阅读说明：本技术 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 (Intracellular transport mediated by HARP1-like polypeptides and application thereof in regulation of biological defense mechanism ) 是由 毛颖波 陈春雨 陈芳艳 刘尧倩 王牧阳 王凌健 陈晓亚 于 2019-06-27 设计创作，主要内容包括：本发明涉及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。首次揭示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,从而其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。(The present invention relates to the intracellular transport mediated by HARP1-like polypeptides and their use in modulating biological defense mechanisms. The HARP1 and its similar polypeptide (HARP1-like, HL) are disclosed for the first time to have the characteristic of entering cells or tissues through complex structures such as cell walls and cell membranes, so that the HARP1 and its similar polypeptide can be applied to establish a high-efficiency transportation system to help exogenous active molecules enter cells. The HARP1 and similar polypeptides also have effector activity and are capable of reducing plant defense responses.)

1. A method of introducing an exogenous active molecule into a cell or tissue, the method comprising:

(1) connecting the exogenous active molecule with HARP1 or conservative variation polypeptide thereof to obtain a connection product;

(2) contacting the ligation product of step (1) with a cell or tissue, whereby the exogenous active molecule is introduced into the cell or tissue.

2. The method of claim 1, wherein the exogenously active molecule is introduced into the cytoplasm or nucleus of the cell.

3. The method of claim 1, wherein said exogenously active molecule comprises: polypeptides, nucleic acids, toxins, compounds.

4. The method of claim 1, wherein said cells or tissues comprise: animal cells or tissues, plant cells or tissues, microbial cells.

5. A method of reducing the defence response capability of a plant to injury, characterised in that the method includes: treating plants with HARP1 or a conservative variant thereof; or plants transformed with a gene encoding HARP1 or a conservative variant polypeptide thereof.

6. The method of claim 5, wherein the HARP1 reduces the plant's ability to respond to defense against injury by inhibiting the expression or activity of a jasmonate signaling pathway gene or polypeptide.

7. The method of claim 6, wherein the jasmonate signaling pathway comprises: jasmonate response genes, protease inhibitor genes, secondary metabolic synthesis related transcription factors, and synthases.

8. The method of claim 1 or 5, wherein HARP1 or a conservative variant polypeptide thereof comprises:

(a) a polypeptide having an amino acid sequence shown in SEQ ID NO. 2 or SEQ ID NO. 4;

(b) a polypeptide which starts from any one of amino acids 21 to 39 of SEQ ID NO. 2 and ends at amino acid sequences 119 to 122 of SEQ ID NO. 2; or a polypeptide which starts from any one of amino acids 21-38 of SEQ ID NO. 4 and ends at amino acid sequences 118-121 of SEQ ID NO. 4;

(c) a polypeptide which is formed by substituting, deleting or adding one or more amino acid residues to the polypeptide (a) or (b) and has the function of the polypeptide (a) or (b);

(d) a polypeptide having an amino acid sequence which is 40% or more identical to the amino acid sequence of the polypeptide (a) or (b) and having the function of the polypeptide (a) or (b); or

(e) A tag sequence is added to the N or C terminal of the polypeptide (a) or (b) or (C) or (d), or a signal peptide sequence is added to the N terminal of the polypeptide (b) or (C) or (d).

9. The method according to claim 8, wherein in (b), the sequence of the polypeptide starts at any one of amino acids 22 to 38, 23 to 37, 24 to 38, 25 to 37, 26 to 36, 27 to 35, 28 to 34, 29 to 33, 30 to 31 or 30 to 32 in SEQ ID NO. 2 and ends at position 120 or 121 in SEQ ID NO. 2; or starting from any of the amino acids at positions 22-37, 23-36, 24-35, 25-34, 26-33, 27-32, 28-31 or 29-30 of SEQ ID NO. 4 and ending at positions 119 and 120 of SEQ ID NO. 4.

10. The method of claim 1 or 5, wherein the HARP1 or conservative variant polypeptide thereof is from: lepidopteran insects; preferably, the lepidopteran insects include insects of the family noctuidae.

11. The method of claim 10, wherein said lepidopteran insect comprises: plutella xylostella, spodoptera exigua (s.exigua), heliothis virescens (h.virescens), spodoptera frugiperda (s.frugiperda), cutworm (a.ipsilon), spodoptera littoralis (M contourata), trichoplusia ni (t.ni).

12. The method of claim 5, wherein said plant comprises:

plants that can be taken by lepidopteran insects;

plants in which a damage defense response mechanism exists;

malvaceae plant, Solanaceae plant, Brassicaceae plant, and Gramineae plant.

Use of HARP1 or a conservative variant polypeptide thereof for introducing an exogenous active molecule into a cell or tissue.

14. The use as claimed in claim 13, wherein the exogenously active molecule is introduced into the cytoplasm or nucleus of the cell.

15. The use according to claim 13, wherein the exogenous active molecule is introduced into the cell or tissue by HARP1 or a conservative variant thereof by linking the exogenous active molecule to HARP1 or a conservative variant thereof.

Use of HARP1 or a conservative variant polypeptide thereof, for reducing the defence response capability of a plant to injury.

