Hrpx protein growth-promoting peptides and their applications
By optimizing the amino acid sequence design of the Hrpx protein, Hrpx-1, Hrpx-8, Hrpx-9 and Hrpx-10 growth-promoting peptides were developed, solving the problems of low efficiency and insufficient thermal stability of the Harpin protein in plant growth regulation, and achieving a more efficient plant growth promotion effect.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- HEBEI MONBAND WATER SOLUBLE FERTILIZER CO LTD
- Filing Date
- 2023-07-18
- Publication Date
- 2026-06-30
Smart Images
Figure CN117106026B_ABST
Abstract
Description
Technical Field
[0001] This invention relates to growth-promoting peptides and their applications. Background Technology
[0002] Harpin proteins are a class of proteins belonging to a protein family, mostly found in Gram-negative bacteria. They were first discovered by Dr. Wei Zhongmin in *Brwinia amylovora*, a Gram-negative bacterium, and are encoded by the *hrp* gene. Current research indicates that these proteins are relatively stable at high temperatures, exhibiting thermostability; they readily react with some proteases, such as proteinase K; and they are rich in glycine, with some Harpin proteins containing small amounts of cysteine. Besides inducing pathogenic responses in susceptible host plants, and triggering allergic reactions and growth-promoting effects in resistant and non-host plants, Harpin proteins can also induce many desirable plant traits, such as increased yield and quality, and enhanced plant immunity. Summary of the Invention
[0003] The purpose of this invention is to provide Hrpx protein growth peptides that have superior performance compared to full-length Harpin protein.
[0004] The Hrpx protein growth-promoting peptide of this invention is Hrpx-1, which can promote plant growth; the amino acid sequence of Hrpx-1 is shown in SEQ ID NO: 1.
[0005] Another Hrpx protein growth-promoting peptide of the present invention is Hrpx-8, which can promote plant growth; the amino acid sequence of Hrpx-8 is shown in SEQ ID NO: 2.
[0006] Another Hrpx protein growth-promoting peptide of the present invention is Hrpx-9, which can promote plant growth; the amino acid sequence of Hrpx-9 is shown in SEQ ID NO: 3.
[0007] Another Hrpx protein growth-promoting peptide of the present invention is Hrpx-10, which can promote plant growth; the amino acid sequence of Hrpx-10 is shown in SEQ ID NO: 4.
[0008] The application of Hrpx protein growth-promoting peptide as a plant growth regulator, with Hrpx-1 being the active ingredient in plant growth regulators.
[0009] The application of Hrpx protein growth-promoting peptide as a plant growth regulator, with Hrpx-8 being the active ingredient in plant growth regulators.
[0010] The application of Hrpx protein growth-promoting peptide as a plant growth regulator, with Hrpx-9 being the active ingredient in plant growth regulators.
[0011] The application of Hrpx protein growth-promoting peptide as a plant growth regulator, with Hrpx-10 being the active ingredient in plant growth regulators.
[0012] The Arabidopsis seeds soaked with Hrpx-1, Hrpx-8, Hrpx-9, and Hrpx-10 of this invention exhibited stronger roots and more branches than the control group, with significantly higher average root length and average fresh weight (*P<0.05, **P<0.01). Furthermore, the average root length and fresh weight of seeds treated with Hrpx-1, Hrpx-8, Hrpx-9, and Hrpx-10 were significantly better than the control group and also greater than those treated with Hrpx. The Hrpx protein growth-promoting peptide of this invention is superior to the full-length Hrpx protein and exhibits greater thermal stability. Attached Figure Description
[0013] Figure 1 This is a diagram showing the analysis results of the amino acid sequence of the Hrpx protein using the ExPASy protein hydrophilicity / hydrophobicity prediction tool in Specific Implementation Method 1; where amino acid residues above the threshold line are hydrophobic and amino acid residues below the threshold line are hydrophilic.
[0014] Figure 2 This is a diagram showing the results of online transmembrane region analysis of the Hrpx protein using TMHMM Server in Specific Implementation Method 1.
