High-yield single-mating cordyceps militaris strain and cultivation method thereof

Abstract

The invention relates to the technical field of biology, in particular to a high-yield single mating type cordyceps militaris strain and a cultivation method thereof. The strain is a single mating fruiting type (MAT1-2) cordyceps militaris high-yield strain, the preservation number of the strain is CGMCC No.41163, the strain does not contain MAT1-1 genes, and the preservation unit is the China General Microbiological Culture Collection Center. The cultivation method of the bacterial strain comprises the following steps: pairing and hybridizing a bacterial strain which has a mating type of MAT1-1, can independently produce mushrooms and does not produce ascocysts and a MAT1-2 type bacterial strain which cannot independently produce mushrooms to obtain sporocarp with the ascocysts and ascospores, collecting the hybridized sporocarp by a hooking and suspending method to obtain the ascospores, coating and diluting the ascospores, selecting a monoascospore bacterial strain of the ascospores, and culturing the monoascospore bacterial strain. And carrying out mating type identification by using a molecular marker to obtain the MAT1-2 type cordyceps militaris strain LDSYMS-15 capable of independently fruiting. The technical scheme has the beneficial effects that the yield is high, the economic benefit is good, the market demand is wide, and the agricultural prospect is large, and meanwhile, the new strain obtained by the method not only can be used for large-scale production of cordyceps militaris, but also has important scientific research value in the aspect of genetic breeding theoretical research of cordyceps militaris.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, in particular to a high-yield single-mating type Cordyceps militaris strain and a cultivation method thereof. Background Art

[0002] Cordyceps militaris, also known as northern cordyceps, belongs to the kingdom Fungi, subphylum Ascomycota, class Ascomycetes, order Hypocreales, family Claviceps in fungal taxonomy. It is a widely distributed and widely studied model species of the genus Cordyceps. As a traditional precious Chinese medicinal material, Cordyceps militaris has been recorded in many Chinese medicine books. Cordycepin and adenosine are the main active ingredients of Cordyceps militaris, which have multiple pharmacological effects such as antiviral and tumor, improving heart and brain blood circulation, and inhibiting the release of central neurotransmitters. At the same time, Cordyceps militaris contains very rich nutrients, including three major nutrients: sugar, fat and protein, as well as a variety of amino acids and more than 30 trace elements. It can be seen that artificial cultivation and production of Cordyceps militaris has great medicinal and nutritional value.

[0003] As a secondary heterothallic fungus, Cordyceps militaris requires the coexistence of two mating type genes, MAT1-1 and MAT1-2, to reproduce sexually. However, the genetic diversity and instability of Cordyceps militaris always lead to strain degeneration and unstable yield in large-scale production, which has seriously restricted the development of the Cordyceps militaris industry. The selection of high-yield, stable-yield and high-quality strains has become a major issue in the large-scale production of Cordyceps militaris. Hybrid breeding is one of the important means of directional breeding for artificial cultivation of Cordyceps militaris. Only by continuously developing and breeding new strains with excellent traits can the vigorous development of the Cordyceps militaris industry be guaranteed and economic benefits be extended.

[0004] In the large-scale production of Cordyceps militaris, most Cordyceps militaris strains are double-mating strains. However, during the preservation and propagation process, the strains are easily degraded due to the imbalance of mating type ratio, resulting in a decrease in yield or even no mushroom production, which seriously affects the production stability. Single-mating fruiting strains are more stable and not easy to degenerate, and have been used for large-scale production. However, the single-mating fruiting strains discovered and reported so far are all MAT1-1 type. Therefore, our company's technicians have specially developed the MAT1-2 single-mating Cordyceps militaris strain that can stably produce mushrooms. This strain has high yield, strong propagation stability, and fast mycelial growth rate. It has broad agricultural prospects and economic benefits. Summary of the invention

[0005] The technical problems to be solved by this technical solution are:

[0006] To achieve the above purpose, the present invention creates a technical solution: a high-yield single-mating Cordyceps militaris strain and a cultivation method thereof, wherein the strain is a single-mating mushroom-producing type (MAT1-2) Cordyceps militaris high-yield strain, whose preservation number is CGMCC No.41163, does not contain the MAT1-1 gene, and is classified as Cordyceps militaris. The preservation unit is the General Microbiology Center of the China Microbiological Culture Collection Administration, and the preservation unit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing. The preservation date is February 2, 2024.

[0007] The strain cultivation method comprises the following steps: pairing and hybridizing a strain with a mating type of MAT1-1, which can produce mushrooms alone and does not produce ascocarps, and a strain of the MAT1-2 type that cannot produce mushrooms alone, to obtain fruiting bodies with ascocarps and ascospores, collecting ascospores from the hybrid fruiting bodies by a hook hanging method, spreading and diluting the ascospores, selecting a single ascospore strain for cultivation, and performing mating type identification by using molecular markers, to obtain a MAT1-2 type Cordyceps militaris strain LDSYMS-15 that can produce mushrooms alone.

[0008] The specific steps include:

[0009] 1) Starting strain: The starting strain is a single-mating fruiting strain with a smooth fruiting body surface and no ascocarp, containing only the MAT1-1 gene, and a double-mating fruiting strain with abundant ascocarp on the fruiting body surface and containing both the MAT1-1 and MAT1-2 genes.

