Application and method of geotrichum galactose yeast NBG001 in improvement of alkaloid accumulation in lycoris radiata

Abstract

The invention provides application and method of geotrichum galactose yeast NBG001 in improving accumulation of alkaloids for lycoris radiata traditional Chinese medicine, and belongs to the technical field of agricultural production, the geotrichum galactose yeast NBG001 is preserved in China Center for Type Culture Collection on July 7, 2025, and the preservation number is CCTCC NO: M 20251547. By inducing the expression of signal molecules and key genes in the lycoris radiate, the simultaneous accumulation of galanthamine, lycorine and narcissus in the lycoris radiate is remarkably improved.

Description

TECHNICAL FIELD

[0001] The application provides application and method of Galactomyces geotropicalis NBG001 in improving alkaloid accumulation in Lycoris radiata, and belongs to the technical field of agricultural production. BACKGROUND

[0002] Alkaloids in Lycoridae are a kind of unique secondary metabolites only existing in Lycoridae plants, and have various medicinal activities. Among them, galanthamine as a representative component has been widely used in the treatment of Alzheimer's disease in the form of hydrobromide; lycoramine has significant anti-tumor, anti-viral, antibacterial and anti-inflammatory activities; and narcissus cyclavasine exhibits strong anti-tumor activity. In Asian regions, alkaloids in Lycoridae mainly rely on extraction from Lycoris radiata ( Lycoris radiata ), but the content of alkaloids in Lycoris radiata is extremely low, only about 60 grams of total alkaloids can be extracted from 15 kilograms of dried bulbs of Lycoris radiata, which leads to great difficulty in extraction and consumption of a large amount of organic solvents, and at the same time, strong corrosive acid and alkali waste liquid is produced. Therefore, how to efficiently promote the accumulation of medicinal alkaloids in Lycoris radiata is the key to reduce the production cost and further promote the development and utilization of Lycoris radiata. However, there is still a lack of practical and efficient induction strategy.

[0003] In recent years, studies have shown that plant endophytic fungi play an important role in promoting the synthesis of secondary metabolites in host plants, which can promote the accumulation of specific active ingredients by secreting induction factors, inducing plant defense response, activating synthesis key genes and other ways. It is expected to break through the current bottleneck of low accumulation efficiency and high extraction cost of alkaloids. SUMMARY

[0004] The application provides a kind of Galactomyces geotropicalis NBG001 in improving alkaloid accumulation in Lycoris radiata and method of application, can significantly improve the accumulation of a variety of alkaloids in Lycoris radiata.

[0005] To solve the above technical problems, the application provides a kind of Galactomyces geotropicalis NBG001 in improving alkaloid accumulation in Lycoris radiata and application, the Galactomyces geotropicalis NBG001, on July 7, 2025, is preserved in China typical culture preservation center, and the preservation number CCTCC NO: M 20251547.

[0006] Further, the alkaloids include galanthamine, lycoramine and narcissus cyclavasine.

[0007] Further, the Galactomyces geotropicalis NBG001 improves the accumulation of alkaloids by inducing Lycoris radiata to produce signal molecules and activating synthesis key genes.

[0008] Further, the signal molecules include NO and H2O2.

[0009] Further, the key genes include a gene encoding phenylalanine ammonia-lyase PAL , a gene encoding tyrosine decarboxylase TYDC , a gene encoding oxygen methyltransferase OMT .

[0010] In addition, the application also provides a method for improving alkaloid accumulation in Lycoris radiata, wherein sterile bulbs of Lycoris radiata are induced to grow into complete plants, and pieces of Galactomyces geotrichum NBG001 are inoculated on the Lycoris radiata seedlings; the Galactomyces geotrichum NBG001 is preserved in the China Center for Type Culture Collection on July 7, 2025, and the preservation number is CCTCC NO: M20251547.

[0011] The specific process comprises the following steps: The surface-sterilized bulbs of Lycoris radiata are cut into four pieces in a longitudinal cross shape, and each piece is vertically inserted into an MS medium containing 6 mg / L 6-BA, 0.5 mg / L NAA, 30 g / L sucrose and 10 g / L agar to induce culture and generate bulbs; the generated bulbs are transplanted into an MS medium containing 0.5 mg / L NAA, 30 g / L sucrose and 10 g / L agar, and are cultured in an illumination incubator to induce the formation of small bulbs; after a plurality of small bulbs are formed, the single small bulbs are separated and transplanted into an MS medium containing 0.5 mg / L NAA, 30 g / L glucose and 10 g / L agar to induce the formation of complete plants; and the experiment is performed after 8 weeks of transplantation; in the illumination incubator, the photoperiod is 12 h, the light intensity is 3400 Lx, and the temperature cycle is 25 / 18°C. The pieces of Galactomyces geotrichum NBG001 are inoculated on PDA medium, and are streaked and cultured until single colonies are formed; sterile punchers are used to take pieces of the bacteria with a diameter of 5 mm, which are inoculated on new PDA medium; after 5 days, sterile punchers are used to take pieces of the bacteria with a diameter of 5 mm at the edge of the colonies, the mycelium faces downward, and the pieces of the bacteria are inoculated on the MS medium.

