Saline-alkali tolerant Stropharia rugosoannulata strain and its application

Abstract

The present invention discloses a strain of Stropharia rugosoannulata resistant to salt and alkali and its application, belonging to the technical field of edible fungi. Through screening the salt and alkali tolerance degree of hyphae and the salt and alkali tolerance cyclic domestication technology, the salt-tolerant domesticated strain and the alkali-tolerant domesticated strain are obtained; using the salt-tolerant domesticated strain and the alkali-tolerant domesticated strain as parents for protoplast fusion, and through re-screening with a salt and alkali culture medium, a strain of Stropharia rugosoannulata resistant to salt and alkali is selected and bred, which has important ecological and economic significance for the development and utilization of the reserve cultivated land resources in saline-alkali land.

Description

Technical Field

[0001] The present invention relates to the technical field of edible fungi, and particularly relates to a strain of salt-tolerant Stropharia rugosoannulata and its application. Background Art

[0002] Stropharia rugosoannulata, also known as wrinkled globe mushroom, belongs to Basidiomycota, Agaricomycetes, Agaricales, Strophariaceae, and Stropharia (Huang Nianlai, 1995). Stropharia rugosoannulata grows singly, in clusters or in groups, medium to large, and a single mushroom cluster can weigh several kilograms. It is hemispherical and then flattened. If there is water on the mushroom cap, it will be a bit sticky. The whole young mushroom is white with some small protrusions on it. As it grows, its mushroom cap will gradually turn reddish-brown, reddish-brown or dark brown, with bright colors, and turn brown or grayish-brown after maturity. During the growth process of Stropharia rugosoannulata, the mushroom cap is covered with fibrous scales, and as it grows, the fibrous scales gradually disappear. The flesh of Stropharia rugosoannulata is very thick and white (Jin Yinhui, 2020). Stropharia rugosoannulata contains various bioactive substances such as amino acids and polysaccharides, has a very good taste, and is known as the "meat among vegetables". It contains all the essential amino acids for the human body (Wang Xiaowei et al., 2007). The content of inorganic salts in Stropharia rugosoannulata is mostly higher than that of edible fungi such as Lentinula edodes, Tremella fuciformis, Auricularia auricula-judae, and Pleurotus eryngii (Wang Fengfang, 2002). Currently, 18 elements related to human health and life activities have been identified. The numerous nutritional components of Stropharia rugosoannulata are an important chemical substance basis for exerting biological activities (Yan Qingxiang et al., 2019; Wang Xiaowei et al., 2007). The research results show that the flavonoid compounds of Stropharia rugosoannulata can suppress the activity of Escherichia coli (Chen Junshen et al., 2010).

[0003] The total area of saline-alkali land in China is 99.13 million hm 2 , accounting for about 10% of the national land area (Wei Xiaobin, 2013), and is mainly distributed in the northwest, north, northeast and coastal areas (Yang Zhen et al., 2015). The saline-alkali land in China has a large area, wide distribution, and complex and diverse formation reasons (Shang Zhenfang et al., 2019). In severely saline-alkali areas, plants can hardly survive. Soil salinization is both a global ecological problem and an important factor restricting the germination of plant seeds, the growth and development of plants (Zhang Xincao et al., 2020).

[0004] At present, there are mainly two ways to develop and utilize saline-alkali land: one is to rely on engineering and technical measures to improve the soil, but the cost is huge, and it is greatly restricted by the lack of fresh water resources; the other is to cultivate salt-tolerant varieties to improve the salt tolerance of plants. This method is the main direction of agriculture and forestry in the world today, with less investment and quick results (Peng Rusheng, 2011). Therefore, identifying and selecting crop varieties suitable for growth in saline-alkali land has great ecological and economic significance.

[0005] At present, there are few studies on cultivating edible fungi in saline-alkali land. In the previous research, the inventor investigated the cultivation of Stropharia rugosoannulata in coastal saline-alkali soil and the improvement effect of its mushroom residue returning to the field on saline-alkali soil, and found that coastal saline-alkali soil with a salt content of <3‰ had no significant effect on the cultivation of Stropharia rugosoannulata; however, coastal saline-alkali soil with a salt content >3‰ significantly reduced the yield of Stropharia rugosoannulata. Therefore, it is urgent to cultivate new varieties of Stropharia rugosoannulata that can tolerate high salinity and alkalinity. Summary of the Invention

[0006] In view of the above-mentioned prior art, the object of the present invention is to provide a salt-alkali-tolerant Stropharia rugosoannulata strain and its application. The present invention obtains salt-tolerant domesticated strains and alkali-tolerant domesticated strains through screening the salt-alkali tolerance degree of mycelia and salt-alkali tolerance cyclic domestication technology; using the salt-tolerant domesticated strain and the alkali-tolerant domesticated strain as parents for protoplast fusion, and through re-screening with a saline-alkali culture medium, a salt-alkali-tolerant Stropharia rugosoannulata strain is selected, which has important ecological and economic significance for the development and utilization of reserve cultivated land resources in saline-alkali land.

