connection is an electrostatic adsorption connection or a receptor ligand reaction.
  • the electrostatic adsorption connection includes, but is not limited to, an electrostatic connection between the cell transmembrane carrier and the nucleic acid molecule.
  • the receptor-ligand reaction refers to that a receptor and a ligand that can be specifically matched are connected to a HARP1 polypeptide and an exogenous active molecule, respectively.
  • the high specificity of the receptor and the ligand realizes the The polypeptide is linked to an exogenous active molecule. Such as the specific match between biotin and avidin.
  • the present invention can promote the entry of exogenous active molecules into cells and cell nuclei through complex structures such as cell walls and cell membranes. Therefore, a variety of exogenous active molecules can be applied to link with the HARP1 type polypeptide to form a complex.
  • the present inventors found that the Venus-HARP1 fusion protein expressed in prokaryotic cells can enter the cell, and a part of the fusion protein is localized in the cell nucleus. This shows that the HARP1 polypeptides in insects can enter the cells to function through the multiple barriers such as cell walls and cell membranes.
  • the types of cells or tissues suitable for mediating the introduction of foreign proteins with the HARP1 polypeptide of the present invention are broad-spectrum, and can be a variety of biological cells, including plant cells or tissues, animal cells or tissues, and microbial cells (including Its culture) and so on.
  • the inventors also found that the HARP1-like polypeptide in Spodoptera exigua has a similar function to HARP1. These findings indicate that HARP1 and its similar peptides can be used as a transport system to help foreign active molecules enter cells by means of protein fusion and other means, thereby changing the properties, structure, characteristics or status of organisms.
  • connection products of the HARP1 type polypeptides and exogenous active molecules enter various cells from the wound.
  • the connection product of the HARP1 polypeptide and the exogenous active molecule moves rapidly and in multiple ways in leaf tissue.
  • the invention provides a method for reducing the defense response ability of a plant to damage, which comprises: treating a plant with a HARP1 type polypeptide; or transforming a gene encoding a HARP1 type polypeptide into a plant.
  • the ability to reduce the plant's defense response to damage includes: reducing the production of toxic defense substances in the plant.
  • the HARP1 type polypeptide exerts a regulatory effect by affecting a defense response mechanism in a plant, and the defense response mechanism includes a mechanism based on a jasmonate signal pathway.
  • the inventors have found that the HARP1 type polypeptide significantly inhibits the expression or activity of a jasmonate signaling pathway gene or polypeptide.
  • the jasmonate signal pathway includes: Jasmonate (JA) response genes, such as TAT1, VSP2, and MYC2. These genes are significantly inhibited after HARP1 type peptides are processed or overexpressed. It is well known in the art that many important plant defense substances are regulated by the JA pathway. Therefore, the weakening of JA responses by HARP1 polypeptides will necessarily reduce the production of toxic defense substances in plants.
  • HARP1 polypeptides have significant effector activity in vitro or in vivo.
  • an expression unit for example, an expression vector or a virus
  • a gene encoding a HARP1 polypeptide is cloned into an appropriate vector by a conventional method, and the recombinant vector with a foreign gene is introduced into a plant that can express the HARP1 polypeptide.
  • the plant cell is caused to express a HARP1 type polypeptide.
  • the plant cells can be regenerated into plants to obtain plants overexpressing HARP1 type polypeptides.
  • the primer information used in the present invention is shown in Table 1.
  • HARP1 plant-induced cotton bollworm's mouth organ secreted protein
  • the inventors obtained the nucleic acid sequence and protein sequence of HARP1. According to the sequence information, primers were synthesized and the HARP1 gene was obtained by PCR amplification. Analysis of the protein by using Blast and by means of AlignX software.
  • the inventors found that the protein contains a signal peptide at the N-terminus, which may have a role in helping the protein to be secreted into the mouth organs ( Figures 1 to 2).
  • the HARP1 nucleotide sequence (SEQ ID NO: 1) is as follows:
  • amino acid sequence of HARP1 (SEQ ID NO: 2) is as follows:
  • Cotton bollworms of 4th instar were selected, and tissues and organs such as midgut and salivary glands were taken. RNA was extracted by Trizol method. Cotton bollworm actA3b was used as internal reference gene for quantitative analysis. It was found that HARP1 was highly expressed in the midgut at the transcriptome level and very low in the salivary glands (Figure 3A).
  • HARP1 protein in order to detect the expression level of HARP1 protein in the intestine, intestinal fluid, and mouth organ secretions of cotton bollworm, the inventors first extracted the total intestinal protein of the 4th instar cotton bollworm with 50mM Tris-HCl (pH 9.0), and simultaneously extracted the cotton boll The intestinal fluid and mouth organ secretions of the insects were extracted with an equal volume of chloroform together with the total intestinal protein solution, washed with 75% ethanol, and finally dissolved in water. An equal amount of protein sample was added to each well in 15% protein gel, and then detected with HARP1 antibody. At the protein level, HARP1 is most abundantly expressed in the saliva of cotton bollworm, and HARP1 protein can be induced by secondary compounds such as gossypol ( Figure 3B).
  • the inventors also investigated the abundance of HARP1 protein in the bollworm's mouth organ secretions from different foods (artificial feed, phenol-free cotton, phenol-cotton, and Arabidopsis thaliana). The results showed that different plants can also induce the abundance of HARP1 protein in the saliva of cotton bollworm ( Figure 3C). The abundance of HARP1 protein in the saliva of cotton bollworms fed with phenol cotton or Arabidopsis was significantly higher.
  • Example 3 HARP1 has obvious distribution in the damaged parts of plant leaves after feeding by cotton bollworm
  • the mouth organ secretions will contact the plant wounds.
  • the cotton bollworm of the fourth instar was selected, and the artificial feed containing gossypol was fed in advance, and then it was fed on the Arabidopsis leaves which grew for 3 weeks.
  • the leaves of Arabidopsis thaliana fed by cotton bollworm were quickly transferred to the FAA fixative solution, vacuum-extracted to be completely immersed in the FAA, and fixed with the fixative solution for 4 hours. as comparison. After that, the fixed leaves are dehydrated with a series of gradient ethanol solutions, and then rehydrated with a series of gradient ethanol solutions.
  • Example 4 Prokaryotic purified protein can attenuate plant response to mechanical damage
  • the HARP1 gene sequence was amplified by PCR and introduced into BamH ⁇ and HindIII before digestion of the HARP1 start codon and stop codon, respectively.
  • Site the HARP1 was introduced between pET32a multicloning sites BamH1 and HindIII, to obtain a recombinant expression vector carrying the target fragment, which is called pET32a / HARP1.
  • the procedure is as follows: The bacterial cells are collected and resuspended in 15 ml of Lysis buffer (50 mM Tris-Cl, pH 8.5, 100 mM NaCl, 10 mM imidazole). After crushing under high pressure, the supernatant was centrifuged and the packed 1ml Ni-NTA resin was used. The protein was eluted with 2 ml of Elution buffer (50 mM Tris-Cl, pH 8.5, 100 mM NaCl, 250 mM imidazole), and replaced with 20 mM Tris-Hcl Buffer, pH 8.5. Using the Bradford method, bovine serum protein was used as a reference. The concentration was quantified.
  • Venus protein was expressed and purified in E. coli BL21 (DE3) (Venus sequence was amplified from pCAMBIA1302 ( http://www.cambia.org/daisy/bios/585.html ) vector) as Control for subsequent experiments.
  • a second pair of true leaves of Arabidopsis thaliana growing for about 3 weeks was selected for mechanical damage treatment, and prokaryotic purified HARP1 protein and Venus protein (control) were applied to the mechanically damaged area. After 4 hours, samples were taken.
  • Trizol method to extract RNA from Arabidopsis leaves, and using S18 gene in Arabidopsis leaves as internal reference genes for quantitative analysis of related genes, it was found that, compared with Venus protein treatment, HARP1 protein can significantly inhibit JA in Arabidopsis leaves Response genes (including TAT1, VSP2, MYC2) to mechanical damage (Figure 5A).
  • the exogenous HARP1 fusion protein can enter plant cells and is partially localized in the nucleus
  • the Venus-HARP1 (5 ' ⁇ 3') fusion sequence was prepared and amplified by recombinant PCR method, and the BamHI and HindIII restriction sites were introduced before the start and stop codons of Venus-HARP1, respectively.
  • Venus- HARP1 was introduced into the multiple cloning sites of pET32a between BamHI and HindIII to obtain a recombinant expression vector carrying the target fragment, which is called pET32a / Venus-HARP1.
  • the Venus-HARP1 protein was expressed and purified in E. coli BL21 (DE3), and the Venus protein was also used as a control for subsequent experiments.
  • the second true leaf of Arabidopsis thaliana was selected for mechanical damage treatment, and then placed in 20 mM Tris-Hcl buffer containing the prokaryotic purified fusion protein Venus-HARP1 and the control Venus.
  • the protein content was adjusted in advance by the Bradeford method (1 mg / ml). ). After 1 hour of incubation, it was washed 3-4 times with PBST solution (1 ⁇ PBS + 0.1% Tween 20 + 1% BSA).
  • DAPI 4,6-diamidino-2-phenylindole
  • the treated leaves were imaged under a laser confocal scanning microscope (Olympus FV3000) and analyzed with software Olympus cellSens (version Dimension 1.18).
