Dendrobium nobile endophytic fungus and application thereof

Abstract

The invention discloses a dendrobium nobile endophytic fungus for producing dendrobine and application of the dendrobium nobile endophytic fungus. The method aims at solving the problems that in an existing plant, the dendrobine content is low, and the culture period is long. The dendrobium nobile endophytic fungus is fusarium 689, and is preserved in the China General Microbiological Culture Collection Center (CGMCC) on May 21, 202 5, and the preservation number is CGMCC 3.27824. The method for producing dendrobine by using the strain comprises the following steps: carrying out dark culture on preserved hyphae in an artificial climate box at 25 DEG C for 12 hours, then picking up the hyphae, inoculating the hyphae into a new solid PDA culture medium, carrying out dark culture at 25 DEG C for several days, picking up the hyphae, inoculating the hyphae into a 100ml liquid PDB culture medium, culturing in a culture bottle in a dark environment at the speed of 120 rpm and the temperature of 25 DEG C for about 30 days, and separating fermentation liquor from thalli (dried), so as to obtain the dendrobine. And detecting dendrobine from the thalli. The strain has good antioxidant enzyme activity and can also be applied to production of dendrobine through microbial fermentation.

Description

Technical Field

[0001] The invention belongs to the technical field of plant growth and secondary metabolism regulation, and particularly relates to an endophytic fungus of Dendrobium nobile and an application thereof. Background Art

[0002] Dendrobium nobile (Dendrobium mobile) is a traditional and precious Chinese medicinal material. Its slow natural growth and low content of active ingredients have severely hampered its industrial development and utilization. Dendrobium alkaloids, a key medicinal component of D. nobile, possess numerous pharmacological effects, including anticancer, antioxidant, anti-inflammatory, and blood sugar regulation. Currently, there is a significant demand for research and production of dendrobium alkaloids. However, D. nobile has a low reproduction rate under natural conditions, its wild resources are scarce, and the quality of cultivated products varies greatly. Therefore, extracting dendrobium alkaloids directly from the plant is difficult to meet demand. Current approaches to improving the quality of D. nobile and promoting the accumulation of its active ingredients include breeding new varieties, changing cultivation methods, optimizing fertilization schedules, and adding hormone elicitors. However, these methods suffer from long production cycles, limited effectiveness, poor sustainability, and soil compaction.

[0003] Numerous studies have shown that endophytic fungi can produce similar or identical secondary metabolites as their host plants. These endophytic fungi can promote the growth of host plants and the accumulation of secondary metabolites, making them of great research value. Therefore, utilizing endophytic fungi to produce plant secondary metabolites will be a hot topic for future research. However, few studies have reported on the application of endophytic fungi to improve the yield and quality of Dendrobium nobile. Therefore, the study of an endophytic fungus that can increase dendrobine production in D. nobile is of great significance for improving the yield and quality of D. nobile and accelerating the development of the D. nobile industry. Summary of the Invention

[0004] The present invention aims to provide an endophytic fungus of Dendrobium nobile, which can be used more conveniently to obtain dendrobine, thereby solving the problem in the prior art that directly extracting dendrobine from Dendrobium is difficult to meet large-scale demand and has low acquisition efficiency.

[0005] The present invention relates to an endophytic fungus of Dendrobium nobile, wherein the nucleotide sequence of the endophytic fungus of Dendrobium nobile is shown as SEQ ID No. 1.

[0006] The endophytic fungus of Dendrobium nobile, classified and named: Fusarium sp., was deposited in the General Microbiology Center of China Culture Collection Administration of Microorganisms on May 21, 2025, with the deposit number: CGMCC No.3.27824.

[0007] Dendrobium is a compound with multiple pharmacological activities and has a wide range of applications in medicine, health products, and other fields. However, traditionally, dendrobium is mainly obtained by extraction from plants such as Dendrobium nobile, which not only consumes a large amount of plant resources but also has low extraction efficiency. The endophytic fungus of the present invention provides an effective way to solve this problem. The present invention not only helps to save Dendrobium resources but also has significant advantages in improving the synthesis efficiency of dendrobium.

