Exciton peptide and application thereof in dendrobium cultivation and propagation

Abstract

The invention provides elicitor peptide and application thereof in dendrobium culture and propagation, and belongs to the field of bioengineering. The amino acid sequence of the elicitor peptide is as shown in SEQ ID NO: 1. The elicitor peptide is used in a primary bud induction stage, a subculture multiplication stage and a rooting stage in dendrobium officinale culture and propagation, and especially has a good effect in dendrobium officinale culture and propagation application. According to the tissue culture method disclosed by the invention, by optimizing explant treatment, optimizing a culture medium formula and adding the elicitor peptide, the problems of low propagation efficiency, long culture period and the like in the existing tissue culture technology of the dendrobium nobile are effectively solved; the bud ratio of the explant can be obviously improved, the primary culture period is shortened, the subculture multiplication coefficient is greatly enhanced, the multiplication process is accelerated, and the root development of the dendrobium seedlings is promoted. According to the method, the overall efficiency and quality of tissue culture and rapid propagation of dendrobium nobile are remarkably improved, the propagation bottleneck is solved, the production period is shortened, and stable and efficient technical support is provided for industrialization.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology and relates to an elicitor peptide and its application in the cultivation and propagation of Dendrobium. Background Technology

[0002] Dendrobium, a plant of the genus Dendrobium in the family Orchidaceae, is listed as a superior herb in the "Shennong's Classic of Materia Medica". It has the effects of nourishing the stomach and promoting the production of body fluids, nourishing yin and clearing heat. Modern research has further confirmed its role in immune regulation and anti-oxidation. It is widely used in the fields of health preservation, food and cosmetics, and also has ornamental value.

[0003] However, the incomplete development of Dendrobium seeds leads to an extremely low natural germination rate. Coupled with over-harvesting and ecological damage, wild resources are on the verge of depletion, and the plant has been listed as a key protected species in China. To alleviate the pressure on seedling supply and meet the growing market demand, tissue culture technology has become the core means to solve the Dendrobium seedling problem.

[0004] Currently, although Dendrobium tissue culture has made some progress in seed germination, explant induction and proliferation, and culture medium optimization (such as improving seed germination rate under aseptic conditions and promoting growth with specific hormones or natural extracts), significant technical bottlenecks still exist: Insufficient propagation efficiency: The budding rate induced by explants, the proliferation coefficient of clustered buds or pseudobulbs (PLBs) are limited, making it difficult to support large-scale production; The cultivation cycle is too long: the initial budding, subculture, and rooting stages take a long time, and the overall cycle from explant to seedling is extended, which restricts production efficiency. Culture medium dependence and instability: Existing technologies are highly dependent on hormone combinations, resulting in poor seedling growth uniformity and uneven quality. At the same time, the effects of natural extracts fluctuate greatly, increasing the difficulty of optimizing culture media.

[0005] In conclusion, facing the dual pressures of Dendrobium resource protection and industrial development, there is an urgent need to develop efficient and stable Dendrobium tissue culture additives and optimized culture media to overcome the aforementioned bottlenecks. Summary of the Invention

[0006] The purpose of this invention is to address the aforementioned problems in existing technologies by proposing an elicitor peptide and its application in the cultivation and propagation of Dendrobium.

[0007] The objective of this invention can be achieved through the following technical solution: an elicitor peptide, the amino acid sequence of which is shown in SEQ ID NO:1, specifically: LAQNQREEIRSKSTAGSGQGQTN. The nucleotide sequence is shown in SEQ ID NO:2, specifically: TCGCACAAAATCAACGCGAAGAGATACGCAGCAAGTCCacggcgggcagcgggcaggggcAAACGAAT.