  • the Arabidopsis seedlings which were grown for about 7 days, were mechanically damaged at the roots. After the seedlings were treated by immersion, they were placed under a laser confocal scanning microscope to observe the hypocotyl part. It was also found that the prokaryotic purified The Venus-HARP1 protein can enter plant cells and is mainly localized in the nucleus, while the externally added Venus protein does not substantially enter plant cells (Figure 6C, D).
  • Example 6 The transiently expressed HARP1 protein of tobacco is localized in the nucleus of plant cells
  • HARP1 was introduced into the plant expression vector YUKHS-GFPL (the backbone is pCambia 1300.
  • 35S Promoter was added between the multicloning site EcoR1 and Sac1. Sac1 and Kpn1 were added to the GFP sequence), the resulting vector was called 35S: GFP-HARP1 expression vector, and the original vector YUKHS-GFPL was called 35S: GFP expression vector.
  • Example 7 HARP1 has no obvious growth inhibitory effect on plants, and no over-expressing plants have obvious phenotypes.
  • the inventors also used the PCR method to add BamH1 and Sal1 restriction sites at both ends of the HARP1 sequence, and introduced HARP1 into the plant expression vector YUKHS-6MYC (the backbone is pCambia1300, and 35S was added between the multicloning sites EcoR1 and Sac1). promoter, adding a 6MYC tag sequence between Kpn1 and Sma1), and the resulting vector is called a 35S: 6MYC-HARP1 expression vector.
  • Floral dip was used to transform Arabidopsis plants.
  • the specific process is as follows: the obtained vectors are transferred into Agrobacterium GV3101 by freeze-thaw method, and single colonies are picked and cultured to obtain 200 ml of bacterial solution as described above. After centrifugation at 5000 rpm for 10 minutes, the bacterial weight was suspended in 250 ml of a 5% sucrose solution containing 0.02% Silwet L-77. The flower bud part of the plant is immersed in the bacterial solution for 1 minute, placed flat in a plastic pot, moisturized, and protected from light. After 14 to 20 hours, it is normally cultivated in a greenhouse until flowering and seeding.
  • the T 0 generation seeds were washed with 75% ethanol for 5 minutes, treated with 20% bleached water (White Cat Company, Shanghai) for 15 minutes, washed with sterile water 3 to 4 times, and spread on 1 / 2MS solid medium (containing 50 ⁇ g / ml Hygo), placed in an Arabidopsis incubator for 1 week, picked green resistant seedlings and transplanted them into nutrient soil for growth. After 4 weeks, leaves with similar growth were used to extract total protein. Western methods were used. The 6MYC antibody (purchased from Xunsheng) was used to detect the gene expression level of transgenic plants.
  • Example 8 HARP1 is widely present in Lepidoptera insects. HARP1-like polypeptides have similar functions to HARP1
  • HARP1 similar peptides of the Lepidoptera insect Through further sequence alignment and further collection of HARP1 similar peptides of the Lepidoptera insect, the inventors obtained diamondback moth, S. exigua, H. virescens, and Spodoptera frugiperda (S.frugiperda), A.ipsilon, M.configurata, T.ni, and other HARP1 similar proteins of Lepidoptera. Further, the present inventors found that HARP1 polypeptides are widely present in Spodoptera insects, and analysis by Mega software (Tamura et al., 2011) and AlignX software show that HARP1 polypeptides have high Conservative (Figure 8).
  • the inventors obtained the HARP1 similar protein REPAT38 in Spodoptera exigua, and used the same method to construct it into the pET-32a vector, and expressed and purified REPAT38 in E. coli BL21 (DE3). Protein and Vennus protein.
  • the REPAT38 nucleotide sequence (SEQ ID NO: 3) is as follows:
  • the Venus-REPAT38 fusion sequence was prepared and amplified by recombinant PCR method.
  • the Venus-REPAT38 (5 ' ⁇ 3') start codon and stop codon were introduced into the restriction sites of BamHI and SalI, respectively.
  • -REPAT38 is introduced between the pET32a multiple cloning sites BamHI and SalI to obtain a recombinant expression vector carrying the target fragment, which is called pET32a / Venus-REPAT38.
  • the Venus-REPAT38 protein was expressed and purified in E. coli BL21 (DE3), and the Venus protein was also used as a control for subsequent experiments.
  • the concentration and method of Venus-REPAT38 fusion protein to treat Arabidopsis thaliana and tobacco were the same as in Example 5.
  • the Arabidopsis seedlings which were grown for about 7 days were treated with mechanical damage to their cotyledons. After the seedlings were treated by immersion, they were placed under a laser confocal scanning microscope to observe that the Venus-REPAT38 protein purified by prokaryotes could Entered into Arabidopsis cells, while the externally added Venus protein did not enter Arabidopsis cells ( Figure 10A, B).
  • Tobacco leaves that were grown for about two weeks were selected, and the small tobacco leaves were pierced with a pipette tip. After the tobacco leaves were treated by immersion, they were placed under a laser confocal scanning microscope to observe that the prokaryotic purified Venus-REPAT38 was found.
  • the Venus-HARP1 fusion protein can enter tobacco leaf cells, while the externally added Venus protein does not basically enter tobacco cells ( Figure 10C, D).
  • Method steps Use the method of Examples 5 and 8 to select Arabidopsis leaves that grow for about three weeks, poke the Arabidopsis small disc leaves with a pipette tip, treat the leaves by immersion, and place them in a laser confocal scan Observe the fluorescence signal near the wound under a microscope.
  • Example 9-1 The leaves treated in Example 9-1 were further enlarged and observed under a laser confocal scanning microscope. During the observation, a fixed focal plane was selected and an interval-free time-series scan was performed. The fluorescence signal changes in the observation area within 30 seconds of shooting happening.
  • Venus-HARP1 can enter Arabidopsis leaf tissue from the wound. HARP entering the tissue space can cross the epidermal cell wall and cell membrane, can shuttle between mesophyll cells and spaces, and can enter cells along the cell space. Internally, it can also be secreted from the cell to the outside of the wound, which indicates that it can move in multiple ways quickly.
  • Example 10 HARP1 enters plant cells with broad spectrum
  • Venus-HARP1 can enter cotton cells in the cotton and solanaceae of the Malvaceae family from the wound, indicating that HARP1 can enter different plant tissues, and that it has a broad spectrum into plant cells or tissues.
  • the transport system constructed by HARP1 can be applied to a variety of plants.
  • Example 11 Removal of the N-terminal partial sequence can significantly improve the efficiency of protein entry into plant cells
  • HARP1 is modified to remove 10, 15, 20, and 25 amino acids at its N-terminus after removing its signal peptide, or remove 5 amino acids at its C-terminus.
  • the test sequence is as follows:
  • HARP1 Different truncated HARP1s have different efficiency in plant cells.
  • HARP1 with N-terminal truncated 10 amino acids is more likely to enter plant tissues from wounds, which greatly improves the efficiency of protein entry into plant cells.
  • C-terminally truncated 5 or N-terminally truncated 20 or more amino acids inhibit HARP1 from entering plant tissues, suggesting that the protein sequence of HARP1 will affect its entry into cells.
  • the delivery efficiency of this vector can be further improved.
  • Example 12 HARP1 protein has the ability to infect animal cells
  • Venus-HARP1 was expressed prokaryotically, and the fusion protein was purified. The fusion protein was incubated with Drosophila S2 cells, and Venus expressed in prokaryotic cells was used as a control for parallel experiments.
  • Venus-HARP1 fusion protein was incubated with human lung cancer cell A549, and Venus expressed in prokaryotic cells was used as a control for parallel experiments.
  • Venus-HARP1 fusion protein was incubated with human embryonic kidney cells 293T, and Venus expressed in prokaryotic cells was used as a control for parallel experiments.

