EPFL8b peptide, its applications, and methods for promoting plant regeneration

Abstract

This invention relates to the field of plant regeneration, and provides an EPFL8b polypeptide, its application, and a method for promoting plant regeneration. The amino acid sequence of the EPFL8b polypeptide is shown in SEQ ID NO: 1, and it has the function of promoting plant regeneration, which can solve the problem of the lack of cross-species applicable technical means for promoting plant regeneration in the prior art.

Description

Technical Field

[0001] This invention relates to the field of plant regeneration, and more specifically, to an EPFL8b polypeptide, its application, and a method for promoting plant regeneration. Background Technology

[0002] In the field of plant biotechnology, in vitro regeneration technology for solanaceous crops, represented by tomatoes, and many other crops faces multiple challenges, including low regeneration efficiency, abnormal sensitivity to the proportion of plant growth hormones, and strong genotype dependence. These problems directly restrict the success rate of genetic transformation, prolong the transformation cycle, and affect the stability of the transformation system.

[0003] One of the main strategies for overcoming these obstacles is to optimize the hormone-inducing system, attempting to promote callus regeneration efficiency by adjusting the ratio of cytokinin to auxin. However, the effectiveness of this method is limited by the hormone response characteristics of the crop variety itself, making it difficult to apply to all types and lacking broad applicability and adaptability. Another approach is to utilize specific transcription factors, such as WUSCHEL and CUC2, to regulate the initiation and differentiation of plant stem cells through gene overexpression. While this can improve regeneration efficiency to some extent, the process is complex, requires gene transformation technology, is time-consuming, and its effects are often concentrated in the later stages of the regeneration process, with relatively limited direct regulatory power on the early initiation of stem cells.

[0004] Therefore, existing plant regeneration systems still suffer from technical bottlenecks such as low regeneration efficiency, sensitivity to hormone ratios, and strong dependence on varieties. In particular, there is a lack of a regeneration-promoting technology that can be easily implemented and is applicable across species, especially for solanaceous crops such as tomatoes and other difficult-to-transform crops. Summary of the Invention

[0005] The main objective of this invention is to provide an EPFL8b polypeptide, its application, and a method for promoting plant regeneration, in order to solve the problem of the lack of cross-species applicable technical means for promoting plant regeneration in the prior art.

[0006] To achieve the above objectives, according to a first aspect of the present invention, an EPFL8b polypeptide is provided, the amino acid sequence of which is shown in SEQ ID NO: 1.

[0007] To achieve the above objectives, according to a second aspect of the present invention, an agent for promoting plant regeneration is provided, the agent containing the above-mentioned EPFL8b polypeptide.

[0008] To achieve the above objectives, according to a third aspect of the present invention, a culture medium for promoting plant regeneration is provided, the culture medium containing the above-mentioned EPFL8b polypeptide for promoting plant regeneration or the above-mentioned preparation for promoting plant regeneration.

[0009] Furthermore, the aforementioned culture media include bud induction medium or callus induction medium.

[0010] To achieve the above objectives, according to a fourth aspect of the present invention, the use of the above-mentioned EPFL8b polypeptide for promoting plant regeneration in the preparation of products for promoting plant regeneration is provided.

[0011] Furthermore, the product includes a formulation or a culture medium. To achieve the above objectives, according to a fifth aspect of the invention, a method for promoting plant regeneration is provided, the method comprising: applying the aforementioned polypeptide or the aforementioned formulation for promoting plant regeneration to cultured plant tissue during plant regeneration culture.

[0012] Furthermore, the cultured plant tissue is in the bud primordia induction stage or the callus induction stage.

[0013] Further, the application method includes any of the following: i) applying the above-mentioned EPFL8b peptide for promoting plant regeneration or the above-mentioned preparation for promoting plant regeneration to the cultured plant tissue; ii) applying the above-mentioned EPFL8b peptide for promoting plant regeneration or the above-mentioned preparation for promoting plant regeneration to the regeneration medium in which the cultured plant tissue is inoculated; the regeneration medium includes shoot induction medium and / or callus induction medium; iii) inoculating the cultured plant tissue into a regeneration medium containing the above-mentioned EPFL8b peptide; the regeneration medium includes shoot induction medium and / or callus induction medium.

[0014] Further, the method includes: I) inoculating the cultured plant tissue into a bud induction medium for culture; wherein the bud induction medium is the aforementioned medium, and / or, during the culture process, applying the aforementioned EPFL8b polypeptide for promoting plant regeneration or the aforementioned preparation for promoting plant regeneration to the cultured plant tissue; II) inoculating the cultured plant tissue into a callus induction medium for a first culture to obtain callus tissue; and then inoculating the callus tissue into a bud induction medium for a second culture; wherein the callus induction medium and / or the bud induction medium are each independently selected from the aforementioned medium, and / or, during the first culture and / or the second culture, applying the aforementioned EPFL8b polypeptide for promoting plant regeneration or the aforementioned preparation for promoting plant regeneration to the cultured plant tissue.

[0015] Furthermore, the cultured plant tissues are derived from dicotyledonous or monocotyledonous plants.

[0016] Furthermore, dicotyledonous plants include tomatoes or peppers; monocotyledonous plants include wheat.

[0017] Furthermore, the cultured plant tissues include one or more of the following: callus, cotyledons, embryo, root, stem, leaf, or flower.

[0018] Furthermore, the working concentration of the above-mentioned polypeptides or preparations for promoting plant regeneration is 0.5-5000 μg / L.

[0019] To achieve the above objectives, according to a sixth aspect of the present invention, a method is provided. EPFL8b The application of genes in regulating plant regeneration includes: EPFL8b Gene editing, through upregulation EPFL8b The expression of gene-encoded proteins promotes plant regeneration; or... EPFL8b Gene editing involves downregulating genes. EPFL8b The expression of gene-encoded proteins inhibits plant regeneration; EPFL8b The nucleotide sequence of the gene is shown in SEQ ID NO: 6.

[0020] By applying the technical solution of this invention, the EPFL8b polypeptide of this application, when used to promote plant regeneration, enhances the regeneration capacity of explants or callus tissues after application, including promoting the formation of callus tissue, bud primordia, and organs in explants. Therefore, the application of the polypeptide of this application can further improve plant regeneration efficiency and shorten the culture cycle, demonstrating significant application value and industrialization prospects for overcoming the bottleneck of crop tissue culture regeneration, improving genetic transformation efficiency, and accelerating the process of crop molecular breeding. Attached Figure Description

[0021] The accompanying drawings, which form part of this application, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an undue limitation of the invention. In the drawings:

[0022] Figure 1 This diagram illustrates the expression of stem cell niche marker genes during regeneration using RNA sequencing analysis in Example 1 of this application. Figure 1 In the text, the error bars represent the mean ± standard deviation (n=3 biological replicates, and the meaning of "n" in this application specification is the same as here).