[0008] The present invention relates to an endophytic fungus isolated from Dendrobium nobile, which can produce a high secondary metabolite, dendrobine, through an optimized cultivation method. Specifically, the hyphae of the Dendrobium nobile endophytic fungus are cultured in the dark at 25°C for several hours, the hyphae are picked up and inoculated into a new solid PDA culture medium, and after being cultured in the dark at 25°C for several days, the hyphae are picked up and inoculated into a liquid PDB culture medium. The culture is carried out in the dark at 120 rpm and 25°C for 25 to 35 days, and the fermentation liquid is separated from the fungus body to obtain the dendrobine from the fungus body.

[0009] Preparation of the potato solid culture medium (PDA): fresh peeled potato pieces are placed in boiling water and kept boiling for 30 minutes. The water is filtered, 20g of glucose and 12.5g of agar powder are added, and the volume of the culture medium is adjusted to 1L with potato extract. The pH value is 5.6±0.2. After the culture medium is packaged, it is sterilized (121°C, 30 minutes) for later use.

[0010] Furthermore, the endophytic fungus of Dendrobium nobile described in the present invention not only produces a high amount of dendrobine but also has oxidase activity, which makes the fungus potentially useful in the field of biocatalysis. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 This is a colony morphology diagram of the endophytic fungus 689 of Dendrobium nobile of the present invention;

[0012] Figure 2 This is a microscopic morphology of the hyphae of the endophytic fungus 689 of Dendrobium nobile of the present invention;

[0013] Figure 3 This is the phylogenetic tree of the endophytic fungus 689 of Dendrobium nobile of the present invention;

[0014] Figure 4 This is the LC-MS spectrum of the dendrobium alkaloid reference substance;

[0015] Figure 5 This is the LC-MS analysis result of the fermentation cell of the endophytic fungus 689 of Dendrobium nobile of the present invention.

[0016] Figure 6 is the LC-MS dendrobine standard curve;

[0017] Figure 7It is the dendrobine content of the fermentation cell of the endophytic fungus 689 of Dendrobium nobile of the present invention. DETAILED DESCRIPTION

[0018] The following is further described in detail through specific implementation methods:

[0019] Prepare culture medium:

[0020] Potato solid culture medium (PDA): Place fresh peeled potato pieces in boiling water and keep boiling for 30 minutes. Filter and add 20g of glucose and 12.5g of agar powder. Use potato extract to adjust the volume of the culture medium to 1L. The pH value is 5.6±0.2. After aliquoting, sterilize (121℃, 30min) and set aside.

[0021] Potato liquid culture medium (PDB): Place fresh peeled potato pieces in boiling water and keep boiling for 30 minutes. Filter and add 20 g of glucose and 0.1 g of chloramphenicol. Use potato extract to adjust the volume of the culture medium to 1 L. The pH value is 5.6 ± 0.2. After aliquoting, sterilize (121°C, 30 minutes) and set aside.

[0022] 1. Isolation of endophytic fungus 689 from Dendrobium nobile

[0023] Fresh stems of Dendrobium nobile were collected and surface-sterilized. Sterilize the stems by adding 20-30 ml of 75% ethanol to a 50 ml centrifuge tube for 30 seconds, shaking the tube several times every 10 seconds. Rinse with sterile water three times, add 20-30 ml of 0.1% mercuric chloride for 1 minute, and rinse with sterile water three to five times. Cut the surface-sterilized stems into 0.5 cm long segments with surgical scissors. Each segment, consisting of four segments, was inoculated into PDA culture medium and incubated in a dark incubator at 25°C.

[0024] After observing the growth of endophytic fungi hyphae, immediately use a sterilized toothpick to pick up the hyphae from the top and inoculate them into new PDA solid culture medium. Repeat the inoculation 3 to 5 times to obtain a single strain. The purified strain is stored in a -4°C refrigerator for a short period of time using the slant preservation method for later use.

[0025] 2. Morphological observation and molecular identification of endophytic fungus 689 from Dendrobium nobile

[0026] The colonies are round and cotton-like, covering the entire culture dish. The front of the colonies is white and light pink. Figure 1 As shown. Microconidia are oval or oblong, with pseudo-heads. Figure 2 shown.

[0027] After amplification of the ITSDNA sequence of the endophytic fungus 689 from Dendrobium nobile, a specific band of 523 bp was obtained, the sequence is shown as SEQ ID No. 1, and submitted to the GenBank database for Blast sequence alignment. The sequence with the highest similarity was screened, and the phylogenetic tree was constructed using MEGA11.0 software. The results showed that it was located in the same branch as Fusarium subglutinans isolate G72L, as shown in Figure 1. Figure 3 , and the homology between the two was 99.22%. Based on morphological and molecular identification, 689 was finally identified as Fusarium.

