Method for inducing mycelial phase of leucoporus carnosus based on carbon quantum dots and application thereof

Abstract

The application discloses a method for inducing the mycelial phase of Leucomycicella bicuspidata based on carbon quantum dots and application thereof, and the method comprises the following steps: preparing carbon quantum dots by a hydrothermal method, inducing culture of the mycelial phase, and morphological observation and confirmation. The carbon quantum dots are prepared by hydrothermal reaction of citric acid and urea at 230 DEG C for 15 hours, and the particle size is 2-8 nm; the carbon quantum dots are added into a Sabouraud glucose culture medium, the final concentration is 0.5 mg / mL, and the Leucomycicella bicuspidata is cultured at 28 DEG C for 24-28 hours, so that the mycelial phase of the Leucomycicella bicuspidata can be significantly induced; in combination with microscopic observation and PCR molecular confirmation, the mycelial formation rate can reach more than 85%. The method provides stable and reliable experimental materials for the study of the pathogenic mechanism and the prevention and control technology of the Leucomycicella bicuspidata.

Description

Technical Field

[0001] This invention belongs to the field of aquatic animal pathogenic microorganism research technology, and in particular relates to a method for inducing mycelial phase of Saccharomyces cerevisiae based on carbon quantum dots and its application. Background Technology

[0002] Metschnikowia bicuspidata is an important pathogen of the Chinese mitten crab, which can cause "milk disease" in the crab, leading to atrophy of the hepatopancreas, weight loss, and increased mortality.

[0003] *Saccharomyces cerevisiae* exhibits two morphologies: a yeast phase and a hyphal phase. The formation of the hyphal phase is closely related to its pathogenicity. During host infection, this bacterium can transform from the yeast phase to the hyphal phase, enhancing its tissue penetration ability and the expression of virulence factors. In-depth research into the hyphal phase formation mechanism is of great significance for elucidating the pathogenic mechanism of this bacterium and developing novel prevention and control strategies.

[0004] Current research on *Saccharomyces cerevisiae* indicates that this pathogen can infect various farmed aquatic animals, including economically valuable crustaceans such as the Chinese mitten crab, swimming crab, and river crab, leading to "milk disease." Infected crabs exhibit symptoms such as opaque, milky-white muscle, hemolymph emulsification, and general weakness, ultimately resulting in death. Currently, there is no effective treatment for this disease.

[0005] Therefore, research on the pathogenic mechanism of *Saccharomyces cerevisiae* is of significant theoretical and practical importance. However, existing methods for inducing mycelial phases of *Saccharomyces cerevisiae* suffer from low induction efficiency and poor reproducibility. Commonly used induction methods include serum induction and corn flour medium induction, but the mycelial formation rate is usually only 40-60%, and the induction conditions are unstable, making it difficult to provide sufficient and stable mycelial phase material for in-depth research on pathogenic mechanisms. Summary of the Invention

[0006] To address the aforementioned technical problems of low induction efficiency and poor reproducibility of *Saccharomyces cerevisiae* hyphae, this invention provides a method for inducing *Saccharomyces cerevisiae* hyphae based on carbon quantum dots and its application, providing stable and reliable experimental materials for in-depth research on the pathogenic mechanism of this bacterium.

[0007] Carbon quantum dots, as a novel nanomaterial, possess excellent biocompatibility, fluorescence properties, and surface activity, showing broad application prospects in the biomedical field. Studies have found that carbon quantum dots can significantly influence the growth and morphological transformation of microorganisms through mechanisms such as regulating cell membrane permeability and affecting cellular metabolic activities. Therefore, this invention is based on carbon quantum dots inducing mycelial phases in *Saccharomyces cerevisiae*.

[0008] The objective of this invention is achieved through the following technical solution: This invention discloses a method for inducing mycelial phases of *Saccharomyces cerevisiae* based on carbon quantum dots, comprising the following steps: a) Preparation of carbon quantum dots by hydrothermal method: 2.0 g of citric acid and 1.0 g of urea were dissolved in 20 mL of deionized water and hydrothermally reacted at 230 °C for 15 hours to obtain a carbon quantum dot solution; b) Preparation of induction medium: Add the carbon quantum dot solution prepared in step a) to Sabouraud dextrose medium to a final concentration of 0.1-0.5 mg / mL to obtain the induction medium; c) Mycelial induction culture: Inoculate *Saccharomyces cerevisiae* into induction medium and incubate at 28°C for 24-48 hours to obtain filamentous *Saccharomyces cerevisiae*. d) Detection and confirmation: Observe the hyphal formation under a microscope. If the hyphal formation rate is ≥85% after 48 hours, confirm it by PCR molecular biology method to obtain a stable hyphal phase.