[0015] Figure 3 This is a diagram showing the results of the online prediction of the secondary structure of the Hrpx protein using NPS@:SOPMA secondary structure prediction in Implementation Method 1.
[0016] Figure 4 This is a predicted diagram of the coiled helix structure of the Hrpx protein according to the present invention;
[0017] Figure 5 This is a predicted diagram of the tertiary structure of the Hrpx protein according to the present invention;
[0018] Figure 6 This is the predicted Hrpx protein signal peptide diagram of the present invention;
[0019] Figure 7 This is a predicted phosphorylation site map of the Hrpx protein in this invention;
[0020] Figure 8 This is a graph showing the results of the Hrpx protein activity assay of this invention;
[0021] Figure 9 These are bar graphs of Arabidopsis roots treated with different concentrations of protein in Example 2;
[0022] Figure 10 This is a comparative diagram showing the effects of Hrpx and multiple peptides on Arabidopsis root growth in Example 2; among them, Figure 10 -a was pre-soaked with purified Hrpx and multiple peptides for 12 h, and then cultured on MS medium for 7 days, with PD 10 Buffer as a negative control; the scale bar represents 1 cm; Figure 10 Root length and fresh weight were measured 7 days after transfer to MS medium; error bars represent the standard error of the mean (n=3);
[0023] Figure 11 This is a diagram showing the effects of Hrpx, Hrpx-B, Hrpx-9, and Hrpx-9B on the root length of Arabidopsis thaliana in Example 2; among them, Figure 11 -a Arabidopsis seeds were soaked in purified protein for 12 h and then cultured on MS medium for 14 d, with PD10 Buffer as a negative control; the scale bar represents 1 cm; Figure 11 Root length was measured 14 days after transfer to MS medium. Error bars represent the standard error of the mean (n=3).
[0024] Figure 12 This is a comparative diagram showing the effects of Hrpx, Hrpx-B, Hrpx-9, and Hrpx-9B on the aboveground parts of Arabidopsis thaliana in Example 2; among them, Figure 12 -a After culturing Arabidopsis seeds in a tissue culture incubator for 7 days, they were transplanted in large quantities to soil and cultured for 30 days, during which time protein was sprayed on them every 5 days; PD 10 Buffer was used as a negative control; Figure 12 -b Measure the blade span length after 30 days; the error bars represent the standard error of the mean (n=3). Detailed Implementation
[0025] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.
[0026] It should be noted that, unless otherwise specified, the embodiments and features described in the present invention can be combined with each other.
[0027] Specific implementation method one: The Hrpx protein in this implementation method has the amino acid sequence shown in SEQ ID NO: 5.
[0028] The Hrpx protein of this invention consists of 137 amino acids, has a relative molecular mass of 13631.48, a theoretical isoelectric point of 4.06, and a molecular formula of C0.06. 557 H 883 N 175 O 215 S5 has a total of 1835 atoms. Among the 137 amino acids in the Hrp protein, glycine (Gly), glutamine (Gln), serine (Ser), and leucine (Leu) are abundant, accounting for 20.40%, 15.30%, 13.90%, and 7.30% of the total amino acids, respectively. There are four positively charged arginine (Arp) and lysine (Lys) amino acids, and eleven negatively charged aspartic acid (Asp) and glutamate (Glu). Because the Hrpx protein is rich in glycine and contains only one cysteine residue, it has high thermal stability. The end of the sequence is glutamine (Gln). Its half-life in mammalian cells in vitro is 30 h; in yeast, it is >20 h; and in E. coli, it is >10 h.
[0029] The amino acid sequence of the Hrpx protein was analyzed using the ExPASy protein hydrophilicity / hydrophobicity prediction tool. The results are as follows: Figure 1 As shown, the hydrophilic amino acids of the Hrpx protein are concentrated between positions 5-44 and 54-89, while the hydrophobic amino acids are concentrated between positions 44-53 and 89-102. The Hrpx protein as a whole is a hydrophilic protein. Its Arg score at position 60 is the lowest (-2.444), indicating the strongest hydrophilicity; Leu at position 49 and Ile at position 50 have the highest scores (1.889), indicating the strongest hydrophobicity.