[0023] Figure 2 The following diagram shows the effect of the EPFL8b peptide in Example 3 of this application on the regeneration of adventitious shoots in tomatoes. Figure 2 Image a shows the morphological regeneration of explants after 8 days of treatment with EPFL8b peptide. Figure 2Figure b shows the statistical chart of the number of adventitious shoots regenerated from explants after 8 days of EPFL8b peptide treatment. Figure 2 The scale bar is 1 cm, n=90 (one-way ANOVA, P<0.05).

[0024] Figure 3 The diagram shows the callus regeneration of Panacea tomatoes after treatment with different concentrations of EPFL8b peptide in Example 4 of this application. Figure 3 Figure a shows representative morphological images of callus from Panalli tomatoes after treatment with different concentrations of EPFL8b. Figure 3 Figure b shows a statistical graph of the number of callus regeneration shoots per explant of Panalli tomato after treatment with different concentrations of EPFL8b protein. Figure 3 The scale ruler is 1 cm, n=30 ( P < 0.05, P < 0.01, P < 0.001).

[0025] Figure 4 The diagram shows the callus regeneration of currant tomato after treatment with different concentrations of EPFL8b peptide in Example 4 of this application. Figure 4 Figure a shows representative morphological images of currant tomato callus after treatment with different concentrations of EPFL8b peptide. Figure 4 Figure b shows a statistical graph of the number of callus regeneration shoots per explant of currant tomato after treatment with different concentrations of EPFL8b peptide. Figure 4 The scale ruler is 1 cm, n=30 ( P < 0.05).

[0026] Figure 5 The diagram shows wheat callus regeneration after treatment with different concentrations of EPFL8b peptide in Example 4 of this application. Figure 5 Figure a shows representative morphological images of wheat callus after treatment with different concentrations of EPFL8 peptide. Figure 5 Figure b shows a statistical graph of the number of callus regeneration shoots per wheat explant after treatment with different concentrations of EPFL8b peptide. Figure 5 The scale ruler is 1 cm, n=30 ( P < 0.05).

[0027] Figure 6 The diagram shows the regeneration of pepper cotyledon explants after treatment with different concentrations of EPFL8b peptide in Example 4 of this application. Figure 6 Image a shows representative morphological figures of pepper cotyledon explants treated with different concentrations of EPFL8b peptide. Figure 6 Figure b shows a statistical graph of the number of regenerated shoots per pepper cotyledon explant after treatment with different concentrations of EPFL8b peptide. Figure 6 The scale ruler is 1 cm, n=33 ( P =0.001).

[0028] Figure 7 Example 5 of this application specification illustrates the exogenous application of EPFL8b peptide to wild-type (WT) and... epfl8b The effect of mutant bud number is shown in the figure. Figure 7 In the middle, 'a' shows the morphological diagrams of regenerated buds from different groups; Figure 7 Figure b shows the statistical chart of the number of regenerated shoots in different groups. Figure 7 The scale bar is 1 cm, n=70 (one-way ANOVA, P<0.05).

[0029] Figure 8 A schematic diagram of the molecular docking prediction structure of ER-EPFL8b in Example 6 of this application specification is shown.

[0030] Figure 9 The diagram shows the results of yeast two-hybrid analysis in Example 6 of this application, verifying the physical interaction between the extracellular domains of EPFL8b and ER / ERL1.

[0031] Figure 10 The diagram shows the results of luciferase complementation imaging in Example 6 of this application, confirming the binding of EPFL8b-nLUC to ER-LRR-CLUC and ER-L1LRR-CLUC in transiently transgenic Tobacco Benedictine leaves. Detailed Implementation

[0032] It should be noted that, unless otherwise specified, the embodiments and features described in this application can be combined with each other. The present invention will now be described in detail with reference to the embodiments.

[0033] Terminology Explanation:

[0034] EPIDERMAL PATTERNING FACTOR-LIKE (EPFL): Epidermal pattern building factors are a family of small, cysteine-rich secreted peptides unique to plants. They typically act as ligands binding to specific receptors, activating downstream signaling pathways and participating in important physiological processes such as plant growth, development, and responses to abiotic stresses. EPFLs are widely involved in important growth and development processes, including leaf, seed, embryo, awn, stomatal, and spike morphogenesis, and play a crucial role in plant responses to drought and cold stress. EPFL8 is a signaling peptide in the EPFL family, and SlEPFL8b refers to tomato EPFL8b.

[0035] DPE stands for "days post excision," which refers to the number of days of culture after excision.

[0036] SIM stands for "Shoot Induction Medium".

[0037] Stem cell niche: refers to the specialized microenvironment in which stem cells exist, which is composed of supporting cells, signaling molecules, tissue structures, etc.

[0038] As mentioned in the background section, the existing technology lacks a regeneration-promoting technique that can be added exogenously, is easy to operate, and is applicable across species. Based on this, this application discovers a novel polypeptide EPFL8b that can be used in the tissue regeneration of various crops, promoting the efficiency of plant tissue regeneration and shortening the regeneration culture cycle. Therefore, this application proposes a series of protection schemes.

[0039] In a first typical embodiment of this application, an EPFL8b polypeptide that promotes plant regeneration is provided, the amino acid sequence of which is shown in SEQ ID NO: 1.

[0040] SEQ ID NO: 1: LGSSPPKCVNRCKGCRPCIATLVIPPHHTNKGLKTITSREDEGYYLLSWKCKCGSKYYHP.

[0041] Optionally, the nucleotide sequence encoding the above-mentioned polypeptide and the nucleotide sequence of the signal peptide are as shown in SEQ ID NO: 6 (i.e., EPFL8b The gene sequence Solyc07g008205 is shown, where the underlined 180 nucleotides are the nucleotide sequence encoding the above-mentioned polypeptide.

[0042] SEQ ID NO: 6: ATGGCTTCAACCATTAATATCTACTCAAAAAGACTAATCATTGTGATCCTCATCTATTCTTTCACATACTTGCATTTATATCAGGAAGAAATGAAAGGAAGCAAATGAGAATGGTGATG TTGGGATCAAGT CCACCAAAGTGTGTTAATAGATGCAAAGGATGCAGACCTTGCATTGCTACACTTGTTATTCCTCCACATACAAACA AAGGTTTAAAAACAATAACATCTAGAGAAGATGAAGGCTATTATTCTCCTTTCATGGAAATGCAAATGTGGATCAAA GTATTATCATCCTTGA .

