Application of BrVCaBL protein and functional peptide fragment thereof in improving high-temperature tolerance of Chinese cabbage crops

Abstract

The invention discloses an application of a BrVCaBL protein and a functional peptide fragment thereof in improving the high-temperature tolerance of Chinese cabbage crops, and solves the technical problem of improving the high-temperature tolerance of plants. In particular to polypeptide or pharmaceutically acceptable salt or solvate thereof. The polypeptide contains at least one of oligopeptide X1 and oligopeptide X2; the amino acid sequence of the short peptide X1 is AIAPAAEN, and the amino acid sequence of the short peptide X2 is EEVVAAEV. When the polypeptide is applied to plants, the high-temperature tolerance of the plants can be improved, and the polypeptide can be applied to industrial production.

Description

Technical Field

[0001] The present invention specifically relates to the application of BrVCaBL protein and its functional peptide segments in improving the high temperature tolerance of cabbage crops. Background Art

[0002] Cabbage is a cool-loving crop and is extremely susceptible to high temperature stress during its growth. High temperature stress can increase the transpiration rate of cabbage, reduce the photosynthetic rate, cause the leaves to turn yellow and curl, or even die, resulting in serious yield reduction or even crop failure. Therefore, improving the heat tolerance of cabbage is of great research significance. The high temperature tolerance of cabbage can be quantified by observing the degree of leaf wilting and measuring the chlorophyll content. In addition, the content of some metabolites can also be used as a measure of heat tolerance. For example, Verbruggen et al. reported that proline, as an osmotic regulating substance, plays an important role in the process of cabbage cells adapting to heat stress. Generally, the proline content of heat-sensitive varieties is lower than that of heat-resistant varieties (Verbruggen et al., 2008). In addition, there are also a large number of studies that use soluble sugar and malondialdehyde content to measure the heat tolerance of cabbage. The soluble sugar content is proportional to the heat tolerance, while the malondialdehyde content is inversely proportional to the heat tolerance (Bita & Gerats, 2013; Chen & Li, 2016; Zhang et al., 2022).

[0003] Improving plant heat resistance by applying exogenous substances is simple to operate and has rapid effects, and has broad application prospects in actual production. Zhang Mingshun et al. reported that exogenous application of 15mmol / LCaCl2 solution can slow down the increase of membrane permeability, the degradation of chlorophyll and the accumulation of malondialdehyde, increase the content of proline and soluble protein, and enhance the heat resistance of lily (Zhang Mingshun et al., 2019). Huang Li et al. reported that exogenous application of melatonin can improve the heat resistance of cabbage and reduce the bitterness caused by high temperature. The present invention found a heat-resistant gene BraA06g009510.3C in the cabbage heart, encoding a vacuolar calcium ion binding protein named BrVCaBL. A high-concentration His-BrVCaBL protein solution can be obtained by prokaryotic purification. The leaves of the cabbage plants are evenly sprayed with 10μM BrVCaBL protein solution at 1mL / plant / day, which can significantly alleviate the wilting and chlorosis of the leaves caused by 45°C high temperature stress, promote leaf growth, increase the content of proline and soluble sugar, reduce the accumulation of malondialdehyde, and improve the tolerance of cabbage plants. Further, two key functional peptides were found in the BrVCaBL protein. After synthesis, they were applied in the same way, which can also significantly improve the heat resistance of cabbage. How to obtain polypeptides or short peptides that can improve the heat resistance of cabbage crops is a technical problem faced by people in this field. Summary of the invention

[0004] The technical problem solved by the present invention is to provide a plurality of peptide segments, and to improve the high temperature tolerance of cabbage crops by applying the peptide segments to cabbage crops.

[0005] In order to solve the above technical problems, the present invention provides a polypeptide or a pharmaceutically acceptable salt or solvate thereof.

[0006] The polypeptide contains at least one of a short peptide X1 and a short peptide X2;

[0007] The amino acid sequence of the short peptide X1 is AIAPAAEN, and the amino acid sequence of the short peptide X2 is EEVVAAEEV.

[0008] As described above, or a pharmaceutically acceptable salt or solvate thereof.

[0009] The amino acid number of the polypeptide is no more than 142.

[0010] As described above, or a pharmaceutically acceptable salt or solvate thereof.

[0011] The polypeptide is selected from any one of the following:

[0012] 1) The short peptide X1 as claimed in claim 1;

[0013] 2) The short peptide X2 according to claim 1;

[0014] 3) A polypeptide having an amino acid sequence as described in SEQ ID NO: 2 or positions 36 to 177 of SEQ ID NO: 2.

[0015] Sequence 2 is as follows:

[0016] MGSSHHHHHHSSGLVPRGSHMASMTGGQQMGRSEFMATVENEQAIAPAAENVEVPTKTVEEPEVTEQPEKVVATTE SAPAPAAVTEEESEAPVVETSKDVVVEEAEEKEEEAEKETEEPKVEEEEEKTENPAVVEEPKEEEKEEETETPAVVEEEE KKTEAEEVVAAEEVAAEKAEE.

[0017] As described above, the polypeptide or its pharmaceutically acceptable salt or solvate. The polypeptide is a polypeptide obtained by performing at least one of the following modifications:

[0018] A1) a polypeptide obtained by connecting an amino terminal protecting group to the amino terminal of the polypeptide and / or connecting a carboxyl terminal protecting group to the carboxyl terminal of the polypeptide;

[0019] A2) a polypeptide obtained by adding amino acid residues to the amino terminus and / or carboxyl terminus of the polypeptide;

[0020] A3) A polypeptide obtained by linking a fatty acid to the amino terminus and / or carboxyl terminus of the polypeptide.

[0021] In the present invention, the amino terminal protecting group or the carboxyl terminal protecting group may be any one of acetyl, amino, maleyl, succinyl, tert-butyloxycarbonyl or benzyloxy or other hydrophobic groups or macromolecular carrier groups; the carboxyl terminal of the cyclic polypeptide of the present invention may contain a carboxyl terminal protecting group, and the carboxyl terminal protecting group may be any one of amino, amide, carboxyl, or tert-butyloxycarbonyl or other hydrophobic groups or macromolecular carrier groups.

[0022] In order to solve the above technical problems, the present invention provides a pharmaceutical composition.

[0023] It comprises any of the above peptide compounds or a pharmaceutically acceptable salt or solvate thereof, and a pharmaceutically acceptable carrier or excipient.