[0007] Specifically, the present invention relates to the following technical solutions:

[0008] In the first aspect of the present invention, a strain of Stropharia rugosoannulata is provided, named M23. This strain was deposited on August 26, 2022 at the China General Microbiological Culture Collection Center (abbreviated as CGMCC, address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing), and its deposit number is: CGMCC No. 40286.

[0009] The Stropharia rugosoannulata M23 of the present invention has the following characteristics:

[0010] (1) Salt-alkali tolerance: The strain can normally grow and produce mushrooms under saline-alkali conditions with a salt content of 0.6% and a pH of 8.2 or above.

[0011] (2) High yield: High mushroom yield and high proportion of grade I mushrooms.

[0012] The Stropharia rugosoannulata claimed in the present invention includes the strain corresponding to CGMCC No. 40286; it also includes the offspring produced by the reproduction of the strain and having the same genetic and / or morphological traits as the strain.

[0013] In the second aspect of the present invention, there is provided the use of the Stropharia rugosoannulata with the preservation number of CGMCC No. 40286 as a parent in breeding.

[0014] In the above use, the breeding is the cultivation of salt-tolerant and alkali-tolerant varieties of Stropharia rugosoannulata.

[0015] In the third aspect of the present invention, there is provided the mycelium or spores obtained by culturing the Stropharia rugosoannulata with the preservation number of CGMCC No. 40286.

[0016] In the fourth aspect of the present invention, there is provided the fruiting body obtained by cultivating the Stropharia rugosoannulata with the preservation number of CGMCC No. 40286.

[0017] In the fifth aspect of the present invention, there is provided the use of any one of the following genes (1)-(10) in regulating the salt-tolerant and alkali-tolerant properties of the Stropharia rugosoannulata with the preservation number of CGMCC No. 40286:

[0018] (1) The gene ID is Cluster-678.1430;

[0019] (2) The gene ID is Cluster-678.2416;

[0020] (3) The gene ID is Cluster-678.2674;

[0021] (4) The gene ID is Cluster-678.3571;

[0022] (5) The gene ID is Cluster-678.5957;

[0023] (6) The gene ID is Cluster-678.617;

[0024] (7) The gene ID is Cluster-678.7799;

[0025] (8) The gene ID is Cluster-678.1586;

[0026] (9) The gene ID is Cluster-678.3333;

[0027] (10) The gene ID is Cluster-678.5714.

[0028] Advantages of the present invention:

[0029] The Stropharia rugosoannulata M23 of the present invention has excellent salt and alkali tolerance performance and can normally produce mushrooms under high salt and high alkali conditions; moreover, the proportion of first-class mushrooms is significantly higher than that of the prior art, that is, the quality of the Stropharia rugosoannulata produced under saline-alkali conditions is better than that of the existing Stropharia rugosoannulata varieties. It has important significance for the development and utilization of saline-alkali land and the improvement of the planting quality of Stropharia rugosoannulata. Description of the drawings

[0030] Figure 1 : Antioxidant enzyme activities in the mycelia of salt-alkali tolerant strains and control strains under salt-alkali mixed stress.

[0031] Figure 2 : Trehalose content in the mycelia of salt-alkali tolerant strains and control strains under salt-alkali stress.

[0032] Figure 3 : Statistics of the number of differentially expressed genes.

[0033] Figure 4 : Venn diagram of the number of differentially expressed genes among different comparison groups of Stropharia rugosoannulata.

[0034] Figure 5 : GO enrichment statistics of differentially expressed genes in the transcriptome of Stropharia rugosoannulata; A: M23_H vs. M23; B: SM_H vs. SM.

[0035] Figure 6 : KEGG metabolic pathway enrichment statistics of differentially expressed genes in the transcriptome of Stropharia rugosoannulata; A: M23_H vs. M23; B: SM_H vs. SM.

[0036] Figure 7 : Verification of transcriptome data and qRT-PCR data; A: M23_H vs. M23; B: SM_H vs. SM. Detailed implementation manners

[0037] It should be noted that the following detailed description is illustrative and is intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the technical field to which the present application belongs.