[0043] As mentioned above, existing technologies for regenerating crops such as tomatoes suffer from bottlenecks such as low efficiency, strong hormone dependence, and poor system stability. This indicates that existing regeneration methods cannot achieve the effects of exogenous addition, simple operation, and cross-species applicability. This application addresses this by exogenously adding the synthesized polypeptide (i.e., EPFL8b peptide, hereinafter referred to as EPFL8b peptide, all of which belong to the plant regeneration-promoting polypeptides mentioned in this application) to the plant regeneration culture system. This can promote the establishment and stabilization of stem cell niches in callus tissue or plant regenerated tissue, thereby increasing the number of shoot primordia in explant regeneration and the regeneration efficiency of the plant.

[0044] This application demonstrates that when the EPFL8b peptide is exogenously applied to the explant culture system within a plant tissue regeneration system, it can promote the formation of bud primordia and organ regeneration in the explant callus tissue, thereby improving regeneration efficiency and shortening the crop culture cycle. Furthermore, when the EPFL8b peptide of this application is used in plant regeneration culture, plant regeneration can be achieved under stable conditions without complex gene transformation steps or fine-tuning of hormone ratios. This provides a theoretical and practical basis for overcoming the bottleneck of in vitro regeneration in Solanaceae crops and even a wider range of crops, improving genetic transformation efficiency, opening up a new path for plant in vitro regeneration technology, further promoting the development of crop molecular breeding, and possessing application potential and broad industrialization prospects.

[0045] In a second typical embodiment of this application, a formulation for promoting plant regeneration is provided, the formulation containing the aforementioned EPFL8b polypeptide.

[0046] The reagent containing the aforementioned peptide in this application can meet the needs of the plant biotechnology field for improving the regeneration efficiency of crops in vitro. Especially when facing long-standing bottlenecks such as low regeneration efficiency, hormone dependence, and genotype limitations, by directly applying the reagent containing the aforementioned EPFL8b peptide exogenously to the plant regeneration system, the regeneration-promoting regulatory effect of the peptide can be exerted, promoting the regeneration and formation of callus tissue, bud primordia, and organs. This eliminates the need for complex genetic engineering operations, avoids dependence on refined hormone regulation, simplifies the plant tissue regeneration process, and further shortens the plant regeneration cycle.

[0047] In addition to the aforementioned polypeptides, the above-mentioned formulations may optionally include buffer solutions, stabilizers, isotonic modifiers, excipients, surfactants, preservatives, or solvents, which can be flexibly selected by those skilled in the art according to actual needs.

[0048] In a third typical embodiment of this application, a culture medium for promoting plant regeneration is provided, the culture medium containing the above-mentioned EPFL8b polypeptide or the above-mentioned preparation for promoting plant regeneration.

[0049] In a preferred embodiment, the culture medium includes a bud induction medium or a callus induction medium.

[0050] In addition to retaining standard nutrients and plant growth regulators, the aforementioned culture medium also contains the aforementioned polypeptides or preparations that promote plant regeneration. When the aforementioned polypeptides or preparations are in the culture medium, the explants (cultured plant tissues) can, during the culture process, absorb the components of the culture medium and the aforementioned polypeptides, thereby stimulating the establishment and stabilization of stem cell niches in callus tissue, and thus increasing the generation rate of shoot primordia and the efficiency of the entire regeneration process.

[0051] The form in which the aforementioned EPFL8b peptide exists in plant regeneration systems can be flexibly adjusted by those skilled in the art according to actual needs. Whether used as an independent regeneration agent or as a key component in the culture medium, the aforementioned peptide can achieve the effects described in this application.

[0052] In a fourth typical embodiment of this application, the above-mentioned EPFL8b polypeptide is provided for use in the preparation of a product for promoting plant regeneration.

[0053] In a preferred embodiment, the product includes a culture medium or formulation.

[0054] In a fifth typical embodiment of this application, a method for promoting plant regeneration is provided, the method comprising: applying the above-mentioned EPFL8b polypeptide or the above-mentioned preparation to cultured plant tissue during the plant regeneration culture process.

[0055] In a preferred embodiment, the cultured plant tissue is a tissue in the bud primordia induction stage or a tissue in the callus induction stage.

[0056] The bud primordium induction stage and the callus induction stage are relatively effective intervention times for plant regeneration. By applying the above-mentioned EPFL8b polypeptide or the above-mentioned preparation to the cultured plant tissues during the bud primordium induction stage, the efficiency of tissue regeneration can be improved, and more bud primordia can be formed, thereby making the entire plant tissue regeneration process more stable and efficient.

[0057] The EPFL8b peptide of this application can be applied during the above-mentioned plant regeneration culture process in any of the following ways:

[0058] i) Apply the above-mentioned peptide for promoting plant regeneration or the above-mentioned preparation for promoting plant regeneration to the above-mentioned cultured plant tissue.

[0059] ii) The above-mentioned peptide for promoting plant regeneration or the above-mentioned preparation for promoting plant regeneration is applied to the regeneration medium in which the above-mentioned cultured plant tissue is inoculated; the above-mentioned regeneration medium includes shoot induction medium and / or callus induction medium.

[0060] iii) Inoculate the cultured plant tissue into a regeneration medium containing the EPFL8b peptide described above; the regeneration medium includes shoot induction medium and / or callus induction medium.

[0061] The target of the aforementioned polypeptides, namely cultured plant tissues, includes, but is not limited to, explants to be regenerated, callus tissue, or bud primordia.

[0062] In a preferred embodiment, the method includes: I) inoculating cultured plant tissue into a bud induction medium for culture; wherein the bud induction medium is the aforementioned medium, and / or, during the culture process, applying the aforementioned EPFL8b polypeptide for promoting plant regeneration or the aforementioned preparation for promoting plant regeneration to the cultured plant tissue;

[0063] II) The cultured plant tissue is inoculated into a callus induction medium for a first culture to obtain callus tissue; the callus tissue is then inoculated into a shoot induction medium for a second culture; wherein the callus induction medium and / or the shoot induction medium are each independently selected from the above-mentioned media, and / or, the above-mentioned EPFL8b polypeptide for promoting plant regeneration or the above-mentioned preparation for promoting plant regeneration is applied to the cultured plant tissue during the first culture and / or the second culture.

[0064] For plant tissues with short culture cycles (e.g., tomato and pepper tissues), the aforementioned polypeptide can be applied on days 6-15 from the initial culture stage. Even after transfer to the next culture medium, no further addition of the polypeptide is necessary. For plant tissues with longer culture cycles (e.g., wheat), the aforementioned polypeptide needs to be applied on days 6-15 of the initial culture stage, with additional additions as needed based on actual growth. When transferring to the next culture medium, the polypeptide should also be added upon transfer. The regeneration culture cycle for different plants is a timeframe for applying the EPFL8b polypeptide or the aforementioned preparation, which can be reasonably estimated by those skilled in the art based on experience and existing literature. Those skilled in the art can adjust this flexibly according to actual needs.

