Application of turpentine in cultivation of poria cocos and cultivation method and composition of poria cocos and ganoderma lucidum

Abstract

The application belongs to the technical field of traditional Chinese medicinal material cultivation, and particularly relates to application of pine oil in cultivation of Poria cocos and a cultivation method and composition of Poria cocos and Ganoderma lucidum. The composition is composed of sawdust, pine oil and auxiliary materials in a mass ratio of 56:0.1:44. The auxiliary materials are composed of cottonseed hulls, gypsum, sucrose and corn flour in a mass ratio of 40:1:1:2. The application proves that the pine oil can replace pine wood for cultivation of Poria cocos in a bag material, which influences the formation of sclerotia by affecting the expression of genes related to nutrient utilization and biomass synthesis in Poria cocos mycelium, genes related to AMPK and MAPK signal pathways, and the growth and development of Poria cocos mycelium by participating in the transportation and oxidative stress of secondary metabolites. Therefore, the pine oil added in sawdust can replace pine wood for cultivation of Poria cocos sclerotia, so that the pine wood resources can be protected, and the cultivation of Poria cocos sclerotia is not limited by pine wood. The application provides a new direction and means for cultivation of Poria cocos, and has important significance for cultivation of Poria cocos.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of traditional Chinese medicinal material cultivation, and particularly relates to application of pine oil in cultivation of poria cocos and a poria cocos cultivation method and composition. BACKGROUND

[0002] Poria cocos is a traditional Chinese medicinal material with a long history of use. It contains various chemical active components, including polysaccharides, triterpenes, sterols, volatile oils, proteins, etc. There are more than 200 kinds of Chinese patent medicines using poria cocos sclerotia as raw materials, with a compatibility rate of more than 80%, a huge market demand, and medicinal values of antioxidant, antitumor, immune regulation, diuresis, anti-inflammatory and liver protection.

[0003] At present, pine wood (chips), pine needles, etc. are needed as materials for cultivation of poria cocos, and the cultivation of poria cocos is restricted by factors such as pine resource protection and growth cycle of pine trees. Therefore, it is urgent to find a material that can replace pine wood for cultivation of poria cocos sclerotia to break through the limitation of pine wood on cultivation of poria cocos sclerotia and reduce the dependence on pine resources in the process of cultivation of poria cocos. Pine oil is the main difference material of pine trees and other trees, and the main chemical components are monoterpene compounds such as α-pinene and β-pinene. Literature analysis shows that among the pine trees of Pinus subgenus, such as Pinus yunnanensis, Pinus massoniana and Pinus tabulaeformis, the content of monoterpene compounds in the metabolic products is the highest. However, there is no relevant patent or literature material to study whether pine oil in pine wood is a key factor for formation of poria cocos sclerotia.

[0004] In the prior art, the patent for invention with publication number CN102934586B discloses a method for low-carbon and high-yield cultivation of poria cocos, which uses pine stumps under the forest to cultivate poria cocos naturally, and combines with a main root inoculation method to cultivate poria cocos, so as to solve the "bacteria-forest contradiction" in production of poria cocos. The patent for invention with publication number CN113711848B discloses a method for cultivation of poria cocos, which uses mixed soil for cultivation of poria cocos, and the raw materials of the mixed soil include sandy soil and crushed pine appendages or manure. Although the patent effectively improves the quality of poria cocos, it depends on pine segments and needs to be restricted by pine resource protection. SUMMARY

[0005] Therefore, one of the purposes of the present application is to provide a composition for bag material cultivation of poria cocos. The present application researches and finds that pine oil in pine wood is a key factor for promoting formation of poria cocos sclerotia, and can be used to replace pine wood for cultivation of poria cocos, so as to effectively reduce the dependence on pine resources in the process of cultivation of poria cocos.

[0006] To achieve the above-mentioned purposes, the present application adopts the following technical solutions:

[0007] The composition for cultivating Poria cocos bag material is composed of sawdust, turpentine and auxiliary materials; the mass ratio of the sawdust, turpentine and auxiliary materials is 56:0.1:44.

[0008] Further, the auxiliary materials are composed of cottonseed hulls, gypsum, sucrose and corn flour; the mass ratio of the cottonseed hulls, gypsum, sucrose and corn flour is 40:1:1:2.

[0009] The second object of the present application is to provide an application of the turpentine and / or the composition in cultivating Poria cocos.

[0010] The third object of the present application is to provide an application of the turpentine and / or the composition in promoting the growth and development of Poria cocos mycelium and polysaccharide metabolism.

[0011] To achieve the above objects, the present application adopts the following technical solutions:

[0012] The application of the turpentine and / or the composition in promoting the growth and development of Poria cocos mycelium and polysaccharide metabolism, the concentration of the turpentine is 100mg / L-300mg / L, more preferably 300mg / L.

[0013] The fourth object of the present application is to provide an application of the turpentine and / or the composition in promoting the sclerotium development of Poria cocos.

[0014] To achieve the above objects, the present application adopts the following technical solutions:

[0015] The application of the turpentine and / or the composition in promoting the sclerotium development of Poria cocos, the turpentine promotes the sclerotium development of Poria cocos by regulating the expression of related genes in Poria cocos mycelium; the related genes include AMPK signal pathway related genes, MAPK signal pathway related genes, protein phosphorylation related genes, carbohydrate active enzyme family related genes, polysaccharide synthesis genes, triterpenoid synthesis genes and / or other genes.

[0016] As preferred, the AMPK signal pathway related gene comprises GTP-binding protein ypt2, glycosyltransferase family 3 protein and / or Δ9-fatty acid desaturase protein; the MAPK signal pathway related gene comprises 14-3-3 protein and / or G-alpha domain protein; the protein phosphorylation related gene comprises protein phosphatase 2C (PP2C), alpha-kinase family and / or serine / threonine-protein kinase B-raf; the carbohydrate active enzyme family related gene comprises carbohydrate esterase family 15 protein, carbohydrate esterase family 4 protein, glycoside hydrolase family 5 protein, glycoside hydrolase 12 family protein and / or glycoside hydrolase 16 family protein; the polysaccharide substance synthesis gene comprises galactan 1,4-alpha-galacturonase A and / or UTP-glucose-1-phosphate uridylyltransferase; the triterpenoid substance synthesis gene comprises ergosterol biosynthesis protein 6 and / or terpene synthase; and the other gene comprises C2H2-type zinc finger, subtilisin-like protein, expansin-like protein, MFS, NAD(P)-binding protein, peptidase inhibitor, 5-aminolevulinic acid synthase, fungal fruiting body agglutinin 1a and / or cytochrome P450.

[0017] As preferred, the GTP-binding protein ypt2 has a Gene-id of gene-WOLCODRAFT_111188; the glycosyltransferase family 3 protein has a Gene-id of gene-WOLCODRAFT_27407; the delta 9-fatty acid desaturase protein has a Gene-id of gene-WOLCODRAFT_23420; the 14-3-3 protein has a Gene-id of gene-WOLCODRAFT_69384; the G-alpha domain protein has a Gene-id of gene-WOLCODRAFT_139231; the protein phosphatase 2C has a Gene-id of gene-WOLCODRAFT_142161 and / or gene-WOLCODRAFT_86489; the serine / threonine-protein kinase B-raf has a Gene-id of gene-WOLCODRAFT_164050; the carbohydrate esterase family 15 protein has a Gene-id of gene-WOLCODRAFT_23632; the carbohydrate esterase family 4 protein has a Gene-id of gene-WOLCODRAFT_81480; the glycoside hydrolase family 5 protein has a Gene-id of gene-WOLCODRAFT_112799; the glycoside hydrolase family 12 protein has a Gene-id of gene-WOLCODRAFT_24190; the glycoside hydrolase family 16 protein has a Gene-id of gene-WOLCODRAFT_79265 and / or gene-WOLCODRAFT_21871; the galactoside 1,4-alpha-galacturonidase A has a Gene-id of gene-WOLCODRAFT_177549; the UTP-glucose-1-phosphate uridylyltransferase has a Gene-id of gene-WOLCODRAFT_90597; the ergosterol biosynthesis protein 6 has a Gene-id of gene-WOLCODRAFT_145787; the terpene synthase has a Gene-id of gene-WOLCODRAFT_137597; the C2H2-type zinc finger has a Gene-id of gene-WOLCODRAFT_50037; the subtilisin-like protein has a Gene-id of gene-WOLCODRAFT_27933; the expansin-like protein has a Gene-id of gene-WOLCODRAFT_136006 and / or gene-WOLCODRAFT_23456; the MFS has a Gene-id of gene-WOLCODRAFT_76753 and / or gene-WOLCODRAFT_138211;The Gene-id of the NAD(P) binding protein is gene-WOLCODRAFT_127992; the Gene-id of the alpha-kinase family is gene-WOLCODRAFT_67511; the Gene-id of the peptidase inhibitor is gene-WOLCODRAFT_108759; the Gene-id of the 5-aminolevulinic acid synthase is gene-WOLCODRAFT_24698; the Gene-id of the fungal fruiting body agglutinin 1a is gene-WOLCODRAFT_81600; and the Gene-id of the cytochrome P450 is gene-WOLCODRAFT_28038.

[0018] Further, the turpentine promotes the sclerotium development of Poria cocos by regulating the expression of AMPK signal pathway related genes and MAPK signal pathway related genes in Poria cocos mycelium; the turpentine promotes the up-regulated expression of Δ9-fatty acid desaturase protein and / or G-alpha domain protein, promotes the down-regulated expression of 14-3-3 protein and / or GTP binding protein ypt2, and regulates the expression amount of glycosyltransferase family 3 protein to first decrease and then increase.

[0019] Further, the turpentine maintains the growth and development of Poria cocos mycelium and promotes the sclerotium development of Poria cocos by promoting the expression of carbohydrate active enzyme family related genes, polysaccharide substance synthesis genes, triterpenoid synthesis related genes and / or protein phosphorylation related genes.

[0020] Further, the turpentine promotes the sclerotium formation by affecting the secondary metabolite substance transport and oxidative stress of Poria cocos mycelium through promoting the up-regulated expression of MFS general substrate transport protein and NAD(P) binding protein.

[0021] The fifth object of the present application is to provide a method for cultivating Poria cocos without pine wood, which successfully breaks through the limitation of pine wood on the cultivation of Poria cocos sclerotium and reduces the dependence on pine wood resources in the cultivation process of Poria cocos.