[0010] 2) Isolation of single mating type strains: From the plate culture or slant culture of the starting strain, take 1 cm×1 cm of the mycelium culture of the test strain and gently shake it in 7-10 ml of sterile water for 1-3 minutes to separate the single conidia of the test strain. After filtering with multiple layers of sterilized lens paper, collect the suspension containing conidia. After gradient dilution of 10-1, 10-2, 10-3, 10-4, and 10-5, draw 0.1-0.4 ml of the spore liquid and evenly spread it on the PDA medium, and culture it at 15-25°C in the dark for 3-5 days. After the colonies visible to the naked eye appear on the culture medium, pick a single colony and inoculate it on the PDA medium, and culture it at 15-25°C in the dark for 12-18 days. The mycelium of the test strain has stable fruiting ability.

[0011] 3) Mating type identification of single mating type strains: After the single mating type strains were isolated and cultured, two parent strains were obtained and mating type identification was performed. The two parent strains were the LDSY02 monoconidial strain with the mating type MAT1-1 that could produce mushrooms and the LDMS05 monoconidial strain with the mating type MAT1-2 that could not produce mushrooms alone.

[0012] 4) Pairing and fruiting of single mating type strains: Take two 9mm diameter mushroom blocks of LDSY02-25 and LDMS05-16 respectively, inoculate them into a triangular flask filled with liquid culture medium, and culture them in the dark at 17-21℃ with shaking for 2-4 days to obtain liquid strains; Take 1.5-2.5ml of each liquid strain of LDSY02-25 and LDMS05-16, mix and inoculate them into the same bottle of oat culture medium, place it in a fruiting room at 18-25℃, and culture it in the dark. When the hyphae cover the culture medium and the amount of hyphae at the bottom of the bottle exceeds two-thirds, perform a scratching treatment, and then transfer it to 18-22℃, humidity 80-90% and culture it in light for 40-50 days, the fruiting body matures and monoascospores are formed.

[0013] 5) Isolation and culture of single ascospore strain: Take mature Cordyceps militaris fruiting body, wrap the base of the fruiting body tightly with sterilized cotton thread, hang upside down in a 50ml Erlenmeyer flask filled with 5-10ml sterile water, and place it in a 15-25℃ incubator for 8-12h to collect ascospore suspension, draw 0.05-0.1ml spore liquid and evenly spread it on PDA culture medium, culture it in the dark at 15-25℃ for 1-3d, pick out a single colony, inoculate it on PDA culture medium, and culture it in the dark at 15-25℃ for 8-12d to obtain MAT1-2 type Cordyceps militaris strain LDSYMS-15.

[0014] 6) The mating type identification of a single colony and the fruiting experiment verification, the mating type identification method of the single colony is to screen a high-yield strain LDSYMS-15 from the obtained single colony, extract the genomic DNA of LDSYMS-15, and design specific primers according to the sequences of the Cordyceps militaris mating type genes MAT1-1 (no.AB194982.1) and MAT1-2 (no.AB084257.1) in the NCBI database, MAT1-1F: 5'-gagcctactatggaaccc-3'; MAT1-1R: 5'

[0015] -caggactgataccagcaaa-3'; MAT1-2F: 5'-gcatcaacccatttgtgaaagttct-3'; MAT1-2R: 5'-cctgtcataatggtgctgt-3', PCR amplification, amplified fragments: MAT1-1 is 427bp, MAT1-2 is 719bp, the amplified products are detected by electrophoresis, the strains with a single gene band of MAT1-1 gene or MAT1-2 gene are single mating type strains, and the strains with both bands are double mating type strains. The mushroom fruiting experimental verification method is to weigh the fresh weight of the fruiting body and calculate its bioconversion rate, and the calculation formula of the bioconversion rate (V, %) is: V = (G1 / G2) × 100% In the formula: G1 is the fresh weight of the fruiting body of Cordyceps militaris, G2 is the dry weight of the culture medium, and the dry weight is recorded after the fruiting body is placed in an oven at 50°C for drying.

[0016] Furthermore, the method for picking a single colony in steps 2 and 5 is as follows: hang a single mature fruiting body upside down in the middle of a sterile and dry 50 ml conical flask, put it in a 15-25° C. incubator to collect ascospores ejected from the fruiting body, carefully remove the hanging fruiting body after 8-12 hours, absorb 7-10 ml of sterile water and place it in a conical flask, gently shake it, collect the ascospore liquid, and after five gradient dilutions of 10-1, 10-2, 10-3, 10-4, and 10-5, absorb 0.1 ml of the spore suspension and evenly spread it on the PDA culture medium, culture it in the dark at 15-25° C. for 3-5 days, observe signs of spore germination, and after colonies visible to the naked eye appear on the culture medium, pick a single colony, inoculate it on the PDA culture medium, and culture it in the dark at 15-25° C.

[0017] Furthermore, the mating type identification in the steps 3 and 6 adopts a novel plant genomic DNA extraction kit to obtain genomic DNA from the mycelium and adopts a PCR reaction system (20 μL): Mix 10 μL, 0.5 μL of each forward and reverse primer, 1 μL of template DNA, 8 μL of ddH2O, and the PCR reaction conditions are: 94°C pre-denaturation for 5 min, 94°C denaturation for 30 s, 56°C annealing for 30 s, 72°C extension for 30 s, for a total of 30 cycles; 72°C fill-in for 10 min.