[0038] Term description:

[0039] Fruiting body: It is the spore-bearing structure of higher fungi, that is, the fruit body, which is composed of organized mycelium.

[0040] Hypha: A single tubular filament, which is the structural unit of most fungi.

[0041] Mycelium: Many hyphae gather together to form the vegetative body of fungi, that is, the mycelium.

[0042] In order to enable those skilled in the art to more clearly understand the technical solutions of this application, the technical solutions of this application will be described in detail below in conjunction with specific embodiments. If the specific test conditions are not specified in the embodiments, they are usually in accordance with conventional conditions or the conditions recommended by reagent companies; the reagents, consumables, etc. used in the following embodiments can be obtained through commercial channels without special instructions.

[0043] Example 1: Breeding of salt-tolerant Stropharia rugoso-annulata strains

[0044] 1. Test strains:

[0045] Twelve Stropharia rugoso-annulata strains collected in the laboratory were used as test strains, and the specific information is shown in Table 1.

[0046] Table 1: Test strains

[0047]

[0048] 2. Salt tolerance screening and domestication:

[0049] (1) Salt tolerance screening:

[0050] Twelve Stropharia rugoso-annulata strains were subjected to salt tolerance screening tests. The NaCl gradients were set as: 0.3%, 0.5%, 0.7%, 0.9%, 1%. The salt-containing medium was prepared by adding 5 NaCl gradients to the PDA medium in turn, as shown in Table 2. After the medium was sterilized, it was poured into a 9 cm diameter plate. After cooling and solidifying, 12 discs (diameter 4 mm) of the same age at the edge of the colonies cultured for 15 days were inoculated, with 3 replicates for each treatment; after culturing in the dark at 26 °C for 12 days, the colony diameter was measured and recorded by the cross method with a ruler, the hyphal growth rate was calculated, and the colony color and growth trend were observed. Strains with a hyphal growth rate not lower than 2 mm / d were selected and inoculated into a medium with a higher salt gradient, and so on. Finally, salt-tolerant alternative strains (D2, L4, R10, S20, XW) were selected.

[0051] Note: 2 mm / d is a reference value that is 1 / 3 slower than the growth rate of the hyphae of ordinary Stropharia rugoso-annulata.

[0052] Table 2: Composition of media with different salt contents (1% = 1 g / 100 mL)

[0053]

[0054] Note: 1% NaCl means adding 1 g of NaCl to 100 mL of PDA.

[0055] (2) Salt tolerance cyclic domestication:

[0056] The selected salt-tolerant candidate strains (D2, L4, R10, S20, XW) were subjected to cyclic domestication at the highest salt concentration for 5 times. They were inoculated onto the PDA medium containing 1% NaCl, and each treatment was repeated 3 times. After culturing for 12 d, the mycelial growth rate was measured and calculated, and the colony color and growth trend were observed. Then, they were inoculated onto the PDA medium without salt and cultured for 7 d to recover the mycelia, and this cycle was repeated 5 times.

[0057] Finally, strain L4 that could tolerate a certain concentration of NaCl was domesticated as the salt-tolerant domesticated strain of Stropharia rugosoannulata, and it was re-numbered as YL4.

[0058] 3. Alkaline tolerance screening and domestication:

[0059] (1) Alkaline tolerance screening:

[0060] The alkaline tolerance screening test was carried out on 12 strains of Stropharia rugosoannulata. The pH gradient was set as: 7.5, 8.5, 9.5, 10.5, 11.5. After the medium was sterilized and through the pH adjustment pre-test, sterile 1 mol·L -1 NaOH was added in the sterile operation mode in the ultra-clean workbench. The pH of the PDA medium was adjusted to 7.5, 8.5, 9.5, 10.5, 11.5 in turn, as shown in Table 3. Pour the plates, and after cooling and solidifying, inoculate the mycelial cakes (diameter 4 mm) of the 12 strains when cultured for 15 d. Each treatment had 3 replicates. After culturing for 12 d, use the cross method with a ruler to measure and record the colony diameter, calculate the mycelial growth rate, observe the colony color and growth trend, and select the strains with a mycelial growth rate not lower than 2 mm / d. Then inoculate them into the culture medium with a higher pH gradient, and so on. Finally, the alkaline-tolerant candidate strains (S, S20, S26, JN, XW) were selected.