[0022] To achieve the above object, the present application adopts the following technical solution:

[0023] A method for cultivating Poria cocos without pine wood, which uses the composition and adopts turpentine to replace pine wood for the bag culture of Poria cocos, and obtains Poria cocos through inoculation, planting and digging.

[0024] As a preferred, the method specifically comprises the following steps:

[0025] 1. Bag preparation: adopt the following bag material formula: 56% sawdust, 40% cotton seed hull, 1% gypsum, 1% sucrose, 2% corn flour and 0.1% turpentine, mix in proportion, add about 60% water; bottle, seal, sterilize twice, and prepare the bag.

[0026] 2. Inoculation: inoculate the activated Poria cocos spores into the bottle containing 56% pine sawdust, 40% cotton seed hull, 1% gypsum, 1% sucrose, 2% corn flour, and incubate at 28°C until the mycelium fills the whole bottle, to obtain the corresponding secondary spores. Then inoculate the Poria cocos secondary spores into the bag, incubate at 28°C, until the mycelium fills the whole bag, to obtain the cultivation species.

[0027] 3. Planting: transport the cultured Poria cocos bag to the cultivation site for sowing. When planting, draw a 10 cm opening on the bag, place a piece of fresh Poria cocos as an inducer at the opening, and then cover the soil with a thickness of about 10 cm.

[0028] As preferred, high-pressure intermittent sterilization method is used for sterilization, and the sterilization condition is 121°C for 45 min.

[0029] As preferred, the inoculation process is carried out in a sterile environment.

[0030] As preferred, the site is selected to be a mountain with an altitude of about 1000 m and not facing north, and the soil is sandy with a certain slope.

[0031] As preferred, the land needs to be prepared before cultivation, and the ditch is about 30 cm deep.

[0032] As preferred, the Poria cocos cultivation species is checked for strength and pollution before planting, and the sowing is carried out on a sunny day.

[0033] Further, field management is needed during planting, and the growth of Poria cocos is observed, weeds are removed, and termites are removed to ensure the growth of Poria cocos.

[0034] The beneficial effects of the present application are:

[0035] 1. The present patent proves that turpentine in pine is the key factor for promoting sclerotium formation of Poria cocos.

[0036] 2. The present patent research finds that turpentine is an important factor affecting the growth and development of Poria cocos mycelium and the metabolism of polysaccharide substances, low concentration (≤300mg / L) turpentine promotes the growth of Poria cocos mycelium, high concentration (400-500mg / L) turpentine inhibits the growth of Poria cocos mycelium, and turpentine also promotes the synthesis of Poria cocos polysaccharide.

[0037] 3. The patent research found that turpentine can affect the growth and development of Poria cocos mycelium by affecting the expression of genes related to nutrient utilization and biomass synthesis in Poria cocos mycelium, AMPK and MAPK signal pathway related genes, and secondary metabolite transport and oxidative stress, thereby affecting sclerotia formation. Therefore, adding turpentine to sawdust instead of pine wood for cultivating Poria cocos sclerotia can protect pine resources and break through the limitations of pine wood for Poria cocos sclerotia cultivation. BRIEF DESCRIPTION OF DRAWINGS

[0038] Figure 1 Figure 1 is a comparison chart of the growth rates of Poria cocos 5.78, Poria cocos 28, and Pleurotus ostreatus mycelium under different concentrations of turpentine treatment, with significance P<0.05;

[0039] Figure 2 Figure 2 is a comparison chart of the mycelial morphology of Poria cocos 5.78, Poria cocos 28, and Pleurotus ostreatus mycelium under different concentrations of turpentine treatment;

[0040] Figure 3 Figure 3 is a comparison chart of the growth state of Poria cocos 5.78, Poria cocos 28, and Pleurotus ostreatus mycelium under different concentrations of turpentine treatment;

[0041] Figure 4 Figure 4 is a chart of mycelial biomass in different concentrations of turpentine medium, with significance P<0.05;

[0042] Figure 5 Figure 5 is a standard curve chart of different concentrations of glucose solution;

[0043] Figure 6 Figure 6 is a chart of the mass fraction of Poria cocos mycelial polysaccharide under different concentrations of turpentine treatment, with significance P<0.05;

[0044] Figure 7 Figure 7 is a correlation analysis result chart between samples;

[0045] Figure 8 Figure 8 is a volcano plot of significantly differentially expressed genes under turpentine treatment of Poria cocos mycelium, wherein, Figure 8 A is the differentially expressed transcriptome of Wc5.78-300mg / L VS Wc5.78-0mg / L; Figure 8 B is the differentially expressed transcriptome of Wc5.78-500mg / L VS Wc5.78-300mg / L; Figure 8 C is the differentially expressed transcriptome of Wc5.78-500mg / L VS Wc5.78-0mg / L; each point in the chart represents a gene, the horizontal coordinate represents the logarithmic value of the differential expression fold of a certain gene in two samples, and the vertical coordinate represents the significant negative logarithmic value of the change in gene expression;

[0046] Figure 9A Venn diagram of differentially expressed genes of Poria cocos mycelium under treatment of turpentine, wherein, pink circle represents up-regulated expression genes of each gene expression amount under treatment of turpentine with a concentration of 300 mg / L compared with each gene expression amount under treatment of 0 mg / L turpentine; green circle represents up-regulated expression genes of each gene expression amount under treatment of turpentine with a concentration of 500 mg / L compared with each gene expression amount under treatment of 300 mg / L turpentine; purple circle represents down-regulated expression genes of each gene expression amount under treatment of turpentine with a concentration of 300 mg / L compared with each gene expression amount under treatment of 0 mg / L turpentine; blue circle represents down-regulated expression genes of each gene expression amount under treatment of turpentine with a concentration of 500 mg / L compared with each gene expression amount under treatment of 300 mg / L turpentine;

[0047] Figure 10 A heat map of differentially expressed genes of Poria cocos mycelium under treatment of turpentine, wherein, Figure 10 A is Figure 9 a combination of genes I in the first group; Figure 10 B is Figure 9 a combination of genes II in the second group; Figure 10 C is Figure 9 a combination of genes III in the third group; Figure 10 D is Figure 9 a combination of genes IV in the fourth group;

[0048] Figure 11 A heat map of differentially expressed genes of Poria cocos mycelium under treatment of turpentine, wherein, Figure 11 A is Figure 9 a combination of genes V in the fifth group; Figure 11 B is Figure 9 a combination of genes VI in the sixth group; Figure 11 C is Figure 9 a combination of genes VII in the seventh group; Figure 11 D is Figure 9 a combination of genes VIII in the eighth group;

[0049] Figure 12 A diagram of GO function enrichment analysis results of differentially expressed genes under treatment of turpentine with a concentration of 300 mg / L and 0 mg / L;

[0050] Figure 13 A diagram of GO function enrichment analysis results of differentially expressed genes under treatment of turpentine with a concentration of 300 mg / L and 500 mg / L;

[0051] Figure 14 A diagram of GO function enrichment analysis results of differentially expressed genes under treatment of turpentine with a concentration of 500 mg / L and 0 mg / L;

[0052] Figure 15 A diagram of KEGG metabolic pathway analysis results of differentially expressed genes under treatment of turpentine with a concentration of 300 mg / L and 0 mg / L;

[0053] Figure 16Figure of KEGG metabolic pathway analysis results of differentially expressed genes for turpentine concentration 300mg / L and 500mg / L;

[0054] Figure 17 Figure of KEGG metabolic pathway analysis results of differentially expressed genes for turpentine concentration 500mg / L and 0mg / L;

[0055] Figure 18 Heat map of expression quantity of differentially expressed genes of CAZymes under turpentine treatment;

[0056] Figure 19 Heat map of expression quantity of differentially expressed genes involved in synthesis of polysaccharide compounds of Poria cocos under turpentine treatment;

[0057] Figure 20 Heat map of expression quantity of differentially expressed genes involved in synthesis of triterpenoid substances of Poria cocos under turpentine treatment;

[0058] Figure 21 Heat map of expression quantity of genes involved in development of sclerotia of Poria cocos;

[0059] Figure 22 Figure of detection results of expression quantity of genes of AMPK signaling pathway and MAPK signaling pathway under turpentine treatment, specifically, Figure 22 A- Figure 22 E represent, in turn, figure of detection results of expression quantity of 14-3-3 protein, G-alpha domain protein, GTP-binding protein ypt2, glycosyltransferase family 3 protein and delta-9-fatty acid desaturase protein;

[0060] Figure 23 Figure of detection results of expression quantity of carbohydrate-active enzyme genes under turpentine treatment, specifically, Figure 23 A- Figure 23 E represent, in turn, glycoside hydrolase 16 family protein (gene-WOLCODRAFT_21871), glycoside hydrolase family 16 protein (gene-WOLCODRAFT_79265), carbohydrate esterase family 15 protein, carbohydrate esterase family 4 protein, glycoside hydrolase family 5 protein;

[0061] Figure 24 Figure of detection results of expression quantity of genes of polysaccharide and triterpenoid compound synthesis of Poria cocos under turpentine treatment; Figure 24 A- Figure 24 D represent, in turn, figure of detection results of expression quantity of galactoglycan 1,4-alpha-galacturonase A, UTP-glucose-1-phosphate uridylyltransferase, Methylglutaconyl-CoA hydratase and terpene synthase;

[0062] Figure 25Figure of the expression quantity detection results of the genes related to sclerotium formation development under the treatment of turpentine; Figure 25 A- Figure 25 E represent MFS (gene-WOLCODRAFT_774929), MFS (gene-WOLCODRAFT_138211), NAD(P) binding protein (gene-WOLCODRAFT_127992), protein phosphatase 2C (gene-WOLCODRAFT_86489) and protein phosphatase 2C (gene-WOLCODRAFT_142161) in turn;

[0063] Figure 26 Figure of the sclerotium formation gene regulatory network of P. lucidum under the treatment of turpentine, the arrow direction represents the regulatory direction;

[0064] Figure 27 A is the mycelium growth situation of P. lucidum in the bag under different turpentine concentrations; Figure 27 B is the field cultivation situation of P. lucidum bag;

[0065] Figure 28 A is the mycelium aggregation situation of P. lucidum in the field cultivation for 6 months, B is the inside situation of the mycelium aggregation after being split in half.