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Abstract

提供了一种HARP1类多肽介导的细胞内转运及其在调节生物防御机制中的应用。所述HARP1及其类似多肽(HARP1-like，HL)具有通过细胞壁和细胞膜等复杂结构进入细胞或组织的特性，可被应用于建立高效的运输系统，帮助外源活性分子进入细胞。所述HARP1及其类似多肽还具有效应子活性，能够降低植物防御响应。

Description

HARP1类多肽介导的细胞内转运及其在调节生物防御机制中的应用 技术领域
本发明属于生物技术以及植物学领域，更具体地，本发明涉及利用昆虫HARP1类多肽介导的细胞内转运及其在调节生物防御机制中的应用。
背景技术
植食性昆虫是以植物活体为食的昆虫，根据它们的口器和消化道在构造和功能上的不同，可分为以完整植物器官、组织为食和吸食汁液两大类。植食性昆虫多见于鳞翅目、弹尾目、等翅目、鞘翅目、双翅目等昆虫。根据它们的口器和消化道在构造和功能上的不同，可分为以完整植物器官、组织为食和吸食汁液两大类。很多植食性昆虫只以植物活体为食，很多农业害虫都有这种食性，在农业上造成很大的危害。
植物被昆虫取食后，植物自身可产生一些自体防御，包括直接防御或间接防御。目前已经了解植物体内存在一些防御信号途径来调控植物的自体防御。茉莉酸信号途径是植物体内主要防御信号途径之一，亚麻酸经过十八烷代谢途径，迅速生成信号物质茉莉酸(jasmonic acid)，茉莉酸激活编码蛋白酶抑制剂的基因，产生蛋白酶抑制剂。昆虫诱导植物产生蛋白酶抑制剂需要茉莉酸和乙烯的参与。植物被昆虫取食后，通过系统素或细胞膜信号级联反应，经茉莉酸途径在转录水平上正向调控蛋白酶抑制剂基因的表达，引起蛋白酶抑制剂快速增加，以抵御昆虫取食。
目前本领域中关于植物对病原菌防御反应在细胞水平的研究已取得了许多进展，但是对于植物与昆虫的互作识别的研究仍然有较多困惑，并且较少地能够被运用于农业中，发挥实用价值。因此，本领域还需要进行深入的研究，来切实地将这种植物与昆虫的互作识别应用于植物改良或者病虫害防治等应用中。
此外，本领域中存在一些具有细胞穿膜功能的肽，它们具有携带其它分子穿透细胞、进入到细胞内的能力。一些具有穿膜功能的肽包括：①蛋白衍生肽(protein derived CPPs)，如penetratin、TAT和pVEC等；②模型肽(model peptides)如MAP和(Arg)7等；③设计肽(designed CPPs)如MPG和Transportan等。从其两亲性性质也可将其分为3类：①两亲性CPPs(PaCPPs)， 如MPG、transportan、TP10、Pep-1；②中等两亲性CPPs(SaCPPs)，如penetratin，RL16；③非两亲性CPPs(NaCPPs)，如R9。尽管已经有一些肽的存在，但是本领域中仍然有较多的分子无法藉由上述已知的穿膜肽被引入到细胞内，因此还需要进一步找到更多的、具有普适性的此类肽。
发明内容
本发明的目的在于提供HARP1类多肽介导的细胞内转运的应用；本发明的目的还在于提供HARP1类多肽在调节植物防御机制中的应用。
在本发明的第一方面，提供一种将外源活性分子引入到细胞或组织中的方法，所述方法包括：(1)将外源活性分子与HARP1或其保守性变异多肽连接，获得连接产物；(2)将步骤(1)的连接产物接触细胞或组织，从而外源活性分子被引入到细胞或组织中。
在一个优选例中，所述的外源活性分子被引入到细胞的细胞质或细胞核中。
在另一优选例中，所述的外源活性分子包括：多肽，核酸，毒素，化合物，或它们的组合。所述的多肽或核酸可以是治疗性的多肽或核酸。
在另一优选例中，所述的外源活性分子包括为多肽，包括：功能性多肽(如酶)或结构多肽。较佳地，所述的外源活性分子是能够改变动物、植物或微生物性状或特性的多肽。例如，所述的外源活性分子包括(但不限于)：转录因子，植物防御蛋白，信号分子，RNA结合蛋白，药物分子(如肽或核酸类的分子)等。
在另一优选例中，所述的连接包括：融合，偶联，吸附，耦合或复合。
在另一优选例中，所述的细胞或组织包括：动物细胞或组织，植物细胞或组织，微生物细胞(包括细胞培养物)。
在另一优选例中，所述的动物包括但不限于：人、哺乳动物、昆虫。
在另一优选例中，所述的植物包括但不限于：单子叶植物、双子叶植物；或所述植物包括但不限于：种子植物、蕨类植物、藻类植物、苔藓植物。
在本发明的另一方面，提供一种降低植物对损伤的防御响应能力的方法，所述方法包括：以HARP1或其保守性变异多肽处理植物；或将HARP1或其保守性变异多肽的编码基因转化植物。
在另一优选例中，所述的HARP1通过抑制茉莉酸信号途径基因或多肽的表达或活性，来降低植物对损伤的防御响应能力。
在另一优选例中，所述的茉莉酸信号途径包括包括：Jasmonate(JA)响应基因、蛋白酶抑制剂基因、次生代谢合成相关转录因子及合酶。
在另一优选例中，所述的Jasmonate响应基因包括：TAT1、VSP2、MYC2。
在另一优选例中，所述的蛋白酶抑制剂基因包括：Gh_Sca005135G01、Gh_A10G2353、Gh_D11G1335。
在另一优选例中，所述的降低植物对损伤的防御响应能力包括：减少有毒化防御物质的产生。
在另一优选例中，HARP1或其保守性变异多肽包括：(a)具有SEQ ID NO:2或SEQ ID NO:4所示氨基酸序列的多肽；(b)起始于SEQ ID NO:2中第21～39位中任一氨基酸，终止于SEQ ID NO:2中第119～122位所示氨基酸序列的多肽；或，起始于SEQ ID NO:4中第21～38位中任一氨基酸，终止于SEQ ID NO:4中第118～121位所示氨基酸序列的多肽；(c)由(a)或(b)多肽经过一个或多个(如1-20个，较佳地1-10个；更佳地1-5个；更佳地1-3个)氨基酸残基的取代、缺失或添加而形成的，且具有多肽(a)或(b)功能的多肽；(d)氨基酸序列与(a)或(b)多肽的氨基酸序列有40％以上(较佳地50％以上、60％以上、70％以上或80％以上；更佳地85％以上，90％以上，95％以上或99％以上)相同性，且具有多肽(a)或(b)功能的多肽；或(e)在(a)或(b)或(c)或(d)所述多肽的N或C末端添加标签序列，或在多肽(b)或(c)或(d)的N末端添加信号肽序列后形成的多肽。
在另一优选例中，(b)中，所述多肽的序列起始于SEQ ID NO:2中第22～38位、第23～37位、第24～38位、第25～37位、第26～36位、第27～35位、第28～34位、第29～33位、第30～31位或第30～32位中任一氨基酸，终止于SEQ ID NO:2中第120或121位；或，起始于SEQ ID NO:4中第22～37位、第23～36位、第24～35位、第25～34位、第26～33位、第27～32位或第28～31位或第29～30位中任一氨基酸，终止于SEQ ID NO:4中第119、120位。
在另一优选例中，HARP1或其保守性变异多肽来自于：鳞翅目昆虫；较佳地，所述的鳞翅目昆虫包括夜蛾科昆虫。
在另一优选例中，所述的鳞翅目昆虫包括：小菜蛾，甜菜夜蛾(S.exigua)，烟芽夜蛾(H.virescens)，草地夜蛾(S.frugiperda)，小地老虎(A.ipsilon)，蓓带夜蛾(M configurata)，粉纹夜蛾(T.ni)。
在另一优选例中，所述的植物包括：能被鳞翅目昆虫取食的植物，存在损伤防御响应机制的植物。
在另一优选例中，所述的植物包括(但不限于)：十字花科植物，锦葵科植物，禾本科植物以及茄科植物等。
在本发明的另一方面，提供HARP1或其保守性变异多肽的用途，用于将 外源活性分子引入到植物细胞或组织中。
在另一优选例中，所述的外源活性分子被引入到植物细胞的细胞质或细胞核中。
在另一优选例中，通过将外源活性分子与HARP1或其保守性变异多肽连接，从而由HARP1或其保守性变异多肽将外源活性分子引入到植物细胞中。
在本发明的另一方面，提供HARP1或其保守性变异多肽的用途，用于降低植物对损伤的防御响应能力。
在本发明的另一方面，提供一种分离的HARP1多肽片段，其为：(i)起始于SEQ ID NO:2中第21～39位中任一氨基酸，终止于SEQ ID NO:2中第119～122位所示氨基酸序列的多肽；或，起始于SEQ ID NO:4中第21～38位中任一氨基酸，终止于SEQ ID NO:4中第118～121位所示氨基酸序列的多肽；(ii)由(i)多肽经过一个或多个(如1-20个，较佳地1-10个；更佳地1-5个；更佳地1-3个)氨基酸残基的取代、缺失或添加而形成的，且具有多肽(i)功能的多肽；(iii)氨基酸序列与(i)多肽的氨基酸序列有40％以上(较佳地50％以上、60％以上、70％以上或80％以上；更佳地85％以上，90％以上，95％以上或99％以上)相同性，且具有多肽(i)功能的多肽；或(iv)在(i)或(ii)或(iii)所述多肽的N或C末端添加标签序列，或添加信号肽序列后形成的多肽。