[0012] The application provides the application and method of Galactomyces geotrichum NBG001 in improving alkaloid accumulation in Lycoris radiata, and the Galactomyces geotrichum NBG001 can significantly promote the simultaneous accumulation of galanthamine, lycorine and narcissusin in Lycoris radiata. BRIEF DESCRIPTION OF DRAWINGS

[0013] Figure 1 The content of nitric oxide in the Lycoris radiata plants inoculated with the Galactomyces geotrichum for 3, 7 and 14 days according to the embodiment of the application; Figure 2 The content of hydrogen peroxide in the Lycoris radiata plants inoculated with the Galactomyces geotrichum for 3, 7 and 14 days according to the embodiment of the application; Figure 3 The relative expression levels of key genes for alkaloid synthesis in Lycoris radiata plants 7 days after inoculation with Geotrichum galactosidase according to the embodiment of the present invention; Figure 4 The content of galantamine, lycorine and narcissin in Lycoris radiata leaves, bulbs and roots inoculated with Geotrichum galactosidase in the embodiment of the present invention.

[0014] The deposit information of the Geotrichum galactosidase of the present invention is as follows: Geotrichum galactosidase, classified as Galactomyces geotrichum NBG001, deposited in China Center for Type Culture Collection, with accession number CCTCC NO: M 20251547, and deposit date: July 7, 2025. DETAILED DESCRIPTION

[0015] The present invention will be further described below in conjunction with specific examples. The following examples are only used to more clearly illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention.

[0016] The present invention is further described below with reference to the accompanying drawings and examples. The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are commercially available unless otherwise specified. The experimental materials involved in the following examples are as follows: Geotrichum galactosidase ( Galactomyces geotrichum ) NBG001 strain, derived from the published strain of our laboratory (public patent application number: 2025111688719), was deposited in the China Center for Type Culture Collection on July 7, 2025, with the deposit number: CCTCC NO: M 20251547, and was stored in a glycerol tube at -80℃.

[0017] Example 1 Method for increasing alkaloids in Lycoris radiata by using Geotrichum galactosidase NBG001 strain Surface-sterilized Lycoris radiata bulbs were cut longitudinally and evenly into four pieces. Each piece was inserted vertically into MS medium containing 6 mg / L 6-BA, 0.5 mg / L NAA, 30 g / L sucrose, and 10 g / L agar to induce bulblet formation. After multiple bulblets formed, individual bulblets were separated and transplanted into MS medium containing 0.5 mg / L NAA, 30 g / L glucose, and 10 g / L agar to induce full plants. Experiments were performed 8 weeks after transplanting. Incubation was carried out in a light incubator with a 12-h photoperiod, 3400 Lx light intensity, and a 25 / 18°C (day / night) temperature cycle.

[0018] G. mellulina NBG001 was suspended in 20% glycerol and stored at -80°C. When the culture was recovered, G. mellulina NBG001 liquid was inoculated by loop, streaked on PDA medium at 28°C until single colony formed, and then a 5 mm diameter mycelium disc was punched by sterile puncher and inoculated on new PDA medium. After 5 days, a 5 mm diameter mycelium disc was punched by sterile puncher at the edge of the colony with mycelium facing down and inoculated on MS medium. The Amaryllis plants inoculated with 5 mm diameter PDA disc were used as control group.

[0019] Example 2: Effect of G. mellulina NBG001 strain on the content of signal molecules in Amaryllis Amaryllis plants were inoculated according to the method of Example 1, and after 3, 7 and 14 days of inoculation, the plants were harvested, frozen in liquid nitrogen and stored at -80°C. 0.1 g of plant tissue was accurately weighed and added to 500 μL of phosphate buffer (0.1 M, pH = 7.0-7.4). The tissue homogenate was obtained by grinding at 0°C and 70 HZ for 30 s in a cryogenic grinder, and centrifuged at 3500 rpm for 10 min to obtain the supernatant. The contents of NO and H2O2 in the supernatant were determined. The method for determining NO was as follows: the supernatant was mixed with nitrate color reagent to generate a light red azo compound, and the absorbance was detected at 550 nm. The content of NO in each sample was calculated based on the standard curve constructed based on the concentration and absorbance of sodium nitrite standard. The method for detecting H2O2 was as follows: the supernatant was mixed with ammonium molybdate solution to generate a light yellow complex, and the absorbance was detected at 405 nm. The content of H2O2 in each sample was calculated based on the standard curve constructed based on the concentration and absorbance of H2O2 standard. The detection results are shown in Figure 1 and Figure 2 .