17. An isolated HARP1 polypeptide fragment that is:

(i) a polypeptide which starts from any one of amino acids 21 to 39 of SEQ ID NO. 2 and ends at amino acid sequences 119 to 122 of SEQ ID NO. 2; or a polypeptide which starts from any one of amino acids 21-38 of SEQ ID NO. 4 and ends at amino acid sequences 118-121 of SEQ ID NO. 4;

(ii) a polypeptide which is formed by (i) a polypeptide having a function of the polypeptide (i) through substitution, deletion or addition of one or more amino acid residues;

(iii) (ii) a polypeptide having an amino acid sequence which is 40% identical to the amino acid sequence of the polypeptide of (i) and which has the function of the polypeptide of (i); or

(iv) (iv) a polypeptide formed by adding a tag sequence to the N-or C-terminus of the polypeptide of (i) or (ii) or (iii), or a signal peptide sequence.

18. The polypeptide fragment of claim 17, wherein in (i), the sequence of the polypeptide starts at any one of amino acids 22-38, 23-37, 24-38, 25-37, 26-36, 27-35, 28-34, 29-33, 30-31, or 30-32 of SEQ ID NO:2 and ends at position 120 or 121 of SEQ ID NO: 2; or starting from any of the amino acids at positions 22-37, 23-36, 24-35, 25-34, 26-33, 27-32, 28-31 or 29-30 of SEQ ID NO. 4 and ending at positions 119 and 120 of SEQ ID NO. 4.

19. A method of increasing the efficiency of introducing an exogenous active molecule into a cell or tissue by a HARP1 polypeptide, comprising: truncating the N-terminus or the C-terminus of the HARP1 polypeptide, thereby obtaining a polypeptide starting at any one of amino acids 21 to 39 of SEQ ID NO. 2 and ending at the amino acid sequence shown at positions 119 to 122 of SEQ ID NO. 2; or a polypeptide which starts from any one of amino acids 21-38 of SEQ ID NO. 4 and ends at amino acid sequences 118-121 of SEQ ID NO. 4.

Technical Field

The invention belongs to the field of biotechnology and botany, and particularly relates to intracellular transport mediated by insect HARP1 polypeptide and application thereof in regulation of biological defense mechanism.

Background

Phytophagous insects are insects that feed on living plants and are classified into two categories, namely, whole plant organs and tissues and sap-feeding according to differences in the structures and functions of their mouthparts and digestive tracts. Phytophagous insects are often found in lepidoptera, rhabdomina, isoptera, coleoptera, diptera, and the like. According to the difference of the structure and function of their oral organs and digestive tracts, they can be divided into two categories, i.e. whole plant organs and tissues are used for feeding and sucking juice. Many phytophagous insects only feed on living plants, and many agricultural pests have the feeding property, so that the phytophagous insects cause great harm in agriculture.

After the plant is eaten by insects, the plant can generate some self defense by itself, including direct defense or indirect defense. It is now known that several defense signaling pathways exist in plants to regulate the self-defense of plants. The jasmonic acid signal pathway is one of main defense signal pathways in plants, linolenic acid rapidly generates a signal substance jasmonic acid (jasmonic acid) through an octadecane metabolic pathway, and the jasmonic acid activates a gene for coding a protease inhibitor to generate the protease inhibitor. The induction of plant production by insects requires the involvement of jasmonic acid and ethylene. After the plant is eaten by insects, the expression of a protease inhibitor gene is positively regulated and controlled on the transcription level through a jasmonic acid pathway through systemin or cell membrane signal cascade reaction, so that the protease inhibitor is rapidly increased to resist the eating of the insects.

Many advances have been made in the art regarding the cellular level of plant defense responses to pathogenic bacteria, but the study of plant-insect interaction recognition remains much elusive and less applicable to agriculture, and is of practical value. Therefore, there is a need in the art for further research to reliably apply such interactive recognition between plants and insects to applications such as plant improvement and pest control.

In addition, there are some peptides in the art with cell-penetrating function that have the ability to carry other molecules to penetrate cells and enter cells, some peptides with membrane-penetrating function include ① protein-derived peptides (protein derived CPPs) such as pentatin, TAT and pVEC, etc., ② model peptides (model peptides) such as MAP and (Arg)7, etc., ③ designed peptides (designed CPPs) such as MPG and tranportin, etc. from their amphiphilic nature they can also be classified into 3 types ① amphiphilic CPPs (papcps), such as MPG, tranportin, TP10, Pep-1, ②, and the like, such as pentitin, RL16, ③ non-amphiphilic CPPs (nacpps), e.g., R9., although some peptides already exist, but molecules still in the art cannot be introduced into cells by the above-known membrane-penetrating peptides, and thus more peptides of this kind are needed to be found.

Disclosure of Invention

The invention aims to provide the application of the intracellular transport mediated by the HARP1 polypeptide; the invention also aims to provide the application of the HARP1 polypeptide in regulating the defense mechanism of plants.

In a first aspect of the invention, there is provided a method of introducing an exogenous active molecule into a cell or tissue, the method comprising: (1) connecting the exogenous active molecule with HARP1 or conservative variation polypeptide thereof to obtain a connection product; (2) contacting the ligation product of step (1) with a cell or tissue, whereby the exogenous active molecule is introduced into the cell or tissue.

In a preferred embodiment, the exogenous active molecule is introduced into the cytoplasm or nucleus of the cell.