[0018] Furthermore, the PDA medium preparation method is as follows: 200g of potato is cut into pieces and boiled in 1000ml of water for 10min, 20g of glucose and 20g of agar are dissolved in the potato filtrate, the volume is fixed to 1000ml, the pH is natural, and the mixture is dispensed into 500ml conical flasks;

[0019] The preparation method of the Cordyceps militaris liquid culture medium is as follows: 20g of glucose, 15g of peptone, 5g of yeast powder, 1g of magnesium sulfate, 2g of potassium dihydrogen phosphate, VB10.1g, and natural pH; the prepared culture medium is divided into 500ml conical bottles, each bottle is filled with 150ml, sealed with a special sealing film for edible fungi, sterilized at 121°C for 30min, and naturally cooled to room temperature;

[0020] The oat culture medium preparation method is as follows: 30g of oats and 50g of water are placed in a culture bottle, covered, and completely sterilized in an autoclave at 121°C for 30min, and then naturally cooled to room temperature.

[0021] Furthermore, the preparation steps of the liquid strain of the strain in step 4 are as follows: preparing a liquid culture medium, filling the prepared liquid culture medium into 500 ml conical bottles, each bottle with 100-150 ml, sealing with a special sealing film for edible fungi, sterilizing at 100-121° C. for 20-30 minutes, and naturally cooling to room temperature; inoculating 2-4 pieces of activated Cordyceps militaris strains with a diameter of 9 mm stored on a PDA slant culture medium into each bottle under sterile conditions, and sealing; placing on a rotary shaker, culturing for 2-4 days at a speed of 140 r / min and a temperature of 17-21° C. to obtain Cordyceps militaris liquid strains for standby use.

[0022] Furthermore, the isolation and cultivation of the monoascospore strain in step 5 is as follows: inoculating the Cordyceps militaris liquid seed onto a sterilized oat culture medium under a sterile environment, placing it in a culture room at 20°C-25°C, and culturing it in the dark. After the mycelium covers the bottom of the culture bottle and the bacteria are scratched, the culture is transferred to a light culture room at a temperature of 15-18°C and a humidity of 80-90%, and the fruiting bodies are harvested after they mature.

[0023] The beneficial effects of this technical solution are:

[0024] The present invention obtains the MAT1-2 type mushroom-producing strain for the first time, and its fruiting body yield and quality are better than those of the parent strain, with good grass-producing effect and excellent commercial properties. The MAT1-2 type mushroom-producing strain can not only be used for large-scale production, but also provides valuable materials for the research on genetic breeding of Cordyceps militaris.

[0025] The hybrid breeding method of the present invention is helpful to promote the breeding of excellent varieties of Cordyceps militaris and based on the identification of mating type genes, it explores the pairing and hybrid characteristics of two types of Cordyceps militaris strains, single-mating mushroom-producing type and double-mating mushroom-producing type, provides technical support for the breeding of new varieties of Cordyceps militaris, and serves as a reference for establishing an effective hybrid breeding program for Cordyceps militaris. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 It is a process flow chart of the present invention.

[0027] Figure 2 The fruiting body (A) and mating type identification (B) of the test strain.

[0028] Figure 3 This is a cross-section of the fruiting body after hybridization of single mating type strains.

[0029] Figure 4 This is the gel electrophoresis diagram for mating type identification of Cordyceps militaris strain LDSYMS-15.

[0030] Figure 5 This is a picture of the mycelium morphology of the Cordyceps militaris strain LDSYMS-15 in Comparative Example 1 in PDA culture medium.

[0031] Figure 6 This is a comparison of the fruiting bodies produced by hybridization of single mating type strains and their parental strains.

[0032] Figure 7 This is a morphological diagram of the fruiting body of the Cordyceps militaris strain LDSYMS-15 and its single conidia strain in Comparative Example 2. DETAILED DESCRIPTION

[0033] In order to enable those skilled in the art to better understand the technical solution of the present invention, the present invention is described in detail below in conjunction with the accompanying drawings. The description in this part is only exemplary and explanatory and should not have any limiting effect on the protection scope of the present invention.

[0034] The invention belongs to the technical field of genetic breeding of edible and medicinal ascomycetes, and relates to a single-mating-type mushroom-producing type (MAT1-2) Cordyceps militaris high-yield strain, which has a preservation number of CGMCC No.41163 and is preserved by the General Microbiological Center of China Microbiological Culture Collection Committee. The strain does not contain a MAT1-1 gene and has high stability in passage. The invention also relates to a method for selecting the strain by hybridization, and uses a single-mating-type mushroom-producing type strain and a double-mating-type mushroom-producing type strain of Cordyceps militaris as materials, and conducts experiments such as separation of single-mating-type strains and identification of mating-type genes, hybrid fertility determination, paired mushroom-producing tests, and analysis of agronomic traits of hybrid progeny strains to carry out a discussion on the breeding systems of the two types of strains. For double-mating-type fruiting strains, the single-mating-type strains separated and purified from asexual spores of the same or different parents, after the different mating types are recombined and paired, the agronomic traits will change significantly. The asexual spore separation and combination pairing method is an effective way to select and rejuvenate production strains; for single-mating-type fruiting strains, the tested strains are paired with different mating-type strains to produce mushrooms, which can produce sexual spores and show hybrid fertility, but the consistency of fruiting body morphology is neat and irregular. The neat type can be used for production; the single-mating-type offspring strains obtained after ascospore separation are MAT1-1 fruiting type, MAT1-2 fruiting type and non-fruiting type. There are significant differences in yield traits, so high-yield single-mating-type fruiting strains can be selected through hybrid offspring. The experimental results can guide the production breeding of Cordyceps militaris.

