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CN-122012257-A - Gene coding filamentous fungi and application thereof in slope protection

CN122012257ACN 122012257 ACN122012257 ACN 122012257ACN-122012257-A

Abstract

The invention discloses a gene coding filamentous fungus and application thereof in slope protection, wherein the gene coding filamentous fungus is prepared by knocking out blue light receptor gene sequences wcl1 and wcl2 through Pleurotus ostreatus, and CDS coding sequences of wcl1 and wcl2 are respectively shown as accession numbers KY348758 and MG679810 of an international public database. According to the invention, the blue light receptor gene of Pleurotus ostreatus filamentous fungi is destroyed by gene editing (CRISPR-Cas 9), the growth of fruiting bodies is inhibited, so that a durable natural hydrophobic layer is formed on the surface of a plant growth layer, soil organic matters are increased by metabolizing enzymes, a 'micro reservoir' is constructed in a plant root system area, the plant habitat is effectively reduced while the water evaporation is effectively reduced, and the hypha and the plant root system cooperatively promote the soil shearing resistance to realize the slope stabilization.

Inventors

  • HAN LIJUN
  • Xu Kangru
  • LI JIANGSHAN
  • WENG XIAOLIN
  • GUO HUANQING
  • HUANG GUANGPING
  • Wang Yaodie
  • HE XINGXING

Assignees

  • 中国科学院武汉岩土力学研究所
  • 长安大学

Dates

Publication Date
20260512
Application Date
20260114

Claims (10)

  1. 1. A gene coding filamentous fungus is characterized in that the gene coding filamentous fungus is prepared by Pleurotus ostreatus knocking out blue light receptor genes wcl1 and wcl2, and CDS coding sequences of wcl1 and wcl2 are respectively shown as accession numbers KY348758 and MG679810 of an International publication database.
  2. 2. The method for producing a gene-encoded filamentous fungus according to claim 1, comprising the steps of: Step A1, constructing an RNP system, namely designing and chemically synthesizing specific sgRNA aiming at the wcl1 and wcl2 genes of Pleurotus ostreatus, wherein the sgRNA comprises a guide sequence complementary to a target gene and a general sgRNA framework sequence; Step A2, delivering the RNP system into Pleurotus ostreatus cells to obtain RNP-Pleurotus ostreatus; And A3, obtaining stable genetic mutants, namely culturing the RNP-Pleurotus ostreatus under dark and illumination respectively, screening and verifying transformants, and obtaining the stable genetic mutants by separating and purifying spores to obtain the gene coding filamentous fungi.
  3. 3. The filamentous fungus of claim 1, wherein the RNP system is delivered to Pleurotus ostreatus cells in step A2 by co-culturing the RNP system with Pleurotus ostreatus protoplasts in PEG solution to induce the cell membrane to open pores and allow the RNP system to enter Pleurotus ostreatus cells.
  4. 4. A process for preparing the fungus homogenate, which is characterized by inoculating the gene-coded filamentous fungus of any one of claims 1-3 to PDA culture medium, culturing for 20 days, pulverizing the grown mycelium in aseptic environment, mixing with water to obtain paste, adding deionized water into the paste, stirring in ultrasonic wave device, sieving to remove solid particles, and obtaining the filtrate.
  5. 5. A mycelium composite soil, which is characterized by comprising a mixed matrix and the fungus homogenate of claim 4 planted on the mixed matrix, wherein the mixed matrix comprises wood chips, gram-negative bacteria, soil samples and trace elements.
  6. 6. The mycelium composite soil according to claim 5, wherein the mass ratio of wood chips, gram-negative bacteria, soil samples and trace elements is 30% -40%, 2% -5%, 50% -60%, 1% -5%.
  7. 7. A steaming-infiltration controllable structure layer based on the mycelium composite soil according to any one of claims 5-6 is characterized by sequentially comprising a capillary blocking layer, a capillary water storage layer, an infiltration buffer layer and a mycelium evaporation prevention layer from bottom to top; The capillary stop 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 mycelium anti-evaporation layer is composed of mycelium composite soil according to any one of claims 5 to 6.
  8. 8. Use of a genetically encoded filamentous fungus according to any one of claims 1 to 3, a fungal homogenate according to claim 4, a mycelium composite soil according to any one of claims 5 to 6, and a steam-infiltration controllable structural layer of a mycelium composite soil according to claim 7 for slope protection.
  9. 9. The method according to claim 8, wherein the mycelium composite soil and the grass seeds are uniformly stirred and then laid on the slope top and leveled, and the clay and the grass seeds are uniformly stirred and then laid on the slope and leveled.
  10. 10. The method for confirming the throwing amount of fungus homogenate in the mycelium composite soil, which is characterized by the application of the method according to claim 8, comprises the following steps: Step B1, designing an in-situ shearing and jet erosion coupling model; step B2, planting the mycelium composite soil in the in-situ shearing and jet erosion coupling model, and simulating dynamic change of performance before and after slope protection in situ; And B3, developing an optimization algorithm of the optimal addition amount of the slope protection thalli based on a distribution function, and judging the sensitivity degree of the cohesive force and the internal friction angle to the influencing factors such as the water content, the mycelium content and the mycelium diameter according to the variation coefficient.