In another preferred embodiment, the exogenous active molecule comprises: 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.

In another preferred embodiment, the exogenous active molecule is a polypeptide comprising: a functional polypeptide (e.g., an enzyme) or a structural polypeptide. Preferably, the exogenous active molecule is a polypeptide capable of altering a trait or characteristic of an animal, plant or microorganism. For example, the exogenous active molecules include (but are not limited to): transcription factors, plant defense proteins, signaling molecules, RNA binding proteins, drug molecules (e.g., molecules of peptides or nucleic acids), and the like.

In another preferred embodiment, the connection comprises: fusion, coupling, adsorption, coupling or complexation.

In another preferred embodiment, the cells or tissues comprise: animal cells or tissues, plant cells or tissues, microbial cells (including cell cultures).

In another preferred embodiment, the animals include, but are not limited to: human, mammalian, insect.

In another preferred embodiment, the plants include, but are not limited to: monocotyledons, dicotyledons; or the plants include, but are not limited to: a spermatophyte, a fern, an algae, a bryophyte.

In another aspect of the present invention, there is provided a method of reducing the defence response capability of a plant to injury, the method comprising: treating plants with HARP1 or a conservative variant thereof; or plants transformed with a gene encoding HARP1 or a conservative variant polypeptide thereof.

In another preferred embodiment, the HARP1 reduces the plant's ability to respond to damage by inhibiting the expression or activity of a jasmonate signaling pathway gene or polypeptide.

In another preferred embodiment, the jasmonic acid signaling pathway comprises: jasmonate (JA) response gene, protease inhibitor gene, secondary metabolic synthesis related transcription factor and synthase.

In another preferred embodiment, said Jasmonate response gene comprises: TAT1, VSP2, MYC 2.

In another preferred embodiment, the protease inhibitor gene comprises: gh _ Sca005135G01, Gh _ A10G2353 and Gh _ D11G 1335.

In another preferred embodiment, said reducing the plant's ability to respond to damage by defence comprises: reducing the production of poisoning defense substances.

In another preferred embodiment, HARP1 or conservative variant polypeptides thereof include: (a) a polypeptide having an amino acid sequence shown in SEQ ID NO. 2 or SEQ ID NO. 4; (b) a polypeptide which starts from any one of amino acids 21 to 39 of SEQ ID NO. 2 and ends at amino acid sequences 119 to 122 of SEQ ID NO. 2; or a polypeptide which starts from any one of amino acids 21-38 of SEQ ID NO. 4 and ends at amino acid sequences 118-121 of SEQ ID NO. 4; (c) a polypeptide which is formed by substitution, deletion or addition of one or more (e.g., 1 to 20, preferably 1 to 10; more preferably 1 to 5; more preferably 1 to 3) amino acid residues to the polypeptide (a) or (b) and which has the function of the polypeptide (a) or (b); (d) a polypeptide having an amino acid sequence which is 40% or more (preferably 50% or more, 60% or more, 70% or more, or 80% or more; more preferably 85% or more, 90% or more, 95% or more, or 99% or more) identical to the amino acid sequence of the polypeptide (a) or (b) and having a function of the polypeptide (a) or (b); or (e) a tag sequence is added to the N-or C-terminus of the polypeptide of (a) or (b) or (C) or (d), or a signal peptide sequence is added to the N-terminus of the polypeptide of (b) or (C) or (d).

In another preferred embodiment, (b) the sequence of the polypeptide starts from any one of amino acids at positions 22 to 38, 23 to 37, 24 to 38, 25 to 37, 26 to 36, 27 to 35, 28 to 34, 29 to 33, 30 to 31 or 30 to 32 in SEQ ID NO. 2 and ends at position 120 or 121 in SEQ ID NO. 2; or starting from any of the amino acids at positions 22-37, 23-36, 24-35, 25-34, 26-33, 27-32 or 28-31 or 29-30 of SEQ ID NO. 4 and ending at positions 119 and 120 of SEQ ID NO. 4.

In another preferred embodiment, HARP1 or a conservative variant thereof is derived from: lepidopteran insects; preferably, the lepidopteran insects include insects of the family noctuidae.

In another preferred embodiment, the lepidopteran insects include: plutella xylostella, spodoptera exigua (s.exigua), heliothis virescens (h.virescens), spodoptera frugiperda (s.frugiperda), cutworm (a.ipsilon), pelargonium virgata (Mconfigurata), and spodoptera littoralis (t.ni).

In another preferred embodiment, the plant comprises: plants that can be fed by lepidopteran insects have an injury defense response mechanism.

In another preferred embodiment, the plant includes (but is not limited to): cruciferous plants, malvaceae plants, gramineae plants, solanaceae plants, and the like.

In another aspect of the invention, there is provided the use of HARP1, or a conservatively variant polypeptide thereof, for introducing an exogenous active molecule into a plant cell or tissue.

In another preferred embodiment, said exogenously active molecule is introduced into the cytoplasm or nucleus of a plant cell.

In another preferred embodiment, the exogenous active molecule is introduced into the plant cell by HARP1 or a conservative variant polypeptide thereof by linking the exogenous active molecule to HARP1 or a conservative variant polypeptide thereof.

In another aspect of the invention, there is provided the use of HARP1 or a conservative variant polypeptide thereof for reducing the defense response of a plant to injury.