[0024] In the above, the drug may be a pesticide. The drug also includes a pharmaceutically acceptable carrier or excipient. The carrier or excipient includes, but is not limited to, a water-soluble carrier material (such as polyethylene glycol, polyvinyl pyrrolidone, organic acid, etc.), a poorly soluble carrier material (such as ethyl cellulose, cholesterol stearate, etc.). Among them, water-soluble carrier materials are preferred. These materials can be made into a variety of dosage forms, including but not limited to tablets, capsules, dripping pills, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, freeze-dried powder injections, etc. It can be a common preparation, a sustained-release preparation, a controlled-release preparation, and various microparticle delivery systems. In order to make a unit dosage form into a tablet, various carriers known in the art can be widely used. Examples of carriers include diluents and absorbents, such as starch, dextrin, calcium sulfate, lactose, mannitol, sucrose, sodium chloride, glucose, urea, calcium carbonate, kaolin, microcrystalline cellulose, aluminum silicate, etc.; wetting agents and binders, such as water, glycerol, polyethylene glycol, ethanol, propanol, starch slurry, dextrin, syrup, honey, glucose solution, acacia, at least one of the sugar alcohol fatty acid esters, sodium lauryl sulfate, methyl cellulose, ethyl cellulose, etc.; disintegration inhibitors, such as sucrose, tristearin, cocoa butter, hydrogenated oil, etc.; absorption promoters, such as quaternary ammonium salts, sodium lauryl sulfate, etc.

[0025] Use of any of the above described polypeptides or pharmaceutically acceptable salts or solvates thereof and / or the above described pharmaceutical compositions in improving plant high temperature tolerance or preparing products for improving plant high temperature tolerance.

[0026] In the above application, the high temperature tolerance index is at least one of wilting rate, leaf fresh weight, aboveground fresh weight, chlorophyll content, proline content, soluble sugar content and malondialdehyde content.

[0027] In the above, the improvement of plant high temperature tolerance can be at least one of reducing wilting rate, increasing leaf fresh weight, increasing aboveground fresh weight, increasing chlorophyll content, increasing proline content, increasing soluble sugar content and reducing malondialdehyde content.

[0028] In the above, the leaf may be the sixth true leaf.

[0029] In order to solve the above technical problems, the present invention provides a method for improving the high temperature tolerance of plants.

[0030] The method comprises the step of applying any of the above polypeptides or pharmaceutically acceptable salts or solvates thereof and / or the above pharmaceutical composition to a plant.

[0031] In the above, the mode of application is foliar application. The application concentration of the short peptide X1 is 5μM-1mM. Preferably, the application concentration is 10μM. The application concentration of the short peptide X1 is 5μM-1mM. Preferably, the application concentration is 10μM. The application concentration of the polypeptide whose amino acid sequence is described in Sequence 2 is 5μM-1mM. Preferably, the application concentration is 10μM.

[0032] In the above, the method further comprises the step of applying the above-mentioned polypeptide or its pharmaceutically acceptable salt or solvate and / or the above-mentioned pharmaceutical composition to the plant 3 days before the plant is placed in a high temperature environment.

[0033] In order to solve the above technical problems, the present invention provides a plant cultivation method.

[0034] The method comprises a plant grown using the above method.

[0035] In any of the above uses or methods, the plant is any of the following:

[0036] B1) Cruciferous vegetables;

[0037] B2) Brassica plants;

[0038] B3) Chinese cabbage or choy sum.

[0039] In the above, the cabbage can be the Chinese cabbage variety Baiyang. The Chinese cabbage heart can be the Chinese cabbage heart variety Shengnong Lvbao Liuye 70-day Chinese cabbage heart.

[0040] In the above, the Chinese cabbage may be Chinese cabbage grown to 5 weeks old. The Chinese cabbage heart may be Chinese cabbage grown to 4 weeks old.

[0041] In the above, the temperature of the high temperature tolerance may be 40° C.-45° C., and the tolerance time of the high temperature tolerance may be 3 days or 7 days.

[0042] Beneficial Effects

[0043] The present invention discloses the application of BrVCaBL protein and its functional peptide segment in improving the high temperature tolerance of cabbage crops, and solves the technical problem of improving the high temperature tolerance of plants. Specifically disclosed is a polypeptide or a pharmaceutically acceptable salt or solvate thereof, wherein the polypeptide contains at least one of a short peptide X1 and a short peptide X2; the amino acid sequence of the short peptide X1 is AIAPAAEN, and the amino acid sequence of the short peptide X2 is EEVVAAEEV. The polypeptide is applied to plants to improve the high temperature tolerance of plants, and can be used in industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 This is a Coomassie blue detection image of prokaryotic purified His-BrVCaBL protein. Marker: prestained protein standard of indicated size (Bio-Rad, Precision Plus Protein Dual Color Standards, #1610374); KD: kilodalton, molecular mass unit.

[0045] Figure 2 This is a graph showing the mass spectrometry purity of synthetic peptides (provided by Beijing Liuhe BGI Genomics Co., Ltd.).

[0046] Figure 3 This is a diagram showing that exogenous application of His-BrVCaBL enhances the heat resistance of Chinese cabbage; A shows the phenotypes before and after spraying diluted elution buffer (control) and His-BrVCaBL protein solution (experimental) at 45°C; B shows the statistics of leaf wilting rate; C shows the statistics of leaf fresh weight; D shows the statistics of chlorophyll content; E shows the statistics of proline content; F shows the statistics of soluble sugar content; G shows the statistics of malondialdehyde content.

[0047] Figure 4 To enhance the heat tolerance of Chinese cabbage by exogenous application of His-BrVCaBL; A, phenotypes were photographed before and after spraying diluted elution buffer (control) and His-BrVCaBL protein solution (experimental) at 45°C; B, leaf wilting rate statistics; C, leaf fresh weight statistics; D, chlorophyll content statistics; E, proline content statistics; F, soluble sugar content statistics; G, malondialdehyde content statistics.

[0048] Figure 5 This figure shows that exogenous application of BrVCaBL key functional peptide enhances the heat resistance of Chinese cabbage; A, Chinese cabbage plants sprayed with diluted elution buffer (control), His-BrVCaBL protein solution and peptide solution, and phenotypes were photographed before and after 45°C high temperature treatment; B, leaf wilting rate statistics; C, leaf fresh weight statistics; D, chlorophyll content statistics; E, proline content statistics; F, soluble sugar content statistics; G, malondialdehyde content statistics. DETAILED DESCRIPTION

[0049] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0050] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0051] The following examples were processed using SPSS11.5 statistical software, and the experimental results were expressed as mean ± standard deviation. One-way ANOVA test was used, and P < 0.05 (*) indicated a significant difference, P < 0.01 (**) indicated a very significant difference, and P < 0.001 (***) indicated an extremely significant difference.

[0052] In the present invention, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In addition, the protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, immunology and laboratory operation procedures used herein are terms and routine procedures widely used in the corresponding fields. At the same time, in order to better understand the present invention, the definitions and explanations of the relevant terms are provided below.