[0035] A high-yield single-mating type Cordyceps militaris strain and a cultivation method thereof. The strain cultivation method comprises the following steps: pairing and hybridizing a strain with a mating type of MAT1-1, which can produce mushrooms alone but does not produce ascocarps, and a strain with a MAT1-2 type that cannot produce mushrooms alone, to obtain fruiting bodies with ascocarps and ascospores, collecting ascospores from the hybrid fruiting bodies by a hook hanging method, spreading and diluting the ascospores, selecting the single ascospore strain for cultivation, and performing mating type identification by using molecular markers, to obtain the MAT1-2 type Cordyceps militaris strain LDSYMS-15 that can produce mushrooms alone.

[0036] The specific steps include:

[0037] 1) Starting strain: The starting strain is a single-mating fruiting strain with a smooth fruiting body surface and no ascocarp, and only contains the MAT1-1 gene. It is a strain selected and preserved by this unit, numbered LDSY02; and a double-mating fruiting strain with abundant ascocarp on the fruiting body surface and both MAT1-1 and MAT1-2 genes. It is a strain selected and preserved by this unit, numbered LDMS05.

[0038] 2) Isolation of single mating type strains: Take 1cm×1cm of the mycelial culture of the test strain from the plate culture or slant culture of the two strains, shake gently in 10ml sterile water for 1min, collect the suspension (containing conidia) after filtering through 2 layers of sterilized lens paper, and after gradient dilution of 10-1, 10-2, 10-3, 10-4, 10-5, take 0.1ml of spore liquid and spread it evenly on PDA medium, and culture it at 25℃ in the dark for 3 days. After the colonies visible to the naked eye appear on the culture medium, pick a single colony, inoculate it on PDA medium, and culture it at 25℃ in the dark for 12 days.

[0039] Among them, the preparation method of PDA medium, which is a solid plate medium, is as follows: 200g of potato is cut into pieces and boiled in 1000ml of water for 10 minutes, 20g of glucose and 20g of agar are dissolved in the potato filtrate, the volume is made up to 1000ml, the pH is natural, and it is divided into 500ml conical bottles.

[0040] Among them, the test strain is a Cordyceps militaris strain obtained by tissue separation and purification, which has a stable mushroom-producing ability and is preserved in the Strain Collection Center of Ludong University after ITS sequence identification (see Table 1 for details).

[0041] Table 1 Name and source of tested Cordyceps militaris strains

[0042]

[0043] 3) Mating type identification of single mating type strains: After the isolation and cultivation of single mating type strains, two parent strains were obtained, one from the LDSY02 monoconidial strain, with a mating type of MAT1-1, capable of producing mushrooms but without producing ascocarps and ascospores, numbered LDSY02-25, and the other from the LDMS05 monoconidial strain, with a mating type of MAT1-2, unable to produce mushrooms alone, numbered LDMS05-16.

[0044] Among them, the mating type identification of single mating type strains uses a new plant genomic DNA extraction kit to extract genomic DNA from mycelium and PCR reaction system (20μL): Mix 10μL, forward and reverse primers 0.5μL each, template DNA 1μL, ddH2O 8μL. PCR reaction conditions: 94℃ pre-denaturation 5min, 94℃ denaturation 30s, 56℃ annealing 30s, 72℃ extension 30s, a total of 30 cycles; 72℃ fill 10min. Mating type gene primer design reference: Feng Yujie. Research on the degradation mechanism of Cordyceps militaris and key greenhouse cultivation technology in Xinjiang [D].

[0045] 4) Pairing and fruiting of single mating type strains: Take two 9mm diameter mushroom blocks of LDSY02-25 and LDMS05-16 respectively, inoculate them into a triangular flask filled with liquid culture medium, and culture them in the dark at 17-21℃ with shaking for 2-4 days to obtain liquid strains; Take 1.5-2.5ml of each liquid strain of LDSY02-25 and LDMS05-16, mix and inoculate them into the same bottle of oat culture medium, place it in a fruiting room at 18-25℃, and culture it in the dark. When the hyphae cover the culture medium and the amount of hyphae at the bottom of the bottle exceeds two-thirds, perform a scratching treatment, and then transfer it to 18-22℃, humidity 80-90% and culture it in light for 40-50 days, the fruiting body matures and monoascospores are formed.

[0046] The liquid seed preparation steps of the strain are as follows: preparing a Cordyceps militaris liquid culture medium, wherein the formula of the liquid culture medium is as follows: 20 g of glucose, 15 g of peptone, 5 g of yeast powder, 1 g of magnesium sulfate, 2 g of potassium dihydrogen phosphate, 0.1 g of VB1, 1000 ml of water, and natural pH; the prepared culture medium is divided into 500 ml conical bottles, each bottle is filled with 150 ml, sealed with a special sealing film for edible fungi, sterilized at 121 DEG C for 30 min, and naturally cooled to room temperature; under sterile conditions, 2 pieces of activated Cordyceps militaris strain LDSYMS-15 with a diameter of 9 mm stored on a PDA slant culture medium are introduced into each bottle, and the bottle is sealed; the bottle is placed on a rotary shaker, and cultured for 4 days at a speed of 140 r / min and a temperature of 21 DEG C to prepare liquid seeds of Cordyceps militaris for standby use.

[0047] The preparation steps of oat culture medium, i.e. Cordyceps militaris cultivation medium, are as follows: 30g of oats and 50g of water are placed in a culture bottle, covered, and completely sterilized in an autoclave at 121°C for 30min, and naturally cooled to room temperature.