在另一优选例中，(i)中，所述多肽的序列起始于SEQ ID NO:2中第22～38位、第23～37位、第24～38位、第25～37位、第26～36位、第27～35位、第28～34位、第29～33位、第30～31位或第30～32位中任一氨基酸，终止于SEQ ID NO:2中第120或121位；或，起始于SEQ ID NO:4中第22～37位、第23～36位、第24～35位、第25～34位、第26～33位、第27～32位或第28～31位或第29～30位中任一氨基酸，终止于SEQ ID NO:4中第119、120位。
在本发明的另一方面，提供一种提高HARP1多肽将外源活性分子引入到细胞或组织中的效率的方法，包括：对HARP1多肽的N端或C端进行截短，从而获得起始于SEQ ID NO:2中第21～39位中任一氨基酸，终止于SEQ ID NO:2中第119～122位所示氨基酸序列的多肽；或，起始于SEQ ID NO:4中第21～38位中任一氨基酸，终止于SEQ ID NO:4中第118～121位所示氨基酸序列的多肽。
本发明的其它方面由于本文的公开内容，对本领域的技术人员而言是显而易见的。
附图说明
图1、蛋白的序列分析发现HARP1在N端具有信号肽。图示HARP1蛋白的全长序列，下划线字体表示HARP1蛋白在N端具有的信号肽序列。
图2、HARP1蛋白的信号肽预测。
图3、HARP1蛋白在棉铃虫口器中大量积累。A：HARP1基因在棉铃虫各组织器官的表达分析。Foregut：前肠；Midgut：中肠；Fat body：脂肪体；Malpighian tubule：马氏管；Ovary：卵巢；Salivary gland：唾液腺。
B：HARP1在棉铃虫中肠(Midgut)；肠液(Gut fluid)及口器分泌物(OS)中的蛋白水平。Gossypol：棉酚，+表示棉铃虫取食含有棉酚的人工饲料，-表示棉铃虫取食不含棉酚的人工饲料。CBB：考马斯亮蓝染色，用于蛋白上样量的测定。
C：取食不同食物的棉铃虫口器分泌物中HARP1蛋白的丰度。AD：人工饲料；GL；无酚棉；GD：有酚棉；AT：拟南芥。
图4、HARP1在被棉铃虫取食后的植物叶片损伤部位有明显分布。图中Mechanical wounding表示人为的机械损伤，Insect wounding表示植物取食造成的伤口。HARP1在叶片上的信号表现为暗紫色，箭头所指部分为HARP1信号较强区域，标尺表示100μm。
图5、原核纯化的HARP1蛋白能够削弱植物对机械损伤的响应。A：原核表达纯化的HARP1蛋白处理拟南芥叶片伤口后，相较于对照Venus蛋白，HARP1蛋白处理后，JA响应基因TAT1、VSP2、MYC2的诱导水平均明显降低。
B：原核表达纯化的HARP1蛋白处理棉花叶片伤口后，相较于对照Venus蛋白，HARP1蛋白处理后，棉花中三个蛋白酶抑制剂基因的诱导水平均明显降低。
图6、Venus-HARP1融合蛋白能够进入植物细胞并部分定位于细胞核中。A：使用原核纯化的Venus-HARP1融合蛋白及Venus蛋白处理拟南芥叶片伤口处。实验发现，仅有Venus-HARP1融合蛋白在拟南芥叶片伤口处能够检测到，而Venus蛋白几乎检测不到。标尺代表500μm。
B：对Venus-HARP1蛋白信号位点(图A)进一步分析发现，部分蛋白可以进入细胞核中。DAPI染料用于细胞核染色。峰图表示箭头部分荧光强度分布。标尺代表5μm。
C：使用原核纯化的Venus-HARP1融合蛋白及Venus蛋白处理拟南芥根部伤口处。实验结果表明，仅有Venus-HARP1融合蛋白能够在拟南芥下胚轴以及 上部叶片检测到信号，而Venus蛋白几乎检测不到。标尺代表500μm。
D：对处理拟南芥根部伤口的材料观察下胚轴，发现部分Venus-HARP1蛋白可以进入细胞核中，而Venus蛋白却未观察到这一现象。标尺代表10μm。
图7、烟草瞬时表达的HARP1蛋白定位于植物细胞核中。35S：GFP-HARP1(GFP-HARP1)以及35S：GFP(GFP)载体顺转入烟草叶片，2天后使用激光共聚焦显微镜观察，标尺代表100μm。
图8、HARP1在鳞翅目昆虫中广泛存在，并在夜蛾科具有较高的相似性。
A：HARP1蛋白广泛分布于鳞翅目昆虫中且在夜蛾科昆虫中更为保守。鳞翅目昆虫中HARP1蛋白系统进化树的构建采用Mega软件完成。
B：夜蛾科昆虫中HARP1及其相似蛋白的序列比对分析。序列比对使用软件Vector NTI Advance中的Align X，采用软件中的Clustal W方法进行。
图9、HARP1类似多肽REPAT38具有与HARP1类似的功能。原核表达纯化的REPAT38蛋白处理植物伤口后，相较于对照Venus蛋白，REPAT38处理后，JA响应基因TAT1、VSP2、MYC2的诱导水平均明显降低。
图10、Venus-REPAT38融合蛋白能够进入拟南芥和烟草细胞。
A：使用原核纯化的Venus-REPAT38融合蛋白及Venus蛋白处理伤处理后的拟南芥，实验显示，仅有Venus-REPAT38融合蛋白能在拟南芥叶片和下胚轴检测到信号，而Venus蛋白几乎检测不到。标尺代表250μm。
B：对融合蛋白处理后拟南芥材料观察下胚轴，Venus-REPAT38融合蛋白能在下胚轴细胞内检测到，而Venus蛋白几乎检测不到。标尺代表50μm。
C：使用原核纯化的Venus-REPAT38融合蛋白、Venus-HARP1融合蛋白及Venus蛋白处理烟草叶片伤口处。Venus-REPAT38和Venus-HARP1融合蛋白能在烟草叶片伤口处及叶片内部检测到信号，而Venus蛋白几乎检测不到。标尺代表250μm。
D：对处理烟草叶片伤口的材料观察内部细胞，Venus-REPAT38和Venus-HARP1融合蛋白能在叶片细胞内检测到，而Venus蛋白几乎检测不到。标尺代表50μm。
图11A～F、Venus-HARP1从伤口处进入细胞及在叶片组织中快速多方式运动。
图12、HARP1进植物细胞的广谱性或多样性研究。
A：生长约24周的无酚棉叶片损伤处理后浸泡Venus-HARP1、Venus 4h后，用PBSA清洗2h后荧光显微镜观察伤口附近结果。图中bar的大小都为 200μm。
B：生长约24周的烟草叶片损伤处理后浸泡Venus-HARP1、Venus 4h后，用PBSA清洗2h后荧光显微镜观察伤口附近结果。图中bar的大小都为200μm。
图13、去除N端部分序列能够显著提高蛋白进入植物细胞的效率。A～G分别为Venus-HARP1；Venus-HARP1δC5(去除C端的5aa)；Venus-HARP1δN10(去除信号肽后N端的10aa)；Venus-HARP1δN15(去除信号肽后N端的15aa)；Venus-HARP1δN20(去除信号肽后N端的20aa)；Venus-HARP1δN25(去除信号肽后N端的25aa)；Venus。
图14、HARP1蛋白具有进入动物细胞的能力，并有部分定位细胞核。
图15、HARP1蛋白具有进入人细胞的能力，图示Venus-HARP1进入A549细胞，并部分进入细胞核。
A：A549细胞浸泡Venus-HARP1、Venus 4h后荧光显微镜观察结果。
B：为A中浸泡Venus-HARP1的细胞进一步放大观察结果。
C：为A中浸泡Venus-HARP1的细胞DAPI染色后观察结果。图中bar的大小如图所示。
图16、Venus-HARP1进入293T细胞，并部分进入细胞核。
A：293T细胞浸泡Venus-HARP1、Venus 4h后荧光显微镜观察结果。
B：为A中浸泡Venus-HARP1的细胞进一步放大观察结果。
C：为A中浸泡Venus-HARP1的细胞DAPI染色后观察结果。图中bar的大小如图所示。
具体实施方式
本发明人经过深入的研究，发现HARP1及其类似多肽(HARP1like，HL)具有通过细胞壁和细胞膜等复杂结构进入细胞以及细胞核的特性，从而其可被应用于建立高效的运输系统，通过蛋白融合等手段，帮助外源活性分子，进入生物体的细胞或组织中，从而改变生物体的性状、特性或状态。所述的HARP1及其类似多肽还具有效应子活性，能够降低植物防御响应，从而减少有毒化防御物质的产生。
HARP1及其类似多肽
植食性昆虫与植物在长期进化过程中建立了复杂的互作信号网络，Jasmonate(JA)是植物主要的防御激素。前期，本发明人发现昆虫口器分泌物可 以干扰植物的防御反应，本发明人从中分离得到一个蛋白，称为HARP1蛋白。HARP1保守性变异多肽例如但不限于来自：小菜蛾，甜菜夜蛾(S.exigua)，烟芽夜蛾(H.virescens)，草地夜蛾(S.frugiperda)，小地老虎(A.ipsilon)，蓓带夜蛾(M.configurata)，粉纹夜蛾(T.ni)等鳞翅目昆虫的与HARP1具有同源性的蛋白；一个具体的例子如来自甜菜夜蛾的REPAT38。