[0061] Table 3: Composition of culture media with different pH values

[0062]

[0063] (2) Alkaline tolerance cyclic domestication:

[0064] The selected alkali-tolerant alternative strains (S, S20, S26, JN, XW) were subjected to high-pH cyclic domestication 5 times. They were inoculated onto a medium with a pH of 10.5, and each treatment was repeated 3 times. After culturing for 12 days, the colony diameter was measured, and the colony color and growth vigor were observed. Then, they were inoculated onto a PDA medium without alkali and cultured for 7 days to recover the mycelium. This was repeated 5 times. Finally, the strain S20 that tolerated a certain pH was domesticated as the alkali-tolerant domesticated strain of Stropharia rugosoannulata and was re-numbered as JS20.

[0065] 4. Protoplast breeding

[0066] The purpose of protoplast fusion breeding is to combine the advantageous traits of the parents to obtain a stable fusion strain with excellent genetic traits of the parents.

[0067] (1) Mycelium culture:

[0068] Based on the above salt-tolerant and alkali-tolerant domestication and screening results, the salt-tolerant domesticated strain YL4 and the alkali-tolerant domesticated strain JS20 with better salt and alkali tolerance were selected. After activating the strains, a 4-mm-diameter mycelial cake was transferred to the center of a culture medium plate (90 mm in diameter) covered with cellophane and cultured statically in the dark at 26 °C for 6 days. The mycelium of each treatment was scraped and reserved.

[0069] (2) Protoplast preparation, purification and fusion:

[0070] Lywallzyme (Guangdong Institute of Microbiology) was dissolved in a sterile osmotic stabilizer and filtered through a 0.22-μm microporous filter membrane to remove bacteria, and the enzyme solution was prepared. The collected mycelium was washed 3 times with a sterile osmotic stabilizer, and the excess liquid was blotted with a sterile filter paper. The processed Stropharia rugosoannulata mycelium was added to a 2-mL centrifuge tube, and the prepared enzyme solution was added according to the ratio of 1 mL of enzyme solution per 100 mg of mycelium. The centrifuge tube was manually shaken to disperse the mycelium as much as possible. It was enzymolyzed in a constant-temperature shaker at 32 °C for 2 - 4 h, and the shaker speed was set at 80 r / min. The degree of enzymolysis was detected every half hour. 10 μL of the enzymolysis solution was taken and counted with a hemocytometer. When the protoplast concentration reached 10 6 ~10 7 cells / mL, it indicated that the degree of enzymolysis was appropriate. After the enzymolysis was completed, the residual mycelium was filtered off with a disposable 40-μm cell sieve. The filtrate was centrifuged at 3500 r / min for 15 min, the supernatant was discarded, the precipitate was washed twice with a sterile osmotic stabilizer, and resuspended to an appropriate concentration, and the final yield of protoplasts was measured with a hemocytometer.

[0071] The two purified Stropharia rugosoannulata protoplasts were adjusted to 10 6cells / mL, respectively, were heat-inactivated and UV-inactivated. 0.5 mL of each inactivated protoplast was pipetted into a 2 mL sterile centrifuge tube, preheated at 30 °C for 5 min, and mixed with 25% PEG6000 and 0.02 mol / L Ca 2+ The mixed solution was used as a cosolvent, and the pH was adjusted to 8.5. Fusion was carried out at 26 °C for 30 min. The supernatant was discarded by centrifugation, and the cells were washed twice with sterile osmotic stabilizer to remove the toxicity of PEG.

[0072] (3) Breeding of salt-alkali-tolerant protoplast fusion strains:

[0073] Using the salt-tolerant strain YL4 and the alkali-tolerant strain JS20 as parents, 25 fusants were obtained after protoplast fusion and screening of binuclear strains. The 25 fusants were inoculated on the salt-alkali mixed medium (H1 - H12) shown in Table 4, and cultured in the dark at 26 °C to observe the germination of the strains.

[0074] Table 4: Salt-alkali content of the fusant screening medium

[0075]

[0076] The strains germinated on each medium were inoculated onto the same medium again. According to the germination order, the strains on medium H11 were numbered M21 - M32, and the strains on medium H12 were numbered M1 - M18. Each treatment was repeated 3 times. The growth rates of the strains were recorded and compared, and the strains M1, M2, M3, M5, M6, M7, M14, M21, M22, and M23 with faster mycelium growth rate and good growth vigor were selected as the fusion strains for standby.

[0077] The domesticated strains (YL4 and JS20) obtained through salt-alkali domestication, the fusion strains (M1, M2, M3, M5, M6, M7, M14, M21, M22, and M23) obtained through protoplast fusion, and the original strains (L4 and S20) were respectively inoculated onto medium H12. Each treatment was repeated 3 times, and the growth rates, mycelium growth vigor, and colony colors of the strains were recorded and compared. The results are shown in Table 5.