In another aspect of the present invention, there is provided an isolated HARP1 polypeptide fragment that is: (i) a polypeptide which starts from any one of amino acids 21 to 39 in SEQ ID NO. 2 and ends from an amino acid sequence shown in positions 119 to 122 in SEQ ID NO. 2; or a polypeptide which starts from any one of amino acids 21-38 of SEQ ID NO. 4 and ends at amino acid sequences 118-121 of SEQ ID NO. 4; (ii) a polypeptide having the function of the polypeptide (i) which is formed by substitution, deletion or addition of one or more (e.g., 1 to 20, preferably 1 to 10; more preferably 1 to 5; more preferably 1 to 3) amino acid residues to the polypeptide (i); (iii) (ii) a polypeptide having an amino acid sequence which is 40% or more (preferably 50% or more, 60% or more, 70% or more, or 80% or more; more preferably 85% or more, 90% or more, 95% or more, or 99% or more) identical to the amino acid sequence of the polypeptide (i) and having a function of the polypeptide (i); or (iv) a polypeptide formed by adding a tag sequence to the N-or C-terminus of the polypeptide of (i) or (ii) or (iii), or by adding a signal peptide sequence.

In another preferred embodiment, (i) the sequence of the polypeptide starts at any one of amino acids at positions 22-38, 23-37, 24-38, 25-37, 26-36, 27-35, 28-34, 29-33, 30-31 or 30-32 of SEQ ID NO. 2 and ends at position 120 or 121 of SEQ ID NO. 2; or starting from any of the amino acids at positions 22-37, 23-36, 24-35, 25-34, 26-33, 27-32 or 28-31 or 29-30 of SEQ ID NO. 4 and ending at positions 119 and 120 of SEQ ID NO. 4.

In another aspect of the present invention, there is provided a method for increasing the efficiency of introducing an exogenous active molecule into a cell or tissue by a HARP1 polypeptide, comprising: truncating the N-terminus or the C-terminus of the HARP1 polypeptide, thereby obtaining a polypeptide starting at any one of amino acids 21 to 39 of SEQ ID NO. 2 and ending at the amino acid sequence shown at positions 119 to 122 of SEQ ID NO. 2; or a polypeptide which starts from any one of amino acids 21-38 of SEQ ID NO. 4 and ends at amino acid sequences 118-121 of SEQ ID NO. 4.

Other aspects of the invention will be apparent to those skilled in the art in view of the disclosure herein.

Drawings

FIG. 1, sequence analysis of the protein found that HARP1 has a signal peptide at the N-terminus. The full-length sequence of HARP1 protein is shown, and the signal peptide sequence at the N-terminus of HARP1 protein is shown in underlined font.

FIG. 2, signal peptide prediction of the HARP1 protein.

FIG. 3, the protein HARP1 was accumulated in a large amount in the mouthparts of Helicoverpa armigera. A: analysis of the expression of the HARP1 gene in various tissues and organs of Helicoverpa armigera. Foregut: the foregut; midgut: the middle intestine; fat body: a fat body; malpighian tube: a Martensitic tube; ovary: an ovary; salivary gland: salivary glands.

B: HARP1 in the Midgut of cotton bollworm (Midgut); levels of protein in intestinal fluid (Gut fluid) and Oral Secretions (OS). Gossypol: gossypol, + indicates that the bollworm takes the artificial feed containing gossypol, and-indicates that the bollworm takes the artificial feed containing no gossypol. CBB: coomassie blue staining was used for protein loading determination.

C: the abundance of HARP1 protein in the secretion from the mouth of cotton bollworms fed different diets. AD: artificial feed; GL; phenol-free cotton; GD: phenol cotton; AT: arabidopsis thaliana.

FIG. 4 shows that the HARP1 is obviously distributed on the damaged part of the plant leaf after being eaten by the cotton bollworm. In the figure, mechanical wrapping represents artificial mechanical injury, and inst wrapping represents wounds caused by plant feeding. The signals of HARP1 on the leaves appear dark purple, the arrow points to the stronger region of HARP1 signal, and the scale indicates 100 μm.

FIG. 5, prokaryotically purified HARP1 protein was able to attenuate plant response to mechanical damage. A: after the arabidopsis leaf wound is treated by the prokaryotic expression of the purified HARP1 protein, compared with the control Venus protein, the induction levels of JA response genes TAT1, VSP2 and MYC2 are obviously reduced after the HARP1 protein is treated.

B: after the cotton leaf wound is treated by the prokaryotic expression of the purified HARP1 protein, compared with the control Venus protein, the induction levels of three protease inhibitor genes in cotton are obviously reduced after the HARP1 protein is treated.

FIG. 6, Venus-HARP1 fusion protein was able to enter plant cells and localize partially in the nucleus. A: prokaryotic purified Venus-HARP1 fusion protein and Venus protein were used to treat arabidopsis leaf wounds. Experiments found that only the Venus-HARP1 fusion protein was detectable in arabidopsis leaf wounds, whereas the Venus protein was barely detectable. The scale represents 500 μm.

B: further analysis of the Venus-HARP1 protein signal site (panel A) revealed that a portion of the protein could enter the nucleus. DAPI dye was used for nuclear staining. The peak indicates the arrow part of the fluorescence intensity distribution. The scale represents 5 μm.