本发明所述的HARP1多肽(蛋白)或REPAT38多肽(蛋白)还包括它们的保守性变异多肽、片段、衍生物和类似物。如本文所用，术语“保守性变异多肽”、“片段”、“衍生物”和“类似物”是指基本上保持本发明的HARP1多肽或REPAT38多肽相同的生物学功能或活性的多肽。本发明的多肽片段、衍生物或类似物可以是(i)有一个或多个(如1-50个；较佳地1-20个；更佳地1-10个；更佳地1-8个；如5个，3个)保守或非保守性氨基酸残基(优选保守性氨基酸残基)被取代的蛋白，而这样的取代的氨基酸残基可以是也可以不是由遗传密码编码的，或(ii)在一个或多个(如1-30个；较佳地1-20个；更佳地1-10个；如5个，3个)氨基酸残基中具有取代基团的蛋白，或(iii)附加的氨基酸序列融合到此蛋白序列而形成的蛋白等。根据本文的定义这些片段、衍生物和类似物属于本领域熟练技术人员公知的范围。
任何一种HARP1多肽或REPAT38多肽的生物活性片段都可以应用到本发明中。在这里，HARP1多肽或REPAT38多肽的生物活性片段的含义是指作为一种多肽，其仍然能保持全长的HARP1多肽或REPAT38多肽的全部或部分功能。通常情况下，所述的生物活性片段至少保持50％的全长HARP1多肽或REPAT38多肽的活性。在更优选的条件下，所述活性片段能够保持全长HARP1多肽或REPAT38多肽的60％、70％、80％、90％、95％、99％、或100％的活性。
在本发明中，术语“HARP1多肽”指具有HARP1多肽活性的SEQ ID NO:2序列的多肽。该术语还包括具有与HARP1多肽相同功能的、SEQ ID NO:2序列的变异形式。这些变异形式包括(但并不限于)：若干个(如1-50个；较佳地1-20个；更佳地1-10个；如5个，3个)氨基酸的缺失、插入和/或取代，以及在C末端和/或N末端添加或缺失一个或数个(通常为20个以内，较佳地为10个以内，更佳地为5个以内)氨基酸。
在本发明中，术语“REPAT38多肽”指具有REPAT38多肽活性的SEQ ID NO:4序列的多肽。该术语还包括具有与REPAT38多肽相同功能的、SEQ ID NO:4序列的变异形式。这些变异形式包括(但并不限于)：若干个(如1-50个；较佳地1-20个；更佳地1-10个；如5个，3个)氨基酸的缺失、插入和/或取代，以 及在C末端和/或N末端添加或缺失一个或数个(通常为20个以内，较佳地为10个以内，更佳地为5个以内)氨基酸。
编码HARP1多肽或REPAT38多肽或其保守性变异多肽的多核苷酸序列(编码序列)也可以应用到本发明中。编码成熟HARP1多肽或REPAT38多肽的编码区序列可以与SEQ ID NO:1或SEQ ID NO:3所示的序列基本上相同或者是简并的变异体。如本文所用，“简并的变异体”在本发明中是指编码具有SEQ ID NO:2或SEQ ID NO:4的蛋白质，但与SEQ ID NO:1或SEQ ID NO:3所示的编码区序列有差别的核酸序列。
术语“编码基因”可以是包括编码所述多肽的多核苷酸，也可以是还包括附加编码和/或非编码序列的多核苷酸。
上述多核苷酸的变异体也是可用的，其编码与本发明有相同的氨基酸序列的多肽或多肽的片段、类似物和衍生物。此多核苷酸的变异体可以是天然发生的等位变异体或非天然发生的变异体。这些核苷酸变异体包括取代变异体、缺失变异体和插入变异体。如本领域所知的，等位变异体是一个多核苷酸的替换形式，它可能是一个或多个核苷酸的取代、缺失或插入，但不会从实质上改变其编码的多肽的功能。
作为本发明的优选方式，本发明提供了一种HARP1多肽或REPAT38多肽的截短体，其是在HARP1多肽或REPAT38多肽的基础上，去除N端或C端部分序列后获得的多肽。较佳地，所述截短体为去除N端(不包括信号肽)的1～19个，2～18个，3～17个氨基酸后获得的多肽。更具体地例如去除N端(不包括信号肽)4、5、6、7、8、9、10、11、12、13、14、15、16、17、18个氨基酸后获得的多肽。而对于HARP1多肽或REPAT38多肽的C端，一般去除小于5个、小于4个、小于3个或小于2个氨基酸。本发明人意外地发现，所述的截短体能够更为高效地将外源活性分子引入到细胞或组织中。
应理解，虽然本发明的HARP1基因或REPAT38基因分别获自夜蛾科昆虫棉铃虫或甜菜夜蛾，但是获自其它昆虫的与该HARP1基因或REPAT38基因高度同源，如具有40％以上；较佳地50％以上、60％以上、70％以上或80％以上；更佳地85％以上，90％以上，95％以上或99％以上的其它基因也在本发明的范围之内。根据同源比对，本发明人发现HARP1类似多肽(HARP1like，HL)在鳞翅目昆虫中普遍存在，而在夜蛾科中有很高的保守性。
包含所述编码序列的载体，以及用所述的载体或HARP1多肽或REPAT38多肽编码序列经基因工程产生的宿主细胞也包括在本发明中。本领域的技术人 员熟知的方法能用于构建含HARP1多肽REPAT38多肽编码序列和合适的转录/翻译控制信号的表达载体。这些方法包括体外重组DNA技术、DNA合成技术、体内重组技术等。所述的序列可有效连接到表达载体中的适当启动子上，以指导mRNA合成。包含上述的适当编码序列以及适当启动子或者控制序列的载体，可以用于转化适当的宿主细胞，以使其能够表达蛋白质。宿主细胞可以是植物细胞或动物细胞。
细胞内转运的应用
本发明提供了一种将外源活性分子引入到细胞或组织中的方法，包括：(1)将外源活性分子与HARP1类多肽连接，获得连接产物；(2)将步骤(1)的连接产物接触细胞或组织，从而外源活性分子被引入到细胞或组织中。较佳地，所述的外源活性分子被引入到细胞的细胞核中。
如本文所用，所述的HARP1类多肽包括REPAT38等HARP1保守性变异多肽，所述保守性变异多肽包括HARP1、REPAT38等多肽的截短体。
如本文所用，所述的“外源活性分子”又称为“功能性分子”，是指一些具有特定的功能，能够改造生物体(包括动物和植物)的性状、结构、特性或状态的一类分子，例如其能够使植物产生至少一个方面性状的改良，又例如可以使疾病状态的动物或人发生疾病的改善；也可以是一类具有指示或报告功能的分子，能够使得生物体的某些器官、组织和细胞被检测到或被定位，例如荧光蛋白，标签蛋白(如myc，HA，His等)。可以被运用的外源活性分子包括：多肽，核酸，化合物(如一些激素)。
本发明的HARP1类多肽可以将外源活性分子引入到细胞或组织中，特别是能够进入到人细胞，这在临床疾病治疗领域将有很大的应用前景。临床上，需要引入外源活性分子(如作为药物)来实施治疗的疾病是较多的。例如肿瘤，可以以HARP1类多肽携带抑制分子或毒素到肿瘤细胞中，从而实现抑制肿瘤的目的。例如一些酶或蛋白功能低下相关的疾病，可以以HARP1类多肽携带活性的酶或蛋白进入到细胞或组织中，来实现缓解或治疗此类疾病的目的。
所述的人细胞可以包括但不限于：肿瘤细胞、体细胞、胚胎细胞。所述体细胞包括但不限于：成纤维细胞、生殖细胞、骨髓细胞、血液细胞等。本发明实施例中论证了人肺癌细胞，人胚胎肾细胞以及动物细胞如果蝇细胞，植物细胞，但是应理解，适用于本发明的技术方案的细胞不限于实施例中所列举的。
本发明所述的HARP1类多肽与外源活性分子的连接方式可以为共价连接 或非共价连接。所述的连接包括：融合，偶联，吸附，耦合或复合等。所述的“连接”为“操作性连接”，即两个或多个分子之间具有功能性的空间排列。应理解，只要能够保留HARP1类多肽及外源活性分子的功能、保留良好的穿透细胞膜及体组织屏障的效果，任何连接方式均可包含在本发明中。共价连接通常以形成共价键的方式将两个分子进行连接。而一些非共价连接(不形成共价键)例如偶联、吸附、结合等也可应用。
作为本发明的优选方式，所述的外源活性分子为多肽，包括：功能性多肽(如酶)或结构多肽。当外源活性分子为多肽时，其与HARP1类多肽进行融合，获得融合蛋白。所述的HARP1类多肽与外源活性分子之间可以直接相连接，或者通过多肽连接子(连接肽)连接。所述的连接子例如包括1-30个氨基酸；较佳地为1-20个氨基酸；例如15、10、8、6、5、4、3、2、1个氨基酸。连接肽的设置基本上不影响HARP1与外源活性分子发挥穿透细胞膜及体组织屏障效果，也不影响外源活性分子的功能。
HARP1类多肽与外源活性分子之间的连接，若以肽键进行连接，则根据需要，外源活性分子可以位于HARP1类多肽的氨基端，也可以位于HARP1类多肽的羧基端。
作为本发明的选择方式，所述的外源活性分子可以是RNA或DNA，例如其是一种能够在生物体内产生针对某一基因具有同源性干扰效应的RNA分子。
作为本发明的选择方式，所述的HARP1类多肽可以通过氨基、羧基或巯基等化学反应实现与外源活性分子的连接，包括但不限于所述多肽与多聚物之间的连接，所述多肽在脂质体或纳米粒子表面的共价修饰、酯化反应、硫化反应等。
所述的非共价连接为静电吸附连接或受体配体反应。所述的静电吸附连接包括但不限于所述细胞穿膜载体与核酸分子之间的静电连接。所述的受体-配体反应指的是分别在HARP1类多肽和外源活性分子上连接在可以特异性匹配的受体与配体，通过受体与配体的高度专一性，实现所述多肽与外源活性分子的连接。如生物素与亲和素之间的专一性匹配。