[0066] Above Figures 22-25 In the above, the horizontal coordinate RNA-Seq is the gene expression quantity in the transcriptome data, and the RT-qPCR is the gene expression quantity determined by real-time fluorescent quantitative PCR; the vertical coordinate expression quantity value is obtained by initializing the expression quantity value of each gene, the expression quantity at the turpentine concentration of 0 mg / L is initialized and assigned as 1, the gene expression quantity at the concentrations of 300 mg / L and 500 mg / L changes with the expression quantity at 0 mg / L, and the expression quantity change trends of the three concentrations are unchanged; the Duncan test in the SPSS18 software is used for single factor variance analysis of the gene expression quantity, and P<0.05.

[0067] Figure 1 、 Figure 4 、 Figure 6 、 Figures 22-25The marked letters in ABCD are used to mark the significance of difference between different turpentine oil concentration treatments of each sample using single factor variance analysis. The marked letters of each column are different, representing that there is significant difference between them, and the same letter in the mark represents no significant difference (P < 0.05). The specific marking rules are as follows: first, arrange the average numbers of each treatment from large to small from top to bottom, then mark the letter a after the largest average number, and compare the average number with the following average numbers in turn, and mark the same letter a if there is no significant difference, until the average number which is significantly different from it is marked with the letter b; then take the average number marked with the letter b as the standard, compare it with each average number above it, and mark b again if there is no significant difference, until there is a significant difference; in this way, mark until the smallest average number is marked and compared. DETAILED DESCRIPTION

[0068] The technical solutions of the present application will be described further and completely in combination with specific examples. Obviously, the described examples are only some of the examples of the present application, not all. Therefore, all other examples obtained by those skilled in the art based on the examples in the present application without creative labor are within the protection scope of the present application.

[0069] In the examples of the present application, two Poria cocos varieties, Poria cocos 28 (preservation number: ACCC51641) and Poria cocos 5.78 (preservation number: ACCC50864), are from Chongqing Institute of Chinese Medicine; the Pleurotus ostreatus (Po) strain is purchased from the market and preserved in the laboratory after organization separation.

[0070] In the examples of the present application, Taq DNA polymerase, Phanta ® Max Super-Fidelity DNA Polymerase and ClonExpress ®MultiS One Step Cloning Kit was purchased from Nanjing Nvzhan Biotechnology Co., Ltd.;OminiPlant RNA Kit (Dnase I), HiFiScript gDNA Removal cDNA Synthesis Kit and UltraSYBR Mixture (Low ROX) were purchased from Kangwei Century Biotechnology Co., Ltd.;Glycerol, anhydrous ethanol, sodium chloride, glucose, sodium hydroxide were purchased from Chengdu Kelong Chemical Co., Ltd.;Agar powder and GenGreen nucleic acid dye were purchased from Beijing Dingguochangsheng Biotechnology Co., Ltd.;dNTPs were purchased from Beijing Bomeide Gene Technology Co., Ltd.;DNA polymerase Klenow Fragment was purchased from Thermo Fisher Scientific;pMDTM19-T Vector Cloning Kit, RrimeScript TM RT reagent Kit and DNA marker (2000) were purchased from TaKaRa;Agarose was purchased from BIOWEAST;Phenol, concentrated sulfuric acid and Tween 80 were purchased from Araldin;Turpentine was purchased from Macklin;Vanillin, glacial acetic acid and oleanolic acid were purchased from Chengdu Kelong Chemical Reagent Factory.

[0071] In the embodiment of the present application, the configuration method of PDA solid culture medium is: fresh potato 200g, glucose 20g, agar powder 20g, water 1L, natural pH, 121℃ high pressure sterilization for 25min, and then it is obtained. The configuration method of PDA liquid culture medium is: fresh potato 200g, glucose 20g, water 1L, natural pH, 121℃ high pressure sterilization for 25min, and then it is obtained.

[0072] In the embodiment of the present application, the configuration method of turpentine mother liquor is: 100mg, 200mg, 300mg, 400mg and 500mg of turpentine are weighed respectively;Tween 80: turpentine = 1:4, mix evenly, add water to 100mL, and then turpentine mother liquor with concentrations of 1000mg / L, 2000mg / L, 3000mg / L, 4000mg / L and 5000mg / L are prepared.

[0073] In the embodiment of the application, the reagent configuration method is: ①1 mol / L NaOH solution: 40 g of NaOH is dissolved in water, and then diluted to 1 L. ②5% phenol solution: 5 g of phenol is dissolved in water at 65 DEG C, and then diluted to 100 mL. ③5% vanillin-glacial acetic acid: 5 g of vanillin is dissolved in glacial acetic acid, and then diluted to 100 mL. ④50x TAE electrophoresis buffer: 242 g of Tris Base, 100 mL of 0.5 mol / L EDTA with pH of 8.0, 57.1 mL of acetic acid, and water to 1 L. ⑤ Lactic acid carbinol cotton blue staining solution: 10 g of carbinol, 10 mL of lactic acid (specific gravity 1.21), 20 mL of glycerol, 10 mL of distilled water, and 0.02 g of cotton blue are weighed, carbinol is added to distilled water and heated to dissolve, then lactic acid and glycerol are added, finally cotton blue is added and dissolved to obtain the lactic acid carbinol cotton blue staining solution.

[0074] In the embodiment of the application, the strain activation is as follows: using a inoculation shovel, 0.5*0.5 cm fungus blocks of Poria cocos strain (Poria cocos 28, Poria cocos 5.78) and Pleurotus ostreatus strain are cut from the slant test tube culture medium, and then transferred to sterilized PDA plates (10 cm in diameter) to be activated and cultured in a 28 DEG C constant temperature incubator, and then placed in a 4 DEG C refrigerator for storage.

[0075] In the embodiment of the application, the transcriptome sequencing is completed by Beijing Nuoweiziyuan Technology Co., Ltd.; and the microbioinformatics website is http: / / www.bioinformatics.com.cn / plot_basic_3_color_volcano_plot_086.

[0076] In the embodiment of the present application, the extraction of total RNA of Poria cocos mycelium and the synthesis method of cDNA are as follows: (1) material treatment: inoculate the mycelium on PDA medium containing different concentrations of turpentine, cultivate in a constant temperature incubator at 28 DEG C for 7 days, scrape off the mycelium, put it in a 2 mL centrifuge tube, write a label, cool rapidly with liquid nitrogen, and store at -80 DEG C. (2) RNA extraction and quality detection: use OminiPlant RNA Kit (Dnase I) kit to extract total RNA of all samples, and the specific operation steps are described in the reference manual. Electrophoresis is used to determine the quality of the extracted RNA. Observe whether the 18s RNA and 28s RNA bands are bright and clear, and whether there is a trailing phenomenon. At the same time, use a ultramicro spectrophotometer to measure the RNA concentration. (3) cDNA synthesis: based on the measured RNA concentration, use DNase I, RNase-free kit and HiFiScript gDNA Removal cDNASynthesis Kit reverse transcription kit to reverse transcribe all the obtained RNA. The preparation system in this process is carried out on ice. The obtained cDNA product is placed in a -20 DEG C refrigerator for storage, and is ready for subsequent tests.

[0077] In the embodiment of the present application, the real-time fluorescent quantitative PCR method is as follows: (1) quantitative primer design: according to the transcriptome sequencing results, screen the genes, find the coding sequence of Poria cocos gene, use the quantitative primer design principle, and use Primer Premier5 software to design the corresponding gene quantitative primer. According to the stress conditions of turpentine in this test, the internal reference gene is GAPDH. (2) The selected genes, primer names, primer sequences and optimal annealing temperatures in this test are shown in Table 1. (3) Gene expression detection: use RT-qPCR to analyze the relative expression level of the extracted cDNA at the optimal annealing temperature. All samples are set with 3 technical repeats, and a negative control is set at the same time. Finally, according to the Ct value of the internal reference gene of each treatment sample, use the 2-ΔΔCt method to calculate the relative expression amount, and perform data processing and analysis. Use OriginPro 2023 (learning version) to perform data statistical processing and drawing, and use SPSS software to perform significant difference analysis on the test data results.

[0078] Table 1

[0079]

[0080] Example 1. Study on the influence of turpentine on the growth and development of Poria cocos mycelium

[0081] (1) Mycelium growth rate determination and morphological observation: Poria cocos and Pleurotus ostreatus mycelial blocks were inoculated on PDA medium containing different concentrations of turpentine, and the specific steps were as follows: Different concentrations of turpentine mother liquor were added to the prepared PDA solid medium, diluted 10 times, and prepared into PDA medium with final concentrations of 100 mg / L, 200 mg / L, 300 mg / L, 400 mg / L and 500 mg / L. After sterilization, it was divided and poured into plates. After the mycelium was activated, a 5mm diameter puncher was used to punch the mycelium surface along the mycelium edge, and the same age mycelial blocks were inoculated in the center of the PDA plate containing different concentrations of turpentine, and placed in a 28°C constant temperature incubator for culture until the mycelium grew throughout the plate. Observe the mycelial growth, measure the mycelial growth length using the cross method, count and record the data, calculate the mycelial growth rate, and determine the growth trend of Poria cocos and Pleurotus ostreatus mycelium under the stimulation of different concentrations of turpentine. To ensure the accuracy of the test, each group was repeated 6 times. The SPSS software single factor analysis of variance Duncan method was used to analyze the significant difference of the test data results, and OriginPro 2021b (learning version) was used for data statistical processing and drawing. The formula for calculating the mycelial growth rate is: , wherein V represents the mycelial growth rate; n represents the culture time; d 1n represents the mycelial diameter 1 cultured for n days; d1 represents the mycelial diameter 1 before treatment. The above mycelium was used as the observation material, and the mycelium treated with different concentrations of turpentine was picked up with an inoculation needle and placed in lactic acid carbonic acid cotton blue staining solution to prepare a fungal mycelium preparation for microscopic examination. The mycelial morphology was observed and recorded.