[0048] 5) Isolation and culture of single ascospore strain: Take mature Cordyceps militaris fruiting body, wrap the base of the fruiting body tightly with sterilized cotton thread, hang upside down in a 50ml Erlenmeyer flask filled with 5-10ml sterile water, and place it in a 15-25℃ incubator for 8-12h to collect ascospore suspension, draw 0.05-0.1ml spore liquid and evenly spread it on PDA culture medium, culture it in the dark at 15-25℃ for 1-3d, pick out a single colony, inoculate it on PDA culture medium, and culture it in the dark at 15-25℃ for 8-12d to obtain MAT1-2 type Cordyceps militaris strain LDSYMS-15.

[0049] Among them, the isolation and cultivation of the monoascospore strain is to inoculate the Cordyceps militaris liquid seed into the sterilized oat culture medium under a sterile environment, place it in a 20℃-25℃ culture room, and culture it in the dark. After the mycelium covers the bottom of the culture bottle and the bacteria are scratched, it is transferred to a light culture under the conditions of temperature 18℃ and humidity 80-90%, and harvested after the fruiting bodies are mature.

[0050] Among them, the method for picking a single colony is as follows: hang a single mature fruiting body upside down in the middle of a sterile and dry 50ml conical flask, put it in a 25℃ incubator to collect the ascospores ejected from the fruiting body, carefully remove the hanging fruiting body after 12 hours, absorb 10ml of sterile water and put it in a conical flask, gently shake it, collect the ascospore liquid, and after five gradient dilutions of 10-1, 10-2, 10-3, 10-4, and 10-5, absorb 0.1ml of spore suspension and evenly spread it on the PDA culture medium, culture it at 25℃ in the dark for 3 days, observe signs of spore germination, and after visible colonies appear on the culture medium, pick a single colony, inoculate it on the PDA culture medium, and culture it at 25℃ in the dark.

[0051] 6) The mating type identification of a single colony and the fruiting experiment verification, the fruiting experiment verification method is to weigh the fresh weight of the fruiting body and calculate its bioconversion rate, the calculation formula of the bioconversion rate (V, %) is: V = (G1 / G2) × 100% In the formula: G1 is the fresh weight of the fruiting body of Cordyceps militaris, G2 is the dry weight of the culture medium, and the dry weight is recorded after the fruiting body is placed in an oven at 50°C for drying. The mating type identification method of a single colony is to screen a high-yield strain LDSYMS-15 from the obtained single colony, and use a new plant genomic DNA extraction kit to extract the genomic DNA of LDSYMS-15. According to the sequences of the mating type genes MAT1-1 (no.AB194982.1) and MAT1-2 (no.AB084257.1) of Cordyceps militaris in the NCBI database, design specific primers MAT1-1F: 5'-gagcctactatggaaccc-3'; MAT1-1R: 5'

[0052] -caggactgataccagcaaa-3'; MAT1-2F: 5'-gcatcaacccatttgtgaaagttct-3'; MAT1-2R: 5'-cctgtcataatggtgctgt-3', PCR amplification, amplified fragment: MAT1-1 is 427bp, MAT1-2 is 719bp, PCR reaction system (20μL): Mix 10μL, forward and reverse primers 0.5μL each, template DNA 1μL, ddH2O 8μL, PCR reaction conditions: 94℃ pre-denaturation 5min, 94℃ denaturation 30s, 56℃ annealing 30s, 72℃ extension 30s, a total of 30 cycles; 72℃ fill 10min. The amplified products were detected by electrophoresis. The strains with a single gene band of MAT1-1 gene or MAT1-2 gene were single mating type strains, and the strains with both bands were double mating type strains.

[0053] Finally, a high-yield strain was screened from the obtained single colonies, numbered LDSYMS-15, with a mating type of MAT1-2, no MAT1-1 gene, a conversion rate of 166%, and a cordycepin content of 0.26%. The verified LDSYMS-15 strain can be used for large-scale cultivation of Cordyceps militaris. The strain has good stability in passage and has been continuously passaged for 10 generations without obvious degradation. The following is the comparative data of the verified LDSYMS-15 strain.

[0054] Comparative Example

[0055] Comparative Example 1 Comparison of mycelial growth rate of Cordyceps militaris strain LDSYMS-15

[0056] The mycelial growth rates of the two parent strains (SY 25, MS16) and the monoascospore strains LDSYMS-3 (a MAT1-1 type strain that can produce mushrooms) and LDSYMS-14 (a MAT1-2 type strain that cannot produce mushrooms) obtained by hybridization were measured and compared, as shown in Table 2.

[0057] Table 2. Comparison of mycelial growth rate of strain LDSYMS-15 and other strains

[0058]

[0059] As can be seen from Table 2, LDSYMS-15 has the fastest mycelial growth rate compared with its parent strains (SY 15, MS16) and other monoascosporic strains, and has a significant difference compared with the MAT1-2 type monoascosporic strain LDSYMS-14 that cannot produce mushrooms alone.

[0060] Comparative Example 2 Determination of Stability of Fruiting Capacity of Cordyceps militaris Strain LDSYMS-15

[0061] Under sterile conditions, take a 1 cm × 1 cm bacterial block of the strain LDSYMS-15PDA, place it in a 50 ml conical flask containing 10 ml sterile water, shake it gently for 2-3 min, filter it with two layers of sterile filter paper to obtain an asexual spore suspension, and then filter it for 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5After five gradient dilutions, 0.1 ml of spore suspension was evenly spread on PDA medium and cultured at 25 ° C in the dark. Asexual spores of Cordyceps militaris are easy to germinate on PDA medium. After signs of spore germination are observed, a single colony is picked after visible colonies appear on the medium, and it is inoculated on PDA medium and cultured at 25 ° C in the dark. The Cordyceps militaris strain LDSYMS-15 was cultured in liquid, and the liquid strain was inoculated on oat culture medium for fruiting test to identify the stability of the strain's fruiting ability. As shown in Table 3, the strain LDSYMS-15 has stable fruiting ability and high fruiting body yield, and the dry weight of fruiting bodies per bottle reaches more than 7.5g. The results in Table 4 show that 10 strains were randomly selected from the monoconidial strains isolated from the strain LDSYMS-15 for fruiting test, all of which were able to form fruiting bodies, and the yield was more than 6.5g / bottle, and the highest could reach 8.54g / bottle.