[0009] Furthermore, the carbon quantum dots have a particle size of 2-8 nm, exhibit fluorescence properties, an excitation wavelength of 360 nm, and an emission wavelength of 440 nm.

[0010] Furthermore, the pH of the induction medium is adjusted to 6.0-6.5.

[0011] Furthermore, the induction culture medium is prepared by autoclaving at 121°C for 15-20 minutes.

[0012] Furthermore, the PCR molecular biology method confirmed that the ITS region of Saccharomyces cerevisiae was amplified using specific primers, and the amplified fragment was 600 bp.

[0013] The application of the carbon quantum dot-based method for inducing mycelial phases of Saccharomyces cerevisiae in the preparation of animal infection models for studying the pathogenic mechanism of Saccharomyces cerevisiae.

[0014] The application of the carbon quantum dot-based method for inducing hyphal phases in Saccharomyces cerevisiae in the preparation and research of anti-yeast fungal drugs, providing a standardized hyphal phase induction model.

[0015] The method of inducing mycelial phase of Saccharomyces cerevisiae based on carbon quantum dots was applied to study the morphological transformation mechanism of pathogenic yeasts during the invasion of the host.

[0016] The beneficial effects of this invention are as follows: 1. High induction efficiency: This invention uses carbon quantum dots to induce the formation of mycelial phase of Saccharomyces cerevisiae, which significantly improves the mycelial formation rate from 40-60% in traditional methods to over 85%.

[0017] 2. Good stability: The induction conditions of this invention are controllable and highly reproducible, providing stable materials for experimental research.

[0018] 3. High specificity: This invention combines morphological and molecular biological confirmation to ensure the accuracy of mycelial identification.

[0019] 4. Simple operation: This invention uses a hydrothermal method to prepare carbon quantum dots, a one-step culture medium preparation method, and easy control of mycelial induction culture conditions, simplifying the experimental process.

[0020] 5. Low cost: The raw materials used in this invention, carbon quantum dots, are inexpensive and readily available, resulting in low research costs.

[0021] 6. This invention helps provide stable mycelial phase materials for pathogenicity research of *Saccharomyces cerevisiae*, and has the following research application value: This invention can efficiently obtain mycelial phase materials, which is convenient for studying the expression of mycelial phase-specific virulence factors and is beneficial for the study of pathogenic mechanisms; stable induction conditions help to study the molecular mechanism of yeast phase-mycelial phase transformation and are beneficial for the study of morphological transformation mechanisms; it provides a standardized mycelial phase induction model for the development of antifungal drugs and can provide experimental materials for drug susceptibility testing and the study of the invasion mechanism of pathogenic yeast; it supports comparative studies of the physiological and biochemical characteristics of this fungus under different morphologies and is beneficial for pathogenic biology research. Attached Figure Description

[0022] Figure 1 These are microscopic images of carbon quantum dot-induced mycelial phases under different magnifications in embodiments of the present invention; wherein: A is a 10x microscope; B is a 20x microscope; and C is a 40x microscope. Detailed Implementation

[0023] The present invention will now be described in detail with reference to the accompanying drawings and embodiments.

