Gene coding filamentous fungus and application thereof in slope protection

Abstract

The invention discloses a gene coding filamentous fungus and application thereof in slope protection, the gene coding filamentous fungus is prepared by knocking out blue light receptor gene sequences wcl1 and wcl2 from Pleurotus ostreatus, and the CDS coding sequences of the wcl1 and the wcl2 are respectively shown as the registration numbers KY348758 and MG679810 of an international public database. According to the method, blue light receptor genes of Pleuromyces ostreatus filamentous bacteria are destroyed through gene editing (CRISPR-Cas9), growth of sporocarp of the Pleuromyces ostreatus filamentous bacteria is inhibited, so that a lasting natural hydrophobic layer is formed on the surface of a plant growing layer, enzyme is metabolized, soil organic matter is increased, a'micro reservoir 'is constructed in a plant root system area, the plant habitat is improved while water evaporation is effectively reduced, and the yield of plants is increased. Hyphae and plant root systems synergistically improve soil shear resistance to realize slope stabilization.

Description

Technical Field

[0001] This invention belongs to the field of microbial slope protection technology, and in particular relates to a gene-encoded filamentous fungus and its application in slope protection. Background Technology

[0002] While my country's economy has developed rapidly, the problem of ecological and environmental damage cannot be ignored. Solid waste dumps from coal mining, thermal power generation, and oil exploration are expanding, especially in the arid Yellow River bend area, causing severe damage to the ecological environment. This region currently houses six national-level coal bases, accounting for approximately 40% of the national coal production, and also possesses abundant crude oil resources, playing a vital role in ensuring my country's energy security. However, this region is located in the arid-semi-arid western region, with a fragile ecological environment and poor resistance to disturbance, making it prone to various geological disasters during mining. According to official statistics, energy bases are mainly located in loess and aeolian sand areas, where the ecological risks are severe. Dust from mining sites, soil erosion, loess landslides, subsidence, and ground fissures are commonplace, and desertification is worsening, with an ecological restoration rate of less than 20%. Therefore, vegetation-based slope protection is the preferred ecological slope protection technology.

[0003] However, the poor water retention capacity and vegetation performance of solid waste dumps and loess slopes increase the difficulty of slope protection. Solving this problem requires comprehensive consideration of multiple factors, including soil characteristics, plant selection, water management, and root system architecture regulation. Among these, the hydrophobic, water-retaining, and anti-evaporation properties of the slope soil structure layer are crucial. Currently used ecological porous concrete suffers from disadvantages such as habitat fragmentation, high initial alkalinity, and high carbon footprint, resulting in low plant germination rates and high long-term maintenance costs.

[0004] Therefore, there is an urgent need to develop a new environmentally friendly mycelium hydrophobic-vegetated slope protection technology. This technology uses gene editing (CRISPR-Cas9) to disrupt the blue light receptor gene of the filamentous fungus Pleurotus ostreatus, inhibiting the growth of its fruiting bodies and thus forming a durable natural hydrophobic layer on the surface of the vegetation layer. It also increases soil organic matter by metabolic enzymes, creating a "miniature reservoir" in the plant root zone. This effectively reduces water evaporation while improving the plant habitat. The mycelium and plant roots work together to enhance the soil's shear strength, thereby stabilizing the slope. Summary of the Invention

[0005] The purpose of this invention is to provide a gene-encoded filamentous fungus and its application in slope protection. The mycelium grows continuously in the slope to form a semi-permeable layer, that is, the mycelium forms a hydrophobic layer in the part of the slope surface that is in contact with the air, which prevents water flow from eroding the slope and forms a water-holding layer inside the slope to inhibit soil and water loss.

[0006] To achieve the above objectives, this application adopts the following technical solution: In a first aspect, the present invention provides a gene-encoded filamentous fungus, which is prepared by knocking out the blue light receptor genes wcl1 and wcl2 from Pleurotus ostreatus. The CDS coding sequences of wcl1 and wcl2 are shown in the accession numbers KY348758 and MG679810 of the international public database, respectively.

[0007] In the above technical solution, the method for preparing the gene-encoded filamentous fungus includes the following steps: Step A1, Constructing the RNP system: Specific sgRNAs are designed and chemically synthesized for the wcl1 and wcl2 genes of Pleurotus ostreatus. The sgRNAs contain a guide sequence complementary to the target gene and a universal sgRNA backbone sequence. The sgRNAs are then assembled with Cas9 protein obtained through in vitro expression and purification to form a ribonucleoprotein RNP complex targeting the gene, which is the RNP system. Step A2: The RNP system is delivered into Pleurotus ostreatus cells to obtain RNP-Pleurotus ostreatus; Step A3, obtaining stable genetic mutants: The RNP-Pleurotus ostreatus is cultured under darkness and light respectively, transformants are screened and verified, and stable genetic mutants are obtained by isolating and purifying spores, thus obtaining the filamentous fungus encoded by the gene.