[0082] Results: Mycelial growth rate, morphology, and growth conditions are as follows Figures 1-3The results showed that different varieties of Poria cocos showed different growth under different concentrations of turpentine treatment. Under turpentine treatment, the mycelial growth rate of Poria cocos 5.78 was relatively stable. When no turpentine was added, the mycelial growth rate was the slowest. With the increase of turpentine concentration, the growth rate showed a gradually increasing trend. The mycelial morphology showed no significant difference under different turpentine concentrations. When the concentration of turpentine was in the range of 0-300 mg / L, the mycelium was dense, white and strong, and the growth was uniform. When the concentration was greater than 300 mg / L, the mycelium gradually became weak and the growth was sparse. The growth rate of Poria cocos 28 showed an increasing trend first and then a decreasing trend under different concentrations of turpentine treatment. The growth rate was the fastest when the concentration was 300 mg / L. The growth condition was similar to that of Poria cocos 5.78. When the concentration was less than 300 mg / L, the mycelium was dense, white and strong, and the mycelial diameter was large. When the concentration was greater than 300 mg / L, the mycelium became weak and the growth was slow. With the increase of turpentine concentration, the mycelial morphology gradually became fine. The results showed that a certain concentration of turpentine was helpful for the growth of Poria cocos mycelium, and 300 mg / L was the most suitable concentration for the growth of Poria cocos mycelium. The growth rate of Pleurotus ostreatus mycelium showed a decreasing trend with the increase of turpentine concentration. When the concentration was 300 mg / L, the mycelium showed uneven growth and morphological abnormalities. When the concentration was greater than 300 mg / L, the growth rate of Pleurotus ostreatus mycelium slowed down. Compared with Poria cocos, the mycelium of Pleurotus ostreatus was fine. With the increase of turpentine concentration, breakpoints appeared between the mycelium, and the mycelium became weak, indicating that turpentine would seriously inhibit the growth of Pleurotus ostreatus mycelium.

[0083] (2) Mycelial biomass determination: The mycelial blocks of Poria cocos and Pleurotus ostreatus were inoculated in liquid PDA medium containing different concentrations of turpentine for cultivation. The specific method was as follows: PDA liquid medium was prepared, different concentrations of turpentine stock solution were added, diluted 10 times to obtain the medium containing the target turpentine concentration, and then dispensed. 50 mL of medium was placed in a 250 mL conical flask. To ensure the accuracy of the test, there were 6 replicates in each group. A puncher was used to punch the surface of the activated mycelium to obtain mycelial blocks of the same age, which were inoculated into the liquid medium. Each conical flask was inoculated with one mycelial block and placed in a 28℃ constant temperature incubator for 7 days. The mycelium was collected by filtration and dried at 50℃ to constant weight. The biomass was measured and weighed. The dried mycelium was saved for determination of Poria cocos mycelial polysaccharide content.

[0084] Results: As Figure 4As shown, the Poria cocos mycelium biomass increased first and then decreased with the increase of turpentine concentration, and the mycelium biomass was the most when the turpentine concentration was 200-300 mg / L, and there was no significant difference; the Pleurotus ostreatus mycelium biomass decreased with the increase of turpentine concentration, and the biomass was basically not affected by the turpentine concentration when the concentration was in the range of 0-200 mg / L, at this time the Pleurotus ostreatus mycelium was relatively dense, and the biomass decreased sharply when the turpentine concentration reached 300 mg / L, and there was a significant difference compared with the mycelium biomass treated with low-concentration turpentine, and the biomass basically remained unchanged with the increase of concentration. The mycelium biomass of Poria cocos and Pleurotus ostreatus was consistent with the growth speed and its growth, which indicated that the appropriate concentration of turpentine could promote the growth of Poria cocos mycelium, and Pleurotus ostreatus was not suitable for growing in a certain concentration of turpentine.

[0085] Example 2. Mycelium polysaccharide content determination

[0086] The mycelium polysaccharide content was determined by using the phenol-concentrated sulfuric acid method. The principle is that under the action of sulfuric acid, polysaccharides are hydrolyzed and dehydrated to form sugar aldehyde derivatives, and sugar aldehyde derivatives can form orange yellow compounds with phenol. By comparing the color depth, and taking glucose as a control, a standard curve was prepared to determine the mycelium polysaccharide content, and the specific operation steps are as follows:

[0087] ① Sample treatment: take the dried mycelium, grind and crush, pass through a 60-mesh sieve, accurately weigh 50 mg into a stoppered test tube, add 1 mol / L NaOH solution 2.5 mL, place in 80°C water for ultrasonic extraction for 50 min, filter, accurately pipette 1 mL of the filtrate into a 50 mL volumetric flask, add water to constant volume, and obtain the sample solution.

[0088] ② Preparation of control: dry the glucose in a 105°C drying incubator until the weight is constant, accurately weigh 50 mg into a 50 mL volumetric flask, dissolve in water and constant volume, shake well. Accurately pipette 5 mL into a 50 mL volumetric flask, add water to constant volume, shake well, and obtain a 100 μg / mL control solution. -1

[0089] ③ Determination: accurately measure 0.0 mL, 0.1 mL, 0.2 mL, 0.4 mL, 0.6 mL, 0.8 mL, and 1.0 mL of the control solution, respectively, and place them in stoppered test tubes, add water to constant volume to 1.0 mL, at the same time, measure 1.0 mL of the sample solution, respectively, add 1.0 mL of 5% phenol solution, shake well, then add 5 mL of concentrated sulfuric acid, shake well, place in boiling water for 20 min, take out and place in a cold water bath to cool to room temperature, measure the absorbance at 490 nm, take the absorbance as the vertical coordinate and the sugar content as the horizontal coordinate (μg), obtain the regression equation, and calculate the polysaccharide content of the sample. The formula is as follows: ​In the formula, m1 represents the sugar content in the sample solution obtained from the standard curve, in micrograms (ug); v1 represents the final volume of the sample, in milliliters (mL); m2 represents the sample mass, in grams (g); v2 represents the volume of the sample solution transferred during colorimetric determination, in milliliters (mL); and 0.9 represents the correction factor for converting glucose to dextran.

[0090] Result: As Figures 5-6 As shown. Using a spectrophotometer, the absorbance values ​​of different glucose concentrations were measured at a wavelength of 490 nm, resulting in a standard curve with a slope of 0.00826 and an intercept of 0.01235, with a linear range of 0-100 μg and an R² of 0.993, indicating reliable experimental results. The sample absorbance values ​​were substituted into the standard curve to calculate the polysaccharide concentration, ultimately yielding the polysaccharide content in the samples. Calculations revealed that the polysaccharide content in Poria cocos mycelium initially increased and then decreased with increasing turpentine oil concentration, and the polysaccharide content was consistently higher than that in Poria cocos mycelium without turpentine oil stimulation. The highest polysaccharide content was observed when the turpentine oil concentration was 200-300 mg / L. The polysaccharide content in Pleurotus ostreatus also initially increased and then decreased with increasing turpentine oil concentration, reaching its highest level at 200 mg / L, subsequently decreasing with further increases in turpentine oil concentration. This result is basically consistent with the mycelial growth rate and growth status, indicating that treatment with a certain concentration of turpentine oil can promote the production of polysaccharides from Poria cocos and Pleurotus ostreatus.

[0091] Example 3. Transcriptome analysis of Poria cocos mycelia after turpentine oil treatment

[0092] (1) Transcriptome sequencing analysis: According to the growth and metabolism of Poria cocos 5.78 and Poria cocos 28 mycelium under the treatment of different concentrations of turpentine, Poria cocos 5.78 was selected as the experimental material, and the Poria cocos mycelium cultured with the addition of turpentine at a concentration of 300 mg / L and 500 mg / L was selected as the treatment group, denoted as Wc5.78-300 and Wc5.78-500. The Poria cocos mycelium cultured without the addition of turpentine was used as the control group, denoted as Wc5.78-0. The Poria cocos 5.78 mycelium was inoculated on PDA medium with turpentine at a concentration of 0 mg / L, 300 mg / L and 500 mg / L, and placed in a constant temperature incubator at 28°C for 7 days. The mycelium was scraped and placed in a 2 mL centrifuge tube, labeled, and quickly frozen in liquid nitrogen and stored in a -80°C ultra-low temperature freezer. Then the treated samples were subjected to transcriptome sequencing, and the sequencing platform was illumina NovaSeq 6000. The raw sequence data (reads) obtained were filtered to remove reads with adapters, low quality, and containing undetermined base information to ensure the quality and reliability of the data analysis. Then StringTie (1.3.3b) was used to splice the complete transcriptome. At the same time, to calculate the expression of genes, featureCounts (1.5.0-p3) was used to calculate the number of reads mapped to each gene. According to the number of reads, length and sequencing depth of each gene, the FPKM value of each gene was calculated. The formula for FPKM is: , where C represents the number of reads of the gene, N represents the total number of reads, and L represents the length of the gene.

[0093] Results: After sequencing, low-quality reads were filtered out, and the average output of the samples was 6.86 GB of data. The total number of assembled genes was 14333, of which 12359 were annotated genes, accounting for 86.22% of the total. Among the 7927 unknown protein genes in the protein-coding genes, 4432 were known proteins, accounting for 35.86% of the protein-coding genes. The Q20 (error rate <1%) ratio of the obtained clean reads was about 98.12%, and the Q30 (error rate <0.1%) ratio was about 94.76%, indicating that the sequencing base recognition was reliable, the sequencing quality was good, and the accuracy was high. The sequencing data is shown in Table 2. From the various indicators, the requirements for subsequent transcriptome analysis were met.

[0094] Table 2. Transcriptome sequencing data evaluation table

[0095] sample Total Clean Reads Total Clean Bases Q20(%) Q30(%) GC Content(%) Wc5.78-0-1 43769244 6.57G 98.04 94.55 58.12 Wc5.78-0-2 42920482 6.44G 98.06 94.65 58.06 Wc5.78-0-3 40278064 6.04G 97.25 93.15 57.98 Wc5.78-300-1 47282416 7.09G 98.34 95.16 58.17 Wc5.78-300-2 48245858 7.24G 98.22 94.87 56.69 Wc5.78-300-3 51729628 7.76G 98.1 94.78 58.2 Wc5.78-500-1 45258060 6.79G 98.36 95.21 58.01 Wc5.78-500-2 44093974 6.61G 98.45 95.42 58.22 Wc5.78-500-3 47820972 7.17G 98.3 95.1 58.53

[0096] (2) Correlation analysis between samples:

[0097] To obtain more comprehensive results of gene expression changes of P. fraxinea 5.78 under different concentrations of turpentine treatment, and ensure the accuracy of experimental results, three biological replicates were set for each treatment sample. The Pearson correlation coefficient (R 2 ) was used to analyze the correlation of gene expression between samples. The correlation reflects the similarity of gene expression patterns between samples. The closer the correlation coefficient is to 1, the higher the correlation is. The Encode program suggests that the Pearson correlation coefficient (R 2 ) should be greater than 0.92 under ideal sampling and experimental conditions. However, in specific project operations, the R 2 between biological replicates is generally required to be greater than 0.88. As shown in Figure 7 , the correlation coefficient R 2 between each group of samples is greater than 0.9, indicating that the sample selection of different treatment groups is reasonable, and each sample has high reliability and repeatability.