C: prokaryotic purified Venus-HARP1 fusion protein and Venus protein were used to treat arabidopsis root wounds. Experimental results show that only the Venus-HARP1 fusion protein can detect signals on the hypocotyl and upper leaves of Arabidopsis, but the Venus protein can hardly be detected. The scale represents 500 μm.

D: observation of the hypocotyl of the material treated with root wounds from Arabidopsis thaliana revealed that some of the Venus-HARP1 protein could enter the nucleus, whereas this was not observed with the Venus protein. The scale represents 10 μm.

FIG. 7, the tobacco transiently expressed HARP1 protein localized in the plant nucleus. 35S: GFP-HARP1(GFP-HARP1) and 35S: GFP (GFP) vector was transferred into tobacco leaf lamina, and observed 2 days later using a confocal laser microscope, and the scale represents 100. mu.m.

Fig. 8, HARP1 is widespread among lepidopteran insects and has a high similarity in noctuidae.

A: HARP1 protein is widely distributed among lepidopteran insects and is more conserved among noctuidae insects. Construction of the phylogenetic tree of HARP1 protein in lepidopteran insects was accomplished using Mega software.

B: sequence alignment analysis of HARP1 and similar proteins in insects of the family noctuidae. Sequence alignment was performed using Align X in the software VectorNTI Advance, using the Clustal W method in the software.

FIG. 9, HARP1-like polypeptide REPAT38 has a function similar to HARP 1. After the wounds of the plants are treated by the prokaryotic expression of the purified REPAT38 protein, compared with the control Venus protein, the induction levels of JA response genes TAT1, VSP2 and MYC2 are obviously reduced after the REPAT38 treatment.

FIG. 10, Venus-REPAT38 fusion protein was able to enter Arabidopsis and tobacco cells.

A: experiments using prokaryotic purified Venus-REPAT38 fusion protein and Venus protein to treat wounded Arabidopsis show that only Venus-REPAT38 fusion protein can detect signals in Arabidopsis leaves and hypocotyls, but Venus protein is hardly detected. The scale represents 250 μm.

B: the fusion protein treated Arabidopsis thaliana material was observed for hypocotyls, and the Venus-REPAT38 fusion protein was detectable in hypocotyl cells, whereas the Venus protein was hardly detectable. The scale represents 50 μm.

C: prokaryotic purified Venus-REPAT38 fusion protein, Venus-HARP1 fusion protein and Venus protein are used for treating the wounds of tobacco leaves. Venus-REPAT38 and Venus-HARP1 fusion proteins were able to detect signals at the wound and inside the tobacco leaf, whereas the Venus protein was almost undetectable. The scale represents 250 μm.

D: observation of the interior cells of the material from which the tobacco leaf wound was treated, the Venus-REPAT38 and Venus-HARP1 fusion proteins were detectable within the leaf cells, whereas the Venus protein was barely detectable. The scale represents 50 μm.

FIGS. 11A-F, Venus-HARP1 enter cells from a wound and move rapidly in multiple ways in leaf tissue.

FIG. 12, broad spectrum or diversity studies of HARP1 into plant cells.

A: after the non-phenol cotton leaves growing for about 24 weeks are treated with the injury, the leaves are soaked in Venus-HARP1 and Venus for 4h, and then washed for 2h by PBSA, and the results around the wound are observed by a fluorescence microscope. The bars in the figure are all 200. mu.m.

B: after the tobacco leaves which grow for about 24 weeks are treated by injury, the leaves are soaked in Venus-HARP1 and Venus for 4h, and then washed with PBSA for 2h, and then the results of the vicinity of the wound are observed by a fluorescence microscope. The bars in the figure are all 200. mu.m.

Fig. 13, removal of the N-terminal partial sequence can significantly improve the efficiency of protein entry into plant cells. A to G are Venus-HARP1 respectively; Venus-HARP1 delta C5 (5 aa at the C-terminus removed); Venus-HARP1 delta N10 (10 aa N-terminal after removal of the signal peptide); Venus-HARP1 δ N15 (15 aa from N-terminus after removal of signal peptide); Venus-HARP1 delta N20 (20 aa N-terminal after removal of the signal peptide); Venus-HARP1 δ N25 (25 aa N-terminal after removal of the signal peptide); venus.

FIG. 14, the HARP1 protein has the ability to enter animal cells and has a partial localization of the nucleus.

FIG. 15, the ability of the HARP1 protein to enter human cells, illustrating that Venus-HARP1 enters A549 cells and partially enters the nucleus.

A: the results of fluorescent microscope observation after the A549 cells are soaked in Venus-HARP1 and Venus for 4 h.

B: the observations were further magnified for cells soaked in Venus-HARP1 in A.

C: the results were observed after DAPI staining of cells soaked in Venus-HARP1 in A. The bar size in the figure is shown.

FIG. 16, Venus-HARP1 entered 293T cells and partially into the nucleus.

A: 293T cells were soaked in Venus-HARP1 for 4h and then observed by fluorescence microscopy.

B: the observations were further magnified for cells soaked in Venus-HARP1 in A.

C: the results were observed after DAPI staining of cells soaked in Venus-HARP1 in A. The bar size in the figure is shown.