[0053] The terms "polypeptide", "short peptide", "peptide" and "protein" are used interchangeably herein to refer to polymers of amino acid residues. The term can be applied to amino acid polymers in which one or more amino acid residues are artificial chemical mimetics of the corresponding naturally occurring amino acids, as well as naturally occurring amino acid polymers and non-naturally occurring amino acid polymers. It is used interchangeably herein to refer to polymers of amino acids of any length. The polymer can be linear, cyclic or branched, it can contain modified amino acids, particularly conservatively modified amino acids, and it can be interrupted by non-amino acids. The term also includes modified amino acid polymers such as those that have been modified by sulfation, glycosylation, lipidation, acetylation, phosphorylation, iodination, methylation, oxidation, proteolytic processing, prenylation, racemization, selenoylation, transfer-RNA mediated amino addition such as arginylation, ubiquitination, or any other operation such as conjugation with a labeling component. As used herein, the term "amino acid" refers to natural and / or non-natural or synthetic amino acids, including glycine and D or L optical isomers, as well as amino acid analogs and peptide mimetics. A polypeptide or amino acid sequence "derived from" a specified protein refers to the source of the polypeptide. The term also includes polypeptides expressed by the designated nucleic acid sequence.

[0054] The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to naturally occurring amino acids. Naturally occurring amino acids are amino acids encoded by the genetic code, as well as subsequently modified amino acids, such as hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. In this article, amino acids may be represented by the common three-letter codes or single-letter codes recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Similarly, nucleotides may be represented by their generally accepted single-letter codes.

[0055] The term "kit" refers to any delivery system used to deliver substances. In reaction assays, such delivery systems include systems that store, transport or deliver reaction reagents (e.g., oligonucleotides, enzymes, etc. in appropriate containers) and / or support materials (e.g., buffers, instructions for performing the assay, etc.) from one location to another. For example, a kit comprises one or more housings (e.g., boxes) containing the relevant reaction reagents and / or support materials.

[0056] The terms "include", "including", "have", "contain" and the like are all open terms, meaning including but not limited to.

[0057] The terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of the features. Further, in the description of the present invention, unless otherwise specified, "plurality" means two or more.

[0058] The terms "optionally," "optional," or "optionally" generally mean that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0059] When the term "identity" is used to describe an amino acid sequence or a nucleic acid sequence relative to a reference sequence, the percentage of identical amino acids or nucleotides between two amino acid sequences or nucleic acid sequences is determined by conventional methods, for example, see Ausubel et al., eds. (1995), Current Protocols in MolecμLar Biology, Chapter 19 (Greene Publishing and Wiley-Interscience, New York); and the ALIGN program (Dayhoff (1978), Atlas of Protein Sequence and Structure 5: Suppl. 3 (National Biomedical Research Foundation, Washington, DC). There are many algorithms for aligning sequences and determining sequence identity, including the homology alignment algorithm of Needleman et al. (1970) J. Mol. Biol. 48: 443; the local homology algorithm of Smith et al. (1981) Adv. Appl. Math. 2: 482; the local homology algorithm of Pearson et al. (1988) P. The similarity search method of Proc. Natl. Acad. Sci. 85: 2444; Smith-Waterman algorithm (Meth. Mol. Biol. 70: 173-187 (1997); and BLASTP, BLASTN, and BLASTX algorithms (see Altsch μL et al. (1990) J. Mol. Biol. 215: 403-410). Computer programs that utilize these algorithms are also available and include, but are not limited to, ALIGN or Megalign (DNASTAR) software, or WU-BLAS T-2 (Altsch μL et al., Meth. Enzym., 266:460-480 (1996)); or GAP, BESTFIT, BLAST Altsch μL et al., supra, FASTA, and TFASTA, available in the Genetics Computing Group (GCG) package, Version 8, Madison, Wisconsin, USA; and CLUSTAL in the PC / Gene program provided by Intelligenetics, Mountain View, California.

[0060] Chinese cabbage Baiyang is recorded in the following document: "Zhang Deshuang, Xin Xiaoyun, Zhu Hongbin, Zhang Fenglan, Yu Shuancang, Su Tongbing, Yu Yangjun, Wang Weihong, Zhao Xiuyun, Li Peirong, Lu Guixiang. Creation and application of Chinese cabbage rosette leaf, large leaf angle and multi-tillering materials [J]. Vegetables, 2024, (09): 50-57." The name in this document is Baiyang.

[0061] Shengnong Green Treasure Willow Leaf 70 Days Vegetable Heart is recorded in the following document: "Xin X, Li P, Zhao X, Yu Y, Wang W, Jin G, Wang J, Sun L, Zhang D, Zhang F, Yu S, Su T. Temperature-dependent jumonji demethylase modulates flowering time by targeting H3K36me2 / 3 in Brassicarapa. Nature Communications. 2024 Jun 28; 15(1): 5470." The name in this document is Shengnong Green Treasure Willow Leaf 70 Days Vegetable Heart.

[0062] Example 1. Acquisition of His-BrVCaBL protein and functional peptides

[0063] Obtaining His-BrVCaBL protein:

[0064] 1. Extract total RNA from the leaves of Shengnong Green Treasure Willow Leaf 70-day-old Chinese cabbage, and obtain cDNA through reverse transcription.

[0065] 2. Using cDNA as a template, primers 510EcoRI-F (5'-GGATCCGAATTCATGGCCACCGTTGAGAATG-3') and 510SalI-R (5'-GTCGACGGAGTTACTCTTCGGCCTTCTC-3') were used to amplify the CDS sequence of BraA06g009510.3C (BrVCaBL) gene. The fragment was recovered and connected with the pET28a(+) (Novagen) vector digested with EcoRI / SalI.

[0066] 3. The ligation product was transformed into competent E. coli Trans1-T1 Phage Resistant (Full Gold, CD501-02), spread on LB solid medium containing 50 mg / L kanamycin sulfate, and cultured inverted at 37°C overnight.

[0067] 4. Pick a single colony of E. coli growing on LB solid medium, use the universal sequencing primers T7-promoter and T7-terminator of pET28a(+) vector to perform colony PCR, select a positive single colony that can amplify the CDS fragment size of the BrVCaBL gene, extract the plasmid, and sequence to confirm the inserted fragment. The pET28a-BrVCaBL recombinant vector is obtained. The pET28a-BrVCaBL recombinant vector replaces the fragment between the restriction endonuclease EcoRI and SalI recognition sites of the pET28a vector with sequence 1, and keeps the other nucleotide sequences of the pET28a(+) vector unchanged. The obtained recombinant plasmid is named pET28a-BrVCaBL recombinant vector or recombinant vector pET28a-BrVCaBL.

[0068] 5. The pET28a-BrVCaBL recombinant vector plasmid was transformed into Escherichia coli competent BL21 (full gold, CD901), spread on LB solid medium containing 50 mg / L kanamycin sulfate, and cultured inverted at 37°C overnight.

[0069] 6. Pick a single colony of Escherichia coli grown on LB solid medium, use the universal sequencing primers T7-promoter and T7-terminator of the pET28a(+) vector for colony PCR, and select the positive single colony that can amplify the CDS fragment size of the BrVCaBL gene, which is BL21 / pET28a-BrVCaBL. BL21 / pET28a-BrVCaBL is the Escherichia coli BL21 strain containing the pET28a-BrVCaBL recombinant vector.