[0008] In the above technical solution, in step A2, the specific method for delivering the RNP system into Pleurotus ostreatus cells is as follows: co-incubate the RNP system with Pleurotus ostreatus protoplasts in a PEG solution to induce the cell membrane to open pores, allowing the RNP system to enter the Pleurotus ostreatus cells.

[0009] Secondly, the present invention provides a fungal homogenate, wherein the above-mentioned gene-encoded filamentous fungus is inoculated into PDA medium, and after culturing for 20 days, the growing mycelium is crushed in a sterile environment and mixed with water to form a paste. After adding deionized water to the paste, it is stirred in an ultrasonic device, passed through a sieve to remove solid particles, and the remaining filtrate is the fungal homogenate.

[0010] Thirdly, the present invention provides a mycelial composite soil, which includes a mixed substrate and the aforementioned fungal homogenate colonized thereon, wherein the mixed substrate includes sawdust, Gram-negative bacteria, soil sample and trace elements.

[0011] In the above technical solution, the mass ratio of sawdust, Gram-negative bacteria, soil sample and trace elements is 30%~40%: 2%~5%: 50%~60%: 1%~5%.

[0012] Fourthly, the present invention provides a controllable evaporation-permeability structural layer based on the above-mentioned mycelial composite soil, which includes, from bottom to top, a capillary barrier layer, a capillary water storage layer, an infiltration buffer layer and a mycelial anti-evaporation layer. The capillary barrier layer is composed of fine materials, the capillary water storage layer is composed of coarse particles, the infiltration buffer layer is composed of fine materials, and the mycelial anti-evaporation layer is composed of the aforementioned mycelial composite soil.

[0013] Fifthly, the present invention provides the application of the above-mentioned gene-encoded filamentous fungi, the above-mentioned fungal homogenate, the above-mentioned mycelial composite soil, and the above-mentioned mycelial composite soil evaporation-infiltration controllable structural layer in slope protection.

[0014] In the above technical solutions, the mycelium composite soil is mixed evenly with grass seeds and then laid on the top of the slope and leveled, while the clay is mixed evenly with grass seeds and then laid on the slope surface and leveled.

[0015] In the above technical solution, the method for confirming the amount of fungal homogenate added to the mycelial composite soil includes the following steps: Step B1: Design an in-situ shearing and jet erosion coupled model; Step B2: The mycelium composite soil is implanted into the in-situ shear and jet erosion coupled model to simulate the dynamic changes in slope performance before and after slope protection in situ. Step B3 involves developing an optimization algorithm for the optimal dosage of slope protection bacteria based on a distribution function, and determining the sensitivity of cohesion and internal friction angle to influencing factors such as moisture content, mycelial content, and mycelial diameter based on the coefficient of variation.

[0016] The beneficial effects of this invention are as follows: (1) The novel microbial slope protection material of this invention is a gene-encoded filamentous fungus. It has multiple functions and is suitable for application in various geological slopes. The hyphae grow continuously in the slope to form a semi-permeable layer, that is, the hyphae form a hydrophobic layer in the part of the slope surface that is in contact with the air, which prevents water flow from eroding the slope and forms a water-holding layer inside the slope to inhibit soil and water loss. For solid waste dumps such as oil mines and fly ash, the phenol oxidase secreted by Pleurotus ostreatus can adsorb petroleum hydrocarbons and heavy metal elements. The growth cycle of filamentous fungi can be up to one year, and after maturity, it can be biodegraded to promote the formation of soil aggregates, enrich the soil and promote vegetation growth. Compared with traditional chemical hydrophobic agents, the higher soil moisture content has less impact on the hydrophobic effect induced by the hyphae, and can even form a hydrophobic layer under saturated conditions.

[0017] (2) The in-situ shear and jet erosion coupled model of the present invention restores the geological conditions of the slope, analyzes the ability of the slope to resist external loads and water erosion under natural conditions, examines the reinforcement effect of longitudinal anchoring and transverse support of mycelium composite soil, reduces or avoids soil sample disturbance caused by indoor test sampling, and reduces test data deviation.