[0020] As can be seen from Figure 1 , there was no significant difference in the content of NO between the control group MOCK and the NBG001 inoculated group on the 3rd day after inoculation. However, the content of NO in the Amaryllis plants of the NBG001 inoculated group was significantly higher than that in the control group on the 7th and 14th day after inoculation. Therefore, the inoculation of G. mellulina NBG001 significantly induced the burst of NO in Amaryllis, and NO is one of the markers of the activation of plant defense response, which can activate downstream signals and promote the synthesis of alkaloids in Amaryllidaceae.

[0021] As can be seen from Figure 2It can be seen that after 3, 7 and 14 days of inoculation, the H2O2 content in the Lycoris radiata plants in the NBG001 treatment group was significantly higher than that in the control group plants inoculated with PDA discs, indicating that the inoculation of Galactomyces NBG001 significantly induced the burst of H2O2 in Lycoris radiata. H2O2 is also one of the signs of activation of plant defense response. It can synergize with NO to activate downstream signals and promote the synthesis of Amaryllidaceae alkaloids.

[0022] Example 3 Effect of Geotrichum galactosidase NBG001 strain on the expression of key genes for alkaloid synthesis in Lycoris radiata The inoculated Lycoris radiata was treated according to the method of Example 1. Seven days after inoculation, the NBG001-inoculated and control plants were harvested and divided into leaves, bulbs, and roots with a sterile knife. After quick freezing in liquid nitrogen, the plants were stored at -80°C. The total RNA of each tissue of Lycoris radiata was extracted using RNAprep Pure Plant Total RNA Extraction Kit. The total RNA of each tissue of Lycoris radiata was extracted using NanoDrop TM RNA was quantified using a 1000 micro-spectrophotometer. 1 μg of total RNA was treated with DNase and reverse transcribed into cDNA. ® Premix Ex Taq TM Perform qRT-PCR reaction to TIP41 As an internal reference gene, 2 –ΔΔCT The relative expression levels of key genes in alkaloid biosynthesis were calculated using the method; the focus was on three key genes in the biosynthesis pathway of Amaryllidaceae alkaloids: PAL (phenylalanine ammonia lyase), TYDC (tyrosine decarboxylase), OMT (oxygen methyltransferase), the specific primer information is shown in Table 1, and the test results are shown in Figure 3 shown.

[0023] Table 1 Primers used in qRT-PCR Primer name Primer sequence Sequence number 5'-GTGATGTTGTTGTTGAGGAGGCGA-3' SEQ ID NO. 1 5'-TAGGAGGACCAAGCAAGGAGGTGC-3' SEQ ID NO. 2 5'-ACCATCGTCTCTGTTTCATCTGTC-3' SEQ ID NO. 3 5'-TTGCTTCTCTCATTCTCATCCGTA-3' SEQ ID NO. 4 5'-GGGAGGTGACTGAGAAACATGAAT-3' SEQ ID NO. 5 5'-CCAAAGCGGTTGTGAGCAGAGAAT-3' SEQ ID NO. 6 5'-AATGTGCAAGCAGGGCTAGTAA-3' SEQ ID NO. 7 5'-GCAACCATCCAAAGTTTAACTGCT-3' SEQ ID NO. 8 from Figure 3 It can be seen that compared with the control group plants inoculated with PDA discs, the NBG001-treated group had PAL The expression levels of the genes were significantly increased, reaching 4.96 and 3.61 times of the control, respectively; TYDC The expression of the gene was significantly increased, reaching 2.45 times that of the control group; OMT The expression levels of the genes were also significantly increased, reaching 1.66 and 1.46 times that of the control group, respectively. This shows that the galactomycete NBG001 can effectively increase the expression levels of key genes in Lycoris radiata.