[0078] Table 5: Results of salt-alkali-tolerant strain screening

[0079]

[0080] It can be seen from Table 5 that the domesticated strains and the fusion strains are better than the initial strains, indicating that domestication and fusion improve the salt-alkali tolerance of the strains. The strains M6, M21, and M23 with faster growth rate, better growth vigor, and higher stability were selected as the salt-alkali-tolerant strains and were used for cultivation verification.

[0081] 5. Mushroom fruiting verification:

[0082] The selected salt-tolerant strains (M6, M21, and M23) and domesticated strains (YL4 and JS20) were used for soil covering cultivation. The SM (a nationally certified variety) was used as the control strain, and four saline-alkali soils with different saline-alkali contents (see Table 6) were selected as the soil covering materials, with ordinary cultivated soil as the control (ck), and each treatment was repeated 3 times.

[0083] Table 6: Soil covering materials with different saline-alkali contents

[0084]

[0085]

[0086] The fruiting cultivation material (by weight percentage: 45% rice husk, 38% broad-leaved tree miscellaneous sawdust, 10% corn cob, 5% garden soil, 2% quicklime, water content 75%) was mixed according to the formula and then subjected to high-temperature and high-pressure sterilization (121 °C, 160 min). A perforated plastic frame with a length of 30 cm, a width of 20 cm, and a height of 17.5 cm was used. There were 4 holes with a diameter of 8 cm at the bottom of the frame, and 6 holes with a diameter of 8 cm on each side of the frame. First, 5 cm of saline-alkali soil (saline-alkali soil numbered ①) was placed in the frame. The sowing method of 3 layers of fruiting cultivation material and 2 layers of strains was adopted. Each frame was filled with about 1 kg of cultivation material. The material was spread and sown in the frame for cultivation. After 10 days of mycelium cultivation, 2 cm of soil was covered, and each treatment had 3 replicates.

[0087] The cultivation experiment was carried out in a winter warm shed. After the mycelium was fully grown, water was poured to induce fruiting. The air humidity during the fruiting period was about 90%, and the ventilation volume was increased. If miscellaneous fungi such as Penicillium, Aspergillus, Mucor, and Discomycetes, Coprinus, and slime molds appeared during the mycelium growth period, they should be removed in time, and a small amount of lime was sprinkled. At the same time, yellow boards were hung to reduce the damage of mushroom flies, flying insects, and mushroom mosquitoes.

[0088] When the fruiting bodies were basically mature, they should be harvested in time. The appropriate harvesting time was in the evening. During harvesting, the yields of mushrooms at all levels and the changes in temperature and humidity were counted. After each flush of mushrooms was harvested, watering was stopped for about a week, and then watering was carried out to induce fruiting after the mycelium recovered. Only the first and second flushes of mushrooms were harvested in the fruiting experiment, resulting in a relatively low overall fruiting yield in the experiment, but there was comparability among different treatments.

[0089] The grading standards for the fruiting bodies of Stropharia rugoso-annulata are as follows:

[0090]

[0091] The mycelium growth conditions of each strain are shown in Table 7.

[0092] Table 7: Mycelium growth conditions of each cultivated strain of Stropharia rugoso-annulata

[0093]

[0094] The mushroom fruiting situation and yield statistics of salt-tolerant strains on ordinary cultivated soil are shown in Table 8.

[0095] Table 8: Mushroom fruiting situation and yield statistics of salt-tolerant strains on ordinary cultivated soil

[0096]

[0097] The mushroom fruiting situation and yield statistics of salt-tolerant strains on covered saline-alkali soil ① are shown in Table 9.

[0098] Table 9: Mushroom fruiting situation and yield statistics of salt-tolerant strains on covered saline-alkali soil ① (0.38% salt)

[0099]

[0100] The mushroom fruiting situation and yield statistics of salt-tolerant strains on covered saline-alkali soil ② are shown in Table 10.

[0101] Table 10: Mushroom fruiting situation and yield statistics of salt-tolerant strains on saline-alkali soil ② (0.63% salt)

[0102]

[0103]

[0104] The M23 strain fruited on both covered saline-alkali soil ③ (0.84% salt) and covered saline-alkali soil ④ (1.05% salt), and the other strains did not fruit yet. Therefore, only the soil-climbing time of the strains covering these two saline-alkali soils was recorded, as shown in Table 11.

[0105] Table 11: Soil-climbing time (d) of salt-tolerant strains on saline-alkali soils ③ and ④

[0106]

[0107] Considering comprehensively the salt tolerance and mushroom fruiting performance of the strains, the present invention selects the protoplast fusion strain M23 for biological preservation.