[0018] (3) This invention proposes a new concept and algorithm for "mycelium soil stabilization". It integrates multiple probability distribution function fitting analysis to determine the correlation between mycelium content and mycelium configuration and soil shear strength indexes c and φ, thereby determining the optimal amount of mycelium added to the slope. Considering the three-dimensional mycelium network formed in the slope, which loosens the soil at the fungal-soil interface due to mycelium mucus, the idea of ​​bacterial hybrid fungi is proposed. Through the natural reinforcing matrix, namely sawdust and Gram-negative bacteria, sufficient mechanical strength is provided during mycelium colonization and growth, respectively, to enhance the load-bearing capacity of the composite soil and further improve the slope stability. Compared with the traditional plant root soil stabilization, the concept of mycelium soil stabilization has a wider range of applications, especially for barren slope soil. How to transform soil organic matter to achieve slope greening is the key. Attached Figure Description

[0019] To more clearly illustrate the optimization method or embodiments of the present invention, the accompanying drawings used in the slope protection optimization method will be briefly described below; because Figure 1 , Figure 4 Some experimental setups are symmetrical; only one side is marked here.

[0020] Figure 1 This is an overall structural diagram of the in-situ shearing and jet erosion test device of the present invention; Figure 2 This is a cross-sectional view of the in-situ shear test apparatus of the present invention; Figure 3 This is a top view of the in-situ shear testing device of the present invention; Figure 4 This is a cross-sectional view of the in-situ jet erosion test device of the present invention; Figure 5 This is a schematic diagram of the evaporation structure layer of the present invention; In the diagram: 1. Weighing sensor; 2. Water tank; 3. Water inlet pipe; 4. Support rod; 5. Fixing screw; 6. AGR pipe; 7. Water pipe interface; 8. Jet pipe; 9. Flushing depth probe; 10. Interface nut; 11. PVC transparent fixing plate; 12. Motor; 13. Model box; 14. Threaded push rod; 15-18. Slope adjustment buckle; 19. Slope adjuster; 20. Casters; 21. Telescopic rod; 22. Computer; 23. Motor wiring harness; 24. Lower shear box 25. Connecting rod; 26. Thrust sensor; 27. Data acquisition instrument; 28. In-situ test soil sample; 29. ​​Upper shear box; 30. Upper connecting connector; 31. Displacement sensor; 32. Sensor connecting rod; 33. Data acquisition instrument; 34. Lower connecting connector; 35. Lower shear box; 36. Large fixing screw; 37. Control rod; 38. Upper ring fixing screw; 39. Lower ring fixing screw; 40. Deflection plate; 41. Upper fixing ring; 42. Lower fixing ring; 43. Nozzle plate. Detailed Implementation

[0021] To better illustrate the objectives, technical solutions, and advantages of this invention, the invention will be further described below in conjunction with specific embodiments. This invention can be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the inventive concept to those skilled in the art. This invention will be defined only by the claims.

[0022] Unless otherwise specified, the test methods or experimental methods described in the following examples are conventional methods; unless otherwise specified, the reagents and materials are obtained from conventional commercial sources or prepared by conventional methods.

[0023] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0024] This invention provides a gene-encoded filamentous fungus prepared by knocking out the blue light receptor gene sequences White Collar 1 (wcl1) and White Collar 2 (wcl2) from *Pleurotus ostreatus*. The coding sequences (CDS) of wcl1 and wcl2 are shown in the accession numbers KY348758 (wcl1) and MG679810 (wcl2) of the international public database (https: / / www.ncbi.nlm.nih.gov / orffinder / ), respectively. Its hyphae cannot sense light signals, thereby blocking the light-induced fruiting body development pathway, keeping it in the hyphal growth stage and maintaining its long-term hydrophobicity. The preparation method includes the following steps: Step A1, constructing the RNP system: Design and chemically synthesize specific sgRNAs for the wcl1 and wcl2 genes of Pleurotus ostreatus. The sgRNAs contain guide sequences complementary to the target genes and a universal sgRNA backbone sequence. Assemble the sgRNAs with Cas9 proteins obtained through in vitro expression and purification to form a ribonucleoprotein (RNP) complex targeting the gene, thus obtaining the RNP system.

[0025] Step A2: The RNP system is delivered into Pleurotus ostreatus cells to obtain RNP-Pleurotus ostreatus.

[0026] Preferably, the specific method for delivering the RNP system into Pleurotus ostreatus cells is as follows: co-incubate the RNP system and Pleurotus ostreatus protoplasts in a PEG solution to induce the cell membrane to open pores, allowing the RNP system to enter the Pleurotus ostreatus cells, thus obtaining RNP-Pleurotus ostreatus.