[0098] Example 4. Screening and analysis of differentially expressed genes of P. fraxinea mycelium after turpentine treatment

[0099] To find the gene set whose expression level changes under 0 mg / L, 300 mg / L, and 500 mg / L turpentine treatment, i.e., the differentially expressed genes, and to use them as the basis for subsequent analysis, DESeq2 software was used to analyze the differential expression between two comparison groups. The Benjamini and Hochberg method was used to adjust the P value (padj) to control the false discovery rate. The threshold for significant differential expression was set as padj≤0.05 and |log2FoldChange|≥1. As shown in Figure 8 , compared the gene expression levels of P. fraxinea mycelium treated with 300 mg / L and 0 mg / L turpentine, respectively, there were 319 differentially expressed genes, of which 134 were up-regulated and 185 were down-regulated. Compared the gene expression levels of P. fraxinea mycelium treated with 500 mg / L and 300 mg / L turpentine, respectively, there were 476 differentially expressed genes, of which 197 were up-regulated and 279 were down-regulated. Compared the gene expression levels of P. fraxinea mycelium treated with 500 mg / L and 0 mg / L turpentine, respectively, there were 186 differentially expressed genes, of which 94 were up-regulated and 92 were down-regulated.

[0100] The differentially expressed genes of P. fraxinea mycelium treated with three different concentrations of turpentine were compared pairwise, and the Venn diagram was drawn. As shown in Figure 9 , among the 319 differentially expressed genes of P. fraxinea mycelium treated with 300 mg / L and 0 mg / L turpentine, 134 were up-regulated and 185 were down-regulated. Among the 476 differentially expressed genes of P. fraxinea mycelium treated with 500 mg / L and 300 mg / L turpentine, 197 were up-regulated and 279 were down-regulated. Among the 186 differentially expressed genes of P. fraxinea mycelium treated with 500 mg / L and 0 mg / L turpentine, 94 were up-regulated and 92 were down-regulated. Figure 9A, 13 overlapping genes were screened from the up-regulated genes in A compared with the genes in 0 mg / L turpentine treatment and the up-regulated genes in A compared with the genes in 300 mg / L turpentine treatment. The expression of these 13 genes gradually increased with the increase of turpentine concentration. These 13 genes were set as combination I. Similarly, in Figure 9 B, 12 overlapping genes were set as combination II from the up-regulated genes in B compared with the genes in 0 mg / L turpentine treatment and the down-regulated genes in B compared with the genes in 300 mg / L turpentine treatment. The expression of these 12 genes increased first and then decreased with the increase of turpentine concentration, and the expression was the highest at the concentration of 300 mg / L. Figure 9 The expression of the 2 overlapping genes in C gradually decreased with the increase of turpentine concentration, and they were set as combination III. Figure 9 The expression of the 18 overlapping genes in D decreased first and then increased with the increase of turpentine concentration, and the expression was the lowest at the concentration of 300 mg / L. They were set as combination IV. Figure 9 A- Figure 9 The expression trend of the genes in D that were not repeated was not determined. For example Figure 9 The 121 genes on the left side of A could only be determined to be up-regulated at the turpentine concentration of 0-300 mg / L, and not up-regulated at the concentration of 300-500 mg / L. Therefore, the expression trend at the concentration of 300-500 mg / L may be down-regulated or unchanged. Therefore, the genes in this set were subjected to a Wayne diagram again with combination II, and the results are shown in Figure 9 E, and combination V was set. The expression of the genes in combination V increased first and then remained unchanged with the increase of turpentine concentration. Similarly, as shown in Figure 9 F, 165 genes were set as combination VI, and the expression of the genes gradually decreased with the increase of turpentine concentration. Figure 9 G, 61 genes were set as combination VII, and the expression of the genes gradually increased with the increase of turpentine concentration. Figure 9 H, 80 genes were set as combination VIII, and the expression of the genes gradually decreased and then remained unchanged with the increase of turpentine concentration.

[0101] Example 5. Cluster analysis of P. fraxinus mycelium differential expression genes

[0102] The P. fraxinus mycelium differential expression genes screened in Example 4 were subjected to cluster analysis, and the genes with the same or similar expression trendFigure 9 Cluster analysis was performed on gene sets I, II, III, and IV, and the results are as follows: Figure 10 As shown, combinations I, II, III, and IV are... Figure 10 A, B, C, and D correspond one-to-one. Figure 10 In group A, the expression levels of 13 genes gradually increased with increasing turpentine oil concentration, with significant differences between groups. These 13 genes include 6 cytochrome P450 genes, one 2OG-Fe(II) oxygenase superfamily protein, both possessing oxidoreductase and catalytic activities; 2 carbohydrate family proteins, including one glycoside hydrolase 12 family protein involved in organic matter decomposition and metabolism, possessing catalytic and hydrolytic activities; one carbohydrate esterase family 15 protein; one CP protein; one transport protein; one fungal pheromone precursor; and one fungal hydrophobic protein. Figure 10 In group B, there are 12 genes in total. Their expression levels first increase and then decrease with the increase of turpentine oil concentration, and the expression level is the highest at a concentration of 300 mg / L. These 12 genes include 9 hypothetical proteins with unknown functions, one mitochondrial carrier protein, tRNA-leucine protein, and GMC oxidoreductase. Among them, GMC oxidoreductase has oxidoreductase activity, can bind to cofactors and coenzymes, and participate in carbon metabolism and methane metabolism. Figure 10 The expression levels of two genes in C decreased with increasing turpentine concentration, including a fungal fruiting body lectin 1a and a hypothetical protein with unknown function. Figure 10 The D gene comprises 18 genes. Its expression level initially decreases and then increases with increasing turpentine oil concentration, reaching its lowest level at a concentration of 300 mg / L. It includes one cytochrome P450 protein, one glycoside hydrolase family 16 protein, two ACC oxidases, one zinc finger protein, one peptidase inhibitor protein with serine-type endopeptidase inhibitor activity (acting as a molecular function regulator), one MFS protein involved in transmembrane transport, one NAD-binding domain protein involved in redox and metabolic processes (capable of binding to NAD, small molecules, cofactors, and coenzymes), and one peptidyl prolyl cis-trans isomerase involved in protein folding and cellular processes.

[0103] Further Figure 9 Cluster analysis was performed on gene sets V, VI, VII, and VIII in the dataset, and the results are as follows: Figure 11 As shown, combinations V, VI, VII, and VIII correspond one-to-one with A, B, C, and D. Group V contains 109 genes, of which 57 have unknown functions. The functions of the remaining 52 genes are known. Their gene expression levels initially increase with increasing turpentine oil concentration and then remain constant. Specifically, in the 0-300 mg / L concentration range, gene expression levels increase significantly with increasing concentration, and then in the 300-500 mg / L concentration range, expression levels show no significant change with increasing concentration.Figure 11 A. Among the 52 genes, there are 11 FAD / NAD(P)-binding related proteins, 8 carbohydrate family proteins, 8 cytochrome P450s, 3 transport related proteins, 2 MAPK or AMPK signaling pathway proteins, 2 sterol synthesis related proteins, 2 heat shock proteins, 1 peptidase, 1 amino acid metabolism related protein, and 1 CP protein, etc. There are 165 genes in group VI, among which 63 genes are hypothetical proteins with unknown functions, and 102 genes are known functional proteins. Their expression levels first decreased and then remained unchanged with the increase of turpentine concentration. That is, in the 0-300 mg / L concentration interval, the gene expression levels significantly decreased with the increase of turpentine concentration, and then in the 300-500 mg / L concentration interval, the expression levels did not change significantly with the increase of concentration, as shown in Figure 11 B. Among the 102 genes, there are 25 domain proteins, 10 oxidation-reduction related proteins, 9 hydrolases, 8 cytochrome P450s, 8 carbohydrate family proteins, 7 carbon metabolism related proteins, 6 FAD / NAD(P)-binding related proteins, 4 terpenoid synthesis related proteins, and 4 transport proteins, etc. There are 61 genes in group VII, among which 20 genes are hypothetical proteins with unknown functions, and 41 genes are known functional proteins. Their expression levels first remained unchanged and then increased with the increase of turpentine concentration. That is, in the 0-300 mg / L concentration interval, the gene expression levels did not change significantly with the increase of turpentine concentration, and then in the 300-500 mg / L concentration interval, the expression levels significantly increased with the increase of concentration, as shown in Figure 11 C. Among the 41 genes, there are 7 carbohydrate family proteins, 5 transport related proteins, 5 FAD / NAD(P)-binding related proteins, 4 cytochrome P450s, 4 hydrolase proteins, 4 transcription related proteins, 3 domain proteins, and 1 peptidase, etc. There are 80 genes in group VIII, among which 45 genes are hypothetical proteins with unknown functions, and 35 genes are known functional proteins. Their expression levels first remained unchanged and then decreased with the increase of turpentine concentration. That is, in the 0-300 mg / L concentration interval, the gene expression levels did not change significantly with the increase of turpentine concentration, and then in the 300-500 mg / L concentration interval, the expression levels significantly decreased with the increase of concentration, as shown in Figure 11 D. Among the 35 genes, there are 8 hydrolase proteins, 8 domain proteins, 4 cytochrome P450s, 3 FAD / NAD(P)-binding related proteins, 1 aquaporin, 1 peptidase, 1 terpenoid synthesis related protein, and 1 oxidative phosphorylation related protein, etc.

[0104] Example 6. Functional annotation and enrichment analysis of differentially expressed genes of P. fraxinus mycelium

[0105] The clusterProfile software was used to perform functional enrichment analysis and metabolic pathway analysis on the transcriptome data. The GO database was mainly used for functional analysis, which classified genes according to their essential functions into three categories: biological process, cellular component, and molecular function, thereby limiting and describing the functions of genes and proteins. A padj less than 0.05 was generally used as the threshold for significant enrichment. The KEGG database was used to analyze the metabolic pathways of genes, and some metabolic pathways in which the corresponding genes were involved were found. Then, the differential genes of all comparison combinations were used to make a volcano plot using the microbio.me website, and a Wayne plot and a heat map were made using TBtools to screen differential genes common or unique to the comparison combinations.