Detailed Description

The present inventors have conducted intensive studies and found that HARP1 and its analogous polypeptides (HARP1like, HL) have the property of entering cells and cell nuclei through complex structures such as cell walls and cell membranes, so that it can be applied to establish a highly efficient transportation system, and help foreign active molecules enter cells or tissues of organisms through means such as protein fusion, thereby changing the properties, characteristics or states of organisms. The HARP1 and similar polypeptides also have effector activity and are capable of reducing plant defense responses, thereby reducing the production of toxic defense substances.

HARP1 and similar polypeptides

The phytophagous insects and plants establish a complex interaction signal network in the long-term evolution process, and Jasmonate (JA) is a main defense hormone of the plants. Earlier, the inventors found that insect oral secretion could interfere with the defense response of plants, from which the inventors isolated a protein, called HARP1 protein. HARP1 conservative variant polypeptides such as, but not limited to, those from: proteins having homology to HARP1 of lepidopteran insects such as plutella xylostella, spodoptera exigua (s.exigua), heliothis virescens (h.virescens), spodoptera frugiperda (s.frugiperda), cutworm (a.ipsilon), pelargonium graveolens (m.configura), trichoplusia ni (t.ni); a specific example is REPAT38 from beet armyworm.

The HARP1 polypeptide (protein) or the REPAT38 polypeptide (protein) of the invention also includes conservative variant polypeptides, fragments, derivatives and analogs thereof. As used herein, the terms "conservative variant polypeptide", "fragment", "derivative" and "analogue" refer to a polypeptide that substantially retains the same biological function or activity of the HARP1 polypeptide or the REPAT38 polypeptide of the invention. The polypeptide fragment, derivative or analogue of the present invention may be (i) a protein in which one or more (e.g., 1 to 50; preferably 1 to 20; more preferably 1 to 10; more preferably 1 to 8; e.g., 5, 3) conserved or non-conserved amino acid residues (preferably conserved amino acid residues) are 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 (e.g., 1 to 30; preferably 1 to 20; more preferably 1 to 10; e.g., 5, 3) amino acid residues, or (iii) a protein in which an additional amino acid sequence is fused to the sequence of the protein, etc. Such fragments, derivatives and analogs are within the purview of those skilled in the art in view of the definitions herein.

Any one of the HARP1 polypeptides or biologically active fragments of the REPAT38 polypeptide may be used in the present invention. Herein, the biologically active fragment of a HARP1 polypeptide or a REPAT38 polypeptide is meant to be a polypeptide that still retains all or part of the function of the full-length HARP1 polypeptide or REPAT38 polypeptide. Typically, the biologically active fragment retains at least 50% of the activity of the full-length HARP1 polypeptide or REPAT38 polypeptide. More preferably, the active fragment is capable of retaining 60%, 70%, 80%, 90%, 95%, 99%, or 100% of the activity of the full-length HARP1 polypeptide or REPAT38 polypeptide.

In the present invention, the term "HARP 1 polypeptide" refers to a polypeptide having the sequence of SEQ ID NO. 2 with the activity of a HARP1 polypeptide. The term also includes variants of the sequence of SEQ ID NO. 2 that have the same function as the HARP1 polypeptide. These variants include (but are not limited to): deletion, insertion and/or substitution of several (e.g., 1 to 50; preferably 1 to 20; more preferably 1 to 10; e.g., 5, 3) amino acids, and addition or deletion of one or several (usually up to 20, preferably up to 10, more preferably up to 5) amino acids at the C-terminal and/or N-terminal.

In the present invention, the term "REPAT 38 polypeptide" refers to a polypeptide having the sequence of SEQ ID NO. 4 with the activity of a REPAT38 polypeptide. The term also includes variants of the sequence of SEQ ID NO. 4 that have the same function as the REPAT38 polypeptide. These variants include (but are not limited to): deletion, insertion and/or substitution of several (e.g., 1 to 50; preferably 1 to 20; more preferably 1 to 10; e.g., 5, 3) amino acids, and addition or deletion of one or several (usually up to 20, preferably up to 10, more preferably up to 5) amino acids at the C-terminal and/or N-terminal.

Polynucleotide sequences (coding sequences) encoding HARP1 polypeptides or REPAT38 polypeptides or conservative variations thereof may also be employed in the invention. The coding region sequence encoding the mature HARP1 polypeptide or the REPAT38 polypeptide may be substantially identical to the sequence shown in SEQ ID NO. 1 or SEQ ID NO. 3 or be a degenerate variant. As used herein, "degenerate variant" refers in the present invention to nucleic acid sequences which encode a protein having SEQ ID NO. 2 or SEQ ID NO. 4, but differ from the coding region sequence shown in SEQ ID NO. 1 or SEQ ID NO. 3.

The term "encoding gene" may be a polynucleotide comprising a sequence encoding the polypeptide, or may be a polynucleotide further comprising additional coding and/or non-coding sequences.

Variants of the above polynucleotides are also useful, encoding polypeptides having the same amino acid sequence as the present invention or fragments, analogs and derivatives of the polypeptides. The variant of the polynucleotide may be a naturally occurring allelic variant or a non-naturally occurring variant. These nucleotide variants include substitution variants, deletion variants and insertion variants. As is known in the art, an allelic variant is a substitution of a polynucleotide, which may be a substitution, deletion, or insertion of one or more nucleotides, without substantially altering the function of the polypeptide encoded thereby.