[0027] Step A3, obtaining stable genetic mutants: RNP-Pleurotus ostreatus is cultured under darkness and light respectively, transformants are screened and verified, and stable genetic mutants are obtained by isolating and purifying spores, thus obtaining the filamentous fungus encoded by the gene of this invention.

[0028] Preferably, the specific method for screening and verifying transformants is as follows: RNP-Pleurotus ostreatus protoplasts are spread on regeneration medium and cultured in the dark to regenerate into colonies; genomic DNA of the obtained transformant colonies is extracted, and specific polymerase chain reaction primers are designed for the target sites of the wcl1 and wcl2 genes for amplification, and the amplification products are sequenced and analyzed. By comparing the sequencing results with the wild-type sequence, transformants with insertion, deletion or substitution mutations at the target sites are screened, which are the preliminary gene knockout mutants.

[0029] Preferably, the specific method for obtaining stable genetic mutants by isolating and purifying spores is as follows: the verified preliminary mutant is cultured under suitable sporulation conditions, and its spores are collected; the spore suspension is serially diluted and spread on a plate culture medium, cultured under light conditions, and colonies formed by the germination of single spores are isolated by micromanipulation or limiting dilution method; the genomic DNA of the colonies after single spore isolation is extracted, and the target site mutation of the wcl1 and wcl2 genes is verified again by polymerase chain reaction amplification and sequencing, and its genetic stability is checked; the verified and genetically stable strain is identified as a stable genetic mutant with double knockout of the wcl1 and wcl2 genes, that is, the gene-encoded filamentous fungus of the present invention is obtained.

[0030] The present invention also provides a fungal homogenate, the preparation method of which is as follows: gene-encoded filamentous fungi are inoculated into PDA medium, and after culturing for 20 days, the growing mycelium is crushed in a sterile environment and mixed with water to form a paste. After adding deionized water to the paste, it is stirred in an ultrasonic device, passed through a sieve to remove solid particles, and the remaining filtrate is the fungal homogenate.

[0031] PDA medium was purchased from a reputable chemical reagent manufacturer. Mycelial inoculation was performed using the streak inoculation method, which results in a significantly faster mycelial growth rate compared to the agar cutting method.

[0032] Pleurotus ostreatus is a non-parasitic white-rot fungus. Its mycelial wall is composed of β-glucan, chitin fibers, and proteins. Among them, chitin fibers have high thermal stability and provide rigidity and strength to the mycelium. During mycelial growth, it continuously secretes amphiphilic hydrophobic proteins and phenol oxidase. The hydrophobic proteins have charged residues on one side, creating a discontinuous hydrophobic region on one side, which can form a semi-permeable layer in the soil, achieving the goal of controllable evaporation and infiltration. Phenol oxidase can act as a biocatalyst, effectively activating aromatic hydrocarbons to generate free radicals, permanently oxidizing pollutants. At the same time, it removes common heavy metal elements through a combination of biosorption and catalysis, in the order of adsorption degree: Cd > Cr > Fe > Cu > Zn. After bio-metabolism and degradation, Pleurotus ostreatus releases nutrients into the soil, transforming them into fertilizers suitable for plant growth.

[0033] Compared with mycelial suspension, fungal homogenate has a higher concentration of fungal biomass and secretions. The preparation method is as follows: mycelia that have grown in PDA medium for about 20 days are aseptically pulverized and mixed with water to form a paste; deionized water is added to the paste (the mixing ratio is 2g of mycelial paste to 1mL of deionized water); the mixture is stirred in an ultrasonic device for 20 minutes, passed through a 2mm sieve to remove larger solid particles, and the remaining filtrate is the fungal homogenate.

[0034] This invention also provides a mycelial composite soil, comprising a mixed substrate and a fungal homogenate colonized thereon. The mixed substrate comprises sawdust, Gram-negative bacteria, soil sample, and trace elements, wherein the mass ratio of sawdust, Gram-negative bacteria, soil sample, and trace elements is 30%~40%:2%~5%:50%~60%:1%~5%.

[0035] The sawdust provides nutrients. Gram-negative bacteria with strength and stiffness-enhancing properties can be used in this invention, including *Xylitolobacterium xylitol*, *Pseudomonas malignans* M47T1, *Pseudomonas putida*, *Pseudomonas fluorescens*, *Acinetobacter calcareae*, *Serratia marcescens*, and *Bacillus pasteurellii*. The soil sample is coarse-grained soil such as sand. Trace elements include phosphorus, potassium, and magnesium, with a total proportion of 1% to 5%.

[0036] This invention also provides the application of fungal homogenate in slope protection. Specifically, it includes: SB1: Based on the slope parameters, design an in-situ shear and jet erosion coupled model.