Description

Gene coding filamentous fungi and application thereof in slope protection Technical Field The invention belongs to the technical field of microbial slope protection, and particularly relates to a gene coding filamentous fungus and application thereof in slope protection. Background The ecological environment damage problem is not neglected while the economy of China is rapidly developed. Solid waste storage yards generated by coal exploitation, thermal power generation and petroleum exploration are increasingly expanded, and particularly in yellow river bending areas in arid areas, the ecological environment is adversely affected. The area is distributed with 6 national coal bases, the coal yield is about 40% of the whole country, the crude oil resources are quite rich, and the area plays an important role in the energy safety supply guarantee of China. However, in the western arid-semiarid region of the region, the ecological environment is fragile, the disturbance resistance is poor, and various geological disasters are easily induced in the mining process. According to official statistics, the energy base is mainly located in loess areas and wind sand accumulation areas, ecological hidden dangers in mining areas are serious, dust raising in storage yards is four, water and soil loss, loess landslide, subsidence and ground cracking are all the same, desertification problems are increasingly aggravated, and ecological restoration rate is less than 20%. In conclusion, vegetation slope protection is the first choice ecological slope protection technology. However, the solid waste storage yard and loess slope soil have weak water storage capacity and poor planting performance, and the slope protection difficulty is increased. The solution of the problem needs to comprehensively consider various factors such as soil characteristics, plant selection, water management, root system configuration regulation and the like. Wherein, the hydrophobic water storage and the evaporation prevention performance of the slope soil structural layer are key. The ecological porous concrete adopted at present has the disadvantages of low germination rate and high long-term maintenance cost of plants due to the disadvantages of ecological environment fragmentation, strong initial alkalinity, high carbon footprint and the like. Therefore, development of a novel environment-friendly hypha hydrophobic-vegetation slope protection technology is needed, blue light receptor genes of Pleurotus ostreatus filamentous fungi are damaged through gene editing (CRISPR-Cas 9), growth of fruiting bodies of the mycelia is restrained, so that a lasting natural hydrophobic layer is formed on the surface of a vegetation layer, soil organic matters are increased through metabolism enzymes, a 'micro reservoir' is built in a plant root system area, plant habitat is improved while water evaporation is effectively reduced, and the mycelia and plant root systems cooperatively promote soil shearing resistance to realize slope fixation and stability. Disclosure of Invention The invention aims to provide a gene coding filamentous fungus and application thereof in slope protection, hyphae continuously grow in a slope to form a semi-permeable layer, namely, mycelia form a hydrophobic layer at the contact part of the slope and air, so that the slope is prevented from being corroded by water flow, and a water-holding layer is formed in the slope to inhibit water and soil loss of the slope. In order to achieve the above purpose, the present application adopts the following technical scheme: In a first aspect, the present invention provides a gene-encoded filamentous fungus, which is prepared by knocking out blue light receptor genes wcl1 and wcl2 from Pleurotus ostreatus, wherein CDS coding sequences of wcl1 and wcl2 are respectively shown as accession numbers KY348758 and MG679810 of an International publication database. In the above technical scheme, the preparation method of the gene encoding filamentous fungus comprises the following steps: Step A1, constructing an RNP system, namely designing and chemically synthesizing specific sgRNA aiming at the wcl1 and wcl2 genes of Pleurotus ostreatus, wherein the sgRNA comprises a guide sequence complementary to a target gene and a general sgRNA framework sequence; Step A2, delivering the RNP system into Pleurotus ostreatus cells to obtain RNP-Pleurotus ostreatus; And A3, obtaining stable genetic mutants, namely culturing the RNP-Pleurotus ostreatus under dark and illumination respectively, screening and verifying transformants, and obtaining the stable genetic mutants by separating and purifying spores to obtain the gene coding filamentous fungi. In the above technical scheme, in the step A2, the specific method for delivering the RNP system into Pleurotus ostreatus cells is that the RNP system and Pleurotus ostreatus protoplast are co-cultured in PEG solution, and the cell membrane is induced to open holes, so that t