[0108] The preservation information of strain M23 is as follows:

[0109] Biological material (strain) referred to: M23;

[0110] Taxonomic name: Stropharia rugosoannulata; it can also be called Stropharia rugosoannulata.

[0111] Preservation institution: General Microbiological Center of China Committee for Culture Collection of Microorganisms

[0112] Abbreviation of the preservation institution: CGMCC

[0113] Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing

[0114] Date of deposit: August 26, 2022.

[0115] Registration number in the deposit center: CGMCC No. 40286

[0116] Example 2: Study on the mechanism of salt and alkali tolerance of the strain Stropharia rugosoannulata M23

[0117] 1. Determination of physiological indexes of the strain Stropharia rugosoannulata with salt and alkali tolerance:

[0118] 1.1 Determination of antioxidant enzyme activity:

[0119] (1) Collection and treatment of mycelium samples

[0120] Select the salt- and alkali-tolerant strain M23 and the control strain SM (national certified strain: Mingda 128), and conduct salt and alkali treatments at different concentrations, as shown in Table 12. Each treatment is repeated 3 times, cultured for 14 days, and the mycelium of each strain is sub-packed into 1.5 mL centrifuge tubes and stored at -80 °C for later use.

[0121] Table 12: Different salt stress treatments of the salt- and alkali-tolerant strain

[0122]

[0123]

[0124] (2) Preparation of crude enzyme solution

[0125] Grind the mycelium of each treatment into powder with liquid nitrogen, weigh it and put it into a 1.5 mL centrifuge tube, and add 1 mL of 50 mM phosphate buffer (pH 7.0). After centrifugation at 12000 g for 10 min at 4 °C, take the supernatant for enzyme activity determination.

[0126] (3) Determination of enzyme activity

[0127] Catalase (CAT): CAT assay kit (ammonium molybdate method) (Nanjing Jiancheng);

[0128] Peroxidase (POD): POD test kit (Nanjing Jiancheng);

[0129] Superoxide dismutase (SOD): T-SOD assay kit (hydroxylamine method) (Nanjing Jiancheng).

[0130] The results are as Figure 1As shown, the results indicate that under the combined saline-alkali stress of 0.5% NaCl and pH 8.5, the SOD and POD contents of the salt-tolerant strain M23 are 1150 U / g and 68 U / g respectively, which are 660 U / g and 17 U / g higher than those before stress. The activities of the two oxidases of the control strain decrease, showing significant differences from those of the control strain, and are 611 U / g and 50 U / g higher than the SOD and POD activities of the control strain respectively.

[0131] The results show that the mycelium of Stropharia rugosoannulata resists salt stress by increasing the activities of antioxidant enzymes, and the salt-tolerant strain has a stronger ability to resist salt stress.

[0132] 1.2 Determination of trehalose content:

[0133] (1) Collection and treatment of mycelium samples are the same as those in part 1.1.

[0134] (2) Content determination

[0135] The high performance liquid chromatography (HPLC) method was used, and the determination method refers to the national standard GB 5009.8 - 2016. 0.1 g of mycelium was added to 1 mL of deionized water, homogenized, extracted overnight, and the supernatant was taken after centrifugation at 8000 r / min for 10 min, and filtered through a 0.45 μm microporous filter membrane. The chromatographic conditions were as follows: Thermo UltiMate 3000 high performance liquid chromatograph, the chromatographic column was HP-Amino (250 mm * 4.6 mm), the mobile phase was 70% acetonitrile, the flow rate was 1 mL / min, the column temperature was 40 °C, the temperature of the differential detector was 40 °C, and the injection volume was 10 μL. Trehalose (Sigma) was used as the standard.

[0136] The results are as Figure 2 shown. Under the combined saline-alkali stress of 0.5% NaCl and pH 8.5 for 14 days, the trehalose contents of the salt-tolerant strain and the control strain SM decreased by 21.3 mg / g and 6.5 mg / g respectively. Without salt stress, there was no significant difference in the trehalose contents of the two strains. It can be seen that the mycelium of Stropharia rugosoannulata is sensitive to the emergency response of saline-alkali stress, and the trehalose content in vivo decreases significantly.

[0137] 2. Transcriptome sequencing analysis

[0138] 2.1 Experimental method:

[0139] (1) Collection and treatment of mycelium samples:

[0140] The specific collection and treatment methods are the same as those in part 1.1.

[0141] (2) Sequencing analysis:

[0142] RNA extraction: Tiangen Polysaccharide and Polyphenol Kit DP441, stored at –80℃ for future use.

[0143] Concentration detection: Nanodrop and Agilent 5400.

[0144] Integrity and purity testing: Agilent 5400.