[0106] The results are as follows:

[0107] (1) GO functional enrichment analysis of Poria cocos mycelium differential expression genes: GO is a comprehensive database for describing gene functions, which can be divided into three parts: biological process, cellular component, and molecular function. GO functional enrichment analysis was performed on the differential expression genes of Wc5.78-300 vs. Wc5.78-0, which were classified into 307 functional entries in biological process, 65 functional entries in cellular component, and 196 functional entries in molecular function. The top ten entries were taken and made into bubble charts, as shown in Figure 12 . In biological process, the most annotated genes were related to organic nitrogen compound biosynthesis process, followed by peptide biosynthesis process, peptide metabolic process, translation process, amide biosynthesis process, and cellular amide metabolic process. In cellular component classification, more genes were annotated to ribosome, cytoplasm, and organelle. In molecular function, the most annotated genes were cofactor binding, followed by iron ion binding, heme binding, tetrapyrrole binding, and coenzyme binding. Similarly, GO functional enrichment analysis was performed on the differential expression genes of Wc5.78-300 vs. Wc5.78-500, which were classified into 265 functional entries in biological process, 40 functional entries in cellular component, and 179 functional entries in molecular function. The top ten entries were taken, as shown in Figure 13 . In biological process, the most annotated genes were related to carbohydrate metabolic process. In cellular component classification, the most annotated genes were related to membrane. In molecular function, the most annotated genes were cofactor binding, followed by metal ion binding. GO functional enrichment analysis was performed on the differential expression genes of Wc5.78-500 vs. Wc5.78-0, which were classified into 293 functional entries in biological process, 69 functional entries in cellular component, and 205 functional entries in molecular function. The top ten entries were taken, as shown in Figure 14The most annotated genes in biological process are related to transmembrane transport, organic nitrogen compound biosynthesis process, followed by peptide biosynthesis, metabolic process, translation process, amide biosynthesis process and its metabolic process. In the classification of cell components, the most annotated is cytoplasm, followed by organelle. In the molecular function, the most annotated is cofactor binding, followed by iron ion binding, heme binding, tetrapyrrole binding, etc. According to these entries, turpentine treatment can significantly affect the organic nitrogen biosynthesis process and peptide biosynthesis process of Poria cocos, as well as the physiological and biochemical processes such as cofactor and iron ion binding, thereby affecting the subsequent sclerotium development.

[0108] (2) KEGG metabolic pathway analysis of Poria cocos mycelium differential expression genes: using KEGG database, the differential expression genes obtained from Poria cocos mycelium treated with turpentine were subjected to metabolic pathway analysis, so as to further understand their biological functions, and systematically analyze the metabolic pathways and complex biological behaviors in cells. The results are shown in Figures 15-17 As shown in the table, the differential expression genes of Wc5.78-300 VS Wc5.78-0 were subjected to KEGG metabolic pathway analysis, and a total of 284 metabolic pathways were annotated. The top 20 classification entries were taken, and the metabolic pathways with more annotations included ribosome, carbon metabolism, amino acid biosynthesis, etc., which were annotated to 64, 53, and 47 genes, respectively, as shown in Figure 15 The differential expression genes of Wc5.78-300 VS Wc5.78-500 were subjected to KEGG metabolic pathway analysis, and a total of 250 metabolic pathways were annotated. The top 20 classification entries were taken, and the metabolic pathways with more annotations were carbon metabolism, amino acid biosynthesis process, etc., which were annotated to 28 and 23 genes, respectively, as shown in Figure 16 The differential expression genes of Wc5.78-500 VS Wc5.78-0 were subjected to KEGG metabolic pathway analysis, and a total of 306 metabolic pathways were annotated. The top 20 classification entries were taken, and the metabolic pathways with more annotations were ribosome, carbon metabolism, endoplasmic reticulum protein processing process, etc., which were annotated to 77, 41, and 32, respectively, as shown in Figure 17 These metabolic pathways provide important references for further exploring the mechanism of turpentine affecting Poria cocos mycelium and sclerotium.

[0109] Example 7. Screening and analysis of sclerotium formation and development related genes

[0110] By collating the transcriptome data, the present application divides the differential expression genes into 8 groups according to the change trend of the expression amount of each gene with the turpentine concentration, analyzes the gene composition, each gene function, metabolic pathway, etc. in each group. The sclerotium of the fungus is a dormant structure formed by the aggregation of the vegetative mycelium under adverse conditions, and it is speculated that the turpentine promotes the formation of the sclerotium by affecting the mycelium, so the genes related to the development of the Poria cocos sclerotium are found from the differential expression genes of the Poria cocos mycelium under the treatment of the turpentine through the references, so as to lay a foundation for speculating the regulation mechanism of the sclerotium formation.

[0111] (1) AMPK signal pathway related genes: the formation of the sclerotium is regulated by the AMPK signal pathway. When the content of ADP and AMP in the cell is relatively increased, the adenylyl cyclase synthesizes cAMP, starts the AMPK signal pathway, and coordinates growth and metabolism. The GTP-binding protein ypt2 (gene-WOLCODRAFT_111188) is mainly involved in the signal transduction mediated by small GTPases and is in the AMPK signal pathway, and its expression amount shows a trend of first decreasing and then being unchanged with the increase of the turpentine concentration, and the expression amount is reduced at 300 mg / L, which may lead to the decrease of the expression level of cAMP in the Poria cocos, and the formation of the sclerotium. The glycosyltransferase family 3 protein (gene-WOLCODRAFT_27407) is involved in the metabolism of sucrose and starch, and its expression amount shows a trend of first decreasing and then being unchanged with the increase of the turpentine concentration; the Δ9-fatty acid desaturase protein (gene-WOLCODRAFT_23420) is a binding protein on the endoplasmic reticulum, and its expression amount is the highest at 300 mg / L, and they are all in the AMPK signal pathway, and it is guessed that they change the cell membrane permeability by participating in the primary metabolism, and affect the formation of the sclerotium.

[0112] (2) Protein phosphorylation related genes: protein phosphorylation is a key step for the formation of the sclerotium, and the protein phosphatase (PPs) promotes dephosphorylation by removing the phosphate groups from the phosphorylated Ser, Thr or Tyr residues, and regulates the cell cycle, metabolism and signal transduction. The gene PP2C (gene-WOLCODRAFT_142161, gene-WOLCODRAFT_86489) has the highest expression amount at 300 mg / L of the turpentine concentration; the serine / threonine-protein kinase B-raf (gene-WOLCODRAFT_164050) is a phosphotransferase that catalyzes the transfer of the phosphate on ATP to the hydroxyl group of the serine / threonine residue, and its expression amount shows a trend of first being unchanged and then increasing with the increase of the turpentine concentration, and it is speculated that the turpentine can induce the protein phosphorylation related genes to regulate the development of the sclerotium.

[0113] (3) MAPK signaling pathway-related genes: cAMP-mediated sclerotium inhibition is also regulated through the MAPK pathway. cAMP activates Rap-1, leading to the inactivation of downstream MAPK and inhibiting sclerotium development. In this experiment, two MAPK signaling pathway genes were identified: the 14-3-3 protein (gene-WOLCODRAFT_69384), whose expression level decreased with increasing turpentine oil concentration; and the G-α domain-containing protein involved in signal transduction (gene-WOLCODRAFT_139231), whose expression level increased with increasing turpentine oil concentration. It is speculated that they promote MAPK by changing their own expression levels, thereby promoting sclerotium development.

[0114] (4) Carbohydrate-active enzyme (CAZymes) family genes: CAZymes play an important role in the degradation of plant cell walls, providing carbohydrates for the growth, development, and reproduction of fungi. Unfavorable environmental stimuli make it difficult for fungi to obtain the external nutrients they need for growth. When the mycelium can no longer sustain growth, sclerotia begin to form. In other words, the formation of Poria cocos sclerotia is closely related to changes in nutrients, accompanied by changes in a large number of carbohydrate-active enzymes. Analysis of the carbohydrate-active enzyme genes in the transcriptome of mycelium treated with turpentine oil in this experiment revealed that the expression levels of 28 CAZymes changed significantly under turpentine oil treatment, such as... Figure 18 As shown, there are 17 GHs, including GH5, GH16, and GH28, consistent with the literature. There are also 5 CEs, 2 CBMs, and 4 GTs. In summary, the overall expression level of CAZymes is relatively low under turpentine stimulation, leading to insufficient nutrients for the hyphae. Only the expression levels of some genes increase to support sclerotium formation and growth, suggesting that these genes are related to sclerotium formation.

[0115] (5) Polysaccharide synthesis-related genes: Pachyman is the main active ingredient in sclerotia of P. lucidum, and the content of polysaccharide affects the medicinal value of sclerotia of P. lucidum. Therefore, it is speculated that the genes related to the synthesis of polysaccharide in P. lucidum mycelium may affect the formation or growth and development of sclerotia of P. lucidum by changing the expression level. In the transcriptome data of this experiment, 7 genes related to this were found, including one galactan 1, 4-alpha-galacturonidase A (gene-WOLCODRAFT_177549) involved in the mutual transformation of pentose and glucuronide, starch and sucrose metabolism; one UTP-glucose-1-phosphate uridylyltransferase (gene-WOLCODRAFT_90597), whose expression level showed a trend of first increasing and then remaining unchanged with the increase of turpentine concentration, and they were involved in the synthesis of sclerotia polysaccharide under the stimulation of appropriate concentration of turpentine, and were speculated to be related to the formation and development of sclerotia, and involved in the nutrition utilization and biosynthesis of sclerotia. Among the polysaccharide synthesis-related genes, there were also three glycoside hydrolase family 16 proteins (gene-WOLCODRAFT_22205, gene-WOLCODRAFT_160308, gene-WOLCODRAFT_136471) containing KRE6 domain, and two phosphoglucomutases (gene-WOLCODRAFT_28782, gene-WOLCODRAFT_130815), but their expression levels showed no significant change under the stimulation of different turpentines. These genes were involved in basic metabolism and were not affected by turpentine, and were involved in the synthesis of sclerotia polysaccharide of P. lucidum mycelium. Their expression level heat map is shown in Figure 19 .