[0030] The transmembrane region of the Hrpx protein was analyzed online using TMHMM Server, and the results are as follows: Figure 2 As shown, Hrpx protein has no transmembrane region and belongs to non-transmembrane protein, which cannot form a corresponding region with Hrpx affinity. This further confirms that Hrpx is a type of protein that is secreted by the type III secretion system.
[0031] The secondary structure of Hrpx was predicted online using NPS@:SOPMA secondary structure prediction; the analysis results are as follows: Figure 3As shown, its random coils are displayed in yellow (55.47%), α-helices in blue (29.93%), β-turns in green (8.03%), and β-sheets in red (6.57%). It can be seen that Hrpx has a large number of random coils. The α-helices of the Hrpx protein account for 29.93%, concentrated in amino acids at positions 39-54 and 90-104.
[0032] The coil-and-coil structure (CC domain) of the Hrpx protein was analyzed online using COILS Server software. The results are as follows: Figure 4 As shown, the results indicate that there is only one possible coiled-coil structure for the Hrpx protein, located at positions 41-54. Combined with the prediction of the secondary structure of the Hrpx protein above, it was found that the coiled-coil structure of the Hrpx protein overlaps with the α-helix region.
[0033] The SWISS-MODEL database was used to search for the template protein XPsB, which is similar to Hrpx, as a template for modeling. However, the similarity was low, only 48.15%. This is because the Hrpx protein contains a large number of random coils, which makes the analysis of its crystallization and tertiary structure extremely difficult. Therefore, there are no proteins in the database with the resolved 3D structures of proteins similar to Hrpx. The resulting tertiary structure prediction model is as follows: Figure 5 As shown, random coils are the predominant secondary structure, while distinct α-helical structures are also visible. Random coils account for 55.47% of the total secondary structures, and α-helices account for 29.93%, mainly concentrated in amino acids at positions 39-54 and 90-104.
[0034] Signal peptide prediction of the Hrpx protein amino acid sequence was performed online using Signal 4.1 software. The results are as follows: Figure 6 As shown, the C, S, and Y values of Hrpx protein are all less than 0.5, which is below the threshold. Therefore, it is indicated that Hrpx protein has no signal peptide and is a type of protein that does not require a signal peptide to exert its effects. This is consistent with the actual situation, as Hrpx protein itself is a type of protein that acts directly on the cell interior by relying on the type III secretion system.
[0035] The phosphorylation sites of the Hrpx protein were predicted and analyzed using NetPhos 3.1. The results are as follows: Figure 7 As shown, the Hrpx protein is subject to phosphorylation modification, with serine having the most phosphorylation sites and threonine having only one possible phosphorylation site.
[0036] Experiments showed that the Hrpx protein could only maintain its tertiary structure, activity, and stability when the buffer solution consisted of 50 mmol / L Tris, 0.9% NaCl, 20% glycerol, and a pH of 7.5, with a denaturation temperature of 86.9℃.
[0037] The optimal storage temperature for Hrpx protein is 4°C. At 4°C, Hrpx protein does not show significant degradation, and only a small amount of protein remains in the precipitate. At -80°C, although Hrpx protein does not show significant degradation, the amount of protein in the precipitate increases significantly. At 22°C, Hrpx protein shows significant degradation, therefore it is not suitable for storage.
[0038] Specific implementation method 2: The Hrpx protein growth-promoting peptide described in this implementation method is Hrpx-1, which can promote plant growth; the amino acid sequence of Hrpx-1 is shown in SEQ ID NO: 1; the molecular weight is 2132.22, and the isoelectric point is 3.80.