[0065] In the method for promoting plant regeneration in this application, whether the above-mentioned polypeptides or reagents are applied directly to the cultured plant tissues or added to the above-mentioned regeneration culture medium, the regeneration potential of the cultured plant tissues can be stimulated, the regeneration cycle can be shortened, the success rate of genetic transformation can be further improved, and the process of crop molecular breeding can be accelerated.

[0066] In a preferred embodiment, the plant tissue cultured above is derived from dicotyledonous or monocotyledonous plants.

[0067] In a preferred embodiment, the dicotyledonous plant includes tomato or pepper.

[0068] In a preferred embodiment, the monocotyledonous plant includes wheat.

[0069] In a preferred embodiment, the cultured plant tissue includes one or more of the following: callus, cotyledons, embryo, root, stem, leaf, or flower of the plant. The cultured plant tissue, in addition to callus, also includes, but is not limited to, plant tissues cut from the embryo, cotyledons, root, stem, leaf, or flower of the plant.

[0070] The EPFL8b peptide of this application has universal applicability in plant regeneration promotion technology; therefore, the method of this application is applicable to the regeneration of various plants. The aforementioned dicotyledonous plants include, but are not limited to, solanaceous crops such as tomatoes and peppers, while the aforementioned monocotyledonous plants include cereal crops such as wheat. By exogenously applying the EPFL8b peptide or its reagent during the tissue regeneration process of these crops, technical challenges such as low regeneration efficiency and long cycles can be solved, opening up new possibilities for rapid genetic transformation and molecular breeding of food crops.

[0071] In the specific implementation of this application, it is demonstrated that although the small peptide (EPFL8b) of this application is derived from tomatoes, its application in promoting regeneration is not limited to tomatoes and peppers of the same family, but is also applicable to monocotyledonous plants with very distant kinship (such as wheat).

[0072] This application reveals that the core function of the EPFL8b peptide is to induce and maintain a "regenerative stem cell niche" in callus tissue. EPFL8b is specifically enriched in the "signal layer" of callus tissue, transmitting positional signals downstream to maintain stem cell activity. The core of plant in vitro regeneration relies on the reprogramming of callus cell fate and the reconstruction of stem cell centers (i.e., establishing ordered division centers from disordered callus cells). Therefore, the EPFL8b peptide regulates a fundamental, universal structure in plant regeneration, demonstrating broad applicability in plants. As long as plants possess callus-based regeneration capabilities, the EPFL8b peptide can promote adventitious shoot regeneration from callus tissue by inducing and maintaining a "regenerative stem cell niche" within the callus.

[0073] The small peptide (EPFL8b) of this application does not act on a species-specific metabolic pathway, but rather on the highly conserved EPFL-ERECTA ligand-receptor signaling pathway in the plant kingdom. ERECTA family receptors (ER / ERL1), as core receptors regulating plant growth and development, are widely found in angiosperms (including dicotyledons and monocotyledons), and their structure and function are highly conserved evolutionarily.

[0074] Therefore, the small peptides of this application achieve good results across a wide range of species because they act on highly conserved receptor systems in plants and regulate the underlying stem cell construction process essential for plant regeneration. Based on this principle, those skilled in the art can reasonably expect that this technical solution will also be applicable to other plants with similar regeneration systems.

[0075] The above results demonstrate that the EPFL8b peptide of this application can effectively promote adventitious shoot formation of callus tissue in both dicotyledonous plants (including peppers of the same family) and monocotyledonous plants (wheat), exhibiting significant cross-species regeneration activity and functional conservation.

[0076] In a preferred embodiment, the working concentration of the above-mentioned polypeptide or the preparation for promoting plant regeneration is 0.5-5000 μg / L, including but not limited to 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 14 00, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, 4000, 4100, 4200, 4300, 4400, 4500, 4600, 4700, 4800, 4900 or 5000 μg / L.

[0077] In one specific embodiment of this application, in the regeneration system of panna commune tomatoes, the regeneration efficiency was increased by approximately 83% (P<0.001) under the 50 µg / L EPFL8b treatment within the application range of 0.5-5000 µg / L. Under the 50 µg / L EPFL8b treatment, the number of new shoots and the shoot formation rate of tomatoes were significantly higher than the control group. The 0.5 µg / L and 5000 µg / L groups also showed a certain promoting effect. In the regeneration system of currant tomatoes, the regeneration efficiency was increased by approximately 81% (P<0.05) under the 50 µg / L EPFL8b treatment. In the regeneration system of chili peppers, the regeneration efficiency was increased by approximately 89.49% (P=0.001) under the 50 µg / L EPFL8b treatment. In the regeneration system of wheat, the number of regenerated shoots increased by approximately 106% in the 50 µg / L treatment group (P<0.05).

[0078] To elucidate the mechanism of action of the EPFL8b peptide, this application further verified its binding ability to the hypothesized receptors ER (ERECTA) and ERL1 (ERECTA-LIKE 1) in tomato.

[0079] This application uses molecular docking prediction and computer-simulated molecular docking analysis to show that the EPFL8b peptide (ligand) has a high affinity binding free energy to the extracellular domain of the ER protein (receptor), and its binding ability is superior to other EPFL family members (such as EPFL4 and EPFL5).

[0080] Yeast two-hybrid (Y2H) assays showed that the EPFL8b peptide interacts physically with both the LRR (leucine-rich repeat) domain of ER and the LRR domain of ERL1.

[0081] Validation by luciferase complementation (LCI) experiments conducted in tobacco leaves further confirmed that the EPFL8b peptide does indeed interact with ER and ERL1 receptor proteins in living cells.

[0082] The above experimental results fully demonstrate that the EPFL8b peptide of this application has the function of significantly promoting the formation of bud primordia and organ regeneration in plant callus tissue, and can significantly improve regeneration efficiency and shorten the culture cycle. The regeneration system based on this peptide can stably achieve efficient regeneration without relying on gene transformation or fine adjustment of hormone ratios, which has important application value and industrialization prospects for breaking through the bottleneck of tissue culture regeneration in tomatoes and other crops, improving genetic transformation efficiency, and accelerating the process of crop molecular breeding.

[0083] In the sixth typical embodiment of this application, a method is provided. EPFL8b The application of genes in regulating plant regeneration, including the application of... EPFL8b Gene editing, through upregulation EPFL8b The expression of gene-encoded proteins promotes plant regeneration; or it can be achieved by downregulating the expression of these proteins. EPFL8b The expression of genes encoding proteins inhibits plant regeneration.

[0084] The aforementioned gene editing includes, but is not limited to, any one or more gene editing techniques known to those skilled in the art, including, but not limited to, CRISPR-Cas9 gene editing technology.