[0037] Furthermore, slope parameters mainly include slope, slope ratio, and slope aspect.

[0038] Furthermore, the in-situ shear device of the coupled model includes a slope adjuster 19, upper and lower shear boxes 28 / 35, a load application device, a monitoring device, eccentric slopes, and a steel track. The device's dimensions (length * height * width) are 0.8m * 0.6m * 0.8m. The slope adjuster 19 is adjustable inclination from 20° to 60°. The shear box diameter * height is 0.3m * 0.15m. The drive end of the load application device connects to the upper shear box 28 to apply thrust, and the monitoring device monitors the soil's resistance to the load.

[0039] The load application device includes a threaded push rod 14 and a motor 12. The threaded push rod 14 is used to connect to the upper shear box 28, and the motor 12 is used to drive the threaded push rod 14 to rotate.

[0040] The monitoring device includes a thrust sensor 25, a displacement sensor 30, and a capacitive sensor (a capacitive sensing unit integrating a motor, sensor, etc.). The thrust sensor 25 is used to obtain the corresponding shear stress caused by the thrust on the soil sample. The displacement sensor 30 is used to monitor whether the upper shear box 28 is displaced relative to the lower shear box 34. The capacitive sensor monitors the stress change of the anchor rod through capacitance fluctuation and then calculates the stress change of the slope.

[0041] Furthermore, the in-situ jet erosion test device for the coupled model includes an AGR tube 6, a scour depth probe 9, a jet tube 8, valves, nozzles, a fixing bracket, a water storage tank 2, a weighing sensor 1, and a data acquisition unit 32. The acrylic tube has a diameter and height of 0.3m * 0.7m, the scour depth probe is a graduated thin rod with a diameter of 6.3cm, the jet tube has a diameter of 0.5cm, and the nozzle has a diameter of 6.4mm. The AGR tube 6 is connected to the shear box via a silicone flexible joint.

[0042] After the test soil was filled into the model, the AGR pipe 6 and the shear box were immediately pressed into the model box 13 by gravity. After curing for 28 days, the test was started. The erosion test was conducted by setting the initial hydraulic head of the jet pipe 8, adjusting the valve to keep the water head height of the jet pipe 8 constant, recording the scouring depth and jet erosion process at a predetermined time under constant water head, and calculating the initial shear stress of the water flow scouring the soil surface according to the following formula.

[0043] In the formula, The initial peak stress (Pa) before scouring is represented; H represents the hydraulic head (m). This indicates the initial height (m) of the nozzle from the soil surface, ranging from 40mm to 120mm.

[0044] SB2: Construct a multi-layered structure of capillary barrier layer, capillary water storage layer, infiltration buffer layer, and mycelial anti-evaporation layer within the model box 13 of the model described in S1, and simulate the dynamic changes in slope performance before and after protection in situ.

[0045] The multi-layer structure consists of, from bottom to top, a capillary barrier layer, a capillary water storage layer, an infiltration buffer layer, and a mycelial anti-evaporation layer. The capillary barrier layer is composed of fine particles (<5mm), the capillary water storage layer is composed of coarse particles (3~16mm), and the infiltration buffer layer is composed of fine particles (<5mm).

[0046] Mycelial anti-evaporation layer: Mix sawdust, Gram-negative bacteria (e.g., xylitol bacteria), soil sample and trace elements (phosphorus, potassium, magnesium) in a weight ratio of 30%~40%: 2%~5%: 50%~60%: 1%~5%, and colonize fungal homogenate on it to form the mycelial anti-evaporation layer.

[0047] Furthermore, based on the carbon source requirements and performance optimization of the composite soil, 30%–40% sawdust is added to provide a carbon source for mycelial growth and enhance its mechanical strength. The sawdust can be selected from furniture production waste. Adding 2%–5% Gram-negative bacteria further improves its strength and stiffness. These bacteria can be extracted from vinegar, but are not limited to vinegar; they can also be extracted from pickling brine, sugarcane bagasse, rotten fruit, and other substances.

[0048] To ensure consistent soil density and void ratio, the composite soil material was filled into the coupled model using a layered filling and compaction method for homogenization planting. Two groups of composite soil containing 7%, 10%, and 20% homogenized slurry, and one group of plain soil for both yin and yang slopes were prepared. In-situ shear tests were conducted after mycelial growth for 28 days.

[0049] SB3: Develop an optimization algorithm for the optimal dosage of mycelium for slope protection based on the distribution function, and determine the sensitivity of cohesion and internal friction angle to influencing factors (moisture content, mycelial content, mycelial diameter) based on the coefficient of variation.