[0028] 3. Liquid fermentation culture of endophytic fungus 689 from Dendrobium nobile

[0029] Basic culture conditions: Stored mycelium was incubated in a 25°C artificial climate chamber in the dark for 12 hours. The mycelium was then picked and inoculated into new solid PDA medium. After 3-5 days of dark incubation in a 25°C artificial climate chamber, the mycelium was picked and inoculated into 100 ml of liquid PDB medium. The culture flasks were incubated in the dark at 120 rpm and 25°C for approximately 30 days. The fermentation broth and the bacterial cells (dried) were separated and stored separately for future use. Treatment group: Fresh Dendrobium stems were washed with distilled water, the water was removed from the plants with filter paper, and the plants were weighed. The plants were cut into small pieces, and 0.1 g of the pieces were added to PDB liquid medium and co-cultured with the endophytic fungus 689 from Dendrobium nobile. The culture flasks were incubated in the dark at 120 rpm and 25°C for approximately 30 days. The fermentation broth and the bacterial cells (dried) were separated and stored separately for future use.

[0030] 4. Analysis of secondary metabolites of endophytic fungus 689 in Dendrobium nobile

[0031] ⑴LC-MS conditions

[0032] BEH C18 (2.1×100mm, 1.7μm), mobile phase: 100% acetonitrile (A)-0.05% formic acid in water (B), flow rate: 1mL / min. Initial temperature: 30°C, increase to 50°C at a rate of 10°C / min, hold for 5min, injection volume: 10uL.

[0033] ⑵ Preparation of standard solution

[0034] Weigh the appropriate mass concentration of dendrobium alkaloid reference substance and prepare it to a mass concentration of 20 ng / mL using 100% chromatography 0.05% formic acid methanol as the reference solution.

[0035] ⑶ Preparation of test solution

[0036] Take 0.1 g of liquid fermentation mycelium of endophytic fungus of Dendrobium nobile, dry it, add 10 ml of 0.05% formic acid in methanol, reflux and evaporate, make the volume to 2 ml, and save it for later use.

[0037] 5. Drawing of the Dendrobiumine Standard Curve

[0038] Accurately weigh 1 mg of dendrobium alkaloid standard powder and dissolve it in 0.05% formic acid in methanol to 2 mL. Dilute to 1, 10, 20, 50, and 100 ng / mL with 0.05% formic acid in methanol. Determine the concentration of dendrobium alkaloids by liquid chromatography-mass spectrometry. Draw a dendrobium alkaloid standard curve based on the peak area of ​​the LC-MS spectrum.

[0039] Experimental results

[0040] LC-MS was used to analyze the fermentation cells of endophytic fungus 689, with dendrobiumine as the reference. The results showed that the same chromatographic peaks appeared in endophytic fungus 689 and the reference at the same retention time. Preliminary identification confirmed that dendrobiumine was present in the fermentation cells of endophytic fungus, and it was speculated that endophytic fungus 689 was the producer of dendrobiumine.

[0041] Liquid chromatography-mass spectrometry was used to determine the peak area of ​​the standard curve and draw the standard curve. Figure 6 .

[0042] By calculating the dendrobium alkaloid content of the control group and the treatment group, the results showed that the content of dendrobium alkaloid in the control group was 246.2±92.15ng / g, and the content of dendrobium alkaloid in the treatment group was 53338.9±16409.2ng / g. The highest yield was 303449.4ng / g, and the yield increased by about 200 times. Figure 7 .

[0043] 5. Antioxidant activity analysis of endophytic fungus 689 from Dendrobium nobile

[0044] ⑴ Preparation of endophytic fungus 689 spore suspension

[0045] Endophytic fungus 689 was cultured on PDA medium at 28°C in the dark for 7 days, inoculated into a 250ml Erlenmeyer flask containing potato liquid medium PDB, and shaken on a shaker for 21 days to prepare endophytic fungus 689 fermentation liquid. The endophytic fungus 689 fermentation liquid was filtered through gauze to remove the mycelium in the fermentation liquid to obtain a spore suspension. The spore count was determined using a hemocytometer and the spore count was adjusted to 1.6×10 6 ml -1 spare.