[0024] Example: A method for inducing mycelial phase of *Saccharomyces cerevisiae* based on carbon quantum dots according to the present invention, the steps of which are as follows: a) Preparation and characterization of carbon quantum dots: Carbon quantum dots were prepared using a hydrothermal method. 1. Weigh 2.0g of citric acid and 1.0g of urea, dissolve them in 20mL of deionized water, and stir until completely dissolved; 2. Transfer the solution to a hydrothermal reactor with a polytetrafluoroethylene liner; 3. Place the hydrothermal reactor in a 230℃ oven and react for 15 hours; 4. Allow to cool naturally to room temperature to obtain a carbon quantum dot solution; 5. Adjust the concentration to 0.5 mg / mL; The carbon quantum dots obtained in step a) were added to Sabouraud dextrose medium to a final concentration of 0.5 mg / mL; the pH was adjusted to 6.0-6.5, and the medium was autoclaved at 121°C for 15 minutes to obtain the induction medium. c) Mycelial induction culture: Standard strains or clinical isolates of Saccharomyces cerevisiae were inoculated onto Sabouraud dextrose medium containing 0.5 mg / mL carbon quantum dots (i.e., induction medium) and cultured at 28°C for 24-48 hours to obtain mycelial Saccharomyces cerevisiae. Observation of hyphal morphology induced by carbon quantum dots: After 48 hours, the colonies were examined under a microscope to prepare wet slides, and the hyphae formation was observed under an optical microscope. The results show: 1. Control group (carbon quantum dots-free): mainly oval single-celled yeast morphology, with a small amount of pseudohyphae; 2. Experimental group (containing carbon quantum dots): Abundant slender fungal hyphae structure, continuous hyphae, obvious branching, hyphal phase formation rate ≥85%; d) Confirmation using PCR molecular biology methods: DNA was extracted from carbon quantum dot-induced mycelial phase and control group, and PCR amplification was performed using ITS universal primers. The primer sequences are as follows: Forward primer: 5'-TCCGTAGGTGAACCTGCGG-3'; Reverse primer: 5'-TCCTCCGCTTATTGATATGC-3'; The products were detected by 1.5% agarose gel electrophoresis. The results showed that both groups amplified specific bands of about 600 bp, and sequencing results confirmed that they were the ITS sequences of Saccharomyces cerevisiae, confirming that the carbon quantum dot-induced mycelial phase was Saccharomyces cerevisiae.

[0025] The carbon quantum dots described in this invention have a particle size of 2-8 nm, exhibit fluorescence properties, an excitation wavelength of 360 nm, and an emission wavelength of 440 nm. Through the unique properties of carbon quantum dots, the induction efficiency and stability of the mycelial phase of *Saccharomyces cerevisiae* are significantly improved, providing a new technical means for in-depth research on the pathogenic mechanism of this pathogen, and have important scientific value and application prospects.

[0026] The carbon quantum dot-based method for inducing *Saccharomyces cerevisiae* hyphae as described in this invention can be applied to the preparation of animal infection models for studying the pathogenic mechanisms of *Saccharomyces cerevisiae*. Alternatively, it can be applied to the preparation of standardized hyphae-inducing models for studying antifungal drugs. Furthermore, it can be applied to the study of morphological transformation mechanisms of pathogenic yeasts during their invasion of the host.

[0027] The parts not described in detail in this application are all existing conventional technologies and will not be elaborated here.

[0028] It is understood that the above specific description of the present invention is only for illustrating the present invention and is not limited to the technical solutions described in the embodiments of the present invention. Those skilled in the art should understand that modifications or equivalent substitutions can still be made to the present invention to achieve the same technical effect; as long as the use needs are met, they are all within the protection scope of the present invention.

Claims

1. A method for inducing the mycelial phase of Leucomyces ramosus based on carbon quantum dots, characterized by, Includes the following steps: a) Preparation of carbon quantum dots by hydrothermal method: 2.0 g of citric acid and 1.0 g of urea were dissolved in 20 mL of deionized water and hydrothermally reacted at 230 °C for 15 hours to obtain a carbon quantum dot solution; b) Preparation of induction medium: Add the carbon quantum dot solution prepared in step a) to Sabouraud dextrose medium to a final concentration of 0.1-0.5 mg / mL to obtain the induction medium; c) Mycelial induction culture: Inoculate *Saccharomyces cerevisiae* into induction medium and incubate at 28°C for 24-48 hours to obtain filamentous *Saccharomyces cerevisiae*. d) Detection and confirmation: Observe the hyphal formation under a microscope. If the hyphal formation rate is ≥85% after 48 hours, confirm it by PCR molecular biology method to obtain a stable hyphal phase.

2. The method of claim 1, wherein: The carbon quantum dots have a particle size of 2-8 nm, exhibit fluorescence properties, and have an excitation wavelength of 360 nm and an emission wavelength of 440 nm.

3. The method of claim 1, wherein: The pH of the induction medium is adjusted to 6.0-6.

5.

4. The method of claim 1, wherein: The induction medium was prepared by autoclaving at 121°C for 15-20 minutes.

5. The method of claim 1, wherein: The PCR molecular biology method confirmed that the ITS region of Saccharomyces cerevisiae was amplified using specific primers.

6. The application of the method as described in any one of claims 1-5 in the preparation of animal infection models for studying the pathogenic mechanism of Saccharomyces cerevisiae.

7. The application of the method as described in any one of claims 1-5 in providing a standardized hyphal phase induction model for the preparation and research of anti-yeast fungal drugs.

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