[0039] Specific implementation method three: The Hrpx protein growth-promoting peptide described in this implementation method is Hrpx-8, which can promote plant growth; the amino acid sequence of Hrpx-8 is shown in SEQ ID NO: 2; the molecular weight is 1840.86, and the isoelectric point is 6.00.
[0040] Specific Implementation Method 4: The Hrpx protein growth-promoting peptide described in this implementation method is Hrpx-9, which can promote plant growth; the amino acid sequence of Hrpx-9 is shown in SEQ ID NO: 3; the molecular weight is 2060.35, and the isoelectric point is 4.86.
[0041] Specific Implementation Method 5: The Hrpx protein growth-promoting peptide described in this implementation method is Hrpx-10, which can promote plant growth; the amino acid sequence of Hrpx-10 is shown in SEQ ID NO: 4; the molecular weight is 2045.34, and the isoelectric point is 4.00.
[0042] SEQ ID NO: 1-4 Hrpx protein growth peptides all showed good solubility in buffer (buffer components: 50 mmol / L Tris, 0.9% NaCl, 20% glycerol, and pH 7.5), with a solubility concentration generally ≤2 mg / mL or ≤5 mg / mL.
[0043] Example 1
[0044] The activity of Hrpx protein was experimentally determined in tobacco and Arabidopsis thaliana, with PD 10 buffer as a negative control. A certain concentration of Hrpx protein was diluted 10-fold and 100-fold and injected into tobacco leaves; the presence of an HR response was observed after 24 hours. Arabidopsis thaliana seeds were soaked in diluted Hrpx protein and cultured on MS medium for 7 days; root length was then measured. Diluted Hrpx protein was sprayed onto Arabidopsis thaliana, and the aboveground growth was observed.
[0045] The activity and growth-promoting function of the purified Hrpx protein were verified by measuring its expression in tobacco and Arabidopsis thaliana, respectively. The results are as follows: Figure 8 As shown, Figure 8 -A shows that obvious allergic reactions occurred at the injection sites of Hrpx protein and Hrpx protein diluted 10 times on tobacco leaves, while no changes were observed in the negative control. Figure 8 -B shows that the root length of Arabidopsis thaliana treated with Hrpx protein increased significantly compared with the negative control treatment, with a root length of about 14.44 mm, while the root length of the negative control treatment was about 10 mm. Figure 8 -C indicates that the aboveground parts of Arabidopsis thaliana sprayed with Hrpx protein grew more vigorously than the negative control, and the results were the same for all four replicate experiments.
[0046] In summary, the purified Hrpx protein is active and can induce the HR response in tobacco leaves. It also has growth-promoting function, which can promote root growth and aboveground growth in Arabidopsis thaliana.
[0047] Example 2
[0048] To investigate the effects of Hrpx protein and four peptides (Hrpx-1, Hrpx-8, Hrpx-9, and Hrpx-10) on root growth, germination, and aboveground vegetative growth in plants, Arabidopsis thaliana was selected as a model plant. To ensure seed survival and prevent contamination from affecting the experimental results, the seeds were sterilized and then cultured on sterile MS medium. The treatment method is as follows:
[0049] (1) Take a certain number of wild-type Col-0 Arabidopsis seeds, divide them into 1.5mL centrifuge tubes, add sterile water and soak for 5min, invert and shake several times during the process, and use a pipette to remove the supernatant and floating seeds.
[0050] (2) Add 1 mL of 70% ethanol to the centrifuge tube, invert and shake to fully disinfect the seeds, continue for 1 min, and then remove the supernatant with a pipette.
[0051] (3) Add 1 mL of sterile deionized water to the centrifuge tube, invert and shake several times, and then remove it with a pipette. Repeat 4 times or more.
[0052] (4) Add 1 mL of 3% (v / v) sodium hypochlorite solution to the centrifuge tube and soak for 10 min, inverting and shaking several times during the process to ensure that the seeds are in full contact with the solution. Use a pipette to remove the supernatant.
[0053] (5) Add 1 mL of sterile deionized water to the centrifuge tube, invert and shake several times, and then remove it with a pipette. Repeat this process at least 4 times.