[0024] Example 4 Effect of Geotrichum galactosidase NBG001 strain on alkaloid accumulation in Lycoris radiata The inoculated Lycoris radiata was treated according to the method of Example 1. 28 days after inoculation, the plants were harvested and separated into leaves, bulbs, and roots with a sterile knife. The plants were dried at 50°C to constant weight and then ground into powder. 1 g of plant powder was accurately weighed, 5 mL of methanol was added, and the plants were extracted at 4°C for 24 h, during which time they were ultrasonically bathed at 70 kHz three times for 30 min each time. Thereafter, the supernatant was obtained by centrifugation at 8000 rpm and 4°C for 10 min. After filtering through a 0.22 μm filter membrane, the contents of galanthamine, lycorine, and narcissin were determined by high-performance liquid chromatography (HPLC). A C18 reversed phase column (4.6 mm × 250 mm × 5 μm) was used, with the mobile phase consisting of (A) 0.05% triethylamine in water and (B) acetonitrile. The gradient elution system was 0-20 min, 10-30% B, 20-35 min, 30-40% B, the flow rate was 1 mL / min, the column temperature was 35°C, and the sample volume was 20 μL. The detection wavelengths for galanthamine, lycorine, and narcissin were 280, 290, and 300 nm, respectively. The results are shown in Figure 2. Figure 4 shown.

[0025] from Figure 4 It can be seen that 28 days after inoculation, the inoculation of Galactomyces NBG001 significantly promoted the accumulation of galantamine in the roots of Lycoris radiata, reaching 6.72 times that of the PDA disc inoculation control; significantly promoted the accumulation of lycorine in the bulbs of Lycoris radiata, reaching 2.20 times that of the control; significantly promoted the accumulation of narcissin in the bulbs and roots of Lycoris radiata, reaching 2.43 and 5.62 times that of the control, respectively.

[0026] The present invention has been disclosed above with preferred embodiments, which are not intended to limit the present invention. Any technical solutions obtained by adopting equivalent replacement or equivalent transformation solutions fall within the protection scope of the present invention.

Claims

1. Use of Galactomyces geotrupus NBG 001 for increasing alkaloid accumulation in Lycoris radiata, characterized in that: The Geotrichum galactomyces NBG001 is preserved in the China Center for Type Culture Collection on July 7, 2025, and the preservation number is CCTCC NO: M20251547.

2. Use according to claim 1, characterized in that: The alkaloids include galanthamine, lycoramine and narcissus cyclamine.

3. Use according to claim 1, characterized in that: The Geotrichum galactomyces NBG001 improves the accumulation of alkaloids by inducing synthesis of signal molecules and activating key genes of synthesis.

4. Use according to claim 3, characterized in that: The signal molecules include nitric oxide NO and hydrogen peroxide H2O2.

5. Use according to claim 3, characterized in that: The key genes include a gene encoding phenylalanine ammonia-lyase PAL , a gene encoding tyrosine decarboxylase TYDC , a gene encoding oxygen methyltransferase OMT .

6. A method for improving the accumulation of medicinal alkaloids in Lycoris radiata, characterized in that: The sterile lycoris bulbs are induced to grow into complete plants, and the Geotrichum galactomyces NBG001 fungus pieces are inoculated on the lycoris seedlings; the Geotrichum galactomyces NBG001 is preserved in the China Center for Type Culture Collection on July 7, 2025, and the preservation number is CCTCC NO: M20251547.

7. The method for improving accumulation of medicinal alkaloids in Lycoris radiata according to claim 6, characterized in that, The specific process includes the following steps: The surface-sterilized lycoris bulbs are cut into 4 pieces in a longitudinal cross shape, each piece is vertically inserted into MS medium containing 6 mg / L 6-benzyladenine, 0.5 mg / L naphthalene acetic acid, 30 g / L sucrose and 10 g / L agar to induce culture, and the bulbs are generated; the generated bulbs are transplanted into Murashige-Skoog medium containing 0.5 mg / L NAA, 30 g / L sucrose and 10 g / L agar, and cultured in a light incubator; after multiple small bulbs are formed, the single small bulbs are separated and transplanted into MS medium containing 0.5 mg / L NAA, 30 g / L glucose and 10 g / L agar to induce into complete plants in a light incubator with a light period of 12 h, a light intensity of 3400 Lx and a temperature period of 25 / 18°C; The Geotrichum galactomyces NBG001 liquid is picked up with an inoculation ring, and streaked on potato dextrose agar medium until single colonies are formed; a sterile puncher is used to take fungus pieces with a diameter of 5 mm, which are inoculated on new PDA medium; after 5 days, a sterile puncher is used to take fungus pieces with a diameter of 5 mm from the edge of the colony, with the mycelium facing down and closely attached to the lycoris plants, which are inoculated on MS medium.

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