[0145] Agilent 2100bioanalyzer: Accurately detect RNA integrity and total amount. (Refer to RNA quality inspection report for specific instruments used). Novo Biotech Co., Ltd. performs library construction and quality inspection, on-machine sequencing, functional annotation and enrichment analysis.

[0146] (3) qRT-PCR verification:

[0147] The accuracy of the gene data screened out by the above transcriptome sequencing was verified by fluorescent quantitative PCR.

[0148] Reverse transcription: Full Gold Reverse Transcription Kit;

[0149] Internal reference: β-actin;

[0150] qPCR primers, primer sequences were designed using Beacon Designer software, see Table 13, and synthesized by Beijing Qingke Biotechnology Co., Ltd.;

[0151] qPCR reaction: Fast Start Universal SYBR Green Master Kit (Rox), iCycleriQ5 real-time quantitative PCR instrument (Bio-Rad, USA);

[0152] Relative gene expression = 2 -ΔΔCt ,log2 fold change=log2(2 -ΔΔCt ).

[0153] Table 13: qRT-PCR primer sequences

[0154]

[0155]

[0156] 2.2 Test results:

[0157] (1) RNA extraction and raw data quality testing

[0158] The extraction of total RNA was relatively complete, and both the RNA concentration and total amount met the quality requirements for establishing a transcriptome library.

[0159] After filtering the raw data, checking the sequencing error rate, and examining the GC content distribution, clean reads for subsequent analysis were obtained. The data summary table is shown in Table 37. The final number of clean reads was approximately 50 to 70 million, and the Q30 of the four groups of samples all reached over 90%, indicating that the probability of the raw data error rate being less than 0.1% was over 90%, and the data quality was high.

[0160] Table 14: Summary of Sample Sequencing Data Quality

[0161]

[0162] Note: Q30: Proportion of bases with Phred value greater than 30; GC: Percentage of the total number of G and C bases in the total number of bases.

[0163] (2) Screening and Identification Results of Differentially Expressed Genes (DEGs)

[0164] According to the threshold conditions DESeq2 padj < 0.05 |log2FoldChange| > 1, the transcriptome data of different treatments of Stropharia rugosoannulata were screened to obtain differentially expressed genes. Divided into 4 comparison groups according to M23_H vs. M23, SM_H vs. SM, M23_H vs. SM_H, M23 vs. SM, and the number of differentially expressed genes between groups was counted. The results showed ( Figure 3 ) that these 4 comparison groups had 515, 1150, 1005, and 1832 differentially expressed genes respectively. It can be seen that there are obvious differentially expressed genes in different strains under saline-alkali stress or not, and these genes may be related to the saline-alkali stress resistance of the mycelium, and the control strain requires more genes to participate in the regulation of the saline-alkali resistant growth of the mycelium. The expression patterns of differentially expressed genes within the groups were counted respectively. M23_H vs. M23, SM_H vs. SM, M23_H vs. SM_H, M23 vs. SM had 215, 561, 229, and 770 genes showing up-regulated expression, and 300, 589, 776, and 1062 genes showing down-regulated expression. It can be seen that the number of down-regulated genes in the two strains under saline-alkali stress changed more significantly than the up-regulated genes, and regardless of whether there is saline-alkali stress, the number of down-regulated genes in the saline-alkali tolerant strain is significantly more than the up-regulated genes compared to the control strain.

[0165] The Venn diagram of differentially expressed genes shows the number of differentially expressed genes between comparison groups and the overlapping relationship of differentially expressed genes between comparison groups. By counting the number of differentially expressed genes between comparison groups, the results showed that there were 139 differentially expressed genes in the mycelium of the saline-alkali tolerant strain and the control strain under salt stress or saline-alkali stress, indicating that these genes may play a key regulatory role in the mycelium's resistance to salt stress or saline-alkali stress ( Figure 4 ).

[0166] (3) Functional annotation and GO classification of differentially expressed genes

[0167] The differentially expressed genes in the two comparison groups of M23_H vs. M23 and SM_H vs. SM were respectively compared with the GO database, and 278 and 685 DEGs were respectively enriched, and were respectively annotated to Biological process, Cellular component, and Molecular function. In these two comparison groups, the differentially expressed genes were more enriched in biological processes and molecular functions. As Figure 5 Shown in A and B, under saline-alkali stress compared with non-saline-alkali stress of the saline-alkali tolerant strain M23, in the biological process classification, the most enriched DEGs were transmembrane transport (40 DEGs) and signal transduction (33 DEGs); in the molecular function classification, the most enriched DEGs were oxidoreductase activity (71 DEGs) and transmembrane transporter activity (44 DEGs); while in the cellular component classification, the cytoskeleton with the most enriched DEGs only had 8. Under saline-alkali stress compared with non-saline-alkali stress of the control strain SM, in the biological process classification, the most enriched DEGs were response to stress (65 DEGs) and catabolic process (54 DEGs); in the molecular function classification, the most enriched DEGs were oxidoreductase activity (153 DEGs); while in the cellular component classification, the most enriched DEGs were extracellular region (71 DEGs).