As a preferred mode of the present invention, the present invention provides a truncation of a HARP1 polypeptide or a REPAT38 polypeptide, which is a polypeptide obtained by removing the N-terminal or C-terminal partial sequence from the HARP1 polypeptide or the REPAT38 polypeptide. Preferably, the truncation is a polypeptide obtained by removing 1-19, 2-18, 3-17 amino acids from 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 from the N-terminus (excluding the signal peptide). Whereas for the C-terminus of the HARP1 polypeptide or the REPAT38 polypeptide, typically less than 5, less than 4, less than 3 or less than 2 amino acids are removed. The present inventors have surprisingly found that the truncation described allows for a more efficient introduction of exogenous active molecules into cells or tissues.

It is to be understood that, although the HARP1 gene or the REPAT38 gene of the present invention is obtained from the noctuidae insect, cotton bollworm or beet armyworm, respectively, it is highly homologous, e.g., has 40% or more, to the HARP1 gene or the REPAT38 gene obtained from other insects; 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 the other genes are also within the scope of the present invention. Based on homology alignment, the present inventors found that HARP1-like polypeptides (HARP1like, HL) are ubiquitous in lepidopteran insects and highly conserved in noctuidae.

Vectors comprising such coding sequences, as well as host cells genetically engineered with such vectors or the coding sequence for the HARP1 polypeptide or the REPAT38 polypeptide are also encompassed by the present invention. Methods well known to those skilled in the art can be used to construct expression vectors containing the coding sequence for the HARP1 polypeptide REPAT38 polypeptide and appropriate transcription/translation control signals. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombinant techniques, and the like. The sequences may be operably linked to a suitable promoter in an expression vector to direct mRNA synthesis. Vectors containing the appropriate coding sequences described above, together with appropriate promoter or control sequences, may be used to transform appropriate host cells to enable expression of the protein. The host cell may be a plant cell or an animal cell.

Use of intracellular transport

The present invention provides a method for introducing an exogenous active molecule into a cell or tissue, comprising: (1) connecting the exogenous active molecule with the HARP1 polypeptide to obtain a connection product; (2) contacting the ligation product of step (1) with a cell or tissue, whereby the exogenous active molecule is introduced into the cell or tissue. Preferably, the exogenous active molecule is introduced into the nucleus of the cell.

As used herein, the HARP1-like polypeptides include HARP1 conservative variants of REPAT38 and the like, including truncations of HARP1, REPAT38 and the like.

As used herein, the term "exogenous active molecule" also referred to as "functional molecule" refers to a molecule that has a specific function and is capable of modifying a trait, structure, property or state of an organism (including animals and plants), for example, by causing a plant to produce at least one improvement in the trait, and for example, by causing an animal or human in a disease state to develop a disease; it may also be a class of molecules with reporter or reporter functions that enable certain organs, tissues and cells of an organism to be detected or localized, such as fluorescent proteins, tagged proteins (e.g., myc, HA, His, etc.). Exogenous active molecules that may be employed include: polypeptides, nucleic acids, compounds (e.g., certain hormones).

The HARP1 polypeptide can introduce exogenous active molecules into cells or tissues, particularly can enter human cells, and has great application prospect in the field of clinical disease treatment. Clinically, many diseases require the introduction of exogenous active molecules (e.g., as drugs) for treatment. For example, the tumor can be inhibited by carrying an inhibitory molecule or toxin to the tumor cells by the HARP1 polypeptide. For example, some diseases related to the hypofunction of enzyme or protein can be alleviated or treated by entering the active enzyme or protein into cells or tissues by using the HARP1 polypeptide.

The human cells may include, but are not limited to: tumor cells, somatic cells, embryonic cells. Such 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 drosophila cells, plant cells are demonstrated in the examples of the present invention, but it is understood that cells suitable for use in the technical scheme of the present invention are not limited to those exemplified in the examples.

The connection mode of the HARP1 polypeptide and the exogenous active molecule can be covalent connection or non-covalent connection. The connection comprises: fusion, coupling, adsorption, coupling or complexing, etc. The term "linkage" is "operative linkage", i.e., a functional spatial arrangement between two or more molecules. It is understood that any connection means can be included in the present invention as long as the functions of the HARP 1-type polypeptide and the foreign active molecule can be maintained, and the good effect of penetrating cell membrane and body tissue barrier can be maintained. Covalent attachment two molecules are typically attached in a manner that forms a covalent bond. While some non-covalent attachment (without formation of covalent bonds) such as coupling, adsorption, conjugation, etc. may also be applied.

In a preferred embodiment of the present invention, the exogenous active molecule is a polypeptide comprising: a functional polypeptide (e.g., an enzyme) or a structural polypeptide. When the exogenous active molecule is polypeptide, it is fused with HARP1 polypeptide to obtain fusion protein. The HARP1 polypeptide and the exogenous active molecule can be directly connected or connected through a polypeptide linker (connecting peptide). The linker comprises, for example, 1-30 amino acids; preferably 1-20 amino acids; e.g. 15, 10, 8, 6, 5, 4, 3, 2, 1 amino acids. The arrangement of the connecting peptide does not basically affect the effect of the HARP1 and the exogenous active molecule on penetrating cell membranes and body tissues and the function of the exogenous active molecule.