[0062] Table 3. Fruiting body yield of Cordyceps militaris strain LDSYMS-15 in three fruiting trials

[0063]

[0064] Table 4. Fruiting body yield of the monoconidial strain of Cordyceps militaris strain LDSYMS-15 in the fruiting test

[0065]

[0066] Test example

[0067] 1. Cultivation and fruiting verification of the test strain and identification of mating type

[0068] like Figure 2 The five tested strains of Cordyceps militaris all have stable fruiting ability, and the fruiting body morphology is obviously different ( Figure 2 A), combined with mating type identification ( Figure 2 B), the fruiting bodies of strains D1 and D2 have smooth surfaces, no ascocarp, and only contain the MAT1-1 gene, which are single-mating fruiting strains; the fruiting bodies of strains S1, S2, and S3 have abundant ascocarp on their surfaces, contain the MAT1-1 and MAT1-2 genes, and are double-mating fruiting strains.

[0069] 2. Isolation of single mating type strains and identification of mating type

[0070] Single conidia of the tested strains were isolated and mating type identification was performed, as shown in Table 5. D1 and D2 each had 48 single mating type strains, all of which were MAT1-1 type; 21 MAT1-1 type strains and 29 MAT1-2 type strains were obtained from strain S1; the number was similar to that of MAT1-1 type strains and MAT1-2 type strains obtained from S3, while the number of MAT1-1 type strains isolated from S2 was relatively small, only 10, and the number of MAT1-2 type strains was relatively large, 55.

[0071] Table 5 Number of single mating type strains of the tested strains

[0072]

[0073] 3. Determination of hybrid fertility of single mating type fruiting strains

[0074] In the double-mating type fruiting strains S1, S2, and S3, the fruiting bodies of the different single-mating type strains can form ascocarps and ascospores, such as Figure 3 Among them, the MAT1-1 type fruiting strain isolated from D1 and D2 was hybridized with the MAT1-2 type strain isolated from S1, S2, and S3, and the fruiting bodies produced ascocarps and ascospores, which could also complete sexual reproduction, indicating that the single mating type strains D1 and D2 have hybrid fertility.

[0075] 3.1 Effects of single-mating strain pairing on agronomic traits of double-mating strains

[0076] The fruiting bodies produced by hybridization of single mating type strains will have significantly different agronomic traits compared with the fruiting bodies of their parental strains. Different single mating type strains from the same test strain will also produce different fruiting body morphology and yield when mated. Figure 6 As shown in the combination S3-34×S3-1 and S3-32×S3-4, the paired strains were all isolated from S3, and the fruiting bodies produced were all medium yellow in color, but the fruiting bodies of S3-32×S3-4 were more slender, with a bioconversion rate as high as (251.10±7.79)%, and an average dry weight of (8.02±0.27) g per bottle, which were 2.5 times and 2.7 times that of the fruiting bodies of S3-34×S3-1, respectively. This indicates that a certain degree of genetic variation will occur during the production of conidia, and agronomic traits will change accordingly. This suggests that on the one hand, the conidia separation and pairing method can be used for production breeding, and on the other hand, the conidia separation and pairing method can also be used to prevent strain degeneration for strain rejuvenation.

[0077] The fruiting bodies of the single mating type strain S2 were short and stout, but after being crossed with the single mating type strains S1 and S3, the fruiting bodies increased in length and their colors also changed. The fruiting bodies of the combination S1-9×S2-25 were light yellow, those of S1-48×S2-3 were medium yellow, those of S2-37×S1-4 were medium yellow, and those of S2-42×S1-16 were orange-yellow. In terms of yield, the fruiting body yield of self-mating pairs of strains S1 and S3 was significantly higher than that of strain S2, among which the fruiting body bioconversion rate of the combination S1-9×S1-4 was 3.8 times that of S2-37×S2-25. However, through hybridization, the fruiting body yield of the hybridized pairing combination of S2 was significantly improved, the bioconversion rate of the combination S1-48×S2-3 was as high as (199.17±6.62)%, the dry weight was (6.60±0.49)g per bottle, and the bioconversion rate of the combination S1-9×S2-25 was (178.27±2.69)%, and the dry weight was (5.55±0.11)g per bottle. The fruiting body of strain S1 had excellent traits, and the fruiting body bioconversion rate produced by hybridization with other single mating type strains was not less than 100%, and the dry weight was not less than 3g per bottle. Therefore, it is an effective means of production breeding to select excellent parent strains, obtain their single mating type strains through isolation and purification, and then perform heterozygous pairing and compare agronomic traits to obtain excellent pairing combinations.