[0168] (4) Analysis of metabolic pathways (KEGG) of differentially expressed genes

[0169] The differentially expressed genes in the two comparison groups of M23_H vs. M23 and SM_H vs. SM were respectively compared with the KEGG database. There were 45 and 158 DEGs in the two groups of differentially expressed genes, which were respectively enriched in 151 and 204 metabolic pathways. The top 20 metabolic pathways with the strongest differential significance were displayed, and the results were as Figure 6A and B. Under saline-alkali stress, according to the number of differentially expressed genes, the salt-tolerant strain M23 was mainly enriched in glycine, serine and threonine metabolism, degradation of aromatic compounds, biosynthesis of unsaturated fatty acids, aminobenzoate degradation, fatty acid metabolism, etc. in turn; the control strain SM was mainly enriched in peroxisome, pyruvate metabolism, glycolysis / gluconeogenesis, regulation of mitophagy–yeast, fatty acid degradation, etc. in turn. The above metabolic processes are of great significance to the salt-alkali stress resistance of mycelia, and it can be seen that the salt-tolerant strain uses fewer DEGs to resist salt-alkali stress than the control strain.

[0170] (5) Salt tolerance-related genes in the transcriptome of Stropharia rugosoannulata

[0171] Based on functional annotation, GO and KOG functional classification, and KEGG metabolic pathway analysis, genes related to salt tolerance in the transcriptome of Stropharia rugosoannulata were further analyzed. By comprehensively analyzing the comparison groups of M23_H vs. M23 and SM_H vs. SM, differentially expressed genes with large up- and down-regulation fold changes, significant expression levels, and successful annotation that were common between strains M23 and SM under salt stress or saline-alkali stress were selected.

[0172] Table 15: 10 significantly differentially expressed genes related to salt-alkali tolerance

[0173]

[0174] Note: Gene ID: is the Gene ID obtained after Corset clustering. Corset (Nadia M Davidson, Alicia Oshlack, 2014), based on Trinity assembly, aggregates transcripts into many clusters according to Shared Reads among transcripts, and then combines the transcript expression levels among different samples and the H-Cluster algorithm to separate the transcripts with differential expression among samples from the original clusters and establish new clusters. Finally, each cluster is defined as a "Gene". This method can aggregate redundant transcripts and improve the detection rate of differentially expressed genes. The official website of Corset is https: / / code.google.com / p / corset-project / .

[0175] As shown in Table 15, under saline-alkali stress, the gene expression levels of enzymes or proteins such as reductase, monooxygenase, β-1,3-glucan-binding protein, protease inhibitor, iron / manganese superoxide dismutase, peroxidase, and acid phosphatase encoded by the mycelium of Stropharia rugosoannulata were up-regulated, while the gene expression levels of enzymes or proteins such as hydroxylase / desaturase asaB, trehalose-6-phosphatase, and protein kri1 were down-regulated. Under the action of these genes, the mycelium of the saline-alkali tolerant strain made a series of stress responses such as antioxidant enzymes and redox to resist the damage caused by saline-alkali stress. It was obvious that the fold change of the up-regulated gene expression of the saline-alkali tolerant strain was greater than that of the control strain, indicating that the self-regulation intensity of the saline-alkali tolerant strain M23 was greater under saline-alkali stress.

[0176] (6) qRT-PCR verification

[0177] The 10 significantly differentially expressed genes screened were detected by fluorescence quantitative PCR to verify the accuracy of the transcriptome sequencing results. The change trend of the gene expression levels of the genes verified by qPCR was basically consistent with that of the transcriptome sequencing results ( Figure 7 ).

Claims

1. A strain of Stropharia rugosoannulata with a preservation number of CGMCC No. 40286.

2. Use of the Stropharia rugosoannulata strain according to claim 1 as a parent in breeding.

3. The use according to claim 2, characterized in that The breeding is for the cultivation of a salt-tolerant variety of Stropharia rugosoannulata.

4. Mycelium and / or spores obtained by culturing the Stropharia rugosoannulata strain according to claim 1.

5. Fruiting bodies obtained by cultivating the Stropharia rugosoannulata strain according to claim 1.

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