[0085] In one specific embodiment of this application, to verify the function of EPFL8b from a reverse genetics perspective, CRISPR-Cas9 gene editing technology was used to create a micro-Tom background in tomato. EPFL8b A null mutant with loss of gene function. Compared to the wild-type (WT) control group, epfl8b The mutant exhibits a severe deficiency in adventitious shoot regeneration ability. Statistical results show that... epfl8b The number of regenerated shoots produced on mutant explants was significantly lower than that of the wild-type control group. Meanwhile... epfl8b When the mutant in vitro plantlets were cultured to day 8 (8 DPE), the above-mentioned EPFL8b peptide was exogenously applied to the culture medium. It was found that the application of the above-mentioned peptide could partially rescue the regeneration defect phenotype of the mutant, and the number of regenerated shoots was significantly increased, which verified that the EPFL8b peptide of this application has the function of promoting plant regeneration.

[0086] Therefore, it can be seen that by studying... EPFL8b After gene editing of the gene (SEQ ID NO: 6, Solyc07g008205), it can be upregulated or downregulated. EPFL8b The expression level of genes-encoded proteins can regulate plant regeneration, obtain different plant types, facilitate research on plant regeneration tissue culture, and provide theoretical and technical support for the creation of new germplasm resources.

[0087] Unless otherwise specified, the reagents used in the embodiments of this application are all commercially available products.

[0088] The beneficial effects of this application will be explained in more detail below with reference to specific embodiments.

[0089] Example 1: Screening of EPFL8b small peptides

[0090] Sequence alignment and functional analysis of tomato EPFL family members identified the regeneration-promoting signal peptide EPFL8b. The screening process for the EPFL8b peptide in this application is as follows:

[0091] Using the regeneration system of tomato Micro-Tom cotyledon segments as the research object, spatial transcriptome sequencing analysis was performed on regenerated callus samples at different time points during the regeneration process. The results are as follows: Figure 1 As shown. Figure 1 This graph shows the expression of stem cell niche marker genes during the regeneration process (0-16 DPE) as analyzed by whole RNA sequencing. The error bars represent the mean ± standard deviation (n=3 biological replicates, other interpretations are the same here). The vertical axis "Expression" refers to the expression level.

[0092] Analysis revealed that the outer layer of the callus primordium initiation site was enriched with members of various small signal peptide families. Furthermore, the expression initiation time of these small peptides was highly synchronized with the formation of the three-layered stem cell niche in the primordium, showing a significant increase primarily during the regeneration phase 8-16 days after bud induction, while the inner layer stem cell marker genes were gradually activated before this point. Therefore, it is inferred that the small peptide signaling in the outer "signal layer" of the primordium is a crucial regulatory factor in maintaining the stem cell niche structure, and based on this, small peptide genes from this layer were selected as key candidate sources for screening.

[0093] Furthermore, focus EPF / EPFL Family, Discovery EPFL8b The expression level continued to increase during the stem cell niche formation and expansion phase 8-16 days after bud induction, while it was almost not expressed in tissues outside the outer layer of the bud primordium initiation site inside the callus.

[0094] Based on the above evidence, it was determined that EPFL8b is a key signal peptide derived from the outer "signal layer" of the callus primordium of tomato regeneration, and its C-terminal mature peptide segment was selected as the effective ingredient in the embodiment of the present invention, and its amino acid sequence is shown in SEQ ID NO: 1.

[0095] Example 2: Preparation of EPFL8b small peptide

[0096] The EPFL8b peptide is the C-terminal mature peptide of the tomato EPFL8b protein, and its amino acid sequence is shown in SEQ ID NO: 1.

[0097] The synthesis was commissioned to Beijing Qingke Biotechnology Co., Ltd. The synthesized EPFL8b peptides all had a purity greater than 85%. The synthesized product, dissolved in TBS buffer, had a concentration of 400 mg / L and was stored at -80℃ in the dark for subsequent experiments to verify its regeneration function in tomato MicroTom tissue culture.

[0098] Example 3: Effect of EPFL8b peptide on adventitious shoot regeneration in tomato

[0099] This embodiment used 5-day-old MicroTom tomato seedlings with uniform growth as experimental material and employed a single-factor, multi-level design to investigate the effect of 50 μg / L EPFL8b peptide on adventitious shoot regeneration in tomatoes. Based on spatial transcriptome sequencing analysis, it was deduced that the optimal treatment time was the 8th day after cotyledon inoculation with SIM. Therefore, in this embodiment, the cotyledons were treated with EPFL8b peptide on the 8th day after cotyledon inoculation with SIM.

[0100] (1) Cotyledon explants and bud induction culture:

[0101] A 5 mm tissue segment from the center of the cotyledon of a MicroTom tomato seedling was used as an explant. The segment was cut along the direction perpendicular to the main vein to form a wound, and then inoculated onto the bud induction medium by contacting the wound with the culture medium.

[0102] Shoot induction medium (SIM): 4.43 g / L MS, 3% sucrose, 1 mg / L zeatin (ZT), 0.1 mg / L auxin (IAA), 3.5% Phytagel, pH 5.8. Culture conditions were 25℃, 16 h light / 8 h dark.

[0103] (2) EPFL8b small peptide treatment

[0104] On day 8 after cotyledonous explants were inoculated into SIM (8 DPE), 50 μg / L EPFL8b solution was dripped into the explant wound; the control group was treated with an equal volume of sterile water. EPFL8b peptide treatment was followed by continued culturing.

[0105] (3) Cultivating observation

[0106] After one week of treatment with EPFL8b peptide, it was observed that the callus in the control group only showed a basal level of regeneration capacity and a small number of adventitious shoots, while the EPFL8b-treated group showed a highly significant promoting effect (regeneration efficiency increased by approximately 1.7 times), with the callus differentiating into a large number of healthy green adventitious shoots, and the number of stem cell niche structures significantly increased (e.g., Figure 2 As shown in Figure a, where, Figure 2 Image a shows the morphology of adventitious buds in tomato callus tissue of the treatment and control groups after 8 days. Figure 2 Figure b is a statistical graph showing the number of adventitious buds regenerated from explants of each treatment after 8 days following EPFL8b treatment. The vertical axis "Number of buds" refers to the number of adventitious buds. The interpretations of the other figures are the same as here.

[0107] (4) Results Analysis

[0108] Sixteen days after EPFL8b peptide treatment, the number of adventitious shoots regenerated from explants in each treatment was counted as an indicator of regeneration rate. Figure 2 As shown in Figure b, the treatment concentration of 50 μg / L showed significant regeneration-promoting activity, and the regeneration rate of explants at this concentration was significantly higher than that of the control group.

[0109] This indicates that the EPFL8b peptide can act as an exogenous signaling molecule to promote the tomato regeneration process, providing an effective way to optimize plant regeneration systems.

[0110] Example 4: Verification Experiment on the Promoting Effect of EPFL8b Small Peptide on the Regeneration of Different Plants

[0111] To verify the role of EPFL8b peptide in different plant regeneration systems and its cross-species functional conservation, this example selected Panacea tomato (Tomato paeonelli) Solanum pennellii ), currant tomatoes ( Solanum pimpinellifolium ),chili( Capsicum annuum L. cv. PC69 ),wheat( Triticum aestivum cv. Using Fielder as the experimental material, a parallel control design was adopted to investigate the effect of exogenous application of EPFL8b peptide on the regeneration efficiency of callus tissue in different plants.