[0078] Table 6 Fruiting body yield of single mating type strains S1, S2 and S3

[0079]

[0080] This study is based on the identification of mating type genes, and studies the pairing and hybridization characteristics of single-mating fruiting strains and double-mating fruiting strains of Cordyceps militaris. When the single-mating fruiting strain is paired with the double-mating strain MAT1-2, fruiting bodies with ascocarps and ascospores can be obtained, showing its hybrid fertility, but the single-mating fruiting strain itself will also produce fruiting bodies. Therefore, in actual production, for single-mating fruiting strains, pairing can also increase the yield, but the uniformity of the fruiting bodies after pairing must be taken into account, and it is necessary to screen suitable pairing strains to increase the yield, ensure uniformity, and ensure its commercial performance.

[0081] In previous production and literature reports, there was only the MAT1-1 type fruiting strain. The present invention separated and obtained the monoascospore strains of the hybrid pairing combination D1-10×S1-16 and D2-25×S1-16, and conducted a separate fruiting test, not only obtaining the MAT1-1 type new fruiting strain, but also obtaining the MAT1-2 type fruiting strain for the first time, whose fruiting body yield and quality are better than the parent strain, with good fruiting effect and excellent commercial properties. The MAT1-2 type fruiting strain can not only be used for large-scale production, but also provide valuable materials for the research on the genetic breeding of Cordyceps militaris.

[0082] It should be noted that, in this article, the terms "include", "comprises" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device. Specific examples are used in this article to illustrate the principles and implementation methods of the present invention. The above examples are only used to help understand the method of the present invention and its core idea. The above is only a preferred implementation method of the present invention. It should be pointed out that due to the limitations of textual expression, there are objectively infinite specific structures. For ordinary technicians in this technical field, without departing from the principle of the present invention, several improvements, modifications or changes can be made, and the above technical features can be combined in an appropriate manner; these improvements, modifications, changes or combinations, or the direct application of the concept and technical solution of the invention to other occasions without improvement, should be regarded as the scope of protection of the present invention.

Claims

1. A high-yield single-mating Cordyceps militaris strain and a cultivation method thereof, characterized in that: The strain is a single mating type mushroom-producing type (MAT1-2) Cordyceps militaris high-yield strain, the deposit number of which is CGMCC No.41163, and the strain does not contain the MAT1-1 gene. The cultivation method of the strain is to pair and hybridize a strain with a mating type of MAT1-1, which can produce mushrooms alone and does not produce ascocarps, and a MAT1-2 strain that cannot produce mushrooms alone, to obtain a fruiting body with ascocarps and ascospores, to collect the ascospores from the hybrid fruiting body by a hook hanging method, to spread and dilute the ascospores, to select the single ascospore strain for cultivation, and to identify the mating type by using molecular markers, to obtain the MAT1-2 Cordyceps militaris strain LDSYMS-15 that can produce mushrooms alone; the method specifically comprises the following steps: 1) Starting strain: The starting strain is a single-mating fruiting strain with a smooth fruiting body surface and no ascocarp, containing only the MAT1-1 gene, and a double-mating fruiting strain with abundant ascocarp on the fruiting body surface and containing both the MAT1-1 and MAT1-2 genes. 2) Isolation of single mating type strains: From the plate culture or slant culture of the starting strain, take 1cm×1cm of the mycelial culture of the test strain and gently shake it in 7-10ml sterile water for 1-3min to separate the single conidia of the test strain. Collect the suspension containing conidia after filtering with multiple layers of sterilized lens paper. After gradient dilution of 10-1, 10-2, 10-3, 10-4, and 10-5, draw 0.1-0.4ml of spore liquid and evenly spread it on PDA medium. Culture it at 15-25℃ in the dark for 3-5d. After visible colonies appear on the culture medium, pick a single colony and inoculate it on PDA medium. Culture it at 15-25℃ in the dark for 12-18d. 3) Mating type identification of single mating type strains: After the single mating type strains were isolated and cultured, two parent strains were obtained and mating type identification was performed. The two parent strains were the LDSY02 single conidia strain with a mating type of MAT1-1 that could produce mushrooms and the LDMS05 single conidia strain with a mating type of MAT1-2 that could not produce mushrooms alone. 4) Pairing and fruiting of single mating type strains: Take two 9mm diameter blocks of LDSY02-25 and LDMS05-16, inoculate them into a triangular flask filled with liquid culture medium, and culture them in the dark at 17-21℃ for 2-4 days to obtain liquid strains; Take 1.5-2.5ml of LDSY02-25 and LDMS05-16 liquid strains, mix and inoculate them into the same bottle of oat culture medium, place them in a fruiting room at 18-25℃, and culture them in the dark. When the hyphae cover the culture medium and the hyphae at the bottom of the bottle exceeds two-thirds, perform a scratching treatment, and then transfer to 18-22℃, 80-90% humidity, and culture them in light for 40-50 days, the fruiting body matures and monoascospores are formed. 5) Isolation and culture of single ascospore strain: Take mature Cordyceps militaris fruiting bodies, wrap the base of the fruiting bodies tightly with sterilized cotton thread, hang upside down in a 50ml triangular flask filled with 5-10ml sterile water, and place it in a 15-25℃ incubator for 8-12h to collect ascospore suspension, draw 0.05-0.1ml spore liquid and evenly spread it on PDA medium, culture it in the dark at 15-25℃ for 1-3d, pick out a single colony, inoculate it on PDA medium, and culture it in the dark at 15-25℃ for 8-12d to obtain MAT1-2 type Cordyceps militaris strain LDSYMS-15; 6) Identification of mating type of single colony and fruiting experiment verification, the fruiting experiment verification method is to weigh the fresh weight of the fruiting body and calculate its bioconversion rate, the calculation formula of the bioconversion rate (V, %) is: V = (G1 / G2) × 100% where: G1 is the fresh weight of Cordyceps militaris fruiting body, G2 is the dry weight of the culture medium, and the dry weight is recorded after the fruiting body is placed in an oven at 50°C for drying.