[0008] The method for isolating and identifying the elicitor peptide sequence is as follows: (a) Identification of elicitor peptide-encoding genes from the Dendrobium genome by sequence alignment; (b) Design specific amplification primer pairs, including an upstream primer and a downstream primer. Upstream primer sequence number: TCGCACAAAATCAACGCG; Downstream primer sequence number: ATTCGTTTGCCCCTGCCC; (c) Using Dendrobium cDNA as a template, PCR amplification was performed using the high-fidelity enzyme 2xPhanta Flash Master Mix (catalog number: P520); (d) The amplification products were detected by electrophoresis and sequenced for verification; The elicitor peptide is prepared by a prokaryotic expression system, a eukaryotic expression system, or by chemical synthesis; in the prokaryotic expression system, prokaryotic Escherichia coli is used; in the eukaryotic expression system, fungal yeast is used. Preferably, in the chemical synthesis method, Fmoc solid-phase synthesis technology is used, employing an Applied Biosystems 431 synthesizer. The specific synthesis steps are as follows: S1 Deprotection: Piperidine is used to remove the Fmoc protecting group of the amino acid to obtain the free amino acid; S2 Activation and Crosslinking: An activator is used to activate the carboxyl group of the next amino acid to obtain the activated amino acid. The activated amino acid reacts with the free amino acid to crosslink and form a peptide bond. In this step, a large amount of ultra-concentrated reagent is used to drive the reaction to complete; S3 Cyclic Extension: Steps S1 and S2 are repeated according to the amino acid sequence of the elicitor peptide until the synthesis is completed; S4 Post-treatment: After the synthesis is completed, TFA solution is first used for elution and deprotection, followed by purification using reversed-phase high-performance liquid chromatography.

[0009] An application of an elicitor peptide in the propagation of Dendrobium officinale, wherein the elicitor peptide is used in the primary shoot induction stage, subgeneration proliferation stage and rooting stage of Dendrobium officinale propagation, especially in the propagation of Dendrobium officinale, with good results.

[0010] Preferably, in the primary bud induction stage, healthy Dendrobium officinale stem tips or stem segments are selected as explants. The explants are then disinfected, and the disinfection process includes the following steps: rinsing with running water, disinfection with 75% alcohol, first sterile water rinsing, disinfection with 1% sodium hypochlorite, and a second sterile water rinsing.

[0011] Preferably, the MS medium is MS medium with catalog number HB8469-5.

[0012] Dendrobium officinale propagation: During the initial shoot induction stage, for the culture medium containing standard MS medium, the following dosages were added: 6-benzylaminopurine: 2 mg / L; naphthaleneacetic acid: 0.5 mg / L; elicitor peptide: 10 nM.

[0013] During the subculture proliferation stage, for culture medium containing standard MS medium, the following dosages were added: 6-benzylaminopurine: 3 mg / L; naphthaleneacetic acid: 0.1 mg / L; elicitor peptide: 10 nM.

[0014] During the rooting stage, for a culture medium containing 1 / 2 standard MS medium, the following dosages were added: indolebutyric acid: 0.5 mg / L; elicitor peptide: 10 nM.

[0015] Culture medium containing standard MS medium: Weigh this product, heat to dissolve in distilled water, autoclave at 116℃ for 30 minutes. Dosage: MS medium: distilled water = 41.74 g : 1000 ml distilled water; Culture medium containing 1 / 2 standard volume of MS medium: Weigh this product, heat to dissolve in distilled water, autoclave at 116℃ for 30 minutes. Dosage: MS medium: distilled water = 20.87 g : 1000 ml distilled water; Compared with existing technologies, this invention effectively solves the problems of low propagation efficiency, long culture cycle, and strong hormone dependence in existing Dendrobium tissue culture technology by optimizing explant treatment, optimizing culture medium formulation, and adding elicitor peptides. Specific beneficial effects include the following aspects: 1. Significantly improves explant germination rate and shortens primary culture cycle: During the primary culture stage, the addition of elicitor peptides significantly improves the germination rate and growth quality of explants, effectively shortening the germination time. Simultaneously, it optimizes the synergistic effect of hormones (such as 6-BA and NAA) in the culture medium, promoting rapid growth and development of bud tissue, thereby reducing the overall time required for primary culture and improving culture efficiency.

[0016] 2. Significantly enhanced subculture proliferation coefficient and accelerated proliferation process: In subculture proliferation culture, the application of elicitor peptides significantly improved the proliferation coefficient and greatly shortened the proliferation time. By optimizing the hormone combination, rapid proliferation was promoted, the time consumed in the culture stage was reduced, and the feasibility of large-scale production was improved.