[0112] (1) Experimental design

[0113] The experimental groups are shown in Table 1.

[0114] Table 1

[0115]

[0116] It should be noted that the explants in tomato shoot induction medium (8 DPE), wheat callus induction medium (14 DPE), and pepper shoot induction medium (8 DPE) were all in the callus induction or shoot primordium induction stage. For different species, it is necessary to observe the callus growth cycle in advance or perform high-throughput transcriptome sequencing of the callus at different time points or determine endogenous [genes / endogenous factors]. EPFL8b The expression levels of key downstream stem cell factors (such as CLV3 and CUC2) are used to determine the timing of bud primordia generation and, consequently, the timing of EPFL8b peptide treatment. These are treatment timings that can be obtained by those skilled in the art through experience or conventional observation and gene analysis.

[0117] (2) Cultivation conditions

[0118] Tomato culture medium: Standard bud induction medium (SIM: 4.43 g / L MS, 3% sucrose, 1 mg / L zeatin, 0.1 mg / L auxin, 3.5% plant gel, pH 5.8) was used, and the culture conditions were 25℃, 16 h light / 8 h dark.

[0119] Wheat culture media: Callus induction medium: 4.43 g / L MS, 3% sucrose, 2,4-D 2.0 mg / L, KT 0.5 mg / L, hydrolyzed casein 500 mg / L, culture conditions: 25℃ in the dark. Shoot induction medium: 4.43 g / L MS, 3% sucrose, IAA 1.0 mg / L, ZT 1.75 mg / L, agar 8 g / L, pH 5.8, culture conditions: 25℃, 16 h light / 8 h dark.

[0120] Pepper culture medium: 4.43 g / L MS callus induction medium, 3% sucrose, 2 mg / L zeatin, 0.1 mg / L auxin, 3.5% plant gel, pH 5.8, culture conditions: 25℃, 16 h light / 8 h dark.

[0121] Cultivation period: 16 days for Panali tomatoes and currant tomatoes, 20 days for peppers, and 8 weeks for wheat. Regularly observe the changes in callus color and morphology, as well as the formation of adventitious buds.

[0122] (3) Results and Analysis

[0123] Results of the tomato (Pannali) system: After applying EPFL8b peptide to the bud induction medium (8 DPE), the number of regenerated buds was counted on day 16. Results are as follows: Figure 3 As shown, Figure 3 Image a shows representative morphological diagrams of tomato callus treated with different concentrations of EPFL8b peptide. Figure 3 Figure b shows the statistical graph of the number of regenerated callus shoots per explant of *Tomato* after treatment with different concentrations of EPFL8b peptide. It can be seen that the callus tissue in the EPFL8b-treated groups was light green in color and had good tissue activity, with a significant promoting effect on adventitious shoot formation. Specifically, the number of adventitious shoots in the 50 µg / L EPFL8b treatment group increased by approximately 83% compared to the control group (P<0.001), and the 0.5 µg / L and 5000 µg / L groups also showed a certain promoting effect. The shoots were normal in morphology and without deformities, indicating that EPFL8b has a stable regeneration-promoting effect in different varieties of *Tomato*.

[0124] Results of the tomato (currant) system: After applying EPFL8b peptide to the bud induction medium (8 DPE), the number of regenerated buds was counted on day 16. Figure 4 As shown, Figure 4 Image a shows representative morphological diagrams of callus from currant tomato treated with different concentrations of EPFL8b peptide. Figure 4 Figure b shows the statistical graph of the number of callus regeneration shoots per explant of Panalli tomato after treatment with different concentrations of EPFL8b peptide. It can be seen that the callus tissue in the EPFL8b treatment group was light green in color, had good tissue activity, and showed a significant promoting effect on adventitious shoot formation. Specifically, the number of adventitious shoots in the 50 µg / L EPFL8b treatment group increased by approximately 81% compared to the control group (P < 0.05), while the 0.5 µg / L and 5000 µg / L groups also showed a certain promoting effect.

[0125] Results of the wheat system: After inoculation with EPFL8b peptide in callus induction medium 14 DPE, callus induction culture was carried out for a total of 6 weeks (EPFL8b peptide was added every two weeks). Then, the callus was transferred to shoot induction medium and cultured for another 2 weeks. The number of adventitious shoots longer than 2 cm was counted. Figure 5 As shown, Figure 5 Image a shows representative morphological diagrams of wheat callus after treatment with different concentrations of EPFL8b peptide. Figure 5 Figure b shows the statistical graph of the number of regenerated shoots per wheat explant callus after treatment with different concentrations of EPFL8b peptide. The number of regenerated shoots in the 50 µg / L EPFL8b treatment group was approximately 106% higher than that in the control group (P<0.05), while the 0.5 µg / L and 5000 µg / L groups also showed some promoting effect. The callus tissue in all treatment groups was intact, uniform in color, and showed no browning. These results indicate that EPFL8b protein still has a regeneration-promoting effect in monocotyledonous plants, demonstrating functional conservation.

[0126] Results of the chili pepper system: EPFL8b peptide was applied when inoculated into callus induction medium 8 DPE, and the number of regenerated shoots was counted on day 20 of total culture. Results are as follows: Figure 6 As shown, Figure 6 Image a shows representative morphological diagrams of pepper cotyledon explants treated with different concentrations of EPFL8b peptide. Figure 6 Figure b shows the statistical graph of the number of regenerated shoots per pepper cotyledon explant after treatment with different concentrations of EPFL8b peptide. Among them, compared with the control group, the 50 µg / L treatment group significantly improved the regeneration efficiency of pepper callus. Specifically, the regeneration efficiency of the 50 µg / L treatment group increased by approximately 89.49% (P=0.001).

[0127] (4) Conclusion: The EPFL8b peptide significantly promoted adventitious shoot formation and regeneration of callus tissue in different plant systems including panna commune tomato, currant tomato, pepper, and wheat. These results indicate that the EPFL8b signaling pathway is highly conserved and has cross-species applicability in various crops. This example further verifies the broad-spectrum regeneration-promoting function of the EPFL8b peptide, laying an experimental foundation for its application in crop regeneration system optimization, genetic transformation efficiency improvement, and molecular breeding.

[0128] Example 5: epfl8b The mutant was constructed and its regenerative function of the EPFL8b peptide was verified.

[0129] This embodiment aims to utilize reverse genetics techniques to... epfl8b The loss-of-function mutant confirmed the essential function of EPFL8b peptide in the regeneration of adventitious shoots in plants.