[0116] (6) Triterpenoid synthesis-related genes: Triterpenoids are also important active ingredients in sclerotium. Triterpenoids are different variants of lanosterol skeleton, which are catalyzed and modified by cytochrome P450, glycosyltransferase and acyltransferase. In the transcriptome data, the present application found 3 genes related to triterpenoid biosynthesis in sclerotium that were significantly changed in expression under the stimulation of different concentrations of turpentine, namely ergosterol biosynthesis protein 6 (gene-WOLCODRAFT_145787), methylglutaryl coenzyme A hydrase (gene-WOLCODRAFT_103953), the expression of which showed a trend of first significant increase and then no significant change with the increase of turpentine concentration, indicating that turpentine would promote the synthesis of triterpenoid precursors at a certain concentration; triterpenoid synthase (gene-WOLCODRAFT_137597) is involved in isoprenoid biosynthesis and metabolic process, lipid metabolism process, and participates in terpenoid skeleton biosynthesis. The expression showed a trend of first no significant change, and then significant decrease in expression with the increase of turpentine concentration, indicating that too high concentration of turpentine would inhibit the biosynthesis of triterpenoid skeleton. In summary, 300 mg / L concentration of turpentine can promote the synthesis of triterpenoids in sclerotium. Cytochrome P450 genes are essential for the formation of cyclic triterpenoid skeleton. The present application predicts cytochrome P450 under the treatment of turpentine concentration, and finds 152 cytochrome P450s, of which 45 show significant changes in expression under the stimulation of different concentrations of turpentine, and 12 genes are co-expressed with triterpenoid synthesis-related genes, indicating that cytochrome P450 genes are related to the synthesis of triterpenoids in sclerotium, and may play a role in the synthesis or modification of triterpenoids. Under the stimulation of 300 mg / L of turpentine, the synthesis of triterpenoids in sclerotium is promoted, and the heat map of the expression of each gene is shown in Figure 20 .

[0117] (7) Other genes: In addition to cAMP-dependent signaling pathway, protein phosphorylation-related genes, MAPK signaling pathway, CAZymes, polysaccharide substances and triterpenoid compound synthesis-related genes involved in sclerotial development, many genes have been proven to be related to sclerotial growth and development. SsSte12 is a direct homolog of Ste12 C2H2-type zinc finger transcription factor. After silencing, abnormal sclerotial development occurs in S. sclerotium, and smaller sclerotia are formed, indicating that Ste12 C2H2-type zinc finger transcription factor is involved in the growth and development of S. sclerotium sclerotia. The deletion of Bcser2 gene in subtilisin affects the formation of sclerotia of Botrytis cinerea, indicating that subtilisin is involved in the formation of sclerotia of Botrytis cinerea. In this experiment, a C2H2-type zinc finger (gene-WOLCODRAFT_50037) and a subtilisin-like protein (gene-WOLCODRAFT_27933) were screened from the transcriptome data. Their expression levels increased first and then remained unchanged with the increase of turpentine concentration, indicating that turpentine can promote their expression and thus promote the growth and development of sclerotia. By analyzing the transcriptome data of the internal and external sclerotia of Grifola frondosa, it was found that the number of NAD- or FAD-related domains was also more than that of other fungi, which was suspected to contribute to faster and better sclerotial growth. In this data, multiple NAD- or FAD-related domain proteins were found, and their expression levels increased first and then increased with the increase of turpentine concentration, suggesting that turpentine treatment promotes their expression and makes the sclerotia grow faster and better. Studies have shown that a CP protein causes filamentous fungal hyphae to lag behind and sclerotia to decrease after silencing, suggesting that CP protein is involved in the growth and development of filamentous fungal hyphae and sclerotia. In this data, an expansin-like protein (gene-WOLCODRAFT_136006) was also found, and its expression level increased with the increase of turpentine concentration, suggesting that it may affect the growth and development of Grifola frondosa hyphae and sclerotia. When comparing the differentially expressed genes between hyphae and sclerotia, researchers found that the expression levels of multiple MFS transport proteins changed significantly, and they suspected that they played a key role in the transport of Grifola frondosa secondary metabolites during sclerotial formation and development, promoting sclerotial development. Under turpentine treatment, the expression levels of MFS transport proteins also increased with the increase of concentration, actively participating in the transport of secondary metabolites, such as gene-WOLCODRAFT_76753 and gene-WOLCODRAFT_138211, suggesting that they are related to sclerotial formation and development.

[0118] In summary, by screening the differentially expressed genes in the transcriptome data, we found the key genes that may be related to sclerotial formation and development, as shown in Table 3. The expression levels of the genes changed with the change of turpentine concentration, as shown in Table 4. Figure 21

[0119] Table 3. Genes involved in the formation and development of Grifola frondosa sclerotia ​

[0120] Gene-id Description GO KEGG gene-WOLCODRAFT_69384 14-3-3 protein - involved in cell cycle, MAPK pathway gene-WOLCODRAFT_111188 GTP-binding protein ypt2 small GTPase-mediated signal transduction AMPK signaling pathway gene-WOLCODRAFT_23420 Δ9-fatty acid desaturase protein - AMPK signaling pathway gene-WOLCODRAFT_139231 G-alpha domain protein signal transduction MAPK signaling pathway gene-WOLCODRAFT_27407 glycosyltransferase family 3 protein - AMPK signaling pathway, starch and sucrose metabolism, gene-WOLCODRAFT_67511 alpha-kinase family protein phosphorylation, protein serine / threonine kinase activity - gene-WOLCODRAFT_142161 protein phosphatase 2C catalytic activity - gene-WOLCODRAFT_86489 protein phosphatase 2C catalytic activity - gene-WOLCODRAFT_164050 serine / threonine-protein kinase B-raf protein phosphorylation, protein kinase activity - gene-WOLCODRAFT_177549 galactan 1,4-alpha-galacturonase A carbohydrate, organic substance metabolic process pentose and glucuronate interconversions, starch and sucrose metabolism gene-WOLCODRAFT_90597 UTP-glucose-1-phosphate uridylyltransferase transferase activity, catalytic activity biological, metabolic process - gene-WOLCODRAFT_23632 Carbohydrate esterase family 15 protein - - gene-WOLCODRAFT_24190 Glycoside hydrolase 12 family protein Organic substance catabolic process, catalytic activity, hydrolase activity - gene-WOLCODRAFT_21871 Glycoside hydrolase 16 family protein - - gene-WOLCODRAFT_81480 Carbohydrate esterase family 4 protein Carbohydrate metabolic process, catalytic activity, hydrolase activity - gene-WOLCODRAFT_79265 Glycoside hydrolase 16 family protein Carbohydrate metabolic process, hydrolase activity - gene-WOLCODRAFT_112799 Glycoside hydrolase 5 family protein Carbohydrate metabolic process, hydrolase activity, catalytic activity - gene-WOLCODRAFT_137597 Terpene synthase Isoprenoid biosynthetic and metabolic process, lipid metabolic process Terpenoid backbone biosynthesis gene-WOLCODRAFT_145787 Ergosterol biosynthesis protein 6 - Steroid biosynthesis gene-WOLCODRAFT_108759 Peptidase inhibitor Serine-type endopeptidase inhibitor activity - gene-WOLCODRAFT_24698 5-Aminolevulinic acid synthase - Glycine, serine and threonine metabolism, porphyrin and chlorophyll metabolism gene-WOLCODRAFT_50037 [C2H2-type zinc finger] Ion binding - gene-WOLCODRAFT_76753 MFS Transmembrane transport - gene-WOLCODRAFT_138211 MFS Transmembrane transport - gene-WOLCODRAFT_127992 NAD(P)-binding protein - - gene-WOLCODRAFT_27933 Subtilisin-like protein Proteolysis, peptidase activity Lysosome gene-WOLCODRAFT_81600 Fungal fruiting body agglutinin 1a - - gene-WOLCODRAFT_28038 Cytochrome P450 Oxidoreductase activity, catalytic activity, heme binding - gene-WOLCODRAFT_136006 CP - - gene-WOLCODRAFT_23456 CP - -

[0121] Example 8. Expression analysis of genes related to sclerotium formation and development in Poria cocos.

[0122] The formation and development of Poria cocos sclerotia are related to genes involved in the AMPK signaling pathway, MAPK signaling pathway, protein phosphorylation, carbohydrate-active enzymes, polysaccharide synthesis, and triterpenoid synthesis. The expression levels of these genes were analyzed using RT-qPCR.

[0123] (1) Expression analysis of genes related to the AMPK and MAPK signaling pathways: To clarify the expression changes of genes related to the AMPK and MAPK signaling pathways under different concentrations of turpentine oil treatment, five genes were screened from the differentially expressed genes in the Poria cocos mycelial transcriptome for RT-qPCR verification. The results are as follows: Figure 22 As shown, the RT-qPCR expression results and transcriptome sequencing data exhibited similar trends. Among them, the 14-3-3 protein and the G-α domain protein are genes in the MAPK signaling pathway. The expression level of the 14-3-3 protein initially decreased and then remained constant with increasing turpentine oil concentration, indicating that turpentine oil treatment reduced its expression level, thus affecting sclerotium formation and development. The G-α domain protein is involved in signal transduction, and its expression level gradually increased with increasing turpentine oil concentration, indicating that turpentine oil can promote the MAPK signaling pathway's participation in sclerotium formation. The other three are genes in the AMPK signaling pathway. The expression level of the GTP-binding protein ypt2 (gene-WOLCODRAFT_111188) initially decreased and then remained constant with increasing turpentine oil concentration, while the expression level of glycosyltransferase family 3 protein (gene-WOLCODRAFT_27407) initially decreased and then increased. The expression level of Δ9-fatty acid desaturase protein (gene-WOLCODRAFT_23420) increased with increasing turpentine oil concentration. The expression trend in RT-qPCR differed from that in the transcriptome, but both showed the lowest expression levels at a turpentine oil concentration of 0 mg / L. This indicates that turpentine oil treatment promotes its expression. The difference in expression trends may be due to experimental errors caused by differences in the measuring instruments and methods used in the two measurements. These results suggest that turpentine oil treatment can affect the expression levels of genes in the AMPK and MAPK signaling pathways, thereby influencing the formation of sclerotia in Poria cocos.

[0124] (2) Analysis of the expression amount of carbohydrate active enzyme family genes: The formation of sclerotia of Poria cocos is closely related to the change of nutrients, and carbohydrate active enzymes can provide carbohydrates for the growth, development and reproduction of fungi by degrading plant cell walls. To explore the effect of CAZymes of Poria cocos mycelium on sclerotia under the treatment of turpentine, five CAZymes genes were selected from the differentially expressed genes for RT-qPCR verification, and the results are shown in Table 2. Figure 23 As shown in Table 2, the expression amount of the remaining four genes showed an increasing trend with the increase of the concentration of turpentine, although the expression trend in RT-qPCR was not completely the same as that in the transcriptome, but the overall trend was similar, indicating that under the treatment of turpentine, it may have an adverse effect on the growth of mycelium, leading to an increase in the expression amount of CAZymes to maintain its own growth and promote the formation of sclerotia.