2. A high-yield single-mating type Cordyceps militaris strain and a cultivation method thereof according to claim 1, characterized in that: The mating type identification in the steps 3 and 6 adopts a novel plant genomic DNA extraction kit to obtain genomic DNA from the mycelium and adopt a PCR reaction system (20 μL): Mix 10 μL, 0.5 μL of each forward and reverse primer, 1 μL of template DNA, 8 μL of ddH2O, and the PCR reaction conditions are: 94°C pre-denaturation for 5 min, 94°C denaturation for 30 s, 56°C annealing for 30 s, 72°C extension for 30 s, for a total of 30 cycles; 72°C fill-in for 10 min.

3. A high-yield single-mating type Cordyceps militaris strain and a cultivation method thereof according to claim 1, characterized in that: The mating type identification method of the single colony in step 6 is as follows: a high-yield strain LDSYMS-15 is screened from the obtained single colony, the genomic DNA of LDSYMS-15 is extracted, and specific primers are designed according to the sequences of the Cordyceps militaris mating type genes MAT1-1 (no.AB194982.1) and MAT1-2 (no.AB084257.1) in the NCBI database, MAT1-1F: 5'-gagcctactatggaaccc-3'; MAT1-1R: 5'-caggactgataccagcaaa-3'; MAT1-2F: 5'-gcatcaacccatttgtgaaagttct-3'; MAT1-2R: 5'-cctgtcataatggtgctgt-3', and PCR amplification is performed, and the amplified fragments: MAT1-1 is 427 bp, and MAT1-2 is 719 bp.

4. A high-yield single-mating type Cordyceps militaris strain and a cultivation method thereof according to claim 1, characterized in that: The preparation method of the PDA culture medium is as follows: 200g of potato is cut into pieces and boiled in 1000ml of water for 10 minutes, 20g of glucose and 20g of agar are dissolved in the potato filtrate, the volume is fixed to 1000ml, the pH is natural, and the mixture is dispensed into 500ml conical bottles.

5. A high-yield single-mating type Cordyceps militaris strain and a cultivation method thereof according to claim 1, characterized in that: The preparation method of the Cordyceps militaris liquid culture medium is as follows: 20g of glucose, 15g of peptone, 5g of yeast powder, 1g of magnesium sulfate, 2g of potassium dihydrogen phosphate, VB10.1g, and natural pH; the prepared culture medium is divided into 500ml conical bottles, each bottle is filled with 150ml, sealed with a special sealing film for edible fungi, sterilized at 121°C for 30min, and naturally cooled to room temperature; The oat culture medium preparation method is as follows: 30g of oats and 50g of water are placed in a culture bottle, covered, and completely sterilized in an autoclave at 121°C for 30min, and then naturally cooled to room temperature.

6. A high-yield single-mating type Cordyceps militaris strain and a cultivation method thereof according to claim 1, characterized in that: The steps of preparing the liquid strain of the strain in step 4 are as follows: preparing a liquid culture medium, filling the prepared liquid culture medium into 500 ml conical bottles, each bottle containing 100-150 ml, sealing the bottles with a special sealing film for edible fungi, sterilizing at 100-121° C. for 20-30 minutes, and naturally cooling to room temperature; inoculating 2-4 pieces of activated Cordyceps militaris strains with a diameter of 9 mm and stored on a PDA slant culture medium into each bottle under sterile conditions, and sealing the bottles; placing the bottles on a rotary shaker, culturing them for 2-4 days at a rotation speed of 140 r / min and a temperature of 17-21° C., and preparing the Cordyceps militaris liquid strains for standby use.

7. A high-yield single-mating type Cordyceps militaris strain and a cultivation method thereof according to claim 1, characterized in that: The isolation and cultivation of the monoascospore strain in step 5 is as follows: inoculating the Cordyceps militaris liquid seed onto a sterilized oat culture medium under a sterile environment, placing the culture chamber at 20°C-25°C for dark cultivation, and after the mycelium covers the bottom of the culture bottle and the culture bottle is scratched, transferring the culture chamber to a light environment at a temperature of 15-18°C and a humidity of 80-90% for cultivation until the fruiting bodies are mature and then harvested.

8. A high-yield single-mating type Cordyceps militaris strain and a cultivation method thereof according to claim 1, characterized in that: The method for picking a single colony in steps 2 and 5 is as follows: hang a single mature fruiting body upside down in the middle of a sterile and dry 50 ml conical flask, put it in a 15-25° C. incubator to collect ascospores ejected from the fruiting body, carefully remove the hanging fruiting body after 8-12 hours, absorb 7-10 ml of sterile water and put it in a conical flask, gently shake it, collect ascospore liquid, and after five gradient dilutions of 10-1, 10-2, 10-3, 10-4, and 10-5, absorb 0.1 ml of spore suspension and evenly spread it on a PDA culture medium, culture it at 15-25° C. in the dark for 3-5 days, observe signs of spore germination, and after colonies visible to the naked eye appear on the culture medium, pick a single colony, inoculate it on a PDA culture medium, and culture it at 15-25° C. in the dark.

9. A high-yield single-mating type Cordyceps militaris strain and a cultivation method thereof according to claim 1, characterized in that: The mycelium of the test strain has stable mushroom-producing ability.

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