[0050] Furthermore, the distribution function is as follows: based on the results of the S1 in-situ shear test, the mean cohesion and internal friction angle of the composite soil are obtained, and the mean water content is calculated; the internal structure of the slope model is synthesized using tomography (CT) technology, and the mycelia are marked by color. The mean mycelial content and mycelial diameter are calculated using ImageJ, thereby obtaining the corresponding standard deviation and coefficient of variation. The sensitivity of cohesion and internal friction angle to influencing factors (water content, mycelial content, and mycelial diameter) is judged based on the coefficient of variation; then, through fitting analysis of normal distribution, gamma distribution, and Weibull distribution functions and KS test for normal distribution, the optimal fitting function of the five physical and mechanical properties of the composite soil is quantitatively determined, and the optimal amount of mycelium is determined to maximize the shear strength of the composite soil.

[0051] Furthermore, the formula for the normal distribution function is as follows: In the formula: and Let x1, x2, ..., x be the mean and standard deviation of the normal distribution, respectively. n This is a set of measured values.

[0052] Integrating f(x) yields x. i Cumulative probability function: Furthermore, the formula for the gamma distribution function is as follows: Furthermore, the formula for the Weil distribution function is as follows: In the formula: β and η are the shape parameter and scale parameter, respectively, whose values ​​can be obtained by the maximum likelihood estimation method; their cumulative probability function is: Furthermore, the KS test method is used to examine the theoretical probability distribution F(x) of the hypothesis and its relationship with the measured sample x. i The cumulative frequency F n The difference between (x) is calculated using the following formula: In the formula: Dn represents a random variable that depends on n; Indicates the significance level as The threshold, The larger the value, the better the corresponding variable fits the theoretical distribution of the distribution function.

[0053] SB4: Propose a method for evaluating the drought resistance and water retention capacity and time effect of slopes, verify the slope protection effect, and further optimize the slope protection scheme.

[0054] Furthermore, the assessment methods for drought resistance and water retention capacity, time effect, and fertilizer release capacity include slope greening test, indoor immersion test, direct shear test, water droplet infiltration test, and soil water potential measurement.

[0055] Further, a greening experiment was conducted: the physical and chemical properties of the soil were measured, mainly including density, porosity, permeability, temperature and humidity, pH value and electrical conductivity; based on the physical and chemical properties of the soil and climate zones, green plants were selected, such as sea buckthorn and tall fescue, which are suitable for planting on loess slopes to prevent wind erosion and stabilize soil; at the same time, plants were planted in plain soil and composite soil model boxes, and the growth of plants and mycelium in the model boxes was observed regularly. After 28 days of growth, the fertilizer release capacity of the composite soil and the plant growth-promoting effect were verified. Furthermore, indoor immersion test: Before the test, a representative soil sample was taken from the model box to prepare the test sample. The test soil sample was dried, and a PVC immersion tube was installed on the soil sample container. Water was slowly injected into the immersion tube. During the water injection process, the water level was kept constant. A water level sensor was used to connect to a computer to observe the moisture content and water level changes. When the soil moisture state was stable, water addition was stopped, the final reading was recorded, and the height of the water above the soil sample was measured to calculate the initial shear stress. The results were compared and analyzed with the in-situ jet erosion test results to verify the rationality of the simulation. Furthermore, indoor direct shear test: A representative soil sample from the model box was taken using a 61.8 mm shear ring for direct shear test. The shear strength value of the indoor direct shear test was calculated based on the Mohr-Coulomb law, and the difference between the shear strength and the in-situ shear test was obtained to verify the rationality of the simulation. Furthermore, the hydrophobicity and duration of the composite soil were evaluated using the water droplet penetration test (WDPT). A 10-microliter droplet of water was released from a height of 5 mm above the soil surface using a pipette, and the time required for the water droplet to completely penetrate into the soil sample was recorded. The test was repeated 5 times for each sample, and the water droplet penetration time of plain soil and composite soil were compared and analyzed. Furthermore, the evaporation resistance of the composite soil was assessed using soil water potential, the soil water suction was measured as a function of soil moisture content using a tensiometer, and the soil water holding capacity was assessed by plotting soil moisture characteristic curves.