[0017] 3. Promotes rooting: During the rooting culture stage, the addition of elicitor peptides significantly improved the rooting rate, promoted the root development of Dendrobium seedlings, improved seedling quality and transplant survival rate, and solved the bottleneck of low efficiency in the rooting stage in existing technologies.

[0018] 4. Overall Optimization of Culture Medium Reduces Hormone Dependence and Improves Stability: This invention reduces excessive reliance on hormones during tissue culture by applying elicitor peptides, thus optimizing the stability and consistency of the culture medium. This avoids the problem of uneven seedling growth and provides an efficient and reproducible technical solution, laying a solid foundation for the large-scale rapid propagation of Dendrobium seedlings.

[0019] In summary, this invention significantly improves the overall efficiency and quality of Dendrobium tissue culture rapid propagation, solves the propagation bottleneck, shortens the production cycle, and provides stable and efficient technical support for industrialization. Attached Figure Description

[0020] Figure 1 This is a purity detection graph for the elicitor peptide in this invention.

[0021] Figure 2 This is a molecular weight verification diagram of the elicitor peptide in this invention.

[0022] Figure 3 This is a schematic diagram showing the comparison of culture results during the optimization of the primary culture medium using orthogonal experiments in this invention.

[0023] Figure 4 This is a schematic diagram showing the comparison of culture results of the optimized primary culture medium with different amounts of elicitor peptides added in this invention.

[0024] Figure 5 This is a schematic diagram showing the comparison of culture results during the optimization of the subculture proliferation medium in the orthogonal experiment of this invention.

[0025] Figure 6 This is a schematic diagram showing the comparison of culture results of optimized subculture proliferation media with different amounts of elicitor peptides added in this invention.

[0026] Figure 7 This is a schematic diagram comparing the culture results of rooting media with different amounts of elicitor peptides added in this invention.

[0027] Figure 8 A schematic diagram illustrating the application of elicitor peptides in the rapid propagation process of Dendrobium officinale tissue culture in this invention. Detailed Implementation

[0028] The following are specific embodiments of the present invention described in conjunction with the accompanying drawings, which further illustrate the technical solution of the present invention. However, the present invention is not limited to these embodiments.

[0029] Example 1: Isolation and identification of the elicitor peptide DoPep1 sequence Sequence alignment was performed using the amino acid sequence of the Arabidopsis homology gene AtPep1 (AT5G64900.1). DoPep1 was isolated and identified from the Dendrobium genome. Upstream primer DoPep1-F and downstream primer DoPep1-R were designed. Using Dendrobium cDNA as a template, the corresponding sequence of DoPep1 was amplified by PCR. The high-fidelity enzyme 2xPhantaFlashMasterMix (catalog number: P520) from Novizan Biosciences was selected for the PCR reaction to ensure the fidelity of the amplification product. After PCR electrophoresis, the PCR product was sent to the company for sequencing. The sequencing result of the PCR product is shown in SEQ ID NO:1, which is the sequence of MsPep1. The amino acid sequence of MsPep1 is shown in SEQ ID NO:1, and the nucleotide sequence is shown in SEQ ID NO:2.

[0030] >DoPep1 SEQ ID NO:1 is: LAQNQREEIRSKSTAGSGQGQTN; >DoPep1 SEQ IDNO:2 is: TCGCACAAAATCAACGCGAAGAGATACGCAGCAAGTCCacggcgggcagcgggcaggggcAAACGAAT.

[0031] Example 2: Synthesis of DoPep1 DoPep1 can be synthesized using microorganisms such as prokaryotic Escherichia coli, fungi, and yeasts, or it can be synthesized using chemical methods.