[0125] (3) Analysis of the expression amount of polysaccharide and triterpenoid synthesis genes: Polysaccharide and triterpenoid are the main active ingredients in Poria cocos sclerotia. To determine the expression amount change of polysaccharide and triterpenoid in Poria cocos under the treatment of turpentine, two genes were selected from the differentially expressed genes for RT-qPCR verification, and the results are shown in Table 3. Figure 24 As shown in Table 3, the expression amount of polysaccharide synthesis genes (gene-WOLCODRAFT_177549, gene-WOLCODRAFT_90597) showed an increasing trend with the increase of the concentration of turpentine, indicating that the treatment of turpentine is beneficial to the synthesis of polysaccharide substances in Poria cocos, and it is speculated that it can promote the formation of Poria cocos sclerotia, thereby affecting the medicinal value of Poria cocos sclerotia. The expression amount of triterpenoid compound synthesis genes (gene-WOLCODRAFT_103953, gene-WOLCODRAFT_137597) showed a trend of first increasing and then decreasing with the increase of the concentration of turpentine, and the expression amount was the highest at the concentration of 300 mg / L, indicating that 300 mg / L may be the best concentration for treating Poria cocos to promote the synthesis of triterpenoid substances.

[0126] (4) Analysis of the expression amount of other genes related to the development of sclerotia: In addition to the above genes, some genes related to the development of sclerotia have been proved by various literature materials, and some genes were selected from the differentially expressed genes, including MFS general substrate transporter, NAD(P) binding protein, PP2C, etc., and the results of RT-qPCR verification are shown in Table 4. Figure 25 As shown in Table 4, the expression amount of them all showed an increasing trend under the treatment of turpentine, indicating that turpentine can promote the expression of MFS general substrate transporter, PP2C and NAD(P) binding protein, thereby promoting the formation of Poria cocos sclerotia.

[0127] In summary, the present study found that the expression levels of AMPK signaling pathway-related genes, MAPK signaling pathway-related genes, protein phosphorylation-related genes, carbohydrate active enzyme family genes, and transport-related protein genes changed significantly with increasing concentrations of turpentine; and confirmed that the above genes are involved in the regulation of sclerotium formation and growth and development, with a specific regulatory network as shown in Figure 26 .

[0128] Example 9. Effect of turpentine on the development of P. lucidum sclerotia

[0129] To investigate the effect of turpentine on P. lucidum sclerotia, five bag material cultivation formulas were designed, in which pine sawdust was used as the main ingredient in the positive control group; no turpentine was added to the wood chip-based formula as the negative control group; and the wood chip formula with 0.05%, 0.1%, and 0.2% turpentine content was used as the experimental group to simulate the pine environment for cultivating P. lucidum. The P. lucidum bag material cultivation formula is shown in Table 4. The P. lucidum bag material cultivation method is as follows: ① Preparation of the fungus bag: mix the components in the formula according to the proportion, add about 60% water, and make sure that the culture medium can be held in the hand with water marks in the finger joints but no water dripping. Then, bottle, seal, and sterilize. Use high-pressure intermittent sterilization method at 121°C for 45 min, repeat the sterilization once after cooling, and complete the fungus bag production. ② Inoculation: inoculate the activated P. lucidum spores into the bottle containing formula 5 medium, place it in a constant temperature incubator at 28°C, and cultivate until the mycelium covers the entire bottle to obtain the corresponding secondary spores. Then, inoculate the obtained P. lucidum secondary spores into the fungus bags of different formulas; inoculate 10 parallel fungus bags for each formula, and cultivate at 28°C to make sure that the mycelium covers the entire fungus bag to obtain the corresponding cultivation species. The inoculation process should be carried out in a sterile environment. ③ Planting: plant the cultivated P. lucidum fungus bags, draw a 10 cm long opening on the fungus bag, place a piece of fresh P. lucidum as an inducer at the opening, and then place the fungus bags in order and cover them with soil with a soil layer thickness of about 10 cm. During this period, field management should be carried out, and the growth of P. lucidum should be observed, weeds and termites should be removed, and the growth of P. lucidum should be ensured. After P. lucidum matures, it can be harvested.

[0130] The growth of P. lucidum in the bag material is shown in Figure 27 As shown in Fig. A, the mycelium of P. lucidum in the fungus bag with 0.1% turpentine content in formula 2 is dense and white, which is basically similar to the mycelium growth in the fungus bag with pine sawdust as the main ingredient in formula 5. However, the mycelium in the fungus bag with 0.05% turpentine content in formula 1 is relatively sparse compared to the mycelium in the fungus bag with 0.2% turpentine content in formula 3, but it is whiter than the mycelium in the fungus bag without turpentine in formula 4, the negative control group. This indicates that the turpentine content in formula 1 is relatively low, and the turpentine content in formula 3 is relatively high, which cannot achieve the best growth state of P. lucidum mycelium. This growth is basically similar to the growth of P. lucidum mycelium treated with turpentine in a culture dish, and the P. lucidum in the fungus bag is not contaminated, which can be used for subsequent cultivation experiments.Figure 27 B is the field cultivation of the fungus bag. The fungus bag of Poria cocos that has grown for 6 months is dug out and no sclerotia has grown, at this time it is still in the third stage of sclerotia formation, and nutrients are being accumulated, which is a storage body tightly wrapped and adhered with nutrients. As shown in Figure 28 The degree of mycelium aggregation is higher, and the mycelium is dense and tightly combined. It is speculated that the optimal concentration of turpentine oil required for the growth of Poria cocos sclerotia is 0.1%, and the mycelium of the fungus bag without turpentine oil grows the worst, with low degree of mycelium clumping, no formation of large area aggregation, and aggregation not tight. Although the mycelium growth is normal, the possibility of aggregation to form sclerotia is smaller than that of other formulations. The mycelium growth of fungus bags with 0.05% and 0.2% turpentine oil is between pine sawdust and 0.1% turpentine oil treatment, with lower aggregation degree and tightness than pine sawdust and 0.1% turpentine oil treatment. The specific situation is shown in Table 5, wherein "*" represents the degree, and the more "*" indicates the better growth. Therefore, the formulation 2 with 0.1% turpentine oil is the most suitable for Poria cocos cultivation, followed by the formulation 1 with 0.05% turpentine oil, and finally the formulation 3 with 0.2% turpentine oil.

[0131] Table 4. Poria cocos bag cultivation formula

[0132] Formulation Wood chips Pine wood chips Cotton seed hulls Gypsum Sucrose Corn meal Turpentine 1 56% 0% 40% 1% 1% 2% 0.05% 2 56% 0% 40% 1% 1% 2% 0.1% 3 56% 0% 40% 1% 1% 2% 0.2% 4 56% 0% 40% 1% 1% 2% 0% 5 0% 56% 40% 1% 1% 2% 0%

[0133] Table 5. Statistical table of mycelium aggregation in Poria cocos field cultivation for 6 months

[0134] Formulation Growth Accumulation Probability of long out nectria Ranking 1 ***** *** *** 3 2 ***** ***** ***** 1 3 ***** *** *** 4 4 **** ** ** 5 5 ***** **** **** 2

[0135] From the growth trend in half a year, it can be seen that adding a certain concentration of turpentine oil in the cultivation of Poria cocos bag can help the formation and growth and development of Poria cocos sclerotia. It can be seen that turpentine oil is the key factor affecting the growth of Poria cocos in pine.

Claims

1. The application of a composition for Poria cocos bag cultivation in promoting the development of Poria cocos sclerotia, characterized in that, The composition comprises sawdust, turpentine oil, and excipients; the mass ratio of sawdust, turpentine oil, and excipients is 56:0.1:44; the turpentine oil promotes the development of Poria cocos sclerotia by regulating the expression of related genes in the mycelium; the related genes include genes related to the AMPK signaling pathway, genes related to the MAPK signaling pathway, genes related to protein phosphorylation, genes related to carbohydrate active enzyme family, genes related to polysaccharide synthesis, genes related to triterpenoid synthesis, and / or other genes.

2. The application according to claim 1, characterized in that, The auxiliary materials consist of cottonseed hulls, gypsum, sucrose, and corn flour; the mass ratio of cottonseed hulls, gypsum, sucrose, and corn flour is 40:1:1:

2.

3. The application according to claim 1, characterized in that, The concentration of the turpentine oil is 100 mg / L-300 mg / L.

4. The application according to claim 1, characterized in that, Application of turpentine oil in promoting the development of Poria cocos sclerotia.

5. The application according to claim 1, characterized in that, The AMPK signaling pathway-related genes include GTP-binding protein ypt2, glycosyltransferase family 3 proteins, and / or Δ9-fatty acid desaturase proteins; the MAPK signaling pathway-related genes include 14-3-3 proteins and / or G-α domain proteins; the protein phosphorylation-related genes include protein phosphatase 2C, α-kinase family proteins, and / or serine / threonine-protein kinase B-raf; the carbohydrate-active enzyme family-related genes include carbohydrate esterase family 15 proteins, carbohydrate esterase family 4 proteins, glycoside hydrolase family 5 proteins, and glycoside hydrolase family 6 proteins. The polysaccharide synthesis genes include galactomannan 1,4-α-galacturonase A and / or UTP-glucose-1-phosphate uridine transferase; the triterpenoid synthesis genes include ergosterol biosynthesis protein 6 and / or terpenoid synthase; the other genes include C2H2-type zinc fingers, subtilisin-like proteins, swelling protein-like proteins, MFS, NAD(P) binding proteins, peptidase inhibitors, 5-aminolevulinic acid synthase, fungal fruiting body lectin 1a and / or cytochrome P450.

6. The application according to claim 1, characterized in that, The turpentine oil promotes the development of Poria cocos sclerotia by regulating the expression of genes related to the AMPK and MAPK signaling pathways in Poria cocos hyphae; the turpentine oil promotes the upregulation of Δ9-fatty acid desaturase protein and / or G-α domain protein, promotes the downregulation of 14-3-3 protein and / or GTP-binding protein ypt2, and regulates the expression of glycosyltransferase family 3 protein by first decreasing and then increasing.

7. The application according to claim 1, characterized in that, The turpentine oil maintains the growth and development of Poria cocos mycelium and promotes the development of Poria cocos sclerotia by promoting the expression of genes related to carbohydrate active enzyme family, polysaccharide synthesis, triterpenoid synthesis and / or protein phosphorylation.

8. The application according to claim 1, characterized in that, The turpentine oil promotes sclerotium formation by upregulating the expression of MFS universal substrate transporter and NAD(P) binding protein, thereby affecting the transport of secondary metabolites and oxidative stress in Poria cocos hyphae.

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