[0056] like Figures 1-3 As shown, the in-situ shear test apparatus of the present invention consists of a model box 13, slope adjustment buckles 15-18 and slope adjuster 19, telescopic rod 21, AGR pipe 6, upper and lower shear boxes 28 / 34, upper and lower connecting joints 29 / 33, threaded push rod 14, motor 12, thrust sensor 25, displacement sensor 30, and data acquisition instrument 26. The telescopic rod connector is fixed to the buckle at the corresponding slope using the slope adjuster 19, and the soil sample is filled and compacted in the model box 13. The upper and lower shear boxes 28 / 34 and two AGR pipes 6 with a total length of 0.7m are fixed using soft silicone connecting joints. Gravity presses the AGR pipes 6 and the upper and lower shear boxes 28 / 34 into the soil layer of the model box 13, ensuring that the compaction degree of the soil sample in the AGR pipe 6 is consistent with that of the soil sample in the model box 13. After 28 days of curing, remove the soil layer from model box 13 down to the top of the lower shear box 34. Use a knife to cut the silicone joint (be careful not to cut into the soil layer), and install the sensor, data acquisition instrument 26, and motor 12. Use motor 12 to drive threaded push rod 14 to measure and collect the displacement and shear force of the in-situ soil sample in real time.

[0057] like Figure 1 and Figure 4 As shown, the in-situ jet erosion test device of the present invention consists of a model box 13, a water storage tank 2, a weighing sensor 1, a water inlet pipe 3, a support rod 4, fixing screws 5, a scour depth probe 9, a PVC transparent fixing plate 11, fixing rings (40 / 41), a deflection plate 39, a control rod 36, and a nozzle plate 42. A hollow PVC transparent fixing plate 11 with a center diameter of 0.55 cm is fixed to the support rod 4 with screws, and the support rod 4 is firmly embedded in the soil layer of the model box 13. A jet pipe 8 is placed on the PVC transparent fixing plate 11, and the upper and lower fixing rings 40 / 41 are further installed, and the deflection plate 39 and control rod 36 are fixed with screws. The water inlet pipe 3 connects the water storage tank 2 and the jet pipe 8, and the water flows through the nozzle plate 42 at the bottom of the jet pipe 8 for scour testing. Before the experiment begins, the control rod 36 is rotated to close the bottom of the jet pipe 8 with the deflection plate 39. After the initial water head stabilizes, the deflection plate 39 is opened to begin the experiment. During the experiment, the scour depth probe 9 continuously monitored the scour depth and calculated the initial shear stress of the water flow scouring the soil surface.

[0058] Application method: (1) Prepare the fungal homogenate of the present invention and store it in a refrigerator at 4°C for later use.

[0059] (2) Based on the actual geological conditions, slope angle, and slope surface of the slope to be maintained, the in-situ simulation experimental conditions were set up. Representative soils of the slope were collected and mixed matrix was prepared according to the proportion in S2. Two groups of 7%, 10%, and 20% were planted in mixed matrix with 10%, 30%, 50%, and 70% water content. One group of plain soil with 10%, 30%, 50%, and 70% water content (soil of the same proportion of mixed matrix) was prepared. The test soil samples were filled into the coupled model according to the porosity and density of the slope soil layer. After the mycelium mixed soil grew for 28 days, in-situ shear test, jet erosion test, and image analysis based on CT scan were carried out to obtain the shear strength of composite soil and plain soil and the initial shear stress of water flow erosion of soil surface under different water contents, average mycelium content and average mycelium diameter.

[0060] (3) Based on the above test results and the S3 distribution function, explore the sensitivity of the cohesion and internal friction angle of the composite soil to the water content, mycelial content and mycelial diameter, and determine the optimal distribution function of the physical and mechanical properties of the five composite soils. Based on the 28-day growth period, use the corresponding optimal function to analyze the optimal amount of mycelium to be placed on the slope, in order to maximize the shear strength of the composite soil and thus increase the slope stability.

[0061] (4) Based on the soil physicochemical properties and climate zones of the slope to be maintained, select suitable slope vegetation and plant it in composite soil and plain soil respectively. Observe the growth of plants in the model box regularly and verify the fertilizer release capacity of composite soil and the plant growth promotion effect after 28 days.

[0062] (5) Finally, select three representative soil samples from the coupled model box and conduct indoor direct shear test, immersion test, water droplet infiltration test and soil water potential measurement. The shear strength of the test soil sample, the height of water erosion of the soil sample and the initial shear stress, the hydrophobicity and duration of the soil sample, and the water holding capacity of the soil are obtained respectively. The slope protection scheme is further optimized by combining the results of the in-situ simulation test.