[0130] (1) epfl8bCreation and identification of mutants

[0131] Materials and Methods: The experimental material was tomato Micro-Tom. CRISPR-Cas9 gene editing technology was used to construct targeted... EPFL8b gRNA vectors for the gene coding region were used. Tomato cotyledonary explants were transformed using Agrobacterium-mediated transformation, and hygromycin B (30 mg / L) was used for screening. Homozygous mutants of the T2 / T3 generation were screened by PCR amplification and DNA sequencing.

[0132] Editing carrier construction:

[0133] ① sgRNA target design

[0134] Based on the tomato genome database, select EPFL8b Two exons in the coding region of the gene (SEQ ID NO: 6, Solyc07g008205) were used as CRISPR / Cas9 editing targets. Two 20 bp sgRNA sequences were designed, each containing a typical PAM sequence (NGG) at its front end, which can effectively induce Cas9 to perform double-strand cutting.

[0135] EPFL8b -sgRNA1 (SEQ ID NO: 2): 5′-CACTTATTC TTTTCAATTAC-3′.

[0136] EPFL8b -sgRNA2 (SEQ ID NO: 3): 5′-TGAAAGGAAGCAAATGAGAA-3′.

[0137] Two sgRNAs were annealed to synthetic oligonucleotides and then inserted into the sgRNA expression cassette of the pCAMBIA1300-Cas9 vector to construct a dual-target gene editing vector.

[0138] Carrier construction method:

[0139] The pCAMBIA1300-Cas9 vector was digested with BsaI.

[0140] The annealed sgRNA oligonucleotide fragments were ligated into a linearized vector;

[0141] Transformation E. coli DH5α was used for screening;

[0142] PCR and sequencing were used to verify positive clones;

[0143] The validated construct was electroporated into Agrobacterium. Agrobacterium tumefaciens GV3101.

[0144] Tomato genetic transformation and mutant acquisition:

[0145] ② Explant preparation and pre-culture

[0146] Select 5-day-old Micro-Tom seedlings, cut about 5 mm of tissue from the center of the cotyledons as explants, inoculate them into PC1 pre-medium, and culture them in the dark at 20°C for 1 day.

[0147] ③ Agrobacterium activation and infection

[0148] The constructed GV3101 strain was cultured in LB liquid medium (containing Rif, Gen, Kan, and AS) until OD. 600 =0.8–1.0;

[0149] Centrifuge at 5000 rpm to remove the supernatant, and wash twice with KC liquid culture medium;

[0150] Resuspended to OD 600 ≈0.1;

[0151] The Agrobacterium suspension was evenly dripped onto the side of the explant wound and left to stand for 10 minutes.

[0152] Aspirate any excess bacterial solution and incubate in the dark at 20°C for 2 days.

[0153] ④ Resistance screening and plant regeneration

[0154] After infection, explants were transferred to 2Z screening medium containing hygromycin B (30 mg / L) or kanamycin (Kan 350 μL / 200 mL). The explants were transferred every 12–15 days until shoot differentiation appeared, at which point they were transferred to 1Z subculture medium for further proliferation. Germinating shoots were then cut and transferred to ENR rooting medium to complete rooting culture.

[0155] The culture conditions were 25℃, 16 h light / 8 h dark.

[0156] Genotyping:

[0157] Obtained from regeneration Genomic DNA was extracted from the plant and used EPFL8b PCR amplification using specific amplification primers:

[0158] epfl8b -F (SEQ ID NO: 4): 5′-ATGGCTTCAACCATTAAT-3′.

[0159] epfl8b -R (SEQ ID NO: 5): 5′-TCAAGGATGATAATACTT-3′.

[0160] PCR amplification conditions:

[0161] 95℃ 3 min → (95℃ 30 s, 58℃ 30 s, 72℃ 30 s) × 35 cycles → 72℃ 5min.

[0162] The amplified products were analyzed by Sanger sequencing. Some strains showed base insertions or deletions at the target site, leading to premature stop codon generation, thus obtaining... epfl8b Loss-of-function mutants. Homozygous mutants in / Fixed by filtering in the generation.

[0163] The formulations of various culture media for plant tissue culture are shown in Table 2.

[0164] Table 2

[0165]

[0166] (2) epfl8b Mutant Regeneration Phenotypic Analysis

[0167] Experimental design: Wild-type (WT) and epfl8b Five-day-old seedling cotyledon segments of the mutant were inoculated onto standard bud induction medium (SIM) and cultured at 25°C under 16h light / 8h dark conditions.

[0168] Results and Analysis (Number of Regenerated Buds): After 16 days of culture, the number of adventitious buds generated on each explant was counted. For example... Figure 7 As shown, wild-type (WT) explants can efficiently regenerate a large number of adventitious buds; in contrast, epfl8b The regenerative capacity of mutant explants was severely impaired, with the average number of regenerated shoots being significantly lower than that of the wild-type control group.

[0169] (3) epfl8b Mutant Reversion Experiment

[0170] Experimental design: To verify that regeneration defects are caused by... EPFL8b Due to the deletion, a mutant revertant group was set up. epfl8b When the mutant in vitro implants were cultured on SIM medium to day 8 (8 DPE), exogenous EPFL8b peptide solution was added dropwise to the medium to bring the final concentration to 50 µg / L.

[0171] Results and Analysis: Figure 7 As shown, Figure 7 Figure 'a' shows the morphological diagrams of regenerated buds from different groups. Figure 7 Figure b shows the statistical chart of the number of regenerated shoots in different groups. It can be seen that, compared with the group without added small peptides... epfl8b mutant group ( epfl8Compared to the group that received EPFL8b peptide on day 8, the recovery group ( epfl8 The number of regenerated shoots (+EPFL8b) increased significantly, and its regeneration ability was partially restored.

[0172] (4) Conclusion

[0173] epfl8b The mutant exhibits a normal phenotype under normal growth conditions, but its in vitro regeneration ability (adventitious shoot formation) is severely inhibited. This regeneration-deficient phenotype can be partially rescued by exogenous application of the EPFL8b peptide. This result strongly demonstrates that the EPFL8b peptide is a key signaling molecule essential for initiating adventitious shoot regeneration in plant callus tissue.

[0174] Example 6: Validation of the binding of EPFL8b small peptide (ligand) to receptor ER / ERL1

[0175] This embodiment aims to verify, through various biochemical and molecular biological methods, the direct interaction between the EPFL8b small peptide as a ligand and its receptors ER and ERL1 in tomato.

[0176] (1) Molecular docking prediction

[0177] Methods: The protein structure of tomato ER (Solyc08g061560) was predicted using tools such as AlphaFold2, and the small peptide structure of EPFL8b (Solyc07g008205) was simulated using SWISS-MODEL. The binding free energy between EPFL8b and the ER receptor was calculated using molecular docking software.