基于本发明的可以促进外源活性分子通过细胞壁和细胞膜等复杂结构进入细胞以及细胞核的特性。因此，多种外源活性分子均可被应用于与所述的HARP1类多肽连接，来构成复合体。
在本发明的具体实施例中，本发明人发现原核表达的Venus-HARP1融合蛋白能够进入细胞内，并有部分融合蛋白定位在细胞核内。这说明昆虫中的 HARP1类多肽可以将与其融合的蛋白，通过细胞壁，细胞膜等多重阻碍，进入到细胞内发挥功能。
适用于以本发明的HARP1类多肽来介导引入外源蛋白的细胞或组织的种类是广谱的，可以是多种生物体细胞，包括植物细胞或组织、动物细胞或组织、微生物细胞(包括其培养物)等。
在本发明的具体实施例中，本发明人还发现，甜菜夜蛾(Spodoptera exigua)中的HARP1类似多肽具有与HARP1类似的功能。这些发现说明，HARP1及其类似多肽可作为一种运输系统，通过蛋白融合等手段，帮助外源活性分子，进入细胞，从而改变生物体的性状、结构、特性或状态。
在本发明的具体实施例中，本发明人观测到，所述HARP1类多肽与外源活性分子连接的连接产物从伤口处进入多种细胞。并且，所述HARP1类多肽与外源活性分子连接的连接产物在叶片组织中快速多方式运动。
调节植物防御响应的应用
本发明提供了一种降低植物对损伤的防御响应能力的方法，包括：以HARP1类多肽处理植物；或将HARP1类多肽的编码基因转化植物。所述的降低植物对损伤的防御响应能力包括：减少植物体内有毒化防御物质的产生。
所述的HARP1类多肽通过影响植物体内的防御响应机制来发挥调控作用，所述的防御响应机制包括基于茉莉酸信号途径的机制。本发明人发现HARP1类多肽显著地抑制茉莉酸信号途径基因或多肽的表达或活性。所述的茉莉酸信号途径包括：Jasmonate(JA)响应基因，如TAT1、VSP2、MYC2，这些基因在HARP1类多肽处理或过表达HARP1类多肽后会受到显著的抑制。很多重要的植物防御物质受JA途径的调控已是本领域公知，因此，HARP1类多肽对JA响应的削弱作用，则必然减少植物体内有毒化防御物质的产生。
本发明人发现，体外表达HARP1类多肽并以之处理后的损伤植物中，JA早期响应基因对机械损伤的应答受到抑制；过表达HARP1类多肽的转基因植物，在受到损伤后，也呈现这种JA早期响应基因对机械损伤的应答的抑制。因此，HARP1类多肽在离体或体内条件下均具有明显的效应子活性。
可以采用本领域人员熟知的多种方法来调节所述的HARP1类多肽的表达。比如可通过一定的途径将携带HARP1类多肽编码基因的表达单位(比如表达载体或病毒等)递送到靶点上，并使之表达活性的HARP1类多肽。作为本发明的一种实施方式，将HARP1类多肽的编码基因通过常规的方法克隆到适当的载 体中，将所述的带有外源基因的重组载体导入到可表达所述HARP1类多肽的植物细胞中，使所述的植物细胞表达HARP1类多肽。可通过将所述植物细胞再生成植物，获得过量表达HARP1类多肽的植物。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件如J.萨姆布鲁克等编著，分子克隆实验指南，第三版，科学出版社，2002中所述的条件，或按照制造厂商所建议的条件。
本发明中用于本实验的引物信息如表1。
表1
Figure PCTCN2019093307-appb-000001
Figure PCTCN2019093307-appb-000002
实施例1、HARP1蛋白序列分析
通过对取食人工饲料和取食拟南芥的棉铃虫口器分泌物的蛋白质组分析，本发明人得到一个受植物诱导的棉铃虫口器分泌蛋白，命名为HARP1。
通过转录组序列比对，本发明人得到HARP1的核酸序列以及蛋白序列。根据序列信息，合成引物，PCR扩增得到HARP1基因。通过使用Blast以及借助Align X软件对该蛋白的分析。
进一步地，通过序列分析，本发明人发现该蛋白在N端含有一个信号肽，可能具有帮助该蛋白分泌到口器中的作用(图1～2)。
HARP1核苷酸序列(SEQ ID NO:1)如下：
Figure PCTCN2019093307-appb-000003
Figure PCTCN2019093307-appb-000004
HARP1氨基酸序列(SEQ ID NO:2)如下：
Figure PCTCN2019093307-appb-000005
实施例2、HARP1蛋白的组织特异性分析
选取4龄的棉铃虫，分别取中肠、唾液腺等组织器官，采用Trizol法提取RNA，以棉铃虫actA3b作为内参基因，进行定量分析。结果发现，转录组水平HARP1在中肠中高表达，而在唾液腺中表达量很低(图3A)。
同时，为了检测HARP1蛋白在棉铃虫中肠、肠液和口器分泌物中的表达水平，本发明人首先用50mM Tris-HCl(pH 9.0)提取4龄棉铃虫中肠的总蛋白，同时提取棉铃虫的肠液及口器分泌物，与中肠总蛋白溶液一起用等体积的氯仿抽提，并用75％乙醇洗涤一遍，最后溶于水中。在15％的蛋白胶中每孔加入等量的蛋白样品，之后用HARP1抗体进行检测。在蛋白水平，HARP1在棉铃虫口水中表达丰度最高，同时HARP1蛋白可以受棉酚等次生化合物诱导(图3B)。
本发明人还考察了取食不同食物(人工饲料，无酚棉，有酚棉，拟南芥)的棉铃虫口器分泌物中HARP1蛋白的丰度。结果显示，不同的植物也可以诱导棉铃虫口水中HARP1蛋白的丰度(图3C)。取食有酚棉或拟南芥的棉铃虫口水中HARP1蛋白的丰度显著更高。
实施例3、HARP1在被棉铃虫取食后的植物叶片损伤部位有明显分布
棉铃虫在取食植物时，口器分泌物会接触植物伤口处。选取四龄初的棉铃虫，通过预先饲喂含有棉酚的人工饲料，然后让其取食生长3周的拟南芥叶片。通过整体的免疫组化方法，将棉铃虫取食的拟南芥叶片快速转移到FAA固定液中，真空抽取至完全沉浸在FAA中，并以固定液固定4小时，同时以人为机械损伤的叶片作为对照。之后，将固定好的叶片利用系列梯度的乙醇溶液进行脱水，然后再使用系列梯度的乙醇溶液进行复水。经过2小时的封闭液(1×PBS， 0.1％Tween 20，1％Albumin，Bovine Serum)封闭后，使用HARP1抗体在4℃孵育过夜，PBST洗涤4次后，经Western Blue碱性磷酸酶底物(Promega)处理后显色，使用显微镜观察。
结果表明，棉铃虫取食后的拟南芥伤口处存在HARP1蛋白，而在人为机械损伤的叶片伤口处不存在信号(图4)。
实施例4、原核纯化的蛋白能够削弱植物对机械损伤的响应
以棉铃虫中肠cDNA为模板，以表1中HARP1表达引物为引物，采用PCR的方法扩增出HARP1基因序列，并在HARP1的起始密码子和终止密码子前分别引入BamHΙ和HindΙΙΙ酶切位点，将HARP1导入pET32a多克隆位点BamHΙ和HindΙΙΙ之间，从而获得携带目的片段的重组表达载体，称为pET32a/HARP1。将pET32a/HARP1转化E.coli BL21(DE3)后，挑取阳性单菌落至2mL LB(含100μg/mL Amp)培养基中，37℃，220rpm培养至对数生长期，取200μl转接入200ml培养基中，大摇至OD＝0.6-0.8，加入IPTG至终浓度为0.5mM，继续16℃诱导培养过夜。依照Ni-NTA Spin Kit手册(Qiagen，Valencia，CA)，纯化带有His-Tag的重组蛋白。过程如下：收集菌体，重悬于15ml Lysis buffer(50mM Tris-Cl，pH 8.5，100mM NaCl，10mM imidazole)。高压破碎后，离心取上清，过装好的1ml Ni-NTA resin。蛋白用2ml Elution buffer(50mM Tris-Cl，pH 8.5，100mM NaCl，250mM imidazole)洗脱，并置换于20mM Tris-Hcl Buffer，pH8.5中，使用Bradeford法，以牛血清蛋白做参照，对蛋白浓度进行定量。采用同样的方法，在E.coli BL21(DE3)中表达并纯化Venus蛋白(Venus序列从pCAMBIA1302( http://www.cambia.org/daisy/bios/585.html)载体中扩增获得)作为后续实验的对照。
选取生长3周左右的拟南芥第二对真叶进行机械损伤处理，同时在机械损伤的部位涂施原核纯化的HARP1蛋白和Venus蛋白(对照)，4小时后，取样。利用Trizol法提取拟南芥叶片RNA，以拟南芥叶片中S18基因作为内参基因，进行相关基因的定量分析，结果发现，相较于Venus蛋白处理，HARP1蛋白能够明显抑制拟南芥叶片中JA响应基因(包括TAT1、VSP2、MYC2)对机械损伤的响应(图5A)。
采用相同的方法，处理棉花刚生长出的第一片真叶。采用CTAB法提取棉花叶片的RNA，以棉花叶片中的His基因作为内参基因进行相关基因的定量分析，结果发现，相较于Venus蛋白处理，HARP1能明显抑制棉花叶片中蛋白酶 抑制剂基因(Gh_Sca005135G01、Gh_A10G2353、Gh_D11G1335)的表达(图5B)。