[0063] (6) Based on the optimized slope protection scheme, it was actually applied to slope protection engineering. First, the proportions of sawdust, Gram-negative bacteria (xylitol bacteria), soil samples, and trace elements in S2, as well as the optimal moisture content, mycelium dosage, and grass seed (30~40g / m²) obtained from the experiment, were combined. 2 The mycelium and grass seeds are added to a mixer truck and thoroughly mixed. Using a low-flow pump (the moisture content of the mycelium-based composite soil is much lower than that of concrete, requiring this type of pump), the mixture is laid on the top of the slope and manually leveled. The clay and grass seeds are then mixed evenly and laid on the slope surface, also manually leveled. After 28 days of growth, a crisscrossing network of mycelium and roots forms in the slope soil. The mycelium improves the slope soil, achieving greening of barren soil while the plant roots further anchor the slope.

[0064] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A gene-encoding filamentous fungus, characterized in that: The gene-encoded filamentous fungus was prepared by knocking out the blue light receptor genes wcl1 and wcl2 from Pleurotus ostreatus. The CDS coding sequences of wcl1 and wcl2 are shown in the accession numbers KY348758 and MG679810 of the international public database, respectively.

2. The gene-encoded filamentous fungus according to claim 1, characterized in that: The method for preparing the gene-encoded filamentous fungus includes the following steps: Step A1, Constructing the RNP system: Specific sgRNAs are designed and chemically synthesized for the wcl1 and wcl2 genes of Pleurotus ostreatus. The sgRNAs contain a guide sequence complementary to the target gene and a universal sgRNA backbone sequence. The sgRNAs are then assembled with Cas9 protein obtained through in vitro expression and purification to form a ribonucleoprotein RNP complex targeting the gene, which is the RNP system. Step A2: The RNP system is delivered into Pleurotus ostreatus cells to obtain RNP-Pleurotus ostreatus; Step A3, obtaining stable genetic mutants: The RNP-Pleurotus ostreatus is cultured under darkness and light respectively, transformants are screened and verified, and stable genetic mutants are obtained by isolating and purifying spores, thus obtaining the filamentous fungus encoded by the gene.

3. The gene-encoded filamentous fungus according to claim 1, characterized in that: In step A2, the specific method for delivering the RNP system into Pleurotus ostreatus cells is as follows: co-incubate the RNP system with Pleurotus ostreatus protoplasts in a PEG solution to induce the cell membrane to open pores, allowing the RNP system to enter the Pleurotus ostreatus cells.

4. A fungal homogenate, characterized in that: The preparation method is as follows: the gene-encoded filamentous fungus described in any one of claims 1-3 is inoculated into PDA medium. After culturing for 20 days, the growing mycelium is crushed in a sterile environment and mixed with water to form a paste. After adding deionized water to the paste, it is stirred in an ultrasonic device, passed through a sieve to remove solid particles, and the remaining filtrate is the fungal homogenate.

5. A mycelial composite soil, characterized in that: The mycelial composite soil comprises a mixed substrate and a fungal homogenate of claim 4 colonized thereon, wherein the mixed substrate comprises sawdust, Gram-negative bacteria, soil sample and trace elements.

6. The mycelial composite soil according to claim 5, characterized in that: The mass ratio of sawdust, Gram-negative bacteria, soil sample, and trace elements is 30%~40%: 2%~5%: 50%~60%: 1%~5%.

7. A controllable evaporation-permeability structural layer based on the mycelial composite soil according to any one of claims 5-6, characterized in that: From bottom to top, it consists of a capillary barrier layer, a capillary water storage layer, an infiltration buffer layer, and a mycelial anti-evaporation layer; The capillary barrier layer is composed of fine materials, the capillary water storage layer is composed of coarse particles, the infiltration buffer layer is composed of fine materials, and the mycelial anti-evaporation layer is composed of the mycelial composite soil according to any one of claims 5-6.

8. The application of the evapotranspiration controllable structural layer of the gene-encoded filamentous fungus of any one of claims 1-3, the fungal homogenate of claim 4, the mycelial composite soil of any one of claims 5-6, and the mycelial composite soil of claim 7 in slope protection.

9. The application according to claim 8, characterized in that: After mixing the mycelium composite soil with grass seeds evenly, lay it on the top of the slope and level it. After mixing the clay with grass seeds evenly, lay it on the slope surface and level it.

10. The application according to claim 8, characterized in that: The method for determining the amount of fungal homogenate added to the mycelial composite soil includes the following steps: Step B1: Design an in-situ shearing and jet erosion coupled model; Step B2: The mycelium composite soil is implanted into the in-situ shear and jet erosion coupled model to simulate the dynamic changes in slope performance before and after slope protection in situ. Step B3 involves developing an optimization algorithm for the optimal dosage of slope protection bacteria based on a distribution function, and determining the sensitivity of cohesion and internal friction angle to influencing factors such as moisture content, mycelial content, and mycelial diameter based on the coefficient of variation.

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