[0032] In the chemical synthesis method, Fmoc solid-phase synthesis technology was employed, and the synthesis was completed using an Applied Biosystems 431 synthesizer. The specific process is as follows: The synthesis consists of the following cycles: Deprotection: The Fmoc-protected column and monomers must have their amino protecting groups removed using an alkaline solvent (piperidine); Activation and cross-linking: The carboxyl group of the next amino acid is activated by an activator. The activated monomer reacts with the free amino group to cross-link, forming a peptide bond. A large amount of ultra-concentrated reagent is used to drive the reaction in this step. Cycling: These two steps are repeated until the synthesis is complete; Elution and deprotection: The elicitor peptide is eluted from the column, and its protecting group is eluted and deprotected using a deprotecting agent (TFA). The synthesized peptide is purified by C18 reversed-phase high-performance liquid chromatography, and the detection results are as follows: Figure 1 As shown, molecular weight verification was performed using a Finnigan L CQ mass spectrometer via direct sample injection. The verification results are as follows: Figure 2 As shown.

[0033] Example 3: Optimization of culture medium during the induction of primary shoots of Dendrobium officinale Optimization of explant treatment: Disinfection of explants: Select healthy, plump stem tips and stem segments free from pests and diseases as explants, rinse with running water for 1 hour, disinfect with 75% alcohol for 30 seconds in a clean bench, rinse with sterile water 3 times, disinfect with 1% sodium hypochlorite for 8 minutes, and rinse with sterile water 5 times.

[0034] Explant treatment: After sterilization, the explants were blotted dry on sterile filter paper, then immersed in primary culture liquid medium, vacuumed for 30-60 minutes, allowed to stand for 1.0-3.0 hours, and then placed on primary culture solid medium. They were cultured in a photoincubator at a temperature of (25±2)℃, light intensity of 1500-2000 lx, humidity of 40%-80% (maintained naturally within the culture flask), and a photoperiod of 12-16 h / d. The induction rate was calculated after 3-4 weeks. Results showed that shoots began to emerge after 10 days of culture, and approximately 70% of the explants successfully sprouted after 20 days.

[0035] Orthogonal experiments were used to optimize the primary culture medium: Based on MS medium, the concentrations of two hormones that significantly affect Dendrobium tissue culture—the plant growth regulators 6-benzylaminopurine (6-BA) and naphthaleneacetic acid (NAA)—were optimized. Two levels were set for each factor: 6-BA concentrations of 1.0 mg / L and 2.0 mg / L; and NAA concentrations of 0.1 mg / L and 0.5 mg / L. An orthogonal experimental design (two factors, three levels) was used to optimize the addition of hormones 6-BA and NAA to the primary culture medium. Four experimental groups were designed, with three replicates per group. After 30 days of primary culture, the budding rate was calculated. The results are shown in Table 1 and [Table data missing]. Figure 3 As shown, in Figure 3 In the following questions, A. Germination status of the first generation culture at 30 days old, B. Average germination rate, C. Germination growth of the first generation culture in media No. 3 and No. 4, and D. Comparison of germination length between media No. 3 and No. 4 of the first generation culture.

[0036] Table 1 Results of orthogonal experiments optimizing the primary culture medium

[0037] The germination rates of experimental groups 3 and 4 were higher, at 89.73% and 94.80% respectively, which were significantly higher than those of experimental groups 1 and 2 (64.21% and 65.75%). This indicates that the germination rates of experimental groups 3 and 4 were significantly higher than those of experimental groups 1 and 2. Further culture and observation revealed that the bud tissue of experimental group 4 grew better, with a length of 1.33 cm. Therefore, the primary culture medium was optimized for experimental group 4 as follows: MS + 2 mg / L 6-BA + 0.5 mg / L NAA.