[0054] (6) Add 1 mL of diluted Hrpx protein and peptide to centrifuge tubes respectively, label them, and soak them in a 4℃ refrigerator for 12 h.
[0055] Hrpx protein and four peptides (Hrpx-1, Hrpx-8, Hrpx-9, and Hrpx-10) were diluted with PD 10 Buffer to three different concentrations of 5 μg / mL, 15 μg / mL, and 30 μg / mL, respectively, and dispensed into sterilized EP tubes. Sterilized Arabidopsis thaliana seeds were soaked in these tubes for 12 h. Afterward, the seeds were sown on prepared MS solid medium on a clean bench and placed in a 4°C environment for 2 days to ensure consistent seed growth, which helps break dormancy and promote growth. The seeds were then transferred to a plant culture room, and root length was measured after 7 days.
[0056] Hrpx protein and multiple peptides (including Hrpx-1, Hrpx-8, Hrpx-9, and Hrpx-10) were diluted to a concentration of 15 μg / mL with PD 10 buffer and dispensed into sterilized EP tubes. Sterilized Arabidopsis thaliana seeds were soaked in the tubes for 12 h. The seeds were then sown on prepared MS solid medium on a clean bench and vernalized at 4 °C for 2 days to ensure consistent seed growth, break dormancy, and promote growth. After that, the tubes were transferred to a plant culture room, and the root length was measured after 7 days.
[0057] Hrpx protein and Hrpx-9 were diluted to 15 μg / mL with PD 10 buffer and aliquoted into sterilized EP tubes. One group was boiled in a water bath for 5 min, while the other group was not heated. The sterilized Arabidopsis seeds were soaked in the tubes for 12 h. The seeds were then sown on prepared MS solid medium on a clean bench and vernalized at 4 °C for 2 days to ensure uniform seed growth, break dormancy, and promote growth. After that, the tubes were transferred to a plant culture room, and the root length was measured after 14 days. Hrpx protein and Hrpx-9 peptide were diluted to 15 μg / mL with PD 10 buffer and aliquoted into sterilized EP tubes. One group was boiled in a water bath for 5 min, while the other group was not heated. The sterilized Arabidopsis seeds were vernalized at 4℃ for 2 days to ensure consistent seed growth, break dormancy, and promote growth. The seeds were then sown on prepared MS solid medium on a clean bench and cultured in a plant culture room for 7 days. After 7 days, the seeds were transplanted into soil for cultivation. During this period, the seeds were sprayed with protein every 5 days. Leaf spread length was measured after 30 days.
[0058] result:
[0059] After culturing Arabidopsis seeds treated with different protein concentrations for 7 days, root length was measured. At a protein concentration of 5 μg / mL, the average root length after different protein treatments was approximately 11 mm, the same as the negative control group treated with buffer solution. This indicates that at a protein concentration of 5 μg / mL, the protein concentration was too low to have a significant growth-promoting effect. At a protein concentration of 15 μg / mL, significant changes in root length were observed after different protein treatments. Roots treated with Hrpx protein reached 14 mm in length, a significant increase compared to the 11 mm after buffer treatment. Roots from Hrpx-1, Hrpx-8, Hrpx-9, and Hrpx-10 all reached lengths exceeding 15 mm, demonstrating a significant growth-promoting effect, even better than that of Hrpx protein. At a protein concentration of 30 μg / mL, all experimental groups of Arabidopsis showed a significant growth-promoting effect. The minimum growth-promoting protein concentration in the Arabidopsis growth-promoting experiment was 15 μg / mL.
[0060] Root length bar graphs after treatment with different concentrations of various proteins are shown below. Figure 9 As shown, when the protein concentration is 5 μg / mL, the root length of Arabidopsis thaliana treated with different proteins tends to be gradual, with no obvious growth-promoting effect. When the protein concentration is 15 μg / mL and 30 μg / mL, compared with the protein treated with 5 μg / mL, the root length of Arabidopsis thaliana increased significantly, and the growth-promoting effect was more obvious and the effect tended to be consistent. Therefore, 15 μg / mL was selected as the lowest growth-promoting concentration.