实施例5、外源的HARP1融合蛋白可以进入植物细胞，并部分定位于细胞核中
采用重组PCR的方法制备、扩增获得Venus-HARP1(5’→3’)融合序列并在Venus-HARP1的起始密码子和终止密码子前分别引入BamHΙ和HindΙΙΙ酶切位点，将Venus-HARP1导入pET32a多克隆位点BamHΙ和HindΙΙΙ之间，从而获得携带目的片段的重组表达载体，称为pET32a/Venus-HARP1。采用实施例4中相同的方法，在E.coli BL21(DE3)中表达并纯化Venus-HARP1蛋白，同时表达Venus蛋白作为后续实验的对照。
选取拟南芥第二片真叶进行机械损伤处理，之后分别置于含有原核纯化的融合蛋白Venus-HARP1及对照Venus的20mM Tris-Hcl Buffer中，蛋白含量事先用Bradeford法调齐(1mg/ml)。孵育1小时后，用PBST溶液(1×PBS+0.1％Tween 20+1％BSA)洗3-4次。随后添加DAPI(4,6-diamidino-2-phenylindole)染料进行细胞核染色。处理后的叶片在激光共聚焦扫描显微镜(Olympus FV3000)下进行成像观察并用软件Olympus cellSens(version Dimension 1.18)进行分析。
结果发现，在拟南芥叶片伤口处外源添加原核纯化的Venus-HARP1蛋白可以进入植物细胞，并有一部分蛋白可以进入细胞核中，而外源添加的Venus蛋白则基本没有进入植物细胞中(图6A，B)。
选取生长7天左右的拟南芥幼苗，对其根部进行机械损伤处理，采用浸泡的方法处理幼苗后，置于激光共聚焦扫描显微镜下对其下胚轴部位进行观察，同样发现，原核纯化的Venus-HARP1蛋白可以进入植物细胞，并主要定位在细胞核中，而外源添加的Venus蛋白则基本没有进入植物细胞中(图6C，D)。
实施例6、烟草瞬时表达的HARP1蛋白定位于植物细胞核中
通过PCR的方法，在HARP1序列的两端分别加入BamH1和Sal1酶切位点，将HARP1导入植物表达载体YUKHS-GFPL(骨架为pCambia 1300，在多克隆位点EcoR1与Sac1间加入35S promoter，在Sac1与Kpn1间加入GFP序列)中，得到的载体称为35S:GFP-HARP1表达载体，原先的载体YUKHS-GFPL称为35S:GFP表达载体。
采用冻融法，将得到的载体分别转入农杆菌GV3101(购自唯地生物)中，挑取单菌落接至3ml LB培养基中(含25μg/ml利福平Rif，100μg/ml庆大霉素Gent，50μg/ml卡那霉素Kan)28℃，220rpm，过夜培养。5000rpm离心10分 钟，菌体重悬于瞬转缓冲液(10mM MES，10mM MgCl 2，150μM乙酰丁香酮)中，调至OD＝0.8，静置3小时后，注射入烟草叶片，2天后，取烟草叶片在激光共聚焦扫描显微镜下进行亚细胞定位观察。
烟草瞬转实验显示，GFP-HARP1在烟草叶片中定位于细胞核中(图7)。
实施例7、HARP1对植物没有明显的生长抑制作用，过表达植物没有明显的表型
本发明人同时通过PCR的方法，在HARP1序列的两端分别加入BamH1和Sal1酶切位点，将HARP1导入植物表达载体YUKHS-6MYC(骨架为pCambia1300，在多克隆位点EcoR1与Sac1间加入35S promoter，在Kpn1与Sma1间加入6MYC标签序列)中，得到的载体称为35S:6MYC-HARP1表达载体。
采用花芽浸泡法(floral dip)进行拟南芥植物的转化。具体过程如下：采用冻融法，将得到的载体分别转入农杆菌GV3101中，如前所述，挑取单菌落并转接培养至得到200ml菌液。5000rpm离心10分钟，菌体重悬于250ml含0.02％Silwet L-77的5％蔗糖溶液中。植株花芽部分在菌液中浸泡1分钟，平放于塑料盆内，保湿，避光，14～20小时后置于温室中正常培养至开花结籽。T 0代种子用75％乙醇洗涤5分钟，20％漂水(白猫公司，上海)处理15分钟，无菌水清洗3～4遍后，铺在1/2MS固体培养基上(含50μg/ml Hygo)，置于拟南芥培养箱中培养1周后，挑取绿色抗性苗移栽到营养土中生长，4周后，取生长情况相似的叶片提取总蛋白，采用Western方法，使用6MYC抗体(购自翊圣公司)检测转基因植物的基因表达水平。
实验检测发现，在获得的HARP1表达量较高的转基因Line中，转基因植物的生长发育相较于野生型没有明显的区别。
实施例8、HARP1在鳞翅目昆虫中广泛存在，HARP1类似多肽具有与HARP1相似的功能
通过进一步的序列比对，以及对鳞翅目昆虫HARP1类似多肽的进一步搜集，本发明人获得了小菜蛾，甜菜夜蛾(S.exigua)，烟芽夜蛾(H.virescens)，草地夜蛾(S.frugiperda)，小地老虎(A.ipsilon)，蓓带夜蛾(M.configurata)，粉纹夜蛾(T.ni)等鳞翅目昆虫的HARP1相似蛋白。进一步本发明人发现HARP1类多肽在夜蛾科昆虫中广泛存在，并且通过Mega软件(Tamura et al.，2011)以及Align X软件的分析，表明HARP1类多肽在夜蛾科昆虫中具有很高的保守性(图8)。
通过PCR的方法，本发明人得到了甜菜夜蛾中的HARP1相似蛋白 REPAT38，并采用相同的方法，将其构建入pET-32a载体中，并在E.coli BL21(DE3)中表达并纯化REPAT38蛋白和Vennus蛋白。
REPAT38核苷酸序列(SEQ ID NO:3)如下：
Figure PCTCN2019093307-appb-000006
REPAT38氨基酸序列(SEQ ID NO:4)如下
Figure PCTCN2019093307-appb-000007
采用如实验例6中相同的方式处理拟南芥第二对真叶，并检测JA早期响应基因的诱导情况。结果发现，甜菜夜蛾中的HARP1相似蛋白REPAT38具有和HARP1相似的功能，均能抑制拟南芥对机械损伤的响应(图9)。
采用重组PCR的方法制备、扩增获得Venus-REPAT38融合序列，并在Venus-REPAT38(5’→3’)的起始密码子和终止密码子前分别引入BamHΙ和SalΙ酶切位点，将Venus-REPAT38导入pET32a多克隆位点BamHΙ和SalΙ之间，从而获得携带目的片段的重组表达载体，称为pET32a/Venus-REPAT38。采用实施例4中相同的方法，在E.coli BL21(DE3)中表达并纯化Venus-REPAT38蛋白，同时表达Venus蛋白作为后续实验的对照。Venus-REPAT38融合蛋白处理拟南芥和烟草的浓度及方法同实施例5。
选取生长7天左右的拟南芥幼苗，对其子叶进行机械损伤处理，采用浸泡的方法处理幼苗后，置于激光共聚焦扫描显微镜下对其进行观察，发现，原核纯化的Venus-REPAT38蛋白可以进入拟南芥细胞，而外源添加的Venus蛋白则基本没有进入拟南芥细胞中(图10A，B)。
选取生长两周左右的烟草叶片，用枪头戳取烟草小圆片叶片，采用浸泡的方法处理烟草叶片后，置于激光共聚焦扫描显微镜下对其进行观察，发现，原核纯化的Venus-REPAT38、Venus-HARP1融合蛋白可以进入烟草叶片细胞，而 外源添加的Venus蛋白则基本没有进入烟草细胞中(图10C，D)。
实施例9、Venus-HARP1从伤口处进入细胞及在叶片组织中快速多方式运动
本实施例中，研究Venus-HARP1进入植物细胞或植物组织的方式及其在细胞或组织内的运动方式。
1、Venus-HARP1从伤口处进入植物细胞
方法步骤：采用如实施例5和8的方法，选取生长三周左右拟南芥叶片，用枪头戳取拟南芥小圆片叶片，采用浸泡的方法处理叶片后，置于激光共聚焦扫描显微镜下观察伤口附近的荧光信号。
结果如图11A～B，Venus-HARP1从伤口处进入拟南芥细胞；而Venus蛋白本身，在不连接HARP1的情况下，则无法进入拟南芥细胞内。
2、Venus-HARP1在叶片组织中快速多方式运动
方法步骤：将实施例9-1中处理后的叶片在激光共聚焦扫描显微镜下进一步放大观察，观察时选取固定焦平面，进行无间隔时间序列扫描，拍摄30秒内观察区域中荧光信号的变化情况。
结果如图11C～F，Venus-HARP1能从伤口处进入拟南芥叶片组织，进入组织间隙的HARP可以跨过表皮细胞壁和细胞膜，能在叶肉细胞和间隙间穿梭，可以沿着细胞间隙进入细胞内，也能从细胞内分泌至伤口外，这表明了其能快速多方式运动。
上述结果提示，HARP1能以外泌体的形式在细胞间扩散。
实施例10、HARP1进入植物细胞具有广谱性
本实施例中，研究HARP1进植物细胞的广谱性或多样性。
生长约4周的无酚棉、烟草叶片，进行损伤处理后，浸泡Venus-HARP1、Venus 4h，用PBSA清洗2h，荧光显微镜观察伤口及其附近区域。