[0038] Optimized primary culture media with different amounts of elicitor peptides: Based on the above optimized primary culture media, 0 nM, 1 nM, 5 nM, and 10 nM elicitor peptides were added, respectively, with the media without elicitor peptides serving as a control. Cultured for 30 days. Specific results are shown in Table 2 and... Figure 4 As shown, in Figure 4 In the middle section, A. the growth of Dendrobium officinale in its first generation culture after the addition of different elicitor peptides, and B. the effect of different amounts of elicitor peptides on the budding length; Table 2 Results of primary culture medium elicitor peptide optimization

[0039] The results showed that the germination rate of Dendrobium explants supplemented with 10 nM elicitor peptide was 98.32%, significantly higher than the control, increasing by 5.87%. Bud length analysis revealed better growth in the 10 nM elicitor peptide-treated buds, with a bud length of 1.69 cm, a 24.85% increase compared to the 1.27 cm length in the 0 nM elicitor peptide-treated buds. Germination time analysis showed that the germination time of Dendrobium explants supplemented with 10 nM elicitor peptide was 8 days, significantly shorter than the 12 days without elicitor peptide supplementation (33.33%). Furthermore, the initial culture time of Dendrobium explants supplemented with 10 nM elicitor peptide was 24 days, significantly shorter than the 30 days without elicitor peptide supplementation (20%). Therefore, the optimal initial culture medium for elicitor peptide supplementation was: Experimental Group 4: MS + 2 mg / L 6-BA + 0.5 mg / L NAA + 10 nM elicitor peptide.

[0040] Example 4: Optimization of culture medium during the subculture propagation stage of Dendrobium officinale Orthogonal experiments were conducted to optimize the subculture medium: Using MS medium as the base, two factors that significantly affected the subculture of Dendrobium tissue culture were selected: the plant growth regulators 6-benzylaminopurine (6-BA) and naphthaleneacetic acid (NAA). Two levels were set for each factor: 6-BA at 2.0 mg / L and 3.0 mg / L; and NAA at 0.05 mg / L and 0.1 mg / L. The experimental results are shown in Table 3 and... Figure 5 As shown, in Figure 5 In the table, A. the growth and proliferation of Dendrobium officinale, and B. the proliferation coefficient of Dendrobium officinale.

[0041] Table 3 Results of orthogonal experiments optimizing the subculture proliferation medium

[0042] Experiment 4 had a higher growth coefficient of 4.66, which was significantly higher than other experimental groups. Therefore, the optimized subculture culture medium was: MS + 3 mg / L 6-BA + 0.1 mg / L NAA.

[0043] Optimized subculture proliferation media with different amounts of elicitor peptide: Based on the above optimized subculture proliferation media, 0 nM, 1 nM, 5 nM, and 10 nM elicitor peptides were added respectively (Table 4), with the media without elicitor peptides serving as a control. The results are shown in Table 4 and... Figure 6 As shown, in Figure 6 In the study, A. the proliferation of Dendrobium officinale with different elicitor peptides was investigated, and B. the effect of elicitor peptides on the proliferation coefficient and bud length.

[0044] Table 4 Results of the optimization experiment of elicitor peptides in subculture proliferation medium

[0045] The proliferation coefficient of the plant with 10 nM elicitor peptide was 5.41, significantly higher than that without elicitor peptide by 23.80%. Statistical analysis of shoot length revealed better shoot growth with 10 nM elicitor peptide, with an average length of 3.59 cm, a significant increase of 31.02% compared to the average length of 2.74 cm with 0 nM elicitor peptide. Furthermore, the proliferation time of Dendrobium explants with 10 nM elicitor peptide was 21 days, significantly shorter than the 30 days without elicitor peptide by 33.33%.

[0046] Example 5: Optimization of rooting culture medium during the rooting stage of Dendrobium officinale Based on 1 / 2 MS medium, single-factor optimization of different concentrations of elicitor peptides was performed, and the culture was carried out for 30 days. The results are shown in Table 5. Figure 7 As shown, in Figure 7 The study included: A) the growth of *Dendrobium* with different amounts of elicitor peptides; B) the rooting rate with different amounts of elicitor peptides; and C) the root length with different amounts of elicitor peptides. The results showed that the rooting rates with 5 nM and 10 nM peptides were 88.33% and 93.33%, respectively, significantly higher than the 81.66% rooting rate without elicitor peptides by 8.17% and 14.29%, indicating that elicitor peptides significantly promoted *Dendrobium* rooting. Simultaneously, root length was measured. The results showed that the root lengths with 5 nM and 10 nM peptides were 2.65 cm and 3.92 cm, respectively, significantly higher than the 2.38 cm root length without peptides by 11.34% and 64.71%, indicating that elicitor peptides significantly promoted *Dendrobium* root growth.