[0070] Sequence 1 is as follows:

[0071] ATGGCCACCGTTGAGAATGAACAAGCGATTGCTCCAGCAGCAGAGAACGTTGAGGTGCCAACAAAGACGGTGGAGGAGCCAGAAGTGACGGAACAGCCGGAAAAAGTCGTCGCCACGACAGAGTCTGCTCCTGCTCCAGCCGCCGTAACAGAAGAAGAATCTGAAGCACCAGTGGTAGAAACGAGCAAAGATGTGGTTGTGGAAGAGGCAGAGGA AAAAGAAGAAGAAGCAGAGAAAGAAACGGAGGAACCAAAGGTAGAAGAGGAAGAAGAGAAGACAGAAAATCCAGCGGTTGTGGAGGAGCCAAAGGAAGAAGAGAAAGAGGAGGAGACTGAAACTCCAGCTGTTGTTGAGGAGGAGGAGAAGAAGACCGAAGCAGAGGAAGTCGTGGCTGCTGAGGAAGTCGCCGCCGAGAAGGCCGAAGAGTAA.

[0072] 2. Prokaryotic protein purification

[0073] 1. Pick 3-5 single colonies (BL21 / pET28a-BrVCaBL) and inoculate them into 3 mL LB liquid medium containing 50 mg / L kanamycin sulfate. Cultivate at 37°C and 220 rpm until OD 600 If the cell culture temperature is greater than 2.0, transfer to 200 mL of LB liquid medium containing 50 mg / L kanamycin sulfate and culture at 37°C and 220 rpm until the OD 600 The p-value was 0.6-0.8, 2 ml of the cells were taken (recorded as the strain before induction, for SDS-PAGE); IPTG (Full Gold, GF101-01) with a final concentration of 0.1 mM was added, and the mixture was induced at 21°C and 140 rpm overnight.

[0074] 2. Centrifuge at 6,000 × g for 10 min to collect 2 ml of cells (recorded as the induced strain for SDS-PAGE). Add 200 μL 1*SDS lysis buffer (50 mM Tris-HCl pH 6.8, 2%

[0075] SDS, 10% glycerol, 0.01 mg / mL bromophenol blue, 12.5 μL / mL β-mercaptoethanol) resuspended in boiling water bath for 5 min, 13,000

[0076] × g centrifugation for 10 min, aspirate the supernatant, and obtain the samples before and after induction ( Figure 1Lanes 1-2 (before and after induction).

[0077] 3. Collect the remaining cells by centrifugation at 6,000 × g for 10 min, add 3 mL of lysis buffer (100 mM Tris-HCl, pH 7.5, 150 mM NaCl, 10 mM PMSF (phenylmethylsulfonyl chloride)) to resuspend, and ultrasonically disrupt for 6 min (output power 270 W, ultrasonic 5 s, interval 5 s), centrifuge at 4 ° C, 13,000 rpm for 40 min, and aspirate the supernatant.

[0078] 4. Take 30 μL of supernatant and add 10 μL of 4*SDS lysis buffer (200 mM Tris-HCl pH 6.8, 8%

[0079] SDS, 40% glycerol, 0.04mg / mL bromophenol blue, 50μL / mL β-mercaptoethanol), then add 360μL 1*SDS lysis buffer to dilute 10 times; take about 10μL of precipitate and add 100μL 1*SDS lysis buffer to resuspend. Boil in water bath for 5min, centrifuge at 13,000×g for 10min, aspirate the supernatant, and obtain the supernatant and precipitate samples ( Figure 1 Lanes 3-4 (supernatant and pellet)).

[0080] 5. Take an affinity nickel column (40724, QIAGEN) containing about 1 ml of filler and first use 10 ml of lysis buffer to equilibrate the filler.

[0081] 6. Add the supernatant obtained by centrifugation (supernatant of the strain after induction) to the affinity nickel column, incubate at 4°C for 3 hours, collect the supernatant, take 30 μL of the supernatant, add 10 μL of 4*SDS lysis buffer, then add 360 μL of 1*SDS lysis buffer to dilute 10 times, boil in water bath for 5 minutes, centrifuge at 13,000×g for 10 minutes, and aspirate the supernatant. This is column penetration ( Figure 1 Lane 5 (through the column).

[0082] 7. Add 10 ml of washing buffer (100 mM Tris-HCl pH 7.5, 150 mM NaCl, 10 mM imidazole) to wash the impurities, collect 30 μL of washing buffer after passing through the column, add 10 μL of 4*SDS lysis buffer, boil in water bath for 5 min, centrifuge at 13,000 × g for 10 min, and aspirate the supernatant. Figure 1 Middle 6 (cleaning)).

[0083] 8. Finally, add 3 ml of elution buffer (100 mM Tris-HCl pH 7.5, 150 mM NaCl, 150 mM imidazole) to elute the target protein. Collect 500 μL of eluted protein in one tube, and collect six tubes of eluted protein (E1, E2, E3, E4, E5 and E6). Figure 1 8-13 lanes), 30 μL of each was added with 10 μL 4*SDS lysis buffer, boiled in a water bath for 5 min, centrifuged at 13,000×g for 10 min, the supernatant was aspirated, and the above-mentioned pre-induction, post-induction, supernatant, precipitate, column, washing and eluted proteins E1, E2, E3, E4, E5 and E6 were subjected to electrophoresis separation by SDS-PAGE (161015, Bio-rad) (220V constant voltage electrophoresis, stopped when the bromophenol blue at the front end of the sample completely ran out of the protein gel), and the protein purity was detected by Coomassie Brilliant Blue staining.

[0084] After induction, the supernatant of the strain contains the His-BrVCaBL protein expressed by the recombinant vector pET28a-BrVCaBL, and the amino acid sequence of the His-BrVCaBL protein is sequence 2. Positions 5 to 10 of sequence 2 are 6×His tags, positions 14 to 19 of sequence 2 are thrombin sites, positions 21 to 31 of sequence 2 are T7 tag tags, and positions 36 to 177 of sequence 2 are BrVCaBL protein (the amino acid sequence is shown in sequence 3).

[0085] Sequence 2 is as follows:

[0086] MGSSHHHHHHSSGLVPRGSHMASMTGGQQMGRSEFMATVENEQAIAPAAENVEVPTKTVEEPEVTEQPEKVVATTE SAPAPAAVTEEESEAPVVETSKDVVVEEAEEKEEEAEKETEEPKVEEEEEKTENPAVVEEPKEEEKEEETETPAVVEEEE KKTEAEEVVAAEEVAAEKAEE.

[0087] Sequence 3 is as follows:

[0088] MATVENEQAIAPAAENVEVPTKTVEEPEVTEQPEKVVATTESAPAPAAVTEEESEAPVVETSKDVVVEEAEEKEEE AEKETEEPKVEEEEEKTENPAVVEEPKEEEKEEETETPAVVEEEEKKTEAEEVVAAEEVAAEKAEE.

[0089] The results are as follows Figure 1 As shown, Figure 1In the figure, the lane before induction is the first lane from the left, the lane after induction is the second lane from the left, the supernatant lane is the third lane from the left, the precipitation lane is the fourth lane from the left, the column lane is the fifth lane from the left, and the washing lane is the sixth lane from the left. Marker is a protein standard marking the molecular weight, and the sizes from top to bottom are 200KD, 150KD, 100KD, 70KD, 50KD, 37KD, 25KD, 20KD and 15KD, and the proportions of E1-E6 are the eluted proteins collected in the above six tubes (E1, E2, E3, E4, E5 and E6 respectively).

[0090] The results showed that 1 mM IPTG could strongly induce the expression of His-BrVCaBL protein in the BL21 / pET28a-BrVCaBL strain, and after ultrasonic disruption of the bacteria, most of the His-BrVCaBL protein remained in the supernatant. After nickel column affinity purification, a high-purity His-BrVCaBL prokaryotic expression protein solution was obtained.

[0091] Acquisition of functional peptides

[0092] The present invention further analyzes and finds two key functional peptides in the BrVCaBL protein, namely the key peptide AN-8 (also known as AN-8 or short peptide X1) and the key peptide AN-9 (also known as AN-9 or short peptide X2). After artificial synthesis, the peptides are diluted to 10 μM with sterile water and sprayed evenly on the leaves of cabbage crops at 1 ml / plant / day. After three days of pretreatment, high temperature treatment is performed, which can significantly reduce the wilting and chlorosis of leaves caused by high temperature stress and significantly improve the high temperature tolerance of cabbage. The specific method is as follows:

[0093] The amino acid sequences of AN-8, EV-9 and AA-11 (AA-11 is a control peptide randomly selected from the BrVCaBL amino acid sequence, also known as a control peptide) were sent to Beijing Liuhe BGI Gene Technology Co., Ltd., where AN-8 and EV-9 are key functional peptides of BrVCaBL, and AA-11 is a control peptide randomly selected from the BrVCaBL amino acid sequence. The peptide dry powder with a purity of 90% was commissioned for synthesis, and the peptide was tested by mass spectrometry.

[0094] The amino acid sequence of the key peptide AN-8 is AIAPAAEN.

[0095] The amino acid sequence of the key peptide EV-9 is EEVVAAEEV.

[0096] The amino acid sequence of the control peptide AA-11 is ATTESAPAPAA.

[0097] Peptides were synthesized on an automated solid phase peptide synthesizer (ResPep SL, Intavis) using 9-fluorenylmethoxycarbonyl (Fmoc) chemistry activated with 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU). Peptides were cleaved from the resin with trifluoroacetic acid (TFA) / triisopropylsilane (TIS) / water / dithiothreitol (DTT) (90 [volume ratio]:5 [volume ratio]:2.5 [volume ratio]:2.5 [mass ratio]) and precipitated with ice-cold diethyl ether.

[0098] The peptides were dissolved in 1:1 Milli-Q water:acetonitrile and purified by reversed-phase HPLC (HPLC) using a semi-preparative HPLC column (Waters) equipped with a polystyrene divinylbenzene column (PolymerX; bead size, 10 μm, 250 mm × 10 mm; Phenomenex). Purity was confirmed by analytical reversed-phase ultra-high performance liquid chromatography (UPLC Aquity) equipped with a UV detector and an analytical C18 column (bead size, 1.7 μm), and the peptide products were characterized by electrospray ionization.

[0099] The results are as follows Figure 2 As shown, AN-8 is the mass spectrum of AN-8, EV-9 is the mass spectrum of AEV-9, and AA-11 is the mass spectrum of AA-11. The mass spectra all show a single peak, indicating that the three synthetic polypeptides are of high purity, have not been degraded, and do not contain other impurities and contamination, and can be used for subsequent experiments.

[0100] Obtaining prokaryotic purified His-BrVCaBL protein

[0101] The eluted proteins in tubes 1 to 5 (E1-E5) prepared above were mixed to obtain a His-BrVCaBL protein solution. The concentration of the protein solution was determined using a microspectrophotometer (OSE-260-25, Tiangen). The protein concentration in the His-BrVCaBL protein solution was diluted to 10 μM using sterile water to obtain a 10 μM His-BrVCaBL protein solution, which was placed on ice and stored at 4°C.

[0102] The elution buffer was diluted with sterile water to make the concentration of the elution buffer the same as that in the 10 μM His-BrVCaBL protein solution, thereby obtaining a control 10 μM His-BrVCaBL protein solution.

[0103] Obtaining synthetic peptide solution:

[0104] The synthesized peptide powders were first dissolved with three volumes of dimethyl sulfoxide (DMSO) to obtain AN-8 solution, EV-9 solution and AA-11 solution. Then, the protein concentrations in the short peptide solutions (AN-8 solution, EV-9 solution and AA-11 solution) were diluted to 2 mM with sterile water to obtain 2 mM AN-8 mother solution, 2 mM EV-9 mother solution and 2 mM AA-11 mother solution, which were stored at 4 °C. Before the treatment experiment, the 2 mM mother solution was diluted to 10 μM with sterile water to obtain 10 mM AN-8 working solution, 10 mM EV-9 working solution and 10 mM AA-11 working solution. .

[0105] Example 2: Exogenous application of His-BrVCaBL protein to enhance the heat resistance of Chinese cabbage

[0106] The Chinese cabbage seeds (Shengnong Green Treasure Willow Leaf 70-day Chinese cabbage) were fully swollen by absorbing water, germinated at 25°C, and sown in nutrient soil (PINDSTRUP, suitable for fruits and vegetables, Denmark), and cultured at 21°C under long-day lighting (16h light / 8h dark) for 4 weeks. The 4-week-old Shengnong Green Treasure Willow Leaf 70-day Chinese cabbage seedlings were randomly divided into two groups of equal number, 32 seeds in each group, namely the experimental group and the control group.

[0107] The experimental group performed the following operations:

[0108] The experimental group was sprayed evenly with 10 μM His-BrVCaBL protein solution at a dosage of 1 mL / plant / day throughout the experiment. After pretreatment (21°C, long day (16 h light / 8 h dark) conditions) for 3 days, the phenotype was recorded by photographing (recorded as before treatment), and the plants were transferred to 45°C, long day (16 h light / 8 h dark) conditions for cultivation for 7 days. The phenotype was recorded by photographing (recorded as 45°C treatment for 7 days), and the degree of leaf wilting was counted; the sixth true leaf of each Chinese cabbage was collected (counting from the first true leaf grown in the order of true leaf growth) and the fresh weight was determined; and the sixth true leaf was randomly mixed into three parts for the determination of malondialdehyde, soluble sugar, proline and chlorophyll contents.

[0109] The control group underwent the following operations:

[0110] The control group experiment was uniformly sprayed with elution buffer diluted 40 times with sterile water at a dosage of 1 mL / plant / day throughout the experiment (the dilution factor was the same as the above-mentioned His-BrVCaBL elution protein solution). After pretreatment (21°C, long day (16 h light / 8 h dark) conditions) for 3 days, the phenotype was recorded by photographing (recorded as before treatment), and the plants were transferred to 45°C, long day (16 h light / 8 h dark) conditions for cultivation for 7 days, and the phenotype was recorded by photographing (recorded as 45°C treatment for 7 days), and the degree of leaf wilting was counted; the sixth true leaf of each Chinese cabbage was collected (according to the growth order of the true leaves, counting from the first true leaf grown), and the fresh weight was determined; and the sixth true leaf was randomly mixed into three parts for the determination of malondialdehyde, soluble sugar, proline and chlorophyll contents.

[0111] Leaf wilting rate statistics:

[0112] Wilting degree evaluation criteria: no symptoms are 0, only local leaves are obviously curled or yellowed and wilted is 0.25, local leaves are obviously curled or yellowed and wilted is 0.5, the whole leaf is wrinkled or most of the leaves are yellowed and wilted is 0.75, and the whole leaf is yellowed and dead is 1. The calculation formula is as follows:

[0113] Plant wilting rate = ∑ wilting rate of each leaf / total number of leaves

[0114] Total wilting rate of treatment = ∑ wilting rate of each plant / total number of plants

[0115] Determination of leaf fresh weight: After the treatment, cut off the sixth true leaf of the plant and weigh the fresh weight immediately.

[0116] Chlorophyll content determination: Cut off the sixth true leaf of the control group and the experimental group, remove the main veins, and randomly divide them into three groups, grind them into powder with liquid nitrogen, take out 0.1g from each group, and use the plant chlorophyll content kit (RXWB0287-96, Quanzhou Ruixin Biotechnology Co., Ltd.) to determine the total chlorophyll content. The calculation formula is as follows:

[0117] Total chlorophyll content (mg / g fresh weight) = = 0.01 × (20.21 × A645 + 8.02 × A663) × 8 ÷ 0.1

[0118] Determination of proline content: The sixth true leaf of the control group and experimental group plants was cut off, and the main veins were removed. Then they were randomly divided into three groups and ground into powder with liquid nitrogen. 0.1 g was taken out from each group, and the proline content was determined using a proline (Pro) content kit (RXWB0160-96, Quanzhou Ruixin Biotechnology Co., Ltd.).

[0119] Determination of soluble sugar content: The sixth true leaf of the plants in the control group and the experimental group was cut off, and the main veins were removed. Then, the plants were randomly divided into three groups and ground into powder with liquid nitrogen. 0.1 g was taken out from each group, and the soluble sugar content was determined using a soluble sugar content (SS) kit (RXWB0386-96, Quanzhou Ruixin Biotechnology Co., Ltd.).

[0120] Determination of malondialdehyde content: The sixth true leaf of the control group and experimental group plants was cut off, and the main veins were removed. Then they were randomly divided into three groups and ground into powder with liquid nitrogen. 0.1 g was taken out from each group, and the malondialdehyde content was sequenced using a malondialdehyde content (MDA) kit (RXWB0005-96, Quanzhou Ruixin Biotechnology Co., Ltd.).

[0121] The control group underwent the following operations:

[0122] The only difference between the control group and the experimental group was that the 10 μM His-BrVCaBL protein solution was replaced by the 10 μM His-BrVCaBL protein solution control solution, and the rest of the operations were the same as those of the experimental group.

[0123] The results are as follows Figure 3 ( Figure 3 The experimental group was the experimental group, and the control group was the control group; Figure 3 A in the middle is the phenotypic photo before treatment, and the phenotypic photo after treatment at 45℃ for 7 days is the phenotypic photo after treatment at 45℃ for 7 days; Figure 3 B is the wilting rate, Figure 3 C in the middle is the fresh weight of leaves, Figure 3 D is the chlorophyll content. Figure 3 E is the proline content, Figure 3 F is the soluble sugar content. Figure 3 G in the figure is the wilting rate (meaning malondialdehyde content). Compared with the control group, the wilting degree of leaves in the experimental group was significantly reduced, the fresh weight and chlorophyll content of the leaves increased, while the proline and soluble sugar contents increased, and the malondialdehyde content decreased, indicating that the exogenous application of His-BrVCaBL protein significantly alleviated the leaf wilting and chlorosis caused by high temperature stress, and improved the high temperature tolerance of Chinese cabbage.

[0124] Example 3: Exogenous application of His-BrVCaBL protein enhances the heat tolerance of Chinese cabbage

[0125] Chinese cabbage seeds (Chinese cabbage variety Baiyang) were fully water-swelled, germinated at 25°C, sown in nutrient soil (PINDSTRUP, suitable for fruits and vegetables, Denmark), and cultured at 21°C under long-day (16h light / 8h dark) for 5 weeks. The 5-week-old Baiyang seedlings were randomly divided into two groups of equal number, 28 in each group, namely the experimental group and the control group.

[0126] The experimental group performed the following operations:

[0127] The experimental group was uniformly sprayed with 10 μM His-BrVCaBL protein solution at a dosage of 1 mL / plant / day throughout the experiment. After pretreatment (21°C, long day (16 h light / 8 h dark) conditions) for 3 days, the phenotype was recorded by photographing (recorded as before treatment), and the plants were transferred to 45°C, long day (16 h light / 8 h dark) conditions for cultivation for 3 days, and the phenotype was recorded by photographing (recorded as 45°C treatment for 3 days), and the degree of leaf wilting was counted; the sixth true leaf of each Chinese cabbage was collected (according to the growth order of the true leaves, counting from the first true leaf grown), and the fresh weight was determined; and the sixth true leaf was randomly mixed into three parts for the determination of malondialdehyde, soluble sugar, proline and chlorophyll contents.

[0128] The control group underwent the following operations:

[0129] In the control group experiment, the elution buffer diluted 40 times with sterile water was evenly sprayed at a dosage of 1 mL / plant / day throughout the experiment (the dilution multiple was the same as the above-mentioned His-BrVCaBL elution protein solution). After pretreatment (21°C, long day (16 h light / 8 h dark) conditions) for 3 days, the phenotype was recorded by taking photos (recorded as before treatment), and the plants were transferred to 45°C, long day (16 h light / 8 h dark) conditions for cultivation for 3 days, and the phenotype was recorded by taking photos (recorded as 45°C treatment for 3 days), and the degree of leaf wilting was counted; the sixth true leaf of each Chinese cabbage was collected (according to the growth order of the true leaves, counting from the first true leaf grown) and the fresh weight was determined; and the sixth true leaf was randomly mixed into three parts for the determination of malondialdehyde, soluble sugar, proline and chlorophyll contents.

[0130] Leaf wilting rate statistics:

[0131] Wilting degree evaluation criteria: no symptoms are 0, only local leaves are obviously curled or yellowed and wilted is 0.25, local leaves are obviously curled or yellowed and wilted is 0.5, the whole leaf is wrinkled or most of the leaves are yellowed and wilted is 0.75, and the whole leaf is yellowed and dead is 1. The calculation formula is as follows:

[0132] Plant wilting rate = ∑ wilting rate of each leaf / total number of leaves

[0133] Total wilting rate of treatment = ∑ wilting rate of each plant / total number of plants

[0134] Determination of leaf fresh weight: After the treatment, cut off the sixth true leaf of the plant and weigh the fresh weight immediately.

[0135] Chlorophyll content determination: Cut off the sixth true leaf of the control group and the experimental group, remove the main veins, and randomly divide them into three groups, grind them into powder with liquid nitrogen, take out 0.1g from each group, and use the plant chlorophyll content kit (RXWB0287-96, Quanzhou Ruixin Biotechnology Co., Ltd.) to determine the total chlorophyll content. The calculation formula is as follows:

[0136] Total chlorophyll content (mg / g fresh weight) = = 0.01 × (20.21 × A645 + 8.02 × A663) × 8 ÷ 0.1

[0137] Determination of proline content: The sixth true leaf of the control group and experimental group plants was cut off, and the main veins were removed. Then they were randomly divided into three groups and ground into powder with liquid nitrogen. 0.1 g was taken out from each group, and the proline content was determined using a proline (Pro) content kit (RXWB0160-96, Quanzhou Ruixin Biotechnology Co., Ltd.).

[0138] Determination of soluble sugar content: The sixth true leaf of the plants in the control group and the experimental group was cut off, and the main veins were removed. Then, the plants were randomly divided into three groups and ground into powder with liquid nitrogen. 0.1 g was taken out from each group, and the soluble sugar content was determined using a soluble sugar content (SS) kit (RXWB0386-96, Quanzhou Ruixin Biotechnology Co., Ltd.).

[0139] Determination of malondialdehyde content: The sixth true leaf of the control group and experimental group plants was cut off, and the main veins were removed. Then they were randomly divided into three groups and ground into powder with liquid nitrogen. 0.1 g was taken out from each group, and the malondialdehyde content was sequenced using a malondialdehyde content (MDA) kit (RXWB0005-96, Quanzhou Ruixin Biotechnology Co., Ltd.).

[0140] The control group underwent the following operations:

[0141] The only difference between the control group and the experimental group was that the 10 μM His-BrVCaBL protein solution was replaced by the 10 μM His-BrVCaBL protein solution control solution, and the rest of the operations were the same as those of the experimental group.

[0142] The results are as follows Figure 4 ( Figure 4 The experimental group was the experimental group, and the control group was the control group; Figure 4 A in the middle is the phenotypic photo before treatment, and the phenotypic photo after treatment at 45℃ for 3 days is the phenotypic photo after treatment at 45℃ for 3 days; Figure 4 B is the wilting rate, Figure 4 C in the middle is the fresh weight of leaves, Figure 4 D is the chlorophyll content. Figure 4 E is the proline content, Figure 4 F is the soluble sugar content. Figure 4G in the figure is the wilting rate (meaning malondialdehyde content). Compared with the control group, the wilting degree of the leaves of the Chinese cabbage in the experimental group was significantly reduced, the fresh weight and chlorophyll content of the leaves increased, while the proline and soluble sugar contents increased, and the malondialdehyde content decreased, indicating that the exogenous application of His-BrVCaBL protein significantly alleviated the leaf wilting and chlorosis caused by high temperature stress, and improved the high temperature tolerance of the Chinese cabbage.

[0143] Example 4: Exogenous application of key functional peptides to enhance the heat resistance of Chinese cabbage

[0144] The protein concentration in the His-BrVCaBL protein solution was diluted to 10 μM using sterile water to obtain a 10 μM His-BrVCaBL protein solution.

[0145] The elution buffer was diluted with sterile water to make the concentration of the elution buffer the same as that in the 10 μM His-BrVCaBL protein solution, thereby obtaining a control 10 μM His-BrVCaBL protein solution.

[0146] The protein concentration in the AN-8 solution was diluted to 10 μM using sterile water to obtain a 10 μM AN-8 solution.

[0147] The protein concentration in the EV-9 solution was diluted to 10 μM using sterile water to obtain a 10 μM EV-9 solution.

[0148] The protein concentration in the AA-11 solution was diluted to 10 μM using sterile water to obtain a 10 μM AA-11 solution.

[0149] The Chinese cabbage seeds (Shengnong Green Treasure Willow Leaf 70-day Chinese cabbage) were fully swollen by absorbing water, germinated at 25°C, sown in nutrient soil, and cultured at 21°C under long-day lighting (16h light / 8h dark) for 4 weeks. The 4-week-old Shengnong Green Treasure Willow Leaf 70-day Chinese cabbage seedlings were randomly divided into 5 groups of equal number, with 32 seeds in each group, namely, 10μM His-BrVCaBL protein solution group, 10μM AN-8 solution group, 10μMEV-9 solution group, 10μM AA-11 solution group and control group.

[0150] The 10 μM His-BrVCaBL protein solution group was operated as follows:

[0151] The experimental group was sprayed evenly with 10 μM His-BrVCaBL protein solution at a dosage of 1 mL / plant / day throughout the experiment. After pretreatment (21°C, long day (16 h light / 8 h dark) conditions) for 3 days, the phenotype was recorded by photographing (recorded as 21°C, four weeks), and transferred to 45°C, long day (16 h light / 8 h dark) conditions for cultivation for 7 days. The phenotype was recorded by photographing (recorded as 21°C, four weeks, 45°C, 7 days), and the degree of leaf wilting was counted; the aerial part of each Chinese cabbage was collected and the fresh weight was determined; the aerial part of the Chinese cabbage was randomly mixed into three parts for the determination of malondialdehyde, soluble sugar, proline and chlorophyll contents.

[0152] The control group underwent the following operations:

[0153] The control group experiment was uniformly sprayed with elution buffer diluted 40 times with sterile water at a dosage of 1 mL / plant / day throughout the experiment (the dilution multiple was the same as the above-mentioned His-BrVCaBL elution protein solution). After pretreatment (21°C, long day (16h light / 8h dark) conditions) for 3 days, the phenotype was recorded by taking photos (recorded as 21°C, four weeks), and transferred to 45°C, long day (16h light / 8h dark) conditions for cultivation for 7 days, and the phenotype was recorded by taking photos (recorded as 21°C, four weeks, 45°C, 7 days), and the degree of leaf wilting was counted; the above-ground part of each Chinese cabbage was collected and the fresh weight was determined; and the sixth true leaf was randomly mixed into three parts for the determination of malondialdehyde, soluble sugar, proline and chlorophyll content. Leaf wilting rate statistics:

[0154] Wilting degree evaluation criteria: no symptoms are 0, only local leaves are obviously curled or yellowed and wilted is 0.25, local leaves are obviously curled or yellowed and wilted is 0.5, the whole leaf is wrinkled or most of the leaves are yellowed and wilted is 0.75, and the whole leaf is yellowed and dead is 1. The calculation formula is as follows:

[0155] Plant wilting rate = ∑ wilting rate of each leaf / total number of leaves

[0156] Total wilting rate of treatment = ∑ wilting rate of each plant / total number of plants

[0157] Determination of above-ground fresh weight: After the treatment, cut off the above-ground part of the plant and weigh the fresh weight immediately.

[0158] Chlorophyll content determination: Cut off the sixth true leaf of the control group and the experimental group, remove the main veins, and randomly divide them into three groups, grind them into powder with liquid nitrogen, take out 0.1g from each group, and use the plant chlorophyll content kit (RXWB0287-96, Quanzhou Ruixin Biotechnology Co., Ltd.) to determine the total chlorophyll content. The calculation formula is as follows:

[0159] Total chlorophyll content (mg / g fresh weight) = = 0.01 × (20.21 × A645 + 8.02 × A663) × 8 ÷ 0.1

[0160] Determination of proline content: The sixth true leaf of the control group and experimental group plants was cut off, and the main veins were removed. Then they were randomly divided into three groups and ground into powder with liquid nitrogen. 0.1 g was taken out from each group, and the proline content was determined using a proline (Pro) content kit (RXWB0160-96, Quanzhou Ruixin Biotechnology Co., Ltd.).

[0161] Determination of soluble sugar content: The sixth true leaf of the plants in the control group and the experimental group was cut off, and the main veins were removed. Then, the plants were randomly divided into three groups and ground into powder with liquid nitrogen. 0.1 g was taken out from each group, and the soluble sugar content was determined using a soluble sugar content (SS) kit (RXWB0386-96, Quanzhou Ruixin Biotechnology Co., Ltd.).

[0162] Determination of malondialdehyde content: The sixth true leaf of the control group and experimental group plants was cut off, and the main veins were removed. Then they were randomly divided into three groups and ground into powder with liquid nitrogen. 0.1 g was taken out from each group, and the malondialdehyde content was sequenced using a malondialdehyde content (MDA) kit (RXWB0005-96, Quanzhou Ruixin Biotechnology Co., Ltd.).

[0163] The 10 μM AN-8 solution group was operated as follows:

[0164] The difference between the 10 μM AN-8 solution group and the 10 μM His-BrVCaBL protein solution group was that the 10 μM His-BrVCaBL protein solution was replaced by the 10 μM AN-8 solution, and the rest of the operations were the same as the experimental group.

[0165] The 10 μM EV-9 solution group was operated as follows:

[0166] The difference between the 10 μM EV-9 solution group and the 10 μM His-BrVCaBL protein solution group was that the 10 μM EV-9 solution was used to replace the 10 μM His-BrVCaBL protein solution, and the rest of the operations were the same as the experimental group.

[0167] The 10 μM AA-11 solution group was operated as follows:

[0168] The difference between the 10 μM AA-11 solution group and the 10 μM His-BrVCaBL protein solution group was that the 10 μM His-BrVCaBL protein solution was replaced by the 10 μM AA-11 solution, and the rest of the operations were the same as the experimental group.

[0169] The results are as follows Figure 5 ( Figure 5The middle control is the control group, AN-8 is the 10 μM AN-8 solution group, EV-9 is the 10 μM EV-9 solution group, AA-11 is the 10 μM AA-11 solution group, and His-510 is the His-510 group; Figure 5 A in the middle is a phenotypic photo, “21°C, four weeks” is a phenotypic photo taken at 21°C for four weeks, and “21°C, four weeks, 40°C, 7 days” is a phenotypic photo taken at 21°C, four weeks, 40°C, and 7 days; Figure 5 B is the wilting rate, Figure 5 C in the middle is the above-ground fresh weight, Figure 5 D is the chlorophyll content. Figure 5 E is the proline content, Figure 5 F is the soluble sugar content. Figure 5 G in the figure is the wilting rate (meaning malondialdehyde content). Compared with the two control groups, the wilting degree of leaves in the three experimental groups was significantly reduced, the fresh weight and chlorophyll content of the leaves increased, while the content of proline and soluble sugar increased, and the malondialdehyde content decreased. This shows that the exogenous application of EV-9 and AN-8 polypeptide solutions has the same function as the exogenous application of His-BrVCaBL protein, which significantly alleviated the leaf wilting and chlorosis caused by high temperature stress and improved the high temperature tolerance of Chinese cabbage.

[0170] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be implemented in a wide range under equivalent parameters, concentrations and conditions without departing from the spirit and scope of the present invention and without the need for unnecessary experimentation. Although the present invention provides specific embodiments, it should be understood that further improvements may be made to the present invention. In short, according to the principles of the present invention, this application intends to include any changes, uses or improvements to the present invention, including changes made by conventional techniques known in the art that depart from the scope disclosed in this application. Applications of some of the basic features may be made within the scope of the following appended claims.

Claims

1. A polypeptide or a pharmaceutically acceptable salt or solvate thereof, characterized in that: The polypeptide contains at least one of a short peptide X1 and a short peptide X2; The amino acid sequence of the short peptide X1 is AIAPAAEN, and the amino acid sequence of the short peptide X2 is EEVVAAEEV.

2. The polypeptide according to claim 1 or a pharmaceutically acceptable salt or solvate thereof, characterized in that: The amino acid number of the polypeptide is no more than 142.

3. The polypeptide according to claim 1 or a pharmaceutically acceptable salt or solvate thereof, characterized in that: The polypeptide is selected from any one of the following: 1) The short peptide X1 as claimed in claim 1; 2) The short peptide X2 according to claim 1; 3) A polypeptide having an amino acid sequence as described in SEQ ID NO: 2 or positions 36 to 177 of SEQ ID NO:

2.

4. The polypeptide or pharmaceutically acceptable salt or solvate thereof according to claim 1, characterized in that: The polypeptide is a polypeptide obtained by performing at least one of the following modifications: A1) a polypeptide obtained by connecting an amino terminal protecting group to the amino terminal of the polypeptide and / or connecting a carboxyl terminal protecting group to the carboxyl terminal of the polypeptide; A2) a polypeptide obtained by adding amino acid residues to the amino terminus and / or carboxyl terminus of the polypeptide; A3) A polypeptide obtained by linking a fatty acid to the amino terminus and / or carboxyl terminus of the polypeptide.

5. A pharmaceutical composition comprising the peptide compound according to any one of claims 1 to 4 or a pharmaceutically acceptable salt or solvate thereof, and a pharmaceutically acceptable carrier or excipient.

6. Use of the polypeptide according to any one of claims 1 to 4 or a pharmaceutically acceptable salt or solvate thereof and / or the pharmaceutical composition according to claim 5 in improving plant high temperature tolerance or in preparing a product for improving plant high temperature tolerance.

7. The use according to claim 6, characterized in that The high temperature tolerance index is at least one of wilting rate, leaf fresh weight, above-ground fresh weight, chlorophyll content, proline content, soluble sugar content and malondialdehyde content.

8. A method for improving high temperature tolerance of plants, characterized in that: The method comprises the step of applying the polypeptide according to any one of claims 1 to 4 or a pharmaceutically acceptable salt or solvate thereof and / or the pharmaceutical composition according to claim 5 to a plant.

9. A method for growing a plant, characterized in that: The method comprises growing a plant using the method of claim 8.

10. The use according to any one of claims 6 to 8 or the method according to claim 9, characterized in that: The plant is any of the following: B1) Cruciferous vegetables; B2) Brassica plants; B3) Chinese cabbage or choy sum.

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