[0046] ⑵ Determination of SOD and POD enzyme activities of endophytic fungus 689

[0047] In this study, the spore suspension of endophytic fungus 689 was used to determine the SOD enzyme activity of the strain using the Shanghai Shenggong SOD assay kit. Principle: SOD (EC 1.15.1.1) is a metalloenzyme widely present in organisms. It is an important oxygen free radical scavenger that can catalyze the dismutation of superoxide anions to produce H2O2 and O2. SOD is not only a superoxide anion scavenger enzyme, but also the main H2O2 generating enzyme. It plays an important role in the biological antioxidant system. Superoxide anions (O2) are produced through the xanthine and xanthine oxidase reaction system, and O2 - Can reduce nitro blue tetrazolium to generate blue formazan, which absorbs at 560nm: SOD removes O2 - , thereby inhibiting the formation of formazan; the darker the blue color of the reaction solution, the lower the SOD activity, and vice versa.

[0048] POD enzyme activity was similarly measured using a spore suspension of endophytic fungi using the Shanghai Sangon POD Assay Kit. Principle: POD (EC 1.11.1.7) is widely present in animals, plants, microorganisms, and cultured cells. It catalyzes hydrogen peroxide to oxidize phenols and amines, thus detoxifying both hydrogen peroxide and phenols and amines. POD catalyzes the oxidation of specific substrates with H₂O₂ and exhibits a characteristic absorbance at 470 nm.

[0049] SOD activity (U / 10 4 cell) = [inhibition percentage ÷ (1-inhibition percentage) x V total] ÷ (150 x V sample + V total) x F = 0.022 x inhibition percentage ÷ (1-inhibition percentage) x F

[0050] Vreaction: total volume of reaction system, 0.2 mL; Vsample: volume of sample added to reaction system, 0.02 mL; Vsample: volume of extract added, 1 mL; Cpr: sample protein concentration, mg / mL; W: sample weight, g; 150: total number of spores, 1.5 million; F: sample dilution factor.

[0051] POD(U / 10 4 cell) = △A x V total = (150 x V sample + V total) ÷ ​​0.01 ÷ T = 14.27 x △A

[0052] Vreaction: total volume of reaction system, 1.07 mL; Vsample: volume of sample added, 0.015 mL; Vsample: volume of extract added, 1 mL; T: reaction time, 1 min; Cpr: sample protein concentration, mg / mL; W: sample mass, g; 150: total number of spores, 1.5 million.

[0053] Experimental results: The spore suspension of endophytic fungus 689 from Dendrobium nobile can efficiently express SOD and POD enzymes within the tested concentration range, with SOD activity of 0.74u / 10 4 cell, POD activity was 4.68u / 10 4 cell,

[0054] In summary, liquid fermentation using endophytic fungus 689 confirmed that endophytic fungus 689 from Dendrobium nobile can not only produce dendrobine, but also significantly increase the yield of dendrobine by changing the culture conditions. It also has good antioxidant enzyme activity, making it an endophytic fungus worthy of further study.

Claims

1. An endophytic fungus of Dendrobium nobile, characterized in that: The nucleotide sequence of the endophytic fungus of Dendrobium nobile is shown as SEQ ID No.

1.

2. The endophytic fungus of Dendrobium nobile according to claim 1, wherein: The endophytic fungus of Dendrobium nobile was deposited in the General Microbiology Center of China Culture Collection Administration on May 21, 2025, with the deposit number: CGMCC 3.27824.

3. Application of an endophytic fungus of Dendrobium nobile according to claim 1 or 2 in producing dendrobine.

4. The use according to claim 2, characterized in that: After culturing the mycelia of the endophytic fungus of Dendrobium nobile at 25° C. in the dark for several hours, the mycelia are picked up and inoculated into a new solid PDA culture medium. After culturing in the dark at 25° C. for several days, the mycelia are picked up and inoculated into a liquid PDB culture medium for culturing. The culture is carried out at 120 rpm and 25° C. in a dark environment for 25 to 35 days, the fermentation liquid is separated from the bacterial body, and the dendrobine is obtained from the bacterial body.

5. Use of an endophytic fungus of Dendrobium nobile according to claim 1 or 2 in increasing dendrobine production.

6. Application of the SOD and POD enzyme activities of the endophytic fungus of Dendrobium nobile according to claim 1 or 2.

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