The linkage between the HARP 1-type polypeptide and the exogenous active molecule may be a peptide bond, and if necessary, the exogenous active molecule may be located at the amino terminus of the HARP 1-type polypeptide or at the carboxy terminus of the HARP 1-type polypeptide.

As an alternative of the invention, the exogenously active molecule may be RNA or DNA, for example an RNA molecule capable of producing in vivo a homologous interference effect against a gene.

As an alternative mode of the invention, the HARP1 polypeptide can be linked to an exogenous active molecule through a chemical reaction such as amino, carboxyl or sulfhydryl, including but not limited to the linkage between the polypeptide and a polymer, covalent modification, esterification, sulfurization of the polypeptide on the surface of a liposome or nanoparticle, and the like.

The non-covalent linkage is electrostatic adsorption linkage or receptor ligand reaction. The electrostatic adsorption connection includes but is not limited to the electrostatic connection between the cell-penetrating vector and the nucleic acid molecule. The receptor-ligand reaction refers to that a receptor and a ligand which can be specifically matched are respectively connected to the HARP1 polypeptide and the exogenous active molecule, and the connection of the polypeptide and the exogenous active molecule is realized through the high specificity of the receptor and the ligand. Such as a specific match between biotin and avidin.

The invention is based on the characteristic of promoting the exogenous active molecules to enter cells and cell nucleuses through complex structures such as cell walls, cell membranes and the like. Thus, a variety of exogenous active molecules can be used to link to the HARP1-like polypeptide to form a complex.

In a specific embodiment of the present invention, the inventors found that a prokaryotic expression of Venus-HARP1 fusion protein could enter the cell and a portion of the fusion protein was localized in the nucleus. This suggests that the HARP1-like polypeptide in insects can function by allowing the protein fused therewith to enter the cell via multiple barriers such as cell wall and cell membrane.

The types of cells or tissues suitable for mediating the introduction of foreign proteins with the HARP 1-type polypeptide of the present invention are broad, and can be cells of various organisms including plant cells or tissues, animal cells or tissues, microbial cells (including cultures thereof), and the like.

In a specific example of the present invention, the present inventors also found that HARP1-like polypeptides in Spodoptera exigua (Spodoptera exigua) have a similar function to HARP 1. These findings suggest that HARP1 and its analogous polypeptides can be used as a transport system to alter the properties, structure, characteristics or state of an organism by means of protein fusion to assist foreign active molecules in entering cells.

In a specific embodiment of the present invention, the present inventors have observed that the ligation product of the HARP1-like polypeptide with an exogenously active molecule is introduced into a variety of cells from a wound site. And, the ligation product of the HARP1-like polypeptide linked to an exogenous active molecule moves rapidly in leaf tissue in multiple ways.

Use for modulating plant defence responses

The invention provides a method for reducing the defense response capability of plants to injury, which comprises the following steps: treating plants with HARP1-like polypeptides; or transforming a plant with a gene encoding a HARP1-like polypeptide. The ability to reduce the defense response of a plant to injury includes: reduce the generation of toxic defense substances in plants.

The HARP1-like polypeptides exert a regulatory effect by affecting defense response mechanisms in plants, including mechanisms based on the jasmonic acid signaling pathway. The inventors found that HARP1-like polypeptides significantly inhibit the expression or activity of jasmonic acid signaling pathway genes or polypeptides. The jasmonic acid signaling pathway comprises: jasmonate (JA) responsive genes such as TAT1, VSP2, MYC2, which are significantly inhibited following treatment with or over-expression of a HARP1-like polypeptide or a HARP1-like polypeptide. Many important plant defense substances are well known in the art to be regulated by the JA pathway, and therefore, the attenuation of the JA response by HARP1-like polypeptides necessarily reduces the production of toxic defense substances in plants.

The inventor finds that the response of JA early response gene to mechanical damage is inhibited in damaged plants which express and treat HARP1 polypeptide in vitro; transgenic plants overexpressing HARP1-like polypeptides, after injury, also exhibit suppression of the response of the JA early response gene to mechanical injury. Thus, HARP1-like polypeptides have significant effector activity under both ex vivo and in vivo conditions.

Various methods well known to those skilled in the art may be employed to modulate the expression of the HARP1-like polypeptide. For example, an expression unit (such as an expression vector or a virus) carrying a gene encoding a HARP 1-type polypeptide can be delivered to a target site in a certain way, and the target site can express the active HARP 1-type polypeptide. In one embodiment of the present invention, a gene encoding a HARP 1-type polypeptide is cloned into an appropriate vector by a conventional method, and the recombinant vector carrying the foreign gene is introduced into a plant cell capable of expressing the HARP 1-type polypeptide, so that the plant cell expresses the HARP 1-type polypeptide. Plants overexpressing HARP1-like polypeptides can be obtained by regenerating the plant cells into plants.

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, for which specific conditions are not noted in the following examples, are generally performed according to conventional conditions such as those described in J. SammBruk et al, molecular cloning protocols, third edition, scientific Press, 2002, or according to the manufacturer's recommendations.

The information of the primers used in the present experiment is shown in Table 1.

TABLE 1