[0047] Table 5. Optimized design and results of rooting culture medium

[0048] In Examples 3-5 above, MS medium with catalog number HB8469-5 was selected.

[0049] Example 6: The culture medium was prepared according to the requirements of Examples 3 to 5, and the elicitor peptide was used in the primary shoot induction stage, subculture proliferation stage, and rooting stage of Dendrobium officinale cultivation. The results are as follows: Figure 8 As shown, in Figure 8 In the given text, A. Primary culture 0 days B. Primary culture 30 days C. Subculture 0 days D. Subculture 60 days E. Rooting culture 0 days F. Rooting culture 30 days

[0050] The specific embodiments described above further illustrate the purpose, technical solution, and beneficial effects of the present invention. It should be understood that the above description is only a specific embodiment of the present invention and is not intended to limit the scope of protection of the present invention. 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 elicitor peptide, characterized in that, The amino acid sequence of the elicitor peptide is shown in SEQ ID NO:

1.

2. The elicitor peptide as described in claim 1, characterized in that, The elicitor peptide is prepared by a prokaryotic expression system, a eukaryotic expression system, or by chemical synthesis.

3. The elicitor peptide as described in claim 2, characterized in that, In the chemical synthesis method, Fmoc solid-phase synthesis technology was adopted using an Applied Biosystems 431 synthesizer. The specific synthesis steps are as follows: S1 Deprotection: Piperidine was used to remove the Fmoc protecting group of the amino acid to obtain the free amino acid; S2 Activation and cross-linking: The carboxyl group of the next amino acid was activated using an activator to obtain the activated amino acid. The activated amino acid reacted with the free amino acid to cross-link and form a peptide bond; S3 Cyclic extension: Steps S1 and S2 were repeated in the order of the amino acid sequence of the elicitor peptide until the synthesis was completed; S4 Post-processing: After the synthesis was completed, TFA solution was first used for elution and deprotection, and then reversed-phase high-performance liquid chromatography was used for purification.

4. The application of an elicitor peptide in the cultivation and propagation of Dendrobium, wherein the elicitor peptide is the elicitor peptide described in any one of claims 1-3, characterized in that, The elicitor peptide is used in the primary shoot induction stage, subgeneration proliferation stage, and rooting stage of Dendrobium cultivation.

5. The application of the elicitor peptide as described in claim 4 in the cultivation and propagation of Dendrobium, characterized in that, Dendrobium specifically refers to Dendrobium officinale. During the primary shoot induction stage of Dendrobium officinale propagation, 6-benzylaminopurine, naphthaleneacetic acid, and elicitor peptides are added to the culture medium containing MS medium. During the subculture proliferation stage of Dendrobium officinale propagation, 6-benzylaminopurine, naphthaleneacetic acid, and elicitor peptides are added to the culture medium containing MS medium. During the rooting stage of Dendrobium officinale propagation, indolebutyric acid and elicitor peptides are added to the culture medium containing MS medium. The MS medium catalog number is HB8469-5.

6. The application of the elicitor peptide as described in claim 5 in the cultivation and propagation of Dendrobium, characterized in that, During the initial shoot induction stage, for the culture medium containing standard MS medium, the following dosages were added: 6-benzylaminopurine: 2 mg / L; naphthaleneacetic acid: 0.5 mg / L; elicitor peptide: 10 nM.

7. The application of the elicitor peptide as described in claim 5 in the cultivation and propagation of Dendrobium, characterized in that, During the subculture proliferation stage, for culture medium containing standard MS medium, the following dosages were added: 6-benzylaminopurine: 3 mg / L; naphthaleneacetic acid: 0.1 mg / L; elicitor peptide: 10 nM.

8. The application of the elicitor peptide as described in claim 5 in the cultivation and propagation of Dendrobium, characterized in that, During the rooting stage, for a culture medium containing 1 / 2 standard MS medium, the following dosages were added: indolebutyric acid: 0.5 mg / L; elicitor peptide: 10 nM.

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