如图12A～B，Venus-HARP1能从伤口处进入锦葵科的棉花和茄科的烟草细胞，表明HARP1能够进入不同的植物组织，其进入植物细胞或组织具有广谱性。
因此，利用HARP1构建的转运体系可适用于多种植物中。
实施例11、去除N端部分序列能够显著提高蛋白进入植物细胞的效率
本实施例中，对于HARP1进行改造，去除其信号肽后N端10、15、20、 25个氨基酸，或去除其C端的5个氨基酸，测试序列如下：
A：Venus-HARP1；
B：Venus-HARP1δC5(去除C端的5aa)；
C：Venus-HARP1δN10(去除N端的信号肽及信号肽后10aa)；
D：Venus-HARP1δN15(去除N端的信号肽及信号肽后15aa)；
E：Venus-HARP1δN20(去除N端的信号肽及信号肽后20aa)；
F：Venus-HARP1δN25(去除N端的信号肽及信号肽后25aa)；
G：Venus。
结果如图13A～G，不同截短的HARP1进植物细胞效率不同，N端截短10个氨基酸的HARP1更易从伤口进入植物组织，大幅提高蛋白进入植物细胞的效率。C端截短5个或N端截短20个及以上的氨基酸抑制HARP1进入植物组织，表明HARP1的蛋白序列会影响其进入细胞。通过利用不同序列的HARP1可进一步提高该载体的递送效率。
实施例12、HARP1蛋白具有侵染动物细胞的能力
原核表达Venus-HARP1，纯化融合蛋白。将该融合蛋白与果蝇S2细胞孵育，以原核表达的Venus做对照，进行平行实验。
孵育4小时后，用PBS洗去细胞表面多余蛋白，用荧光染料DAPI显示细胞核，在显微镜下观察。
结果如图14，发现Venus本身不具备进入S2细胞的能力，而Venus-HARP1能够进入S2细胞，并有部分定位于细胞核。
实施例13、Venus-HARP1进入人源细胞
1、Venus-HARP1进入人肺癌细胞
将Venus-HARP1融合蛋白与人肺癌细胞A549孵育，以原核表达的Venus做对照，进行平行实验。
孵育4小时后，用PBS洗去细胞表面多余蛋白，用荧光染料DAPI显示细胞核，在显微镜下观察，并进行DAPI然后和观察。
结果如图15所示，Venus-HARP1进入A549细胞，并部分进入细胞核；而Venus本身不具备进入细胞的能力。
2、Venus-HARP1进入人胚胎肾细胞
将Venus-HARP1融合蛋白与人胚胎肾细胞293T孵育，以原核表达的Venus做对照，进行平行实验。
孵育4小时后，用PBS洗去细胞表面多余蛋白，用荧光染料DAPI显示细胞核，在显微镜下观察，并进行DAPI然后和观察。
结果如图16所示，Venus-HARP1进入293T细胞，并部分进入细胞核；而Venus本身不具备进入细胞的能力。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (19)

  1. 一种将外源活性分子引入到细胞或组织中的方法，其特征在于，所述方法包括：
    (1)将外源活性分子与HARP1或其保守性变异多肽连接，获得连接产物；
    (2)将步骤(1)的连接产物接触细胞或组织，从而外源活性分子被引入到细胞或组织中。
  2. 如权利要求1所述的方法，其特征在于，所述的外源活性分子被引入到细胞的细胞质或细胞核中。
  3. 如权利要求1所述的方法，其特征在于，所述的外源活性分子包括：多肽，核酸，毒素，化合物。
  4. 如权利要求1所述的方法，其特征在于，所述的细胞或组织包括：动物细胞或组织，植物细胞或组织，微生物细胞。
  5. 一种降低植物对损伤的防御响应能力的方法，其特征在于，所述方法包括：以HARP1或其保守性变异多肽处理植物；或将HARP1或其保守性变异多肽的编码基因转化植物。
  6. 如权利要求5所述的方法，其特征在于，所述的HARP1通过抑制茉莉酸信号途径基因或多肽的表达或活性，来降低植物对损伤的防御响应能力。
  7. 如权利要求6所述的方法，其特征在于，所述的茉莉酸信号途径包括包括：Jasmonate响应基因、蛋白酶抑制剂基因、次生代谢合成相关转录因子及合酶。
  8. 如权利要求1或5所述的方法，其特征在于，HARP1或其保守性变异多肽包括：
    (a)具有SEQ ID NO:2或SEQ ID NO:4所示氨基酸序列的多肽；
    (b)起始于SEQ ID NO:2中第21～39位中任一氨基酸，终止于SEQ ID NO:2中第119～122位所示氨基酸序列的多肽；或，起始于SEQ ID NO:4中第21～38位中任一氨基酸，终止于SEQ ID NO:4中第118～121位所示氨基酸序列的多 肽；
    (c)由(a)或(b)多肽经过一个或多个氨基酸残基的取代、缺失或添加而形成的，且具有多肽(a)或(b)功能的多肽；
    (d)氨基酸序列与(a)或(b)多肽的氨基酸序列有40％以上相同性，且具有多肽(a)或(b)功能的多肽；或
    (e)在(a)或(b)或(c)或(d)所述多肽的N或C末端添加标签序列，或在多肽(b)或(c)或(d)的N末端添加信号肽序列后形成的多肽。
  9. 如权利要求8所述的方法，其特征在于，(b)中，所述多肽的序列起始于SEQ ID NO:2中第22～38位、第23～37位、第24～38位、第25～37位、第26～36位、第27～35位、第28～34位、第29～33位、第30～31位或第30～32位中任一氨基酸，终止于SEQ ID NO:2中第120或121位；或，起始于SEQ ID NO:4中第22～37位、第23～36位、第24～35位、第25～34位、第26～33位、第27～32位或第28～31位或第29～30位中任一氨基酸，终止于SEQ ID NO:4中第119、120位。
  10. 如权利要求1或5所述的方法，其特征在于，HARP1或其保守性变异多肽来自于：鳞翅目昆虫；较佳地，所述的鳞翅目昆虫包括夜蛾科昆虫。
  11. 如权利要求10所述的方法，其特征在于，所述的鳞翅目昆虫包括：小菜蛾，甜菜夜蛾(S.exigua)，烟芽夜蛾(H.virescens)，草地夜蛾(S.frugiperda)，小地老虎(A.ipsilon)，蓓带夜蛾(M configurata)，粉纹夜蛾(T.ni)。
  12. 如权利要求5所述的方法，其特征在于，所述的植物包括：
    能被鳞翅目昆虫取食的植物；
    存在损伤防御响应机制的植物；
    锦葵科植物，茄科植物，十字花科植物，禾本科植物。
  13. HARP1或其保守性变异多肽的用途，用于将外源活性分子引入到细胞或组织中。
  14. 如权利要求13所述的用途，其特征在于，所述的外源活性分子被引入到细胞的细胞质或细胞核中。
  15. 如权利要求13所述的用途，其特征在于，通过将外源活性分子与HARP1或其保守性变异多肽连接，从而由HARP1或其保守性变异多肽将外源活性分子引入到细胞或组织中。
  16. HARP1或其保守性变异多肽的用途，用于降低植物对损伤的防御响应能力。
  17. 一种分离的HARP1多肽片段，其为：
    (i)起始于SEQ ID NO:2中第21～39位中任一氨基酸，终止于SEQ ID NO:2中第119～122位所示氨基酸序列的多肽；或，起始于SEQ ID NO:4中第21～38位中任一氨基酸，终止于SEQ ID NO:4中第118～121位所示氨基酸序列的多肽；
    (ii)由(i)多肽经过一个或多个氨基酸残基的取代、缺失或添加而形成的，且具有多肽(i)功能的多肽；
    (iii)氨基酸序列与(i)多肽的氨基酸序列有40％以上相同性，且具有多肽(i)功能的多肽；或
    (iv)在(i)或(ii)或(iii)所述多肽的N或C末端添加标签序列，或添加信号肽序列后形成的多肽。
  18. 如权利要求17所述的多肽片段，其特征在于，(i)中，所述多肽的序列起始于SEQ ID NO:2中第22～38位、第23～37位、第24～38位、第25～37位、第26～36位、第27～35位、第28～34位、第29～33位、第30～31位或第30～32位中任一氨基酸，终止于SEQ ID NO:2中第120或121位；或，起始于SEQ ID NO:4中第22～37位、第23～36位、第24～35位、第25～34位、第26～33位、第27～32位或第28～31位或第29～30位中任一氨基酸，终止于SEQ ID NO:4中第119、120位。
  19. 一种提高HARP1多肽将外源活性分子引入到细胞或组织中的效率的方法，包括：对HARP1多肽的N端或C端进行截短，从而获得起始于SEQ ID NO:2中第21～39位中任一氨基酸，终止于SEQ ID NO:2中第119～122位所示氨基酸序列的多肽；或，起始于SEQ ID NO:4中第21～38位中任一氨基酸，终止于SEQ ID NO:4中第118～121位所示氨基酸序列的多肽。
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