[0178] Results: The prediction results show (e.g.) Figure 8 As shown in the figure, EPFL8b has a highly efficient binding free energy (e.g., -129.5237 gb) with the ER receptor, and its affinity is significantly stronger than that of ER with EPFL4 (-37.4085 gb) or EPFL5 (-35.4946 gb), indicating that EPFL8b is the preferred ligand of the ER receptor. Figure 8 In the diagram, the left image shows the docking structure of tomato ER (green) and AtERL2 (yellow, PDB: 5XKN), while the middle and right images show the docking structure of EPFL8 (green) and ER (pink) (corresponding to front-middle and side-right images), overlapping with AtEPFL4 (yellow) and AtERL2 (cyan). Table 3 shows the comparison between the binding free energies of ER with EPFL4, EPFL5, and EPFL8b and the binding free energy of AtERL2 with AtEPFL4.

[0179] Table 3

[0180]

[0181] (2) Yeast double hybridization verification

[0182] Methods: The coding sequence of the mature EPFL8b peptide was cloned into the pGADT7 vector (AD, activation domain), and the extracellular LRR domains of the ER and ERL1 receptors were cloned into the pGBKT7 vector (BD, binding domain). The AD and BD recombinant plasmids were co-transformed into Y2HGold yeast strain.

[0183] Result: As Figure 9 As shown, yeast strains expressing EPFL8b-AD and ER-BD, as well as yeast strains expressing EPFL8b-AD and ERL1-BD, can grow normally on SD-Leu-Trp-His-Ade (quadruple deficiency) selection medium, indicating that there is a physical interaction between EPFL8b and the extracellular domains of ER and ERL1.

[0184] (3) Luciferase complementation verification

[0185] Methods: EPFL8b was constructed into the nLUC (luciferase N-terminus) vector, and the LRR domains of ER and ERL1 were constructed into the cLUC (luciferase C-terminus) vector, respectively. The combination of nLUC and cLUC constructs was transiently co-infiltrated into *Nicotiana benthamiana* via *Agrobacterium* injection. N.benthamiana )blade.

[0186] Result: As Figure 10 As shown, tobacco leaves co-expressing EPFL8b-nLUC and ER-cLUC, as well as co-expressing EPFL8b-nLUC and ERL1-cLUC, all showed strong fluorescence signals after being sprayed with the fluorophore substrate, while the negative control group (such as the combination of EPFL8b-nLUC and empty cLUC vector) showed no signal.

[0187] (4) Conclusion

[0188] Molecular docking, yeast two-hybrid assays, and in vivo LCI validation all consistently demonstrated that the EPFL8b peptide, acting as a signal ligand, can specifically bind to the ER and ERL1 receptor proteins in tomato. This result elucidates the molecular mechanism by which the EPFL8b peptide exerts its regeneration-promoting function, namely, by regulating the regeneration process through activation of the ER / ERL1-mediated signaling pathway.

[0189] The ERECTA (ER) family receptors and their mediated MAPK signaling cascade are highly conserved throughout the evolution of angiosperms and are core pathways regulating plant organogenesis and tissue pattern building. Existing research indicates that activating this signaling pathway can restart cell division and maintain stem cell activity, thereby regulating the regenerative capacity of various plants. This example confirms, through molecular docking, yeast two-hybrid, and LCI experiments, that the EPFL8b peptide specifically binds to and activates the ER / ERL1 receptor. Combined with the significant regeneration-promoting effects of EPFL8b in both dicotyledonous (tomato, pepper) and monocotyledonous (wheat) crops demonstrated in Example 5, this strongly supports the conclusion that EPFL8b exerts its effects by activating this highly conserved ER / ERL1 signaling pathway in the plant kingdom. This mechanistic conservation lays the biological foundation for the EPFL8b peptide to serve as a broad-spectrum regeneration regulator applicable to a wide range of crops.

[0190] As can be seen from the above description, the embodiments of the present invention achieve the following technical effects: The EPFL8b regeneration-promoting peptide of this application has the function of significantly promoting the formation of bud primordia and organ regeneration in plant callus tissue, which can significantly improve regeneration efficiency and shorten the culture cycle. Furthermore, the EPFL8b peptide of this application can be exogenously applied to the plant regeneration culture system, and can stably achieve efficient regeneration of explants without relying on gene transformation or fine adjustment of hormone ratios. This has important application value and industrialization prospects for breaking through the tissue culture regeneration bottleneck of tomatoes and other crops, improving genetic transformation efficiency, and accelerating the process of crop molecular breeding.

[0191] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. An EPFL8b polypeptide, characterized in that, The amino acid sequence of the polypeptide is shown in SEQ ID NO:

1.

2. The use of the EPFL8b polypeptide according to claim 1 in the preparation of products for promoting plant regeneration, characterized in that, The product includes formulations.

3. The application according to claim 2, characterized in that, The preparation includes a culture medium.

4. EPFL8b The application of genes in regulating plant regeneration is characterized by, The applications include: for EPFL8b Gene editing, By adjusting the above EPFL8b The expression of gene-encoded proteins promotes plant regeneration; or By lowering EPFL8b The expression of gene-encoded proteins is inhibited, thereby suppressing plant regeneration; Among them, the EPFL8b The nucleotide sequence of the gene is shown in SEQ ID NO: 6, wherein positions 1 to 120 of SEQ ID NO: 6 are nucleotide sequences encoding a signal peptide, and positions 121 to 300 of SEQ ID NO: 6 are nucleotide sequences encoding an EPFL8b polypeptide.

5. A method for promoting plant regeneration, characterized in that, The method includes: applying the EPFL8b polypeptide of claim 1 to the cultured plant tissue during plant regeneration culture.

6. The method according to claim 5, characterized in that, The cultured plant tissue is in the bud primordium induction stage or callus induction stage.

7. The method according to claim 5, characterized in that, The application method includes any one of the following: i) applying the EPFL8b polypeptide of claim 1 or a preparation containing the EPFL8b polypeptide to the cultured plant tissue; ii) Apply the EPFL8b peptide of claim 1 or a formulation containing the EPFL8b peptide to the regeneration medium in which the cultured plant tissue is inoculated; iii) The cultured plant tissue is inoculated into a regeneration medium containing the EPFL8b polypeptide of claim 1; The regeneration medium includes bud induction medium and / or callus induction medium.

8. The method according to claim 5, characterized in that, The cultured plant tissues are derived from dicotyledonous or monocotyledonous plants.

9. The method according to claim 8, characterized in that, The dicotyledonous plants include tomatoes or peppers; The monocotyledonous plants include wheat.

10. The method according to claim 5, characterized in that, The cultured plant tissues include one or more of the following: callus, cotyledons, embryo, root, stem, leaf, or flower.

11. The method according to claim 5, characterized in that, The working concentration of the EPFL8b peptide according to claim 1 is 0.5-5000 μg / L.

Images