[0061] Arabidopsis seeds were soaked in 15 μg / mL Hrpx and several peptides (including Hrpx-1, Hrpx-8, Hrpx-9, and Hrpx-10) for 12 h, and then transferred to MS medium for culture, with PD 10 buffer as a negative control. After 7 days, Arabidopsis seeds soaked in Hrpx, Hrpx-1, Hrpx-8, Hrpx-9, and Hrpx-10 developed stronger roots and more branches than the control group. Figure 10 As shown in the figure, the average root length and average fresh weight were significantly higher than those of the control group (*P<0.05, **P<0.01), while other peptides showed no significant difference compared to the control group. Specifically, the average root length and fresh weight of seeds treated with Hrpx-9 were significantly better than those of the control group, and also greater than those of seeds treated with Hrpx (as shown in the figure). Figure 10 -b).
[0062] Purified Hrpx, heat-treated Hrpx-B (Hrpx diluted to 10 μM with PD 10 buffer and aliquoted into sterile EP tubes, one group subjected to heat treatment by boiling in a water bath for 5 min), Hrpx-9, and heat-treated Hrpx-9B (Hrpx-9 diluted to 10 μM with PD 10 buffer and aliquoted into sterile EP tubes, one group subjected to heat treatment by boiling in a water bath for 5 min) were soaked in Arabidopsis seeds at the same concentration for 12 h. The seeds were then transferred to MS medium for culture, with PD 10 buffer as a negative control. After 14 days, it was observed that compared to the negative control, the root length of Arabidopsis treated with Hrpx, Hrpx-B, Hrpx-9, and Hrpx-9B was significantly longer, while the root length of Arabidopsis treated with Hrpx-B and Hrpx-9B was shorter than that of those treated with Hrpx and Hrpx-9 (e.g., ...). Figure 11 As shown in the figure. By measuring and comparing root lengths, the root length of Arabidopsis thaliana treated with protein was significantly better than that of the negative control, showing a highly significant difference (**P<0.01). After high-temperature heat treatment, the seed growth of protein-treated seeds slowed down, and the average root length also decreased slightly, but the difference was not statistically significant. Figure 11 -b). Arabidopsis seeds were cultured in a tissue culture incubator for 7 days and then transplanted into soil. The seeds were sprayed with purified Hrpx, heat-treated Hrpx-B, Hrpx-9, and heat-treated Hrpx-9B at the same concentration every 5 days, with PD 10 buffer as a negative control. After 30 days, it was observed that the number and size of the aboveground leaves of Hrpx, Hrpx-B, Hrpx-9, and Hrpx-9B were significantly better than the negative control (e.g., ...). Figure 12-a). By measuring leaf length, the average leaf length of Arabidopsis treated with protein was significantly better than the negative control, and this was highly significant (*P<0.05, **P<0.01). Simultaneously, it was observed that the root length of Hrpx-B was significantly shorter than that of Hrpx after high-temperature heat treatment, while there was no significant difference between Hrpx-9 and Hrpx-9B (as shown in -a). Figure 12 -b is shown).
Claims
1. Hrpx protein-promoting growth peptide, characterized in that, The Hrpx protein growth-promoting peptide is Hrpx-1, Hrpx-8, Hrpx-9, or Hrpx-10, which can promote plant growth; the amino acid sequence of Hrpx-1 is shown in SEQ ID NO: 1, the amino acid sequence of Hrpx-8 is shown in SEQ ID NO: 2, the amino acid sequence of Hrpx-9 is shown in SEQ ID NO: 3, and the amino acid sequence of Hrpx-10 is shown in SEQ ID NO:
4.
2. The application of the Hrpx protein growth-promoting peptide according to claim 1 in promoting plant root growth, characterized in that, The plant in question